Biochemistry of malathion resistance in a strain of Cimex lectularius resistant to organophosphorus compounds

Bull. Org. mondSanteO 1971, 45, 795-804 Biochemistry of Malathion Resistance in a Strain of Cimex lectularius Resistant to Organophosphorus Compounds M. FEROZ 1 The penetration of 14C-labelled malathion was found to be similar in two bedbug strains, of which one was susceptible and the other moderately resistant to a range of organo- phosphorus insecticides. The latter strain was especially resistant to malathion and fenchlorphos, but was not resistant to organophosphates or (significantly) to organo- chlorine insecticides. There was no difference in acetylcholinesterase activity or sensitivity, or in total esterase activity (using 1- or 2-naphthyl acetates as substrates). In vitro experi- ments on malathion metabolism showed about 17 % greater degradation in the resistant strain, demethyl malathion and malathion diacid being the main products. As resistance to organochlorine compounds becomes more and more widespread, organophos- phorus pesticides are being more widely used against arthropod pests; but it is evident that arthropods can develop resistance to these chemicals as well. Biochemical research on various arthropods resistant to organophosphorus compounds has indicated that they develop resistance by several mechanisms. For instance, compounds with carboxyester linkages may be broken down by carboxylesterase(s), as in some strains of Musca domestica (Matsumura & Hogen- dijk, 1964), Culex tarsalis (Matsumura & Brown), 1963), Nephotettix cincticeps (Kojima, Ishizuka & Kitakata, 1963), Tribolium castaneum (Dyte & Rowlands, 1968), and Chrysomya putoria (Towns- end & Busvine, 1969). The P-S-C and P-O-C bonds in the molecule are also vulnerable to hydrolytic attack and several insect and acarine strains resistant to organophosphorus compounds have been shown to possess these detoxification mechanisms (Stone & Brown, 1969; Roulston et al., 1969). Dealkylation is another route by which the organophosphorus molecule may be degraded-e.g. in Chilo suppressalis (Kojima et al., 1963). Apart from detoxification, certain strains of Tetranychus urticae and Boophilus microplus have developed insensitive cholinesterase as a defence mechanism against organophosphorus chemicals (Smissaert, 1964; Lee & Batham, 1966). Finally, a rather nonspecific resistance mechanism I London School of Hygiene and Tropical Medicine, Keppel Street, London, England. is a reduction in penetration of pesticides, which has been demonstrated in a number of strains of Musca domestica (Plapp & Hoyer, 1968; Sawicki & Farn- ham, 1969), one strain of Aedes aegypti (Matsumura & Brown, 1961), and one strain of Culex pipiens fatigans (Stone & Brown, 1969). This paper reports biochemical investigations on a strain of Cimex lectularitus resistant to organophos- phorus compounds. MATERIALS AND METHODS Insect strains Two strains of bedbugs, one susceptible to and one resistant to organophosphorus compounds, were used. Their origin and the rearing technique used has been described by Feroz (1969, 1970). The sus- ceptible strain came from a laboratory colony at the Liverpool School of Tropical Medicine, while the resistant strain originated in Israel, following field use of malathion and diazinon. It showed resistance to phosphorothioates (P = S compounds) only and not to phosphates (P=0 compounds), carbamates, or organochlorine compounds. The strain was about x 10 resistant to malathion, a level that did not change in the absence of selection pressure, during the course of this work. Chemicals The common compounds and solvents, mentioned in this paper, were usually of analytical grade. The radioactive malathion (both carbon atoms in the 2771 795 - M. FEROZ succinyl part of the molecule were labelled; specific activity 4.6 mc/mmol) employed in the studies of penetration and of in vitro metabolism was a gift from the World Health Organization. The acetyl- choline iodide used for acetylcholinesterase (AChE) assay was bought from British Drug Houses (BDH). A small sample of pure malaoxon for testing AChE sensitivity was kindly provided by Mr D. G. Row- lands, Pest Infestation Laboratories, Slough, Eng- land. Normal carboxylesterase levels in the two strains were measured by using 1- and 2-naphthyl acetates bought from BDH. Chromatographically homogeneous samples of malathion, malathion monoacid, malathion diacid, and demethyl malathion were presented by the American Cyanamid Company. Diethyl malate was bought from Kodak Ltd., London, and diethyl mercaptosuccinate was synthe- sized in the laboratory. Malic acid and mercepto- succinic acid were made available by the courtesy of Mr G. D. Rowlands. These chemicals were used as reference compounds in studies of the metabolism of 14C-malathion. Penetration of '4C-malathion Adult bedbugs were used in these studies. Indi- vidual bugs were tethered with a nylon thread round the thorax, the end of the thread being secured with adhesive plaster to a piece of cork standing vertically on a cardboard base. Known amounts of radioactive malathion were deposited on the abdominal tergites of batches of 10 bedbugs suspended as described. At predeter- mined intervals after treatment, the tethering threads were cut and the bugs were collected in test-tubes. Immediately, 1.0 ml of hexane was added to the tube and this was shaken gently for 30 seconds. The solvent was pipetted into a scintillation counting vial and the process was repeated with two further lots of solvent. The hexane was evaporated, scintil- lation fluid was added, and the radioactivity was determined as described below to give a measure of the amount of insecticide remaining on the outside of the body of the bedbug. The difference between the amount of insecticide applied and the amount recovered was considered to be the amount that had penetrated into the body of the bedbug. Care was taken that only clean bugs were used. Excrement was sometimes found adhering to the bodies of the bedbugs, especially those from old and crowded cultures, and this could have affected the penetration of insecticide. Esterase assay Enzyme preparation. Suitable bugs were selected and were chilled at -10°C for 30 min; then they were homogenized in 0.067N phosphate buffer (pH 7.2 for AChE assay and 7.0 for other hydrolases) using an all-glass Potter-Elvehjem type homogenizer. The plunger of the homogenizer was driven by an electric motor at 1 000 rev/min and the homogenizer tube was kept immersed in ice-cold water. The total run- ning time of the homogenizer was 1.5 min but the motor was stopped for 1 min after every 30 seconds to allow the homogenate to cool down, so that the heat generated during the process would not denature the proteins. To estimate the hydrolase levels, equal numbers of each sex were always used; however, in some assays with individual bugs, the levels for the two sexes were noted separately. The insects destined for homogenization were isolated as fifth-instar nymphs and used when six- to seven-day-old adults. To minimize difficulties caused by blood in their gut, the bugs were not blood fed during the post-emergence period. AChE activity and senisitivity determinationis. The colorimetric method described by Hestrin (1949) and later improved by Robbins, Hopkins & Roth (1958) was used to estimate the AChE activity in the two strains of bedbugs. Different homogenate concen- trations in 0.5 ml of 0.067M phosphate buffer, pH 7.2, were placed in test-tubes, and 0.25 ml of phosphate buffer of the same concentration and pH was added. To each tube was then added 0.25 ml of 8 x 10-3M acetylcholine iodide in 0.067M phosphate buffer, pH 7.2; thus, the final concentration of the substrate was 2 x 10-3M. The tube was incubated in a metabolic water-bath at 30°C for 30 min. Immediately after incubation, 2.0 ml of a freshly prepared 1: 1 mixture of 3.5M sodium hydroxide and 2.OM hydroxyl- ammonium chloride (both in distilled water) were added by pipette. The reactants in the tube were vigorously swirled and about 3 min later, 1.0 ml of hydrochloric acid solution (prepared by mixing one volume of hydrochloric acid, sp. gr. 1.18, with two volumes of distilled water) was added. On adding 1.0 ml of 0.37M iron(III) chloride solution in 0.1N hydrochloric acid, a brown complex of acetylcholine with hydroxylammonium and iron(III) ions appeared. The contents of the tube were filtered through a Whatman No. 1 filter paper and the optical density of the complex was measured at 500 nm on a spectropho- tometer (Hilger & Watts, London) using 10-12-cm- 796 BIOCHEMISTRY OF MALATHION RESISTANCE IN CIMEX LECTULARIUS wide cells, about 45 min after the addition of iron(III) chloride. The AChE activity was estimated from the difference between the initial amount of acetylcholine and that remaining at the end of the reaction. Controls were run with each set of experi- ments. The procedure used for testing AChE sensitivity was the same as described above, but 0.25 ml of phosphate buffer was replaced by different concen- trations of malaoxon in 0.25 ml of phosphate buffer containing 2% acetone. Malaoxon solutions were freshly prepared every morning. Estimation of esterase activity. Cholinesterase and carboxylesterase activities in the susceptible and resistant bedbugs were determined by the method described by van Asperen (1962). This technique in- volves the hydrolysis of naphthyl acetates to naph- thols, which, after coupling with a chromogen, can be quantitatively measured. Five ml of 1.2 x 10-4M solution of 1- or 2-naphthyl acetate in 0.067 M phosphate buffer, pH 7.0, contain- ing 1.00% acetone, were placed in a Pyrex test-tube and homogenate was added. The total volume of the reactants in the tube was always made up to 6.0 ml with phosphate buffer so that the concentration of the substrate was 1 x 10-4M. The tube was shaken in a water-bath at 27°C, 3 min being allowed for equilibration. After agitation for 30 min, 1.0 ml of aqueous Fast Blue B plus sodium monodecyl (lauryl) sulfate was added. Coloured complexes formed with the naphthols present; with 1-naphthol, a red colour that turned blue and with 2-naphthol a more perma- nent red. After centrifugation for 5 min to remove debris, the optical densities were measured spectro- photometrically at wavelengths of 600 nm for blue and 540 nm for red. Preliminary investigations had indicated that cholinesterase activity on 1- and 2-naphthyl acetates could be suppressed with I x 10-5M physostigmine sulfate; therefore this was added when esterases other than cholinesterase were to be studied. The in vitro metabolism of 14C-malathion Incubation and extraction of metabolites. Small quantities of 14C-malathion in 2-butanone (ethyl methyl ketone) were placed in test-tubes; the solvent was evaporated and 0.1 ml of 100% ethanol was added. The homogenates, which had been prepared in the same way as those for investigating esterase activity, were then introduced. One ml of homo- genate, containing 30 mg of bedbug material (about 4 bugs) was added to each tube and the tubes were incubated at 28°C (shaken in a water-bath and open to the air). The reaction was terminated after 1.5 h by the addition of 0.3 ml of 5% (w/v) trichloroacetic acid (TCA) and the tube was put into a refrigerator at 4°C to allow precipitation of the proteins. The pH was then adjusted to 7.5 with 0.5 N NaOH and 2.0 ml of chloroform were added and the tube was shaken well. The contents of the tube were centrifuged at 3 000 rev/min for 5 min and both phases (i.e., chloroform and water) were removed. The protein precipitate was again extracted with 2.0 ml of 0.067 M phosphate buffer (pH 7.2) and 2.0 ml of chloroform, and the fractions were combined. The precipitate, after it had been washed with 1.0 ml each of methanol and acetone, was discarded. The methanol and acetone were evaporated under reduced pressure and the residue was taken up into chloroform and water fractions. After centrifugation, the chloroform phase was removed to form Fraction I. The pH of the water layer was adjusted to 1.5 or below with SN HC1 and extracted twice with 4.0-ml portions of chloroform and of diethyl ether to give Fraction II, leaving the final water phase (Fraction III) behind. At the start of the work, a different extraction pro- cedure was used, but this did not give good recovery of radioactivity. The difference was that after the termination of the enzymic reaction the homogenate was not allowed to stand with TCA and perhaps any malathion adsorbed on to protein material was not stripped off. Analysis offractions. The solvent present in each fraction was evaporated under reduced pressure and the residues were further analysed by thin-layer chromatography or thin-layer electrophoresis. Stan- dards were run on each chromatogram or electro- pherogram. Only the areas of known compounds were sprayed with colour reagents; portions corre- sponding to the standard compounds were scraped into scintillation counting vials and their radioactivity was determined. Fraction I contained malathion, malaoxon, diethyl malate, and diethyl mercaptosuccinate, which were separated on Silica Gel G with the solvent system (hexane, ethyl acetate, and benzene in 4: 2: 1 ratio, v/v) described by Matsumura & Voss (1964). The plates were activated for 1 hour at 110°C and were pre-run for 3 hours before the samples were spotted on to them. Development of the chromatograms took 18-20 min. After that, the solvent front was marked, the plate was taken out of the tank, and the solvent was evaporated with a hair dryer. 797 M. FEROZ Diethyl malate was located by spraying a fresh 1: 1 mixture of 1.0% (w/v) potassium permanganate and 2.0% (w/v) sodium carbonate in water (Peerebroom, 1960). Thirty minutes after spraying, a yellow spot appeared on a dull yellowish brown background. The spot eventually turned white. Diethyl mercaptosuccinate, malathion, and mala- oxon were detected by spraying 0.5% brilliant green (C.I. 42040) in acetone, followed by exposure to bromine vapour (Abbott, Crosley & Thomson, 1965). The compounds gave stable, bright orange spots on a light-green background. Fraction II contained malathion monoacid, mala- thion diacid, and demethyl malathion, which had hitherto been extremely difficult to separate by thin- layer chromatography using different solvent systems; these have now been successfully and clearly separ- ated by using the technique of thin-layer electro- phoresis. The apparatus and methods used were essentially similar to those of Ritschard (1968). A powerpack built in our laboratories with an output of 0-2 000 V and 0-200 mA with voltage stabilization was used. The test sample, along with known standards, was spotted on to the commercially available precoated silica gel sheet (Eastman Chromagram sheet 6060) in the anodic region (as the compounds in question are anionic) and the sheet and paper wicks were sprayed with 0.05M citrate buffer, pH 3.6. The same buffer was poured into the electrode compartment and the electrophoresis was carried out for 50 min at 980 V and 20 mA. The sheet was then removed and dried in a stream ofhot air. The compounds were made visible as greyish spots with brilliant green and bromine treatment as described above. Malathion diacid was found to be the fastest moving spot and malathion monoacid was the slow- est: demethyl malathion was intermediate. The spots were well separated and could be cut off and put straight into the scintillation vials. Fraction III, containing malic acid and mercapto- succinic acid, was analysed on thin layers of cellulose MN 300G, using propanol, eucalyptol, formic acid, and water (10: 10: 4: 1, v/v) as the mobile phase. Before application of the samples, the plate was activated for 10 min and was then pre-run for 24 hours. When spotting was completed, the plate was introduced into the tank containing the solvent mixture. Development of the chromatogram took 21/2-3 hours. The solvent front was marked and the plate was taken out and dried in a stream of air. The acids were located with alkaline potassium per- manganate. Malic acid gave a light-pink spot that turned white, while mercaptosuccinic acid gave a quickly-fading yellowish pink colour. Assay of radioactivity All radioactivity determinations were carried out using a Packard Tricarb Scintillation Spectrometer (model 3314), operated at 10% gain and 100-1 000 channel width. These settings gave 75% counting efficiency with 14C. Samples were prepared in 5.0 ml of 0.5% (w/v) butyl-PBD [2-(4-biphenylyl)-5-(p-tert-butylphenyl)- 1,3,4-oxadiazole] in toluene. For polar compounds containing 14C (malathion diacid, malathion mono- acid, demethyl malathion, malic acid, and mercapto- succinic acid) 0.5 ml of anhydrous methanol was added prior to the addition of the scintillation fluid. Quenching was checked with an internal standard (14C-hexadecane) and an external standard (Radium- 226). The background never exceeded 10-20 counts/ min. Corrections were made whenever necessary. RESULTS AND DISCUSSION Penetration of "4C-malathion Penetration of "4C-malathion into the adult bed- bugs was studied at two dose levels-0.044 ,ug/bug and 0.088 ,tg/bug-applied in 0.22-tlitre droplets of 2-butanone. The former dose level was about LD,0 for the susceptible strain and the experiments were replicated twice with this dose. The latter dose was approximately the LD50 for susceptible bedbugs and three replicates were performed at this rate of appli- cation. The percentage recoveries (average values) of radioactive malathion at various intervals after treat- ment are set out in Table 1. Penetration of 14C- malathion applied at the rate of 0.088 ,ug/bug could not be studied beyond 6 hours, because some sus- ceptible bugs showed severe signs of poisoning. A comparison of the values after equal intervals shows that the two strains did not differ in penetration of malathion. Plots of log percentage recovery of 14C-malathion against time were linear, suggesting that the amount penetrating at any time was proportional to that present on the surface. Times for 50% penetration calculated from these graphs were: at 0.044 ,ug/bug, 75 min (susceptible) and 78 min (resistant); at 0.088 ,ug/bug, 116 min (susceptible) and 1 16 min (resistant). Decreased penetration has been shown to be a cause of resistance in some resistant strains of the 798 BIOCHEMISTRY OF MALATHION RESISTANCE IN CIMEX LECTULARIUS Table 1. Percentage recoveries of 14C-malathion from susceptible and resistant strains of bedbug Rate of application (hours) 0.044 jig/bedbug 0.088 ,ug/bedbug Susceptible Resistant Susceptible Resistant 0 97.7 96.8 97.1 96.3 1 56.3 52.3 68.7 73.4 2 32.7 36.5 54.0 49.1 4 13.9 16.1 21.2 20.6 6 - 15.1 14.9 8 4.5 7.7 16 trace 2.1 _ 24 trace trace housefly and in a few culicine mosquito strains (Sawicki & Farnham, 1969); Matsumura & Brown, 1961; Stone & Brown, 1969). The mechanism itself provides protection of low order (probably by delay- ing the toxic action of insecticides) but can augment the effect of other resistance mechanisms. Moreover, its action is not necessarily restricted to one com- pound or a class of compounds, but may also extend to other insecticides. There is no evidence for such a mechanism in the resistant bedbugs studied. AChE activity and sensitivity AChE activity in the homogenates of two strains of Cimex is shown in Fig. 1. The relationship be- tween AChE activity and homogenate concentration in the susceptible strain was linear, up to the con- centration of 6 bugs/tube. The fall-off in activity above 6 bugs/tube may have been due to depletion of the substrate. On the other hand, resistant strains did not show such a relationship. Also it can be seen from Fig. 1 that susceptible bedbugs were more active in hydrolysing acetylcholine, especially athigh enzyme concentrations. Thus, the resistant strain, at 6 bugs/ tube, hydrolysed only 64 %Y as much acetylcholine as the susceptible strain under similar conditions. This anomaly may have been due to the presence of a natural inhibitor in the resistant strain, because at low concentrations of homogenate activity it was almost identical in the two strains: dilution of the inhibitor might have taken place in low concen- trations of homogenate of the resistant strain. The sensitivity of AChE to malaoxon was also 2.0 7 1.5 a) 2- I -C 1.0 U 0) E X- 0.5 S 1 2 3 4 5 6 7 8 Bugs/tube Fig. 1. Acetylcholinesterase activity in susceptible (S) and resistant (R) bedbugs. Conditions: substrate concentration, 2 x 10-3M; temperature, 30°C; pH 7.2; incubation time, 30 min. tested in the two strains and the results are shown in Fig. 2. It is clear that the AChE of the resistant strain was not less sensitive to malaoxon; it was, in fact, slightly more sensitive than that of the 100 F- 's 80 t 0 60 V -C c 40 20 0 I 1111 I I I1 I"I 6 5 4 pl (Malooxon) 3 Fig. 2. Sensitivity of acetylcholinesterase to malaoxon in the susceptible (S) and resistant (R) strains of bedbug. Conditions: homogenate concentration, 6 bugs per tube. All other conditions as in Fig. 1. 799 M. FEROZ susceptible strain. From the lines in Fig. 2, I5 values of 3.8 x 10-5M and 6.5 x 10-5M malaoxon for the AChE of the resistant and susceptible bed- bugs, respectively, can be calculated. These values are relatively high and two of the several possible reasons for this are that (1) the malaoxon was added to the homogenate at almost the same time as the acetylcholine, and this could have resulted in competition between the two substrates; or that (2) adsorption of malaoxon on one or more unspecific component of the homogenate was responsible, since whole homogenates were employed. Nevertheless, it appears that decreased sensitivity of AChE is not a cause of resistance in the resistant strain of bedbugs. Toxicological work also supports this finding, since the strain was not resistant to any of the organophosphorus compounds or carbamates tested. In the event of the occurrence of insensitive AChE, some resistance to these compounds would be expected. Comparison of esterase activity in the two strains Esterase activities of the two strains of bedbug on 1-naphthyl acetate (Fig. 3) and 2-naphthyl acetate (Fig. 4) were compared. The susceptible and resis- tant bedbugs did not differ from each other in hydro- lysis of either of the substrates. In this respect, the 0.7 4 0. 0 0- *0 0.5 0.3 0.1 0 I& 0 TE 0.083 0.167 Bug/tube , RE 0.25 Fig. 3. Comparison of esterase activity on 1 -naphthy acetate in resistant (solid circles and squares) and susceptible (open circles and squares) strains. TE = total esterase activity, RE = residual esterase activity, i.e., the activity after the addition of 1 x 1 -5M physostigmine sulfate. Conditions: 1 -naphthyl acetate 1 X 10-4M; temperature, 27°C; pH 7.0; incubation time, 30 min.; final volume of reactants in the tube = 7.0 ml (1.5 O.D. equivalent to 0.2 1tmol of 1 -naphthol). 0.9 G) c]0 -a .2_ 0.0 0.6 0.3 0 RE 0.1 Bug/tube Fig. 4. Comparison of esterase activities to 2-naphthyl acetate in resistant (solid circles and squares) and susceptible (open circles and squares) strains. Con- ditions: 2-naphthyl acetate 1 x 10-4M. All other conditions and abbreviations as in Fig. 3 (1.04 O.D. equivalent to 0.2 ,umol of 2-naphthol). Israel strain of bedbugs resembles the Fresno strain of Culex tarsalis (Matsumura & Brown, 1963), the malathion-tolerant Penang strain of Aedes aegypti (Matsumura & Brown, 1961) and the parathion- resistant Chilo suppressalis (Kojima et al., 1963); but differs from other arthropod strains resistant to organophosphorus compounds in which esterase levels are changed with the advent of resistance. Reduced esterase activity has been demonstrated in several strains of Musca domestica resistant to organophosphorus compounds (van Asperen & Oppenoorth, 1959), the CM strain of Chrysomya putoria (Townsend & Busvine, 1969) and the Lever- kusen-R strain of Tetranychus urticae (Smissaert, 1965). On the other hand, strains of Nephotettix cincticeps, Laodelphax striatellats, and Culex pipiens fatigans resistant to organophosphorus compounds show increased esterase levels (Kojima, Ishizuka & Kitakata, 1963; Ozaki, 1969; Stone & Brown, 1969). As regards substrate specificity, total bedbug esterase was more active on 1-naphthyl acetate than on 2-naphthyl acetate: a situation similar to that found in Musca (van Asperen, 1962). But residual esterases in the presence of physostigmine (pre- sumably carboxylesterase, because the activity was inhibitable by low concentrations of malaoxon) 800 BIOCHEMISTRY OF MALATHION RESISTANCE IN CIMEX LECTULARIUS Table 2. Esterase levels in individual bedbugs of the susceptible and resistant strains Susceptible Resistant SPeCimen Females Males Females Males TEa REb TEa REb TEa REb TEa REb 1 73 43 48 29 61 35 47 29 2 56 30 49 31 63 36 49 30 3 68 37 64 41 63 35 50 29 4 95 58 51 34 66 38 53 30 5 60 32 55 32 68 38 54 32 6 75 43 60 36 71 40 54 33 7 65 35 57 34 76 42 58 35 8 84 53 75 45 81 48 61 36 9 64 37 55 32 95 49 67 39 1 0 67 38 69 36 100 68 78 46 avera9el 71 ] 41 58 35 74 43 57 34 a TE total esterase activity (O.D. x 100). b RE residual esterase activity, i.e., activity after the addition of 1 x 10-5M physostigmine sulfate(OD. x 100). hydrolysed greater quantities of 2- than of 1-naph- thyl acetate, in contrast to the opposite specificity shown by housefly carboxylesterases. Determination of esterase levels in individual bedbugs The extent of the variation in esterase levels was studied in individual bedbugs of the susceptible and resistant strains (either sex) using 1-naphthyl acetate as a substrate. The results are shown in Table 2. It has already been pointed out that there was no difference in the esterase levels of the resis- tant and susceptible strains. The results in Table 2 further confirm this conclusion. The males and females of one strain show almost the same degree of variation as those of the other. Although there was some overlapping between the activities shown by males and females (within each strain), on the average the males exhibited only about 60% of the esterase activity of the females, whether total or residual. Metabolism of'4C-malathion in vitro Table 3 shows the results of detailed analysis of various extracts from homogenates incubated with 2.0 ,ug of malathion. The total recoveries of radio- activity (62% and 59%, respectively, in susceptible and resistant strains) were very low, but since they were almost equal, a comparison of the two strains seems reasonable. It will be seen that although malathion can be broken down at several different linkages the quali- tative pattern of metabolites is similar for both strains. Intact malathion constituted a major portion with both strains, but extracts from the homogenate of the susceptible strain contained about 19% more of it. Malaoxon attained a higher level with the homogenate of the susceptible strain than with that of the resistant strain. In contrast, the hydrolytic products (demethyl malathion, malathion mono- acid, malathion diacid, and diethyl mercaptosucci- nate) were produced in greater quantities with the homogenate of the resistant strain. The amounts of diethyl malate produced by the homogenates of the two strains were about equal. Malic acid could be detected with both strains. Mercaptosuccinic acid was also produced but it could be quantified only with the homogenate of the resistant strain. Since the total recoveries in the above experiments were rather unsatisfactory, the extraction technique was improved. Table 4 shows the percentages of 8 801 M. FEROZ Table 3. Amounts of various metabolites (expressed as percentages of the total recovery) after incubation of 30 mg of homogenate (in 1 ml), from susceptible and resistant bedbugs, with 2,ug 14C-malathion Compound Susceptible Resistantstrain strain malathion 94.198 75.625 malaoxon 0.069 0.044 diethyl malate 0.037 0.039 diethyl mercaptosuccinate 3.440 8.328 malathion monoacid 0.011 3.058 malathion diacid 0.018 0.551 demethyl malathion 2.193 11.973 malic acid Trace Trace mercaptosuccinic acid Trace 0.019 unknowns and tails 0.034 0.363 total recovery 61.79 % 59.33 % Table 4. Amounts of various metabolites (expressed as percentages of the total recovery) after incubation of 30 mg of homogenate (in 1 ml), from susceptible and resistant bedbugs, with 0.8 tug 14C-malathion * Compound Susceptible Resistantstrain strain malathion 12.35 10.17 malaoxon 25.08 9.70 total toxic materials 37.43 19.87 malathion monoacid 0.09 0.17 malathion diacid 2.26 7.91 demethyl malathion 41.93 57.21 diethyl mercaptosuccinate 1.48 3.27 diethylmalate 13.06 8.77 mercaptosuccinic acid 1.35 0.51 malic acid 0.04 0.04 total hydrolytic products 60.22 77.88 unknowns 2.35 2.50 Total recoveries about 90 % i Average of two replicates. in each experiment. metabolites separated from the incubated mixture of 0.8 jug of malathion with homogenate, after the modi- fications. The results resembled those described above qualitatively, but the recoveries were greater (about 90% in each experiment). The amounts of malathion recovered from the homogenates of the two strains were nearly equal; but the homogenates of the susceptible bedbugs contained 18 % more malaoxon than those of the resistant strain (average of two replicates). Thus, on the whole, the toxic compounds (malathion plus malaoxon) were present in higher proportions in the homogenate of the sus- ceptible strain. The hydrolytic products, however, showed a different picture. Four metabolites (mala- thion monoacid, malathion diacid, demethyl mala- thion, and diethyl mercaptosuccinate) were produced in greater quantities in the homogenate of the resis- tant strain than in that of the susceptible strain. Malic acid was almost equal with both strains. Die- thyl malate and mercaptosuccinic acid were present in slightly greater amounts in the homogenate of the susceptible strain. When total hydrolysis with the homogenates of the two strains is compared, that of the resistant bugs is slightly greater; they contained about 17% more radioactivity. The malathion molecule has several bonds that are vulnerable to hydrolytic attack and consequently a variety of metabolites may be produced. Mala- oxon, itself a product of malathion, may undergo parallel degradation. The metabolism of malathion in bedbugs revealed a complex picture. Without studies on the time course of the appearance of various metabolites, it is difficult to decide whether they are the result of primary hydrolysis or secon- dary attack. Of the metabolites found in this work, demethyl malathion and malathion monoacid are the two compounds that seem to be produced by primary hydrolysis. In contrast, malic acid and mercaptosuccinic acid are certainly the consequence of secondary hydrolysis. If malathion diacid is also a secondary product (as it is in many insect species) from malathion monoacid, then the rate ofconversion ofthe latter into the former must be very rapid in bed- bugs, because malathion diacid was the predominant product ofcarboxyesteratic action. Diethyl malate and diethyl mercaptosuccinate may be produced as pri- mary metabolites by cleavage of the P-S-C bonds in malathion or malaoxon, or as secondary products by splitting of these bonds in demethyl malathion. Because the susceptible strain contained more malaoxon and the resistant strain more of the hydro- lytic products, it may be questioned whether resis- 802 BIOCHEMISTRY OF MALATHION RESISTANCE IN CIMEX LECTULARIUS tance is due to differential intoxication or to diffe- rential detoxification. The former possibility seems unlikely, however, because if this were the mecha- nism, resistance to all P=S compounds would be expected, and this is not found. The presence of greater amounts of malaoxon in the susceptible strain may be due to greater availability of malathion for conversion to its oxygen analogue. It is difficult to say whether a difference of 17% (x 1.3) between the two strains in the hydrolysis of malathion is sufficient to explain x 10 malathion resistance of the Israel strain. Biochemical research in the field of insecticide resistance generally indicates that even a slight physiological advantage of a resis- tant strain over its normal counterpart can produce large inter-strain differences when insecticides are tested by surface contact. With this method the final toxicity is determined by several factors and it is only a fraction of the externally applied insecticide that is decisive (Winteringham, 1969). Many studies illus- trate this conclusion. For example, Matsumura & Voss (1964) worked on a x 60 malathion-resistant strain of Tetranychus urticae and found that the amount of malathion it degraded in vitro was only 3 times the amount degraded by a susceptible strain. Townsend & Busvine (1969) studied the in vivo meta- bolism of malathion in the x 168 malathion-resistant CM strain of Chrysomya putoria and compared it with the susceptible CS strain. They observed that the magnitude of the difference between the strains at 2 hours (and this time was critical at the dose level studied) was 4-fold. The amount of malathion de- graded by the resistant G strain of Musca domestica (resistance level x 200) in vitro was 2.5 times the amount degraded by a susceptible strain (Matsumura & Hogendijk, 1964). ACKNOWLEDGEMENTS I am grateful to Professor J. R. Busvine for editing this paper and for supervising its publication in my absence. Mr S. Venkatesan was most helpful with technical advice, in particular in introducing me to the use of electrophoresis for separating the malathion meta- bolites. The work was made possible by a grant from the Government of Pakistan. R1ESUME BIOCHIMIE DE LA RJSISTANCE AU MALATHION CHEZ UNE SOUCHE DE CIMEXLECTULARIUS RtSISTANTE AUX COMPOSt-S ORGANO-PHOSPHORtS On a utilise au cours de cette etude deux souches de punaises de lit, l'une sensible, l'autre resistante aux organo-phosphores, et notamment au malathion (resis- tance: IOx). Dans un premier temps, par application de malathion radioactif (14C-malathion) sur les punaises immobilisees, on a evalue la penetration de l'insecticide en mesurant la quantite restant sur le corps des insectes 'a intervalles determines. Aucune difference de penetration du compose n'a et relevee entre souches normale et resistante. On a ensuite compare in vitro l'activite de diverses enzymes. L'acetylcholinesterase s'est montree egalement active chez les deux souches et egalement sensible a l'action inhibitrice du malaoxon. Quant a l'activite de la cholinesterase et de la carboxylesterase, mesuree par le pouvoir d'hydrolyse des acetates d'a- et de P-naphtyle en presence ou en l'absence de sulfate de physostigmine, elle est apparue sensiblement egale dans les deux cas. Enfin, on a etudie le metabolisme du malathion en incubant des homogenats de punaises sensibles et resistantes et une certaine quantite d'insecticide marque au "C, et en recuperant les differents metabolites par fractionnements successifs. La souche resistante contenait moins de malaoxon et davantage de produits d'hydrolyse. Dans l'ensemble, la degradation du malathion a ete superieure de 17% chez la souche resistante par rapport A la souche sensible. Il semble que ce fait puisse expliquer le mecanisme de la resistance. 803 804 M. FEROZ REFERENCES Abbott, D. C., Crosby, N. T. & Thomson, J. (1965) Proceedings of the Society of Analytical Chemists Con- ference, Nottingham, pp. 121-134 Asperen, K. van (1962) J. Insect Physiol., 8, 401-416 Asperen, K. van & Oppenoorth, F. J. (1959) Entomologia exp. appl., 2, 48-57 Dyte, C. E. & Rowlands, D. G. (1968) J. stored Prod. Res., 4, 155-173 Feroz, M. (1969) Bull. ent. Res., 59, 377-382 Feroz, M. (1970) Toxicological and genetical studies on organophosphorus-resistance in the bed-bug, Cimex lectularius L., Thesis, London University, pp. 165 Hestrin, S. (1949) J. biol. Chem., 180, 249-261 Kojima, K., Ishizuka, R. & Kitakata, S. (1963) Botyu- Kagaku, 28, 17-25 Kojima, K., Ishizuka, T., Shiino, A. & Kitakata, S. (1963) Jap. J. appl. Ent. Zool., 7, 63-69 Lee, R. M. & Batham, P. (1966) Entomologia exp. appl., 9, 13-24. Matsumura, F. & Brown, A. W. A. (1961) Mosquito News, 21, 192-194 Matsumura, F. & Brown, A. W. A. (1963) J. econ. Ent., 56, 381-388 Matsumura, F. & Hogendijk, C. J. (1964) Entomologia exp. appl., 7, 179-193 Matsumura, F. & Voss, G. (1964) J. econ. Ent., 57, 911-917 Ozaki, K. (1969) Rev. Plant. Protec. Res., 2, 1-15 Peerebroom, J. W. C. (1960) J. Chromat., 4, 323-328 Plapp, F. W., Jr & Hoyer, R. F. (1968) J. econ. Ent., 61, 1298-1303 Ritschard, W. J. (1968) In Smith, I., ed., Chromato- graphic and electrophoretic techniques, London, Heine- mann, vol. 2, pp. 147-166 Robbins, W. E., Hopkins, T. L. & Roth, A. R. (1958) J. econ. Ent., 51, 326-329 Roulston, W. J., Schuntner, C. A., Schnitzerling, H. J. & Wilson, J. T. (1969) Aust. J. bio. Sci., 22, 1585-1589 Sawicki, R. M. & Farnham, A. W. (1969) Bull. ent. Res., 59, 409-421 Smissaert, H. R. (1964) Science, 143, 129-131 Smissaert, H. R. (1965) Nature, 205, 158-160 Stone, B. F. & Brown, A. W. A. (1969) Bull. Wld Hlth Org., 40, 401-408 Townsend, M. G. & Busvine, J. R. (1969) Entomologia exp. appl., 12, 243-267 Winteringham, F. P. W. (1969) Ann. Rev. Ent., 14, 409-442