Medical and Veterinary Entomology (1988) 2, 37-46

Reduced susceptibility to permethrin and its relationship to DDT resistance in larvae of Anopheles stephensi

C. A. MALCOLM Research Institute ITAL, P.O. Box 48, 6700 AA Wageningen, The Netherlands

ABSTRACT. Permethrin selection of DDT resistant Anopheles stephensi Liston mosquito larvae produced a reduction in susceptibility to knockdown (2 h exposure) of 17-fold, but only 1.6-fold to kill (24 h exposure). Genetic analysis, incorporating visible mutant markers, was interpreted as indicating that, through multigenic inheritance, several interacting genetic factors were collectively responsible for reduced larval susceptibility to knockdown. These were maintained together only under selection pressure, as the effect was lost quickly in the absence of selection or with outcrossing. The 30-40-fold DDT-resistance found in the parental strain was barely altered by permethrin selection, suggesting no relation- ship with the major source of DDT resistance. This was confirmed in single family studies. Some evidence for an additional tolerance to DDT was found to be associated with reduced larval susceptibility to permethrin.

Key words. Anopheles stephensi, mosquito larvae, larvicides, insecticide resistance, knockdown, cross-resistance, tolerance, DDT-resistance, permet hrin.

Introduction

Studies on resistance to several insecticides in larvae of Anopheles stephemi Liston were initi- ated as part of a programme to develop genetic zontrol methods for this species of mosquito, an important urban malaria vector in the Indian subcontinent. Of particular interest were resist- ance genes which might prove suitable for incor- poration into a genetic sexing system. In the case of pyrethroid-resistance and DDT-resistance, the evidence for a mutual resistance mechanism in Amstephemi (Omer el al., 1980) indicated that both should be studied simultaneously.

Correspondence: Dr C. A. Malcolm, London School of Hygiene and Tropical Medicine, Keppel Street (Gower Street), London WClE 7HT.

Omer et al. (1980) found that larvae of a strain of An.stephemi from Pakistan, initially showing low-level resistance to DDT combined with susceptibility to pyrethroids, exhibited up to 18-fold cross-resistance to permethrin after six generations of larval selection with DDT had produced 144-fold DDT-resistance. Higher levels of cross-resistance, up to 23-fold, to per- methrin were obtained in a sub-colony selected with DDT plus the synergist DMC. As in pre- vious studies (Perry, 1960; Lipke & Chalkley, 1964), no evidence was found for enhanced metabolism of either insecticide in the resistant strains. Neurophysiological studies, however, showed that resistant strains required approx- imately 20 times more permethrin than the susceptible strain to induce an increase in the

37

38 C. A. Malcolm

frequency of miniature excitatory postsynaptic potentials. This suggested a resistance mecha- nism comparable to that controlled by kdr, the major component of DDT/pyrethroid-resist- ance in the housefly (Farnham, 1977). DDT/pyrethroid-resistance has also been shown in three other mosquitoes: Culex quin- quefasciatus Say (Priester & Georghiou, 1980), Anopheles gambiae Giles (Prasittisuk & Cuds, 1982) and Aedes aegypti (L.) (Malcolm, 1983b).

This paper reports an attempt to reproduce a permethrin-resistant strain similar to that studied by Omer et al. (1980) by selection of a DDT resistant strain; the subsequent genetic analysis of the response obtained and its rela- tionship to DDT resistance.

Materials and Methods

Strains of An.sfephensi Liston

Lahore, DDT resistant; derived from thirty- eight females collected in Lahore, Pakistan, August 1980. Lines selected for resistance to permethrin were designated LaPml and LaPm2. When LaPm2 was further selected tor the mutant Stp (striped larva - intermediate domi- nance on chromosome 11; Sakai et al., 1974) the final strain was re-named PmSt (Table 2).

Karachi. DDT resistant; supplied by Interna- tional Centre for Medical Research and Training (ICMRT), Lahore, Pakistan.

Blro. DDT resistant, homozygous for two morphological mutants: Bl. black larva, inter- mediate dominance on chromosome I11 (Akhtar eraf., 1982) and ro, rosy eye, a sex-linked reces- sive (Aslamkhan & Gul, 1979). Supplied by ICMRT, Lahore, Pakistan.

(The above three strains were brought from Pakistan in 1980 by Dr C. van Heemert.)

BARR. DDT and dieldrin resistant; originally from Bangalore, India, supplied by The Entomology Department, London School of Hygiene and Tropical Medicine (LSH&TM), in 1981. LASS. Partly DDT resistant; originally col-

onized from field material col!ected in Lahore, Pakistan, in 1978. Supplied by LSH&TM in 1982, this strain was re-selected for DDT suscep- tibility and the new strain was designated LS (London Susceptible).

Beech. Partly DDT resistant; a laboratory

stock originally held at Beecham's Laboratories. Supplied by LSH&TM in 1982.

insecticides

Permethrin: (3-phenoxybenzyl)-3-(2,2-di- chlorovinyl)-2,2-dimethyl cyclopropane car- boxylate, cis:trans ratio 25:75%; supplied by Wellcome Research Laboratories, Berk- hamsted.

pp'DDT: 2,2-bis (p-chlorophenyl)l,l,l,- trichloroethane; supplied by Aldrich Chemical Co. Ltd.

Larval insecticide bioassay

A modification of the WHO standard larval bioassay (WHO, 1975) was used. Batches of fifty fourth instar larvae were exposed in transparent polypropylene beakers (capacity 550 ml, diame- ter 11 cm, height 7 cm) containing 500 ml of insecticide suspension in de-ionized water. The insecticide suspension was prepared by adding 1 ml of insecticide solution in acetone to 499 ml of de-ionized water to give the desired concen- tration (expressed as mg/l). Tests were scored after 0.5, 1. 2, 4 and 24 h exposure, by gently pushing larvae below the water surface with a glass rod and recording those able to swim back to the surface as being not killed or knocked down. All bioassays were performed in a cons- tant climate room at a temperature of 28+. 1°C and relative humidity of 75fr5%. The scores at 2 and 24 h were analysed by probit analysis (Fin- ney, 1971). Concentrations giving 50% and 95% effective response and EGs values) were calculated for each time of exposure.

Mass insecticide selection of larvae normally involved exposing 800 fourth instar larvae in a white polypropylene tray (40.5 cm long, 31 cm wide, 8.5 cm depth) containing 8 litres of insec- ticide suspension. These conditions maintained the same depth of insecticide suspension and density of larvae in relation to surface area as in the standard bioassay procedure. Larvae cap- able of returning to the surface at the end of the exposure period were removed, rinsed several times in tap water (at ambient temperature), finally placed in conditioned water (i.e. water sieved from an existing larval culture and diluted approximately 1:l with clean tap water) and fed. When the numbers to be selected were lower

Reduced susceptibility to permethrin in An.stephensi 39

than 800, the standard bioassay containers were used.

Rearing

The mosquitoes were reared in a constant cli- mate room held at a temperature of 28fl"C and relative humidity of 75+5%. Photoperiodicity was maintained on a cycle of 12 h light, 12 h dark (LD 12:12 h). Adults were fed on 5% glucose solution and females bloodfed with anaesthetized mice. Eggs collected on filter paper in small bowls of water, were placed in tap water 1-2 cm deep in white plastic trays. Upon hatching, small larvae were provided with a few drops of Liquifry No. 1 (Interpet Ltd, Dorking, Surrey). Larvae from 2 days old were fed with Tetra Mikromin (Tetra Werke, Ulrich Baensch, West Germany).

Results and Discussion

Pilot tests with permethrin on three DDT-resis- tant strains, BARR, Karachi and Lahore, sug- gested very little difference in the levels of susceptibility (Table 1). Lahore showed the greatest heterogeneity of response and was used in the subsequent studies. However, these early results proved difficult to interpret. In the first few hours of treatment a knockdown response was observed: the larvae remained on the bot- tom of the test container unable to return to the surface, but clearly not dead. At 24 h the response normally ranged from severe

knockdown to kill. However, after 24 h some of the larvae were observed to have recovered from the knockdown recorded at 2-4 h, despite con- tinuous exposure.

Comparison of the ECS after 24 h exposure (Table 1) with the susceptible strain values obtained by Omer et al. (1980) suggested that Lahore had little potential for selection for resistance (resistance ratio=l.l), whereas a different interpretation can be placed on results scored after 2 h exposure (resistance ratio=2.77). The difficulty is to decide which time of exposure gives the most meaningful results.

Post-exposure recovery from pyrethroid knockdown is well known for adult mosquitoes (Malcolm & Wood, 1982) and other insects but not, as in this case, where larval exposure to the insecticide continues after knockdown. The possibility that the polypropylene containers were the source of the problem was examined by comparing the responses obtained in white plas- tic yoghurt cartons and glass beakers. The same level of recovery was observed with both alter- xiatives, although overall toxicity was slightly higher with glass. Containers similar to the unwaxed paper cups used by Omer et al. (1980) could not be found, so the project was continued using polypropylene containers. The recovery was not a result of oxidative metabolism of per- methrin, as the synergist piperonyl butoxide (PBO) did not increase the response to per- methrin (Table 1) in either the Lahore strain or DDT susceptible strain, LS (see below).

TABLE 1. Permethrin bioassay results, mg/I.

Strain or cross 2 h exposure 21 h exposure ECso E G Slope Rorsyn' ECSo EC, Slope Rorsyn*

ratio ratio BARR 0.0064 0.0298 1.073 Karachi 0.0062 0.0154 1.815 Lahore 0.0180 0.1010 0.954 Lahore (+PB) 0.0168 0.2154 0.646 LS 0.0065 0.0259 1.190 LS (+PB) 0.0063 0.0310 1.036 Blro 0.0180 0.2870 0.596 BlroDDT 0 . m 0.0260 1.583 PmSt 0.1107 0.8142 0.824 PmStxBlroDDT F, 0.0260 0.1900 0.834 (PmStxB1roDDT)xI-S F2 0.0070 0.0403 0.966 LSx(PmStxB1roDDT) F2 0.0080 0.0390 1.036

PB=piperonyl butoxide (used 1:l with permethrin). R=resistance ratio (EC,/ECSO for LS strain); syn=

0.98 0.0290 0.95 0.0194 2.77 0.0227 1.07* 0.0148 1 .00 0.0207 1.03* 0.0141 2.77 0.0300 1.38 0.0240 17.03 0.0331 4.00 0.0250 1.08 0.0310 1.23 0.0287

:synergist ratio (ECS

0.1OOO 1.334 1.40 0.0362 2.640 0.94 0.1070 1.062 1.10 0.0837 0.948 1.53' 0.0598 1.549 1.00 0.0261 2.684 1.47' 0.1040 1.336 1.45 0.0543 2.008 1.16 0.0840 1.760 1.60 0.6700 1.636 1.21 0.0726 1.962 1.50 0.0637 2.061 1.39

without PB/ECso with PB);

40 C. A. Malcolm

Selection for resistance to permethrin

As the pyrethroid resistance mechanism in An.stephensi reported by Omer et al. (1980) was likened to the kdr (knockdown resistance) mechanism in houseflies (Farnham, 1977), so-called because it conferred resistance to knockdown, larval selection was based on the immediate response to 2 4 h exposure, i.e. the criterion of rapid larval knockdown as distinct from eventual kill. Two permethrin-selected lines of Lahore strain were termed LaPml and LaPm2. Table 2 gives results for both series of selection. In the first series susceptibility decreased markedly after two generations, which then changed very little until a slight increase in susceptibility in the FS generation following the absence of F4 selection, and then again little change in susceptibility in Fh and F7. These observations were confirmed by bioassays using a range of concentrations on the first five generations (results not shown). This strain was lost shortly after selection of the F7 generation, due to unexplained contamination of the insectary.

The results from the second series (Table 2) resembled the first: an initial large decrease in susceptibility followed by little change until a

marked increase in susceptibility following the absence of F4 selection. Clearly, the selection pressure failed to fix any genetic factors con- tributing to reduced susceptibility to per- methrin. Moreover, 4 h larval exposure nor- mally produced not much more knockdown than 2 h exposure, with signs of some recovery within 4 h. Therefore, the shorter exposure period of 2 h was adopted for subsequent generations of larval selection. Since no further reduction of susceptibility was achieved by treatment of F,F, generations with 0.2 mg/l, selection pressure was relaxed to 0.02-0.04 mg/l (Table 2), intended to eliminate only fully susceptible phenotypes.

From generation Fp. line LaPm2 was also selected for Stp (striped larva), a visible morpho- logical mutant controlled by a gene with inter- mediate dominance on chromosome I1 (Sakai et al., 1974). Frequency of Stp in the parental Lahore strain had apparently been maintained independently of permethrin selection. The line was re-named PmSt (Table 2).

Selection for DDT sicsceptibility Strains LASS and Beech each produced a

range of response to DDT (Fig. 2, showing mor- tality resulting from 24 h exposure) which sug-

TABLE 2. Larval knockdown rates from mass selection of fourth instar larvae with permethiin. Strain/line Exposure Knockdown Total 5 Knockdown generation -

Lahore P 0.004 24 205 1600 11.8 LaPrnl F, 0.05 4 1083 1600 67.7

F2 0.1 4 676 800 84.5 F, 0.2 4 1115 1600 69.7 F, nil Fs 0.2 4 1438 1600 89.9 Fb 0.2 4 1406 1600 87.9 F7 0.4 4 2324 2400 96.8

Lahore P 0.1 4 1225 1330 92.1 LaPrnZ F, 0.1 3 1084 1600 67.7

0.2 4 1460 1600 91.2 F2 0.2 4 3007 3200 93.9 F3 0.4 4 3898 4000 97.4 F4 - F5 0.1 2 1511 1600 94.4 F6 0.2 2 1488 1600 93.0 F7 0.2 2 1331 1MjO 83.2 FB 0.2 2 1529 1600 95.5

2 336 800 42.0 Fp 0.02 PmSt F, 0.02 2 513 1000 51.3

F2 0.04 2 2884 2986 96.6 F, 0.04 2 744 1255 59.3 F4 0.04 2 543 1650 32.9

Conc. (mg/l) Time (h) -~

- - - -

- - - -

Reduced susceptibility to peniiethrin in An.stephensi 41

00-

70- - - s - C

0 .K 2 50- e - Y

30-

20-

10-

5-

I 95

-4 1 I I 1 1 1 1 1 I S I S a l 0.1 0.2 0.5 1 2 5 10 2 0 5 0 100

Concentration (rng/lJ

FIG. 1. DDT bioassays, 2 h exposure.

gested that they were not composed purely of homozygous susceptible individuals. In order to obtain maximum DDT susceptibility, the LASS strain was subjected to single family sib selec- tion: fourth instar larvae from forty-three lines were exposed to DDT at 4 mg/l. The ranges of response resulting from 2 h or 24 h exposures were wide and showed non-normal distribution. confirming the heterogeneity of the parental population. Unexposed sibs from the most susceptible lines, giving greater than 75% knockdown in 2 h and 98% or greater kill in 24 h, were selected and combined to form a new strain termed London Susceptible (LS). The LS responses to 2 h (Fig. 1) or 24 h (Fig. 2) exposure to DDT were typical of a homozygous susceptible strain, since a straight log-dose pro- bit line with a relatively steep slope could be fitted to the points. The ECm value of 0.954 mg/l (EC~S, 2.4) was similar to those for susceptible strains reported by Davidson (1958) and Omer et al. (1980), compares favourabiy with Davidson & Jackson (1961) and Rongsriyam & Busvine (1979, but 10-20 times higher than the values reported by Singh & Mohan (1965) and Mohan & Singh (1965).

Relative susceptibiliry of selected lines

Fig. 3 illustrates the change in susceptibility to knockdown (2 h response) by permethrin induced by selection of PmSt, a difference of 17-fold when compared to LS. Fig. 4, however, illustrates the very small change (1.6-fold) in susceptibility to kill (24 h response). Consider- able recovery from knockdown in LS and Lahore at all concentrations tested, particularly the lower ones, contrasts with the increase in response with time of exposure observed with the PmSt strain, which together produces the apparent similarities between the strains after 24 h exposure.

Larval responses of each strain to DDT are shown in Fig. 1 (2 h exposure) and Fig. 2 (24 h exposure). The susceptible LS line showed no signs of recovery between 2 h and 24 h. The resistant strains, however, produced plateaux in the response above approximately 20 mg/l at 24 h or 40 mg/l at 2 h. Some recovery was also observed during exposure ‘at these concentra- tions. Saturation of the insecticide suspension was indicated by an increasing opacity above 5 mg/l and, at high concentrations, crystalliza-

42 C. A. Malcolm

90 951 / - g

z7: 0.1

80-

70-

- g - 50- - .- Y

Concentrat ion (mgl I)

FIG. 2. DDT bioassays, 24 h exposure.

. 0

0!2 ' as' I 1 a $ I ' I ' 10 20 ' ' s'o""' loo Concentrat ion (mgl I)

FIG. 2. DDT bioassays, 24 h exposure.

tion of DDT on the bottom and sides of the con- tainer. The effective increase in concentration above the target concentrations of 20-40 mg/l was probably negligible and variable, depending on exact conditions of temperature and humidity. This would explain the observed levelling off in the graph of mortality against target concentration. It also implies that homo- zygosity for DDT resistance could not be con- firmed directly by larval bioassay, nor could accurate EC, or ECp, values be calculated.

Resistant strains (including Karachi and BARR, not shown in Figs. 1 or 2) all showed similar levels of response to DDT, about 30-40- fold resistance to kill (after 24 h) and 8-10-fold to knockdown (after 2 h). In particular, there was very little difference in DDT response (Figs. 1 and 2) between PmSt (permethrin selected for twelve generations) and Lahore (DDT resistant strain from which PmSt was derived) at either exposure time, indicating that at least the major component of DDT resistance is unrelated to any factors responsible for changes in per- methrin susceptibility (Table 2; Figs. 3 and 4).

A cross between PmSt and LS produced Fl progeny showing an intermediate level of response to pemiethrin, but the overlap with

parental strains meant that continuing the gene- tic analysis without the aid of mutant markers would have yielded little information. Therefore the Blro strain, which was similar to Lahore in its responses to permethrin and DDT (Figs. la), was selected for full permethrin susceptibility. At the same time an attempt was made to examine further any relationship with DDT resistance. Mass selection of Blro, by exposing fourth instar larvae to DDT at 40 mg/l and removing those showing greatest susceptibility to knockdown, was unsuccessful because, although the larvae generally recovered, most of the selected insects died during emergence. The selection procedure was improved by exposing the larvae at 20°C and returning them to 28°C for recovery. The survivors did not produce a line showing different responses from the parent strain. Single family selection was then under- taken using a combination of DDT and permethrin.

The FI progenies of fifty-five single females of Blro were exposed to DDT at 40 mg/l for 24 h. Based on their responses after 2 and 24 h, the families were roughly divided into three groups: (a) showing recovery from knockdown, (b) showing no recovery but a moderate increase in

Reduced susceptibility to permethrin in An.stephensi 43

0

Concentration irnglll

FIG. 3. Permethrin bioassays, 2 h exposure.

9 5

9 0

8 0

7 0

- s -

5 0 - - .- Y

30

2 0

10.

5

C Concentration (mg1 I)

FIG. 4. Permethrin bioassays, 24 h exposure.

44 C. A. Malcolm

response between 2 and 24 h, and (c) showing a marked increase in response between 2 and 24 h. Unexposed members of families from each group were kept and mated amongst themselves.

Three insecticide tests were performed on the Fz larvae of each family: (1) 24 h exposure to DDT at 4 mg/l, the dose which produces 99% kill in the susceptible LS strain; (2) 24 h exposure to DDT at 40 mg/l, with responses scored at 2 and 24 h; (3) 2 h exposure to per- methrin at 0.04 mg/l, the dose which produces 99% knockdown in the most susceptible strains.

The groupings established in the F, were not reproduced in the F2; most families showed a response similar to group b above. Of the fifteen families tested, five showed reduced suscep- tibility to knockdown by permethrin (2 h exposure) coupled with reduced responses to DDT at 4 mg/l (24 h exposure) and at 40 mg/l (2 and 24 h exposure). Two families (Nos. 9 and 43) were fully susceptible to permethrin. but showed relatively low responses to DDT at 4 mg/l. Both were more susceptible than all the other families to DDT at 40 mg/l (both 2 h and 24 h exposures). Most of the families gave a very similar repsonse to 4 mg/l DDT and when taken together with the 40 mg/l results, suggested that most if not all of the families were homo- zygous for the major gene for DDT resistance.

There were no grounds for linking the major source of DDT resistance with reduced suscep- tibility to knockdown by permethrin. A relation- ship between the latter and an additional tolerance to DDT was suggested by slightly lower responses to DDT by larvae of the five families which showed reduced responses to per- methrin but, from direct observations, factors

such as size and precise age of larvae influence the response to DDT and thus a definite correla- tion could not be confirmed.

Unexposed sibs from families 9 and 43, which were susceptible to permethrin but apparently homozygous for DDT resistance, were used to produce a new strain termed BlroDDT. This strain gave a response to permethrin similar to that of the DDT susceptible LS (Figs. 3 and 4). BlroDDT resistance to DDT was slightly less than the parent strain Blro, but still similar to the other resistant strains BARR, Karachi, Lahore and PmSt (Figs. 1 and 2).

Genetic analysis of reduced susceptibility to knockdown by permethrin

PmSt survivors from 2 h exposure to per- methrin at 0.04 mg/l (the dose producing approximately 99% knockdown in fully suscep- tible strains) were crossed reciprocally to BlroDDT. FI larvae from one of the crosses were exposed to a range of concentrations of permethrin, giving an intermediate response between the two parental strains, the range overlapping each considerably (Fig. 5) . Larvae from both reciprocal crosses were exposed to permethrin at 0.04 mg/l and the survivors outcrossed to LS, or backcrossed to BlroDDT. This selection produced a very similar response from both F,s (Table 3) indicating no involve- mcnt of sex-linked factors.

F, survivors were crossed reciprocally with LS, the progeny sorted into the four chromo- some I1 and 111 marker categories and exposed separately to permethrin. Both Bland Stp show intermediate expression in the heterozygote, easily distinguishable from wild type homo-

TABLE 3. Genetic analysis of reduced susceptibility to knockdown. Results of exposure to perrnethrin at 0.04 mg/l. Per- centages of knockdown and kill (numbers tested). MBI, intermedi- ate black phenotype (BI heterozygotes); BI, full black phenotype (BlIBl homozygotes).

Cross %Knockdown, %Kill, Total 2 h 24 h tested

(PmStxBlroDDT) F, 60.3 (752) - 1246

(FlxBlroDDT)B, 96.7 (145) 50.7 (76) 150 BI 98.7 (148) 50.7 (76) 150

(BlroDDTxPmSt)Fl 60.6 (744) - 1228

(BlroDDTxF1)B, MBI 91.3 (137) 50.0 (75) 150 B1 96.7 (145) 51.3 (77) 150

Reduced susceptibility to pentiethrin in An.stephen.si 45

90 -

80-

I 70- s - c I 50- '0 1 0 -

2 * 30-

2 0-

0-

5-

zygotes. The ratio of phenotypes did not differ significantly from the expected 1:1:1:1 on the hypothesis of no linkage. Fig. 5 shows results of permethrin bioassays

(2 h exposure) conducted with larvae from the above crosses (reciprocal data combined). ECm values for 2 h and 24 h exposure (Table 1) were close to those for the susceptible parental LS strain, but the EC&s and slopes were greater, so that the overall response was rather less than intermediate between the F, and the susceptible parent. Although it was not possible to suppress recombination, linkage between the mutant markers and a major factor for permethrin resistance would produce differences in the response to permethrin between the four marker phenotypes (see Malcolm, 1983a). Actually, the graphs obtained were very similar, from which it is inferred that reduced susceptibility to per- methrin is unlikely to be governed by a major genetic factor with clear-cut effects.

Reciprocal backcrosses to BlroDDT gave similar results. The B1 heterozygotes and homo- zygotes can be distinguished and were exposed

951

F2: +Bl*St

BlroDDT/

?'/

/

separately to permethrin at 0.04 mg/l, the con- centration which produces approximately 99% knockdown in a fully susceptible strain. The Stp heterozygotes and homozygotes could not be distinguished in a homozygous Bl background, so any relationships between either Stp genotype and differences in response to permethrin could not be examined. As the F, results at 0.04 mg/l permethrin were very nearly 60% knockdown (Table 3), a result close to 80% knockdown would be expected for the backcross progenies in the case of monofactorial inheritance. In fact the results obtained (Table 3) were very close to complete susceptibility, with no evidence of a significant distortion from a 1:l ratio of Bl phenotypes in the larvae responding, or the lar- vae unaffected. at either the 2 h or 24 h exposure times, thus indicating that chromo- some 111 did not have a specific influence on the results.

The data can be interpreted as showing inheri- tance due to several interacting genes, collec- tively producing reduced susceptibility to knockdown by perrnethrin, greater than the sum

a I , I I I I I I , I I I 1 I I I I

OlOOl 01002 ' ' olods' ' blo1 0.02 olos 0.1 0.2 0.5 1

Concentration (mgll)

FIG. 5. Genetic analysis of reduced susceptibility to knockdown by permethrin (2 h exposure). YzBl indicates intermediate (heterozygous) black larva phenotype; lhSt indicates intermediate (heterozygous) striped larva phenotype.

46 C. A. Malcolm

of their individual effects and maintained by selection pressure, but quickly lost in the absence of selection or when outcrossed. In par- ticular this would explain why the second outcross to LS, and the backcross to BlroDDT produced responses which were much closer to full susceptibility than to the intermediate response of the FI.

These results contrast those of Omer et al. (1980) and clearly demonstrate that the DDT/pyrethroid resistance mechanism found in their strain is not in the strain selected in the present study, nor is it likely to be present in the other two DDT-resistant strains Karachi or BARR. Instead there appears to be a major DDT resistance factor which does not confer cross-resistance to pyrethroids, and additional multigenic factors conferring a very significant reduction in susceptibility t o knockdown by per- methrin and probably a minor reduction in susceptibility to DDT. This situation is much more similar to that reported by Mohan & Singh (1965) and Singh & Mohan (1965).

The absence of a distinct permethrin resist- ance mechanism in the strains studied, par- ticularly Lahore and Karachi, which have been collected relatively recently from widely separ- ate areas of Pakistan provides better prospects for the future use of pyrethroids against An . stephensi.

Acknowledgments

The author is particularly grateful to Martien Visser for technical assistance, ta Dr A. Ringoet, Director of ITAL, for the provision of facilities, and to the members of the Insect Genetics Group for their help and encourage- ment. The author was supported by a SERC/NATO Postdoctoral Fellowship throughout the duration of this work.

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Accepted 8 July 1987