JUNE 1991 Muerruor RESISTANcE IN Sppclos B AND C

RESISTANCEIN CULICIFACIES SIBLING SPECIES B AND C TO MALATHION IN ANDHRA PRADESH AND GUJARAT STATES, INDIA

K. RAGHAVENDRA,I K. VASANTHA,I S. K. SUBBARAO,I M. K. K. PILLAI, AND V. P. SHARMA1

ABSTRACT. Studies conducted in Warangal, Khammam and Mahabubnagar districts in Andhra Pradeshand Surat district in Gujarat have revealedthat A nophelesculicifacies sensu Into (s.1.) populations were resistant to malathion. In the absenceof indoor spraying of malathion in public health programs in the 3 districts of Andhra Pradesh, resistance is attributed to the extensive use of pesticides in agriculture. SpeciesB and C were sympatric in all areassurveyed, an4 both the spe_cieswere resistant to^ -llathiotr. In most of the surveys-cairied out in Mahabubnagar, Khammam and Warangal, levels of resistancewere higher in speciesC than in B. In Mahabubnagardistrict an increasein resistancefrom 5.5to 64% wasobJerved from 1985to 1987in An. culicifaciess.l. The proportionof speciesC waslow in the initial 2 surveys,and in the later surveys the proportion was almost equal to that of speciesB; the resistancelevel was also significantly highei than in speciesB. In Surat district, where resistanceranged from ?4 to 93%,the levelof resistancein the 2 specieswas almostthe same.

INTRODUCTION to Plastnodiuminfection (Subbaraoet al. 1988c). A distinct pattern of distribution of the 4 sibling Anophelesculicifacies sensu lnto (s.1.)is the speciesoccurs in India (Subbaraoet al. 1988a). major vector of in the rural and periur- These findings encouragedus to study the re- ban areasof India and neighboringcountries. In sponsesof sibling speciesto other . India, insecticidessuch as DDT, HCH and mal- The present study was carried out in Surat athion are being sprayed by the National Ma- district in Gujarat and in Warangal, Khammam laria Eradication Programme (NMEP) to inter- and Mahabubnagardistricts in Andrha Pradesh. rupt the transmission of malaria. In spite of the We examined the role of indoor spraying of continuoususe of insecticides,malaria incidence malathion and usageof pesticidesin agriculture remains high in several parts of the country. A in selecting for malathion resistancein An. cul- major factor contributing to the resurgenceof icifacies s.1., and to detect differences in the malaria was the development of resistance to responsesof its sympatric sibling speciesB and insecticidesin the vectorspecies (Sharma 1984), C to malathion. especiallyin An. culicifaciess.l. (Bang 1985).In this species,resistance to DDT and HCH is METHODS reported for most parts of the country and to MATERIALS AND malathion in the statesof Gujarat, Maharashtra In the state of Gujarat, Surat district, and in and Madhya Pradesh. Malathion resistance in the state of Andhra Pradesh,Warangal, Kham- An. culicifaciess.l. in Andhra Pradeshstate is mam and Mahabubnagar districts, 2-3 villages consideredprimarily due to the use of agricul- per district were selectedfor collec- tural pesticides(Subbarao 1979). tions. These were riverine villageswith similar Anophelesculicifacies Giles has been cytotax- ecology.Female An. culicifaciess.l. resting on onomically identified as a complex of 4 sibling the walls, roofs and other objects in human species:A, B (Green and Miles 1980),C (Sub- dwellings and cattle sheds were collected with baraoet al. 1983)and D (Subbaraoet al. 1988a, an aspirator and a flashlight. The mosquitoes Sugunaet al. 1989).In northern India, where were brought to the laboratory within an hour HCH has been sprayedfor the past 10 yearsor of collection in 30 x 30 x 30 cm cloth cages so, the 2 sympatric sibling species,A and B of covered with wet towels. The resistance status the An. culicifaciescomplex, differ in their sus- of field-collectedmosquitoes to malathion was ceptibility to DDT both in the laboratory and determined by exposing An. culicifaciess.l. in field, with speciesA being more susceptiblethan batches of 2O-25 mosquitoesto 5% malathion- speciesB (Subbaraoet al. 1988b).These 2 sib- impregnatedpapers for t h. Adults were held for ling speciesdiffer in their seasonalprevalence a24-hrecovery period before recordingthe mor- (Subbaraoet al. 1987), host feeding patterns talities [World Health Organization (WHO) (Joshi et al. 1988) and in their susceptibilities 1981]. Mosquitoes were also exposed to the WHo-recommended diagnbstic dose for differ- ent intervalsto plot dose-mortalityresponse. To to mal- 1Malaria ResearchCentre, (ICMR), 22-ShamNath study the susceptibility of sibling species Marg,Delhi-110 054. India. athion, semi-gravid females with ovaries in zDepartment of Zoology, University of Delhi, Christophers' stage III were used. Immediately Delhi-110007, India. after insecticideexposure, ovaries were removed JoURNAL oF THE AMERToANMosqutto CoNrnor, Assocretror.r Vor..7,No.2

Table 1. Response of Anophelesculicifacies s.l. and its sibling speciesfrom Andhra Pradeshand Guiarat states to E% malathion impregnatedpapers. ^ Semi-gravidAn. culicit'actes s.l. Predominantsibling All gonotrophic stages species Survivals(%) of of An. c. s.l.* composition** STATE [% survival/no. District treated* sp.F sp{ Chi-square Period o/o of survey (mean + SD)l %B C \/ \, values*** ANDHRA PRADESH Mahabubnagar Feb. 1985 5.5/100 84.9(79) 15.1(14) 0 (0) 0 (0) (6.4+ 2.2) Apr. 1986 23/79 98.3(58) 1.7(1) 23(r7) 0 (0) (23.0+ 17.9) Dec. 1986 58/165 52.3(56) 47.7(51) 24(53) 96 (48) -f 49.82 (59.3 10.5) P < 0.001 Nov. 1987 64/585 50.4 (232) 4e.6 (228) 51.5(r67) 94 (r41) 6.55 (65.0+ 14.2) P < 0.02 Khammam Feb. 1985 53/230 36.3(29) 63.7(51) 32 (25) 65 (46) -f 5.91 (52.7 11.3) P < 0.02 Warangal Dec. 1985 90/225 29 (20) 7r (49) 60 (20) s2 (49) 7.90 (83.4+ 8.2) P < 0.01 Apr. 1986 43/r22 57.8(37) 42.2(27) 100(9) 87.5(8) 0.003n.s. (17.8+ 13.1) Dec. 1986 5r/727 37.7(20\ 62.3(33) 52 (r9) 94 (32) 9.61 (48.8+ 20.5) P < 0.01 Nov. 1987 97/r34 32.7 (r8) 67.3(37) 66.6(3) 88.9(9) 0.41n.s. (96.0+ 2.8) GUJARAT Surat Apr. 1984 87.4163^^^ 6.3(4) 93.7(59) 75 (4) 88 (59) .00015n.s. (87.7+ 5.3) March 1986 93/100 11.5(3) 88.5(23) 100(1) 100(18) (93+ 3.3) Apr. 1987 74/226 42.2(43) 57.8(5e) 73 (15) 93 (15) 0.96n.s. (76.5+ 11.9) Nov. 1987 86/469 33.9 (292) 66.1(569) 94 (50) 97 (77) 0.25n.s. (87.4-+ r5.2) * 20-25 Art culicifacies s.L were treated per replicate. After 1-h treatment, ovaries from semi-gravid fe-ut". were removed to establish the species composition ** in dead and alive mosquitoes. No. in parenthesesare actual number of mosquitoes identified for sibling species.Data include both -treatedand untreated populations. l** Chi-squarevalues tested for responsesof siblingspecies. ^^Semi-gravid femalesin a collection generally uary fio- about 0 to 70Vodepending on the season ^^^Tt?l numtter of mosquitoesidentified for the speciesboth dead and alive mosquitoes. Only semi-gravidmosquitoes were selectivelyexposed.

from both the live and the knocked-downmos- Hunt (1980). The diagnostic inversion geno- quitoes and stored in vials with modified Car- types, recognizableon the polytene chromo- noy's fixative (1:3,glacial acetic acid:methanol) somes for the identification of sibling species, for chromosomepreparation to be used for the were: speciesB-Xab 2gr*h'and speciesC-Xab identification of sibling species.The standard 2c'+ hr (Subbaraoet al. 1983). procedure of holding the mosquitoes for 24 h post-exposure to the insecticide was not fol- RESULTS lowed, as the mosquitoes would have gone be- yond the suitable stagefor speciesidentification. Malathion resistance in An. culicifacies s.l. Polytene chromosomeswere prepared from the was observedin all the collections made in Gu- fixed ovaries using the method of Green and jarat and Andhra Pradesh states, though in JUNE 1991 Mer-lrnroN RESTsTANcErN Spsctns B eNo C 257 varying levels (Table 1). In Andhra Pradesh, DISCUSSION percent survival ranged between 5.5 and 64% in In Surat district, indoor spraying of malathion district and between43 and97% Mahabubnagar was introduced into the Public Health Pro- when exposedto 5% mal- in Warangal district gramme in 1969 to interrupt malaria transmis- papers for t h in bioassay athion-impregnated sion, and in 1973malathion resistancewas re- district, the percent survival tests. In Khammam ported in An. culicifaciess.l. (Rajagopal1977). 1985;in the later surveys' was 537oin February The high percentage of resistance observed in of mosquitoeswere not avail- sufficient numbers the present study further confirms the above tests. Survival able to carry out the bioassay report. In this district, there has beenlittle usage and.93% in Surat district ranged between 74 ofpesticides as there is no cash crop cultivation, (Gujarat). tests, mosqui- In the above bioassay and cereals and millets are cultivated only in after toes were scoredfor mortality immediately Iimited areas. Hence, it seems likely that the were to be the exposureperiod as these selection for resistanceto malathion in An. cul- used for cytological identification of sibling spe- icifaci.ess.l. in these areasis mainly due to indoor cies. However in November 1987 in a few repli- residual spray of malathion in public health cates,mosquitoes were scoredimmediately after programs. The high level of resistanceto mala- the exposureperiod, whereasin others they were thion observedin this specieswas not altered percent scoredafter a 24-h recoveryperiod. The evenwhen malathion was replacedby DDT on survival in replicates with recovery period was an experimental basis in 1986and 1987(NMEP (n: 48% (n: 119),85% (n: 66) and 83.5% directorate, personal communication). 144) in Mahabubnagar, Warangal and Surat In Warangal, Khammam and Mahabubagar districts, respectively, whereas in replicates districts in Andhra Pradesh, which were under without the recoveryperiod, the percent survival a regular spraying of 2 rounds of DDT at I g/ was64, 97 and 86%,respectively. The difference m' since the late 1950s.resistance to malathion in mortalities between the 2 proceduresused is was observedin An. culicifaciess.l. populations; : significantfor Mahabubnagarpopulations (12 i.e.. resistanceto malathion was observedin the = 10.7P < 0.005)and Warangalpopulation (1' absenceof indoor residualspraying. In 1987,3 10.05P < 0.005)whereas it was nonsignificant rounds of malathion were sprayed for the first for Surat (x' : 0.59 n.s.).The percent mortali- time at the rate of 2 g/m2 in Khammam and ties were higher in replicates that were scored Warangal districts. Thus, this study confirms after the recoveryperiod. This could be because the suggestion of Subbarao (1979) that mala- the tests were canied out in the field where thion resistance in An. culicifaciesmight have temperature and humidity could not be con- resulted from the extensive use of pesticides in trolled. agriculture in Andhra Pradesh. For Mahabubnagar populations' LTso and The pesticidesused for the protection of chili LTgo values were 69 min and 656 min, respec- peppers and cotton, the 2 major cash crops in tively, whereasfor Surat populations they were Andhra Pradesh, are mono-crotophos, dime- 109 min and 517 min, respectively. In Kham- thoate, methyl parathion, endosulfan, mala- mam and Warangal, tests for dose-mortality thion, fenthion, cypermethrin, deltamethrin, curye could not be carried out due to nonavail- etc. As agricultural use of malathion is minimal ability of the required number of mosquitoes. in these areas (Directorate of Agriculture, An- For all areasin Andhra Pradesh and Gujarat, dhra Pradesh; personal communication) the speciesB and C were found to be sympatric possible reason for malathion resistance could (Table 1) with the occasionaloccurrence ofother be usage of related compounds with carboxyl- sibling species.The proportion of speciesC was ester bonds. However, An. culicifaciess.l. popu- almost alwayshigher, irrespectiveof the period lations are still susceptibleto other organophos- of survey, in all districts except Mahabubnagar phatesand carbamates,viz, fenthion, fenitroth- (Andhra Pradesh). In Mahabubnagar, during rion and propoxur (unpublisheddata). This sug- the first 2 surveys,the proportion of speciesB geststhat the malathion resistancemechanism was 85-98%, whereas in the later surveys the in An. culicifaciespopulations in the above dis- proportion of speciesC increasedand was al- tricts may be exclusively carboxylesteraseme- most equal to that of speciesB. Malathion re- diated, as has been observedin An. culicifacies sistancewas observedin both species,but the B in Sri Lanka (Herath et al. 1987,Hemingway percent survival of speciesC was found to be et al. 1988),An. stephcnsiListon in Pakistan significantly higher than that of species B in (Hemingway 1983), and An. arabiensis Patton somedistricts of Andhra Pradesh(12 valuesin in the Sudan (Hemingway1985). Table 1). In Surat (Gujarat),no significantdif- The low levels of resistanceobserved in Ma- ference was observedbetween the 2 species(1' habubnagardistrict in Andhra Pradesh in 1985 valuesin Table 1). and its gradual increase in subsequentsurveys, 258 JounNer,oF THEAlrpnrceN Moseurro CoNrRor,AssocreuoN VoL. 7, No. 2

in contrast to the high levels observed in the (Herath et al. 1987).At presentthere is no such surveys in Khammam and Warangal, suggest policy in India. This study illustrates the impor- that the developmentof resistancein Mahabub- tance of careful selectionof pesticidesfor both nagar populations is probably of recent origin. agriculture and public health. In Warangal and Khammam, cash crop culti- vation has been done since the 19b0sand large areas are under cultivation. In Mahabubnagar, ACKNOWLEDGMENTS practice this was introduced in the early 1980s The authors thank Mr. Y. P. Chawla, Mr. in limited areas. Differences in acreagesunder K. B. Masiwal and Mr. H. D. Joshi for assistine cultivation, amount of insecticidesused and du- in field work. The technical assistanceof Mr. ration of selection pressureare common factors R. S. Saini in the identification of the sibline affecting resistance, as has been well docu- speciesis acknowledged. mented in An. albimanusWied. in El Salvador (Georghiouet aI. 1974). REFERENCES In Warangaland Surat districts,the levelsof CITED resistance developed in both the species was Bang,Y. H. 1985.Implications in the controlof ma- high. In Mahabubnagar,where the development laria vectorswith insecticidesin tropicalcountries of resistanceto malathion is a recentphenome- of SoutheastAsia. J. Commun.Dis. 1?:199-218. non, the proportion of speciesC in 2 initial Breeland, S. G., J. W. Kliewer, J. R. Austin and C. W. surveys was low, and hence responseof this Miller. 1970. Observations on malathion resistant speciesto the insecticidecould not be ascer- adults of Anopheles albimanus Wiedemann in coastal El tained. But in the 2 later surveys,the proportion Salvador. Bull. W. H. O.43:627-681. Georghiou, G. P., V. Ariaratnam, H. Ayad of speciesC in the population and B. was almost equal Betzios. 1974. Present status of research on resist- to B, and the percentresistance in speciesC was ance to carbamate and organophosphorous insecti- 96 and 947o, respectively. The high resistance cide in Anopheles albimanus WHO/VBC/?4.508. Ievelsobserved in speciesC comparedwith that Georghiou, G. P., V. Ariaratnam and S. G. Breeland. in speciesB in the later 2 surveys suggestthat 1972. Development of resistance to carbamates and developmentof resistancein speciesC probably organophosphate compounds in Anopheles albi- is at a faster rate than in speciesB. manus in nature. Bull. W. H. O. 46:551-554. Green, C. A. and R. H. Hunt. Since all the study areas have a similar ecol- 1980. Interpretation of variation in ovarian polytene chromosomes of ogy, it may be assumed that in all the areas Anopheles funestus Giles, A. parensis Gillies and A. speciesB could havebeen the predominantspe- aruni (?). 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