Bull. Org. mond. Santg 1964, 30, 57-64Bull. Wld Hlth Org.

The Conversion of DDT to DDE bySome AnophelinesH. LIPKE 1, 2 & JILL CHALKLEY'

The metabolism of DDT has been followed in pure lines of laboratory-reared resistantand susceptible anophelines using gas-liquid chromatography. Relatively large amounts ofDDE were formed in vivo by susceptible strains of Anopheles stephensi, A. quadrimacu-latus and A. gambiae, and by resistant strains of A. stephensi and A. quadrimaculatus.Resistant and susceptible A. albimanus showed no difference in the rate ofDDEproduction.Volatile metabolites of DDT other than DDE were not observed in most of the strainschromatographed. A procedure for the measurement of DDT-dehydrochlorinase in smallnumbers of mosquitos was devised based on conditions developed for the glutathione-dependent DDT-dehydrochlorinase present in resistant houseflies. This method has shownthe enzyme to occur in highest titre in DDT-susceptible A. albimanus and A. stephensi,indicating little or no correlation with resistance to DDT. Major differences in dehydro-chlorination rates and in patterns of resistance between anopheline species have beenobserved. In A. stephensi, these differences extend to the level of strains.

In the housefly, the appearance of high levels ofresistance to DDT is accompanied by the con-comitant development of increased titres ofDDT-dehydrochlorinase (Lipke & Kearns, 1960).Auxiliary mechanisms of resistance in houseflies andother species have been proposed (Bradbury &Winteringham, 1958; Brown, 1960), but conclusiveevidence concerning the significance of thesemechanisms has not been forthcoming. The con-tribution of detoxicating mechanisms to the etiologyof resistance in resistant (R) strains may be assessedby measurement of the conversion of insecticide toinnocuous products in vivo and in vitro, followedby comparison of these same properties in susceptible(S) strains.3 Only where significant differences in therate of degradation are demonstrable between theR and the S can detoxication be assigned a majorrole. The enhanced rate of detoxication in thetreated strains must in addition be detectable by

1 Ross Institute of Tropical Hygiene, London School ofHygiene and Tropical Medicine, London, England.

2 Permanent address: Physiology Division, EdgewoodArsenal, Maryland, USA.

3Throughout this paper R is used to designate DDT-resistance and S to designate DDT-susceptibility. Otherabbreviations used are: DDT, 1,1,1-trichloro-bis-(p-chloro-phenyl) ethane; DDE, 1,1-dichloro-bis-(p-chlorophenyl)ethylene; TDE, 1,1-dichloro-bis-(p-chlorophenyl) ethane;DTB,4,4'-dichloro-alpha-trichloromethylbenzhydrol; EDTA,ethylene diamine tetraacetic acid; GSH, reduced glutathione.

comparison of appropriate enzyme preparationsfrom both populations to minimize behavioural ormortality differences. The degree to which anophe-lines fulfil these conditions has not been establishedand its determination would appear to be a necessaryadjunct to the investigation of other biochemical orbehavioural phenomena that may be operative inthis genus (Micks, 1960; Vargas, 1960; Garms, 1960).

Resistance exhibited by anophelines to DDT,although of considerable practical importance, isgenerally of a lower order than that observed inhouseflies, and is less amenable to detailed bio-chemical investigation. The first report of DDEformation by DDT-treated anophelines (A. quad-rimaculatus), was by Gartrell & Ludvik (1954).This metabolite appears in adult A. sacharovi, A.maculipennis, A. labranchiae, A. stephensi andA. claviger (Perry, 1960). Similar results wereobtained with A. atroparvus (Frontali & Carta, 1959)and A. subpictus (Kalra & Pal, 1959). Other meta-bolically derived products of DDT have not beenreported in anophelines, nor has the successfulconversion of DDT and DDE in vitro. This contri-bution describes the extent of conversion by certainanophelines of DDT to DDE as measured by gas-liquid chromatography. The conversion of DDT toDDE by tissue extracts using the conditions knownto favour this conversion in the housefly is also

1377 -57

H. LIPKE & J. CHALKLEY

reported for some populations. The results showDDE to be the major metabolite of DDT, but failto establish any correlation between insecticidemetabolism in the living animal and resistance or

the ability to degrade DDT to DDE in vitro.

EXPERIMENTAL METHODS AND MATERIALS

OrganismsThe S and R strains of Musca domestica were

kindly furnished by Professor Busvine and Dr Fineof the London School of Hygiene and TropicalMedicine. This species was reared according to thetechnique of Busvine (1959) and represents normaland Swedish strains previously described (Fine, 1961;Lipke & Chalkley, 1962). Anophelines were rearedand tested for susceptibility according to the tech-niques described by Davidson (1958) and Davidson& Jackson (1961) and represent genetically purelines isolated at the Ross Institute over a periodof years (Davidson, 1958; Davidson & Mason,1963). Susceptibility was also measured under theconditions described below with no apparent dis-crepancies between the various modes of treat-ment. Some characteristics of the strains studiedare presented in Table 1, where the treatment periodsconducive to the most favourable demonstration ofdifferences in detoxication rates are presented.

DDTtreatment

Unsexed mosquitos in groups of 50-200, or 10-20houseflies, were subdued at 5°C and transferred toround stainless steel cages 13 cm high and 7 cm indiameter. The sides of the cages were lined with a

double layer of Whatman No. 1 filter-paper, theinnermost of which was treated with a 4% solution ofDDT (Geigy, 99.9% p,p') in Risella oil. The DDTconcentration was 40 mg/l0O cm.2 The base of thecage rested on a pad of moist cotton-wool. Controlgroups were similarly handled, but for the absence oftoxicant. Following recovery of half the populationfrom cold anaesthesia, usually after 5-10 minutes,exposure proceeded for the intervals described inTable 1. The filter-paper was then withdrawn by a

slight displacement of the cage lid and the subjectsheld at -20°C for a few minutes until quiescent.Transfer was effected to Potter-Elvehjem grindersequipped with loose-fitting pestles and containingspectral grade 2,2,4,-trimethyl pentane (1 ml/20mosquitos) and Pyrex glass ground to 200-400 mesh.After homogenization for 2 minutes, initially at300 r.p.m. and finally at 800 r.p.m., the extract was

TABLE IRESISTANCE TO DDT OF ANOPHELINES EXPOSED TOFILTER-PAPER IMPREGNATED WITH 4% DDT IN RISELLA

OIL

Expo- IPer-Speciessand .. Nature of sure centage

strains a Origin resistance time pros-I ~~~~~~~~(mi-tros-

nutes)

A. albimanus

S(PALB) Panama 60 85

S(ITALB) Panama 30 70

R(LLTT) El Salvador DDT, 60 3dieldrin

A. quadri-maculatus

S(QC) S. Carolina, 50 85USA

R(QMY-1) Maryland, USA DDT, 270 10dieldrin

A. gambiae

S(TAV) Kenya 60 80

S(Bobo) Upper Volta Dieldrin 50 50

A. stephensi

S(SS) India 60 50

S(YF) Iran Dieldrin b 60 75

R(IQ) Iraq DDT 120 1

M. domestica

R(Swedish) Sweden DDT, 720 5pyrethrin

a Resistant strains.prefixed by R, susceptible by S.b Formerly R to DDT and dieldrin, reverted to S for DDT.

filtered through No. 1 paper containing 4 g anhydrousNa2SO4 and stored at -20°C following the additionof one drop of refined liquid paraffin to reducevolatilization of the insecticide and metabolites.Housefly extracts contained 5 flies/ml trimethylpentane. External DDT and DDE were removedfrom the ffies prior to grinding by rapid rinsing inacetone.

Gas-liquid chromatographyWe are indebted to the staffof the Shell Woodstock

Agricultural Research Centre, Sittingbourne, Kent,for all the gas chromatographic analyses as well asfor the development of this method of revealing

58

CONVERSION OF DDT TO DDE BY SOME ANOPHELINES 59

submicrogram quantities of insecticides and meta-bolites rapidly and definitively. The procedure hasbeen described in detail by Goodwin et al. (1961).In brief, the extracts are chromatographed on24-inch columns, using both polar and non-polarstationary phases, and detected by an electron-capture ionization detector. The polar column isformed of 1% polyethylene glycol 4000 on 100/120-mesh Celite. The non-polar column consists of2.5% silicone elastomer E301 plus 0.25% Epikoteresin 1001 on 100/120-mesh Celite. Both columnswere operated at 163°C with 02-free N2 as carriergas.The retention times of DDT and the various

analogues examined are shown in Table 2. It canbe seen that the peaks for DDT and DDE and4,4' - dichloro - alpha - (trichloromethyl) benzhydrol(DTB) (Agosin et al., 1961) are well separated onboth columns. The experimental error is ±5%and ±10% for DDT and DDE respectively. Thepresence of interfering material in mosquito extractsat 4.3 minutes on the non-polar column preventsany positive identification of DTB.

All the insecticides and derivatives with theexception ofDDT were gifts from the Rohm & HaasCorporation, Philadelphia, Pa., and were recrystal-

TABLE 2MEAN RETENTION TIMES (IN MINUTES) OF DDT AND ITSANALOGUES FOR GAS-LIQUID CHROMATOGRAPHY a

Column bCompounds

Non-polar Polar

p,p' DDT 14.3 12.9

o,p DDT 10.3 7.3

DDE 7.8 4.1

TDE 7.8 4.1

Dehydrochlorinated TDE 4.1 (major) 3.2 (major)2.7, 7.8 (minor) 1.3 (minor)

4,4'-dichloro-a/pha-(trichloromethyl)-benzhydrol(DBT) 4.3 (major) 3.3

7.8 (minor)

Mosquito extracts c (Risellaoil only) 1.0 (major)

4.3 (minor) 3.2 (minor)

a Minor components represent impurities or decompositionproducts comprising 2 %-10 % of the total.

b See the text and Goodwin et al. (1961) for explanation ofpolar and non-polar columns.

c Mosquitos treated as described in text in absence of DDT.

lized from ethanol before use. Good agreement wasobtained between the results for DDT and DDEobtained from chromatography on the polar andnon-polar stationary phases with all the extractssubjected to chromatography. The results withgas-liquid chromatography were confirmed in thecase of the housefly by reverse-phase paper chromato-graphy of dehydrochlorinase reaction media (Bridges,Harrison & Winteringham, 1956; Mitchell, 1957) andshowed only DDT and DDE to be present.

Assay of DDT dehydrochlorinase

Initial studies using the glass-bead technique ofDDT-dehydrochlorinase assay described by Stem-burg et al. (1954) showed a rate of DDE formationin the R flies of 39 m,umoles/mg protein/90 minutes.No dehydrochlorination was evident with the Sstrain of flies or with any of the mosquitos tested bythis method. It appeared that a more sensitiveassay was required if low levels of enzyme were tobe measured. A technique for the estimation ofDDT-dehydrochlorinase using small amounts oflipoprotein to solubilize DDT has been describedin which the dehydrochlorination proceeded tocompletion (Lipke & Kearns, 1959); the reliabilityof this method has been confirmed by radiometricanalysis.' The lipoprotein method has been modifiedfor mosquito extracts although the extent of reactionis incomplete, probably due to the presence of aninhibitor in the concentrated unclarified lipoproteinpreparations. The dependability of the modificationused with anophelines was established using enzymefrom Musca domestica (R) prepared according toprocedure B of Sternburg et al. (1954) and was alsoverified with the high-speed supemate of houseflyhomogenates, which exhibit lower dehydrochlorinaseactivity.

Preparation of enzyme extracts

From 600 to 1000 anopheline adults were groundwith the aid of glass powder in pre-chilled mortarscontaining 5 ml of homogenization medium. Thecomposition of the medium was as follows: Na andK phosphate buffer, 0.05 M; EDTA, 1.5 x 10-3M;GSH, 3 x 10-3M; bovine serum albumin 0.5%;the mixture was adjusted to pH 7.0. The mash wasfiltered through muslin (previously boiled) andcentrifuged at 5000 g for 15 minutes at 1°C andassayed immediately.

1 Personal communication from Dr R. D. Elliott, Com-municable Disease Center, Savannah, Ga., USA.

H. LIPKE & J. CHALKLEY

Incubation of extractsFor DDT-dehydrochlorinase analysis, lipoprotein

was prepared by the method of Lipke & Kearns(1959, 1960) and dialysed against 4%. Na3 citratecontaining 0.05% EDTA for 24 hours. Aftersaturation with DDT, the material was diluted withan equal volume of citrate and stored at 5°C. Forassay, 0.09 ml of lipoprotein-DDT was added to0.45 ml of enzyme and 0.10 ml of 3 x 10-3M GSH,all held at 0°C under N2. The reaction was initiatedby placing the mixture in a water-bath at 37'C withagitation while gassing was continued. After 120minutes, the reaction was stopped with 0.70 ml ofconcentrated H2SO4 and the products were analysedas described below. Routine controls includedlipoprotein without DDT as well as a replicatewhere the reaction was stopped at time zero.

DDT and DDE analysisIn the preparation of alumina for chromatography,

A1203 (BDH Ltd., Alumina for chromatography)was heated for three hours at 380°C and suspendedin 4 litres of 0.12 N-HCI. The absorbent was washedfree of Cl- with water, suspended three times in 4litres of methanol and heated to 70°C for 12 hours.After washing with petroleum ether (60°C boilingpoint) the alumina was activated by heating to 1 80°Cfor 4 hours. For chromatography, columns of0.9 cm internal diameter containing 5 g of absorbentwere prepared immediately before use. A 1-cmlayer of anhydrous Na2SO4 was placed above thealumina on the columns. No degradation of DDTor DDE was mediated by alumina prepared inthis manner.

Analysis ofenzyme preparationsTo the sulfuric-acid-treated reaction mixture were

added 10 ml of water; the tube was stopperedtightly and held at 100°C for 15 minutes. The digestwas diluted to 50 ml with water and extracted inungreased separatory funnels with 50 ml ofpetroleumether (40°C boiling point) which had previously beenpassed through an alumina column. The aqueousphase was discarded and the ether washed oncewith water, once with 1% NaOH and again withwater until neutral to phenolphthalein. The etherextract was washed with saturated NaCl solutionand filtered through No. 1 paper containing 10 gof anhydrous Na2SO.. After the addition of onedrop of refined paraffin the extract was taken todryness with a gentle stream of air at roomtemperature.

The sample was transferred to the column with5 ml of petroleum ether followed by 30 ml ofpetroleum ether containing 0.5% redistilled ethylether. The first 5 ml of effluent were discarded andthe remainder of the effluent containing essentiallyall of the DDT and DDE was collected and takento dryness after the addition of a drop of paraffin.Each of the preparations was dissolved in 4 ml ofethanol or trimethyl pentane and the absorbancerecorded in a Hilger Uvispek spectrophotometer at230, 241, 245, 250, 260, and 275 m,u. Blank valuesfrom the zero time samples or those devoid of DDTwere substracted, the values were adjusted for equalabsorbance at the isosbestic point for DDT andDDE (241 m,u), and the differences due to DDE at260 mp, were taken as evidence of DDE formation.The shapes of the absorption spectra were identicalwith those obtained using this procedure with variousmixtures of lipoprotein-DDT and lipoprotein-DDEto which enzyme extracts had been added im-mediately following addition of the sulfuric acid.These procedures are modifications of methodsreported by Sternburg & Kearns (1952) and Stern-burg et al. (1954). Data treated in this manner arepresented in the figure, which shows the spectralchanges accompanying DDE formation. It can beseen that the absorbance at 260 m,u due to DDEformation is a function of the incubation time.Evidence that the 260-m,i-absorbing material ob-served in the experiments with mosquitos is DDEis restricted to the general shape of the absorptionspectrum, which is identical with that obtained withauthentic lipoprotein-DDE similarly processed, andto the chromatographic mobility of the reactionproduct on alumina when compared with authenticDDE.

RESULTS

In addition to detecting DDT and congeners,gas-liquid chromatography can reveal the presenceof the large number of volatile ether-soluble organichalides which can be formed by mild or extensivedegradation of the parent compound. Table 3illustrates that detectable amounts of DDE areformed in all the groups analysed, no correlationwith resistance being evident. In the case of A.stephensi, strains S(SS) and R(IQ) form approxi-mately equivalent amounts of DDE, whereas thereverted straia S(YF) produces relatively little DDE.The DDE formed by R(IQ) may be even l:ss thanthat formed by S(SS) during the first 60 minutes oftreatment. A similar pattern was observed with

60

CONVERSION OF DDT TO DDE BY SOME ANOPHELINES 61

ABSORPTION SPECTRA OF REACTION PRODUCTS OF DDT-DEHYDROCHLORINASEa

M. domestica R (SWEDISH) A. stephensi SIYF)1.6 -1.6

1.4 -1.4-

1.2 1.2

1.0 1.0

0.8 .

0.6 0.6-

0.4 \ 240 Minutes 0.4-

120 Minutes

0.2 -10Mnts 0.2-

0 Minutes ~ ~ ~ ~ ~ ~ ~ ~~~~~~~~ ints0 0~~~~~~~

240 250 260 270 240 250 260 270

WAVE-LENGTH (mju)

a The system contains: Na and K phosphates, 0.05 M (pH 7.0); glutathione, 3x 10-3 M; bovine serum albumin, 0.5%, lipoprotein-DDT, 0.09 ml; enzyme extract 0.45 ml, volume 0.65 ml. Incubated at 37°C under N, for indicated interval. Sarrpla- preparationdescribed in text.

A. quadrimaculatus S(QC) and-R(QMY-l), where thedisparity between DDE formation and time of treat-ment is even more pronounced. The ability of 97%of the R(LLTT) A. albimanus to withstand 60 minutesof exposure to DDT (Table 1) is not accompanied bya corresponding increase in DDE production; nosignificant difference in this capacity is evident rela-tive to the S(ITALB) individuals, 70% of which be-come prostrate in 30 minutes. The widespread occur-rence of the DDE-forming mechanism in anophelinesis made more evident by the identification of thisolefin in the three S strains of A. gambiae as well asin a culicine (Culex fatigans) which was also in-cluded in this series. With respect to the R housefly,about 25% of the dose is dehydrochlorinated duringthe interval of treatment with essentially completerecovery.The occurrence of significant amounts of meta-

bolites other than DDE appears unlikely in most ofthe strains examined, since complete removal of the

chlorine from the parent molecule would be requiredto escape detection in the gas chromatogram. Oneexception may be A. albimanus R(LL1T), where apeak in the region of DTB was observed. Exposureof A. stephensi S(YF) for 30 minutes occasionallyproduced a component with mobility similar toDTB. This was lacking in populations treated forlonger periods, e.g., of 60-minutes' duration (Table3). Further characterization of this benzhydrol-likesubstance requires the elimination of an interferingmaterial in the ether extracts which sporadicallyobscured the benzhydrol region of the chromato-grams. Efforts to this end were not successful.Resistance to dieldrin in A. gambiae and M. domes-tica is not accompanied by extensive metabolism ofthe toxicant (Winteringham, Harrison & Davidson; 1

1 Winteringham, F. P. W., Harrison, A. & Davidson, G.(1960) Absorption and metabolism of 14C-labelled aldrin bysusceptible and resistant mosquito larvae. In: WHO Informa-tion Circular on Insecticide Resistance, No. 21 (unpublished).

5

H. LIPKE & J. CHALKLEY

TABLE 3GAS CHROMATOGRAPHIC ANALYSIS OF FLY AND

MOSQUITO EXTRACTS FOLLOWING TREATMENT WITHDDT OR DIELDRIN a

Treatmen Total amountTreatmen recovered (i-g)

Species and strain c Time Un-

Iscide (mi- DDT DDE iden-nutes) tified

A. stephensiS(SS) DDT 60 3.9 0.20 NoneS(YF) DDT 60 9.0 0.02 NoneR(IQ) DDT 120 6.5 0.20 None

A. quadrimaculatusS(QC) DDT 50 3.0 0.20 NoneR(QMY-1) DDT 270 9.4 0.17 None

A. albimanusS(ITALB) DDT 30 1.9 0.02 NoneR(LLTT) DDT 60 2.4 0.03 Hydrol-

like

A. gambiaeS(TAV) DDT 60 12.0 0.30 NoneS(Bobo) DDT 50 2.5 0.20 NoneS(Bobo) b Dieldrin 60 5.0 d None

C. fatigans(Lagos) DDT 60 4.0 0.14 None

M. domestica cR(Swedish)

Internal DDT 1 440 6.0 1.8 NoneExternal DDT 1 440 2.0 0.20 None

a Susceptible (S) insects were sacrificed when prostrate,resistant (R) insects at incipient prostration.

b S(Bobo) strain resistant to dieldrin but not to DDT.CM. domestica recoveries per fly following topical appli-

cation of 10 #Ag DDT.

d Value represents g dieldrin recovered; no metabolitesevident on chromatograms.

Brooks, 1960). These observations are confirmedand have been extended to the dieldrin-resistantstrain of A. gambiae S(Bobo), where only dieldrinwas observed in the volatile fraction which registeredon the detector (Table 3).Table 4 presents the information regarding DDT-

dehydrochlorinase. It can be seen that little or norelation is evident between the presence of a GSH-dependent dehydrochlorinase in cell-free extractsand the degree of resistance. A marked lack ofcorrelation between the ability to dehydrochlor-inate DDT in vivo and the dehydrochlorinaseactivity of the enzyme extracts was obtained. Forexample, the greatest titre of dehydrochlorinase,0.056 ,umole/120 minutes, was observed in A.stephensi S(YF), which formed DDE relatively slowly

TABLE 4DDT METABOLISM BY ANOPHELINE EXTRACTS AND

IN THE INTACT MOSQUITO

DDEformed DDE formedSpecies and strain DD,E formeda D vtr

(Mmole)

A. stephensiS(SS) +++ NoneS(YF) + 0.056R(IQ) +++ None

A. quadrimaculatusS(QC) +++ NoneR(QMY-1) ++ None

A. albimanusS(ITALB) -c 0.046S(PAL) + 0.022R(TTLL) + 0.002

A. gambiaeS(TAV) +++ NoneS(Bobo) +++ c

a Data from Table 3; + = < 0.05 tAg DDE recovered by gaschromatogram; ++ = 0.05-0.19 ug; +++ = > 0.20 ,g.

b Assay conditions as in the figure; incubation period120 minutes. Quantities of DDE less than 0.002 Amole arereported as " None

c No test run.

in vivo. The S(SS) A. stephensi, although capable ofDDE formation in vivo, produced little or no DDEenzymatically. The results with a resistant A.stephensi, strain R(IQ), emphasized the diversity ofresponse in this genus, there being little dehydro-chlorinase in vitro but significant dehydrochlorina-tion in the intact mosquito. No dehydrochlorinasecould be detected in the A. quadrimaculatus or A.gambiae strains, even though significant levels wereobserved in vivo. In A. albimanus high levels ofdehydrochlorinase were associated with susceptibilityrather than with resistance. The S A. albimanusstrains appear somewhat similar to A. stephensiS(YF) with respect to moderate to high dehydro-chlorinase activity coupled to limited dehydro-chlorination in vivo.

DISCUSSION

The evidence for different mechanisms of resistancewithin a genus, first suggested by Bradbury &Standen (1960), constitutes the major trend of thefindings (Table 4); the demonstration of a dehydro-

62

CONVERSION OF DDT TO DDE BY SOME ANOPHELINES

chlorinase in mosquitos is of minor importance.Most of the S strains produced significant amounts ofDDE following treatment. In two cases, A. stephensiand A. albimanus, higher titres of DDT-dehydro-chlorinase characterized the S strain rather than theR strain. The most resistant of the species studied,A. quadrimaculatus, showed no demonstrable DDT-dehydrochlorinase and no relation of resistance todetoxication in vivo. Table 4 presents evidence thatstrains can produce low, intermediate, or high levelsof DDE and still be classified as resistant. Diversityof the same type is observed in the S strains. Theseanomalies can in fact be viewed to the effect thatdehydrochlorination is without major significancein these members of this genus as a mechanism ofresistance.The conversion ofDDT to DTB has been reported

in Culex quinquefasciatus by Agosin et al. (1961)and Dinnamarca et al. (1962). Some decompositionof DTB occurs on the polar and non-polar columnsas well as occasional interference from the mosquitoextracts (Table 2). It appears, however (Table 3),that large accumulations of this metabolite do notoccur in any of the strains tested, although tracesmight be present in A. stephensi S(YF) and A.albimanus R(LLTT).

Consideration of the evidence concerning theanophelines must be preceded by considerablequalification. (1) The chromatographic methodevolved makes no distinction between absorbed andunabsorbed toxicant, and does not allow for differ-ences in pick-up rates. (2) The water-solublefraction of the mosquitos was discarded for theanalyses. (3) Some strains may degrade DDT orDDE to compounds not visible on the chromato-grams which would be overlooked in the absence ofdata accounting for the entire dose received by theinsect. (4) Marked differences (Table 3) in exposuretimes were required in order to accentuate metabolicdifferences with respect to DDT degradation. (5)No detailed investigation of the optimum conditionsfor the enzymic reactions were undertaken. (6)Strain S(YF) of A. stephensi, whose performance in

the various trials was particularly unusual, wasoriginally a DDT- and dieldrin-resistant strainwhich had reverted to S for DDT, but whosedieldrin-resistance was unimpaired. (7) Definitiveidentification of all the metabolites was not attempted.(8) Finally, the method of exposure to a DDTresidue did not allow behavioural patterns to comeinto play (Trapido, 1954).The sensitivity of the gas chromatographic pro-

cedure (about 10-10 g of organic halide) and thesimplicity of sample preparation makes possiblethe determination of the levels of insecticide pick-upand degradation in very small numbers of field-caught anophelines. No effort was made in thesestudies to ascertain the minimum number of mos-quitos that could be analysed by the proceduredescribed, but it would appear that as few as 10mosquitos can be homogenized and chromato-graphed with significant results.One of the objects of this study was to judge the

suitability for field stations of gas chromatographyfor the rapid detection of DDT metabolism inmosquitos from treated areas. Consequently,relatively crude petroleum-ether extracts wereemployed, with the intentional avoidance of clean-upprocedures. The procedure described appearsadequate for the detection of DDE, but the pos-sible occurrence of the benzhydrol in some strainsindicates that some elimination of interfering sub-stances is necessary. The method is capable offurther refinement for the analyses of enzymeincubation mixtures. In this respect, Berger (1961)has recently described a modification of the directspectrophotometric assay for DDT-dehydrochlori-nase that may eliminate the shortcomings of thelipoprotein assay.With the appearance of resistance in anophelines,

considerable discussion has been devoted to similar-ities and differences of resistance patterns character-istic of this genus in relation to other resistantgenera. The present data indicate that modes ofresistance may in fact diverge at the level of strainswithin a species.

ACKNOWLEDGEMENTS

To his colleagues in the Ross Institute the senior authoris especially indebted for their counsel and assistance.Special gratitude is expressed to Professor G. Macdonaldand Mr G. Davidson, who presented the opportunity totake up a US Public Health Service Fellowship and to

utilize the facilities of the Institute. Dr R. A. E. Galley,Research Director of Shell Woodstock AgriculturalResearch Centre, kindly offered the co-operation of thatorganization. Mr Kenneth Elgar and staff of the Ana-lytical Section provided the knowledge and effort to make

63

64 H. LIPKE & J. CHALKLEY

possible the presentation of the chromatographic infor-mation. Much apparatus was made available throughthe kindness of Dr W. E. Ormerod, Dr E. Stickings,Dr J. Busvine, Dr B. Fine, and Miss W. Wall of the

London School of Hygiene and Tropical Medicine. TheUnited States Army Chemical Corps granted leave to thesenior author in support of the anti-malaria campaign ofthe World Health Organization.

RI%Jlu-,SUMI

Chez la mouche domestique, on a observe que I'appa-rition d'une resistance 6lev6e au DDT s'accompagnait detitres accrus de dehydrochlorase du DDT, en presence delaquelle le DDT est transforme en DDE. On a propose,pour preciser le r6le de la detoxication dans les meca-nismes de r6sistance, de comparer le taux de cet enzymechez les souches sensibles et les souches r6sistantes. Dansle cas d'une difference significative, on pourrait consi-d6rer la detoxication comme le mecanisme principalde la resistance. Jusqu'ici, les recherches n'avaientpas porte sur les anopheles. Les auteurs comblent cettelacune.Le metabolisme du DDT a ete etudie par la methode

chromatographique gaz-liquide sur des lign6es puresd'anopheles sensibles ou resistants, e1eves en laboratoire.Des quantites relativement elevees de DDE sont formeesin vivo par des souches sensibles de A. stephensi, A. quadri-

maculatus et A. gambiae, et par des souches resistantes deA. stephensi et A. quadrimaculatus. Les A. albimanus,resistants ou sensibles, ne presentaient pas de differencesdans le taux de production du DDE. Des metabolitesvolatils du DDT autres que le DDE n'ont pas e observ6sdans la plupart des souches soumises a la chromato-graphie. Un proced6 de mesure de la dehydrochlorase duDDT a e mis au point, sur le modele de celui que l'on autilis6 pour le dosage de la dehydrochlorase du DDTdependante du glutathion, chez les mouches domestiquesr6sistantes. L'enzyme existait A un titre maximum chez lesalbimanus et les stephensi sensibles, donc sans relationevidente avec la r6sistance au DDT. On a observe d'im-portantes diffdrences dans le taux de resistance et dans lesschemas de resistance au sein des genres etudies. ChezA. stephensi, ces differences se manifestent au niveau dela souche.

REFERENCES

Agosin, M., Michaeli, D., Miskus, R., Nagsawa, S. &Hoskins, W. M. (1961) J. econ. Ent., 54, 340

Berger, R. S. (1961) The determination of DDT-dehy-drochlorinase, Ithaca, N.Y. (Thesis, Cornell University)

Bradbury, F. R. & Standen, H. (1960) J. Sci. Food Agric.,11, 92

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