Ansari, Jamil A.

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Title Studies on the Isolation and Genetic Nature of SpecificInsecticide Resistance in HousefliesAuthor(s)Ansari, Jamil A.Citation防虫科学 (1969), 34(2): 70-78Issue Date1969-05-31URL http://hdl.handle.net/2433/158574RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityStudies on the Isolation and Genetic Nature of Specific Insecticide Resistance in HousefliesIarnil A. Ansari (Department of Zoology, 'Aligarh Muslim University, Aligarh, India) ReceivedApril 5, 1969. Botyu-Kagaku 34, 70, 1969).9. DDT, T.of-Jvr'J~mmtt-1'.:::cJ~.:::cO)~~c!::.O{:OO)ilffi~ttW. JamilA.Ansari(Department of Zoology, Aligarh Muslim University, Aligarh, India) 43. 4. 5. ~1lJ1t!l~O)~!lJ.llUIl::~iv-r:j[Et/Ltto)-1.:r./-(.:r. Musca domestica nebulo ~"?< 7.>t:1b, DDT c.7"-1-;\1 r ~ ;"-"l'3CliI~i'l;Jtt~ 30ftfitJ:-? tz: 'CO)Ra5l!jj!U~1l'JI~:Xtv-rOO ;j;)I~:j[Etictto) -1 i/-(.:r.j;q(}Gn tz, I:.O):j[Etlctt -1 x.r« .:r.1~~~tto)-1 .:r./-(.:r. L'lJ:! v3CM~Wl!& l>, DDT ~ 7.> ~'I;:t; 7" -1 -;\1 r .);"-L'~ttv~M~ N~!IJ.~~'Cn~n:j[EMttO)~M~N~.N~MO)3CMI~ J: ~. DDT :j[EMttl;:t;!l!-~.:rI~ J: 7.> ;I z> T ;\lill~L'~ ~. 7" -1 -;\1 r ~ ;.,-:j[Ebtt'l:~.~0)~.:r~J:~m~3n, OO;~~9~~.1bGn~~~~.IntroductionThe failure of DDT to acheive effective houseflycontrol due to the development of resistance hasled to the introduction of some newer insecticides.But the housefly which has already developedresistance against DDT was able to developresistance to subsequent insecticides which suc-ceeded it, producing thereby the so-called 'Multipleinsecticide resistant strain'. Occurrence of sucha strain has been reported by Schoof et al (1951),Hess (1952), Keiding (1953) and Goodwin-Baileyet al (1954). from various parts of the world.Nearly everywhere the development of resistancehas progressed to a marked degree, more or lessat the verge of immunity. Such a developmentof multiple insecticide resistance by a single strainof housefly to a number of chemically unrelatedcompounds poses a serious problem for the insectcontrol in general and the housefly control inparticular with regard to the progress of humanhealth and welfare. To overcome such a menacingproblem a knowledge of the type of inheritanceto a certain insecticide is very essential as itmay offer valuable informations during controloperations. The fact that predictions or interpre-tations of rates of development or regression ofresistance are untenable without any under-standing of the mode of inheritance or thecharacter of resistance, is another aspect related insolving this problem. Furthermore, a quantitativeanalysis of the relative frequency of the genesfor inheritance of resistance is obtained by theknowledge of the phenotypic response of the70various genotypes to a certain insecticide.There are enough published data on the in-heritance of DDT and Dieldrin resistance in thehousefly, but all are very divergent. Bruce andDecker (1950) reported a polygenic factor respon-sible for the hereditary transmission of DDT-resistance. Harison (1951) on the other hand founda single gene inheritance for DDT resistance.Keiding (1953) supported the view of Harison. Adominant gene for resistance to DDT has beenreported in houseflies by Lichtwardt (1956).Dominant gene responsible for DDT-resistancehave also been reported by Oshima and Hiroyoshi(1956). Milani (1957) has proposed that DDT-resistance in the housefly may be controled by asingle gene which is partially dominant for thecharacteristic. There are also contrary reportson the inheritance of resistance to Dieldrin in thehousefly. Resistance to Dieldrin has been shownto be monofactorial by Georghiou, March andPrinty (1963), Lanna (1963), Milani(1963), Guneidyand Buswine (1964). Whereas Abdullah (1961),Rahman and Khan (1964) published results whichshow that inheritance of Dieldrin resistance ispolygenic.Under these circumstances revaluation, however,of certain of the study is very essential. It is,therefore, not unlikely that repitition of the studyutilizing homogeneous strains of the housefly anda full range of closely spaced dosages, may yieldmore complete data which will necessitate reap-praisal of the previous interpretations. With thispurpose the present study has been undertaken.It deals with the development of a multipleW ill n·~.f: m 34 ~-I1resistant strain in the laboratory so as to providematerial for the isolation of specific resistantstrains for genetical studies.Material and MethodThe housefly, Musca domestica nebulo was usedas the test insect. Multiple insecticide resistantstrain was raised from the parent susceptiblestock by the selection pressure of DDT andDieldrin. The stock culture of the susceptiblehouseflies has never been exposed to any insecticide.AIl the strains utilized in the experiment werereared on milk soaked cotton at a temperature2S"±1"C and humidity between 70-S096. Theinsecticides used were pure DDT and Dieldrinobtained from the SheIl International ChemicalCompany, London. The formulations used wereprepared in Acetone.The experimental technique as adopted for thedevelopment of multiple resistant strain mainlyconsisted of topical application of the insecticideto the thorax of the houseflies and recording theirpercentage mortality after 24hours. The 4-dayold flies unmated of each sex were exposed toDDT and Dieldrin in alternate generations. Theconcentration of the insecticide was graduallyincreased to obtain a kiII of SO-9096. In the 30thgeneration when resistance appeared to havefairly stabilized, LCo• value was determined withthe help of log-concentration regression lines. Forcomparision susceptible strain was also similarlytested.Isolation of specific resistant strain was carriedout by a combination of back-cross and selectivebreeding. Multiple resistant flies were crossedwith those of the susceptible, and the progenywas divided into two colonies. One of the colonywas selected for resistance to DDT and the otherfor resistance to Dieldrin. After the selectionpressure the surviving males from respectivecolonies were back-crossed with the susceptiblefemales and the progeny was selected again. Theprocess continued for six back generations and ineach generation LCo• of the houseflies selectedfor resistance, together with their cross-tolerancewas determined.Inheritance studies were made by single pairsof reciprocal cross-mating of male and femaleparents of susceptible and isolated DDT andDieldrin resistant strains. A portion of the F 1generation thus obtained was inbred to produceF, generation and the rest were used to measuretheir response to graded doses of the insecticides.AIl the individuals of F2 generation were testedfor their level of resistance in the same manner.The concentrations used were sufficient to coverthe complete range of susceptibility from the LCo•with the most susceptible strain to nearly anLCo• with the most highly resistant strain.For convenience the various strains of thehouseflies will be reffered to hereafter as S forsusceptible strain, Multi-R, DDT-R and Dld-Rrespectively for Multiple, DDT and Dieldrinresistant strains.Results and DiscussionDevelopment of multiple resistant strain:LCo• values of Multi-R and S houseflies havebeen determined and presented in the Table (I).Resistant ratio indicates that the process ofdevelopment of resistance has greatly intensifiedby submitting the houseflies to DDT and Dieldrinin alternate generations. The houseflies havedeveloped enough resistance to be utilized forexperimental purposes.Isolation of specific strains:LCo• values of parents, hybrid and offspringfrom backcrosses after selection of hybrids withDDT and Dieldrin have been presented in theTable (1) and illustrated in Figures (1,2,3 and 4).The F1 progeny of the reciprocal crosses betweenthe Multi-R and S strain were identical with eachother as regard to their DDT and Dieldrinresistance, which was intermediate between thatof their parents. Either group of the hybridprogeny after being exposed to selection pressurefrom DDT to Dieldrin, the male survival wasback-crossed with female susceptible strain givingtwo first back-groups under DDT":pressure andtwo first back-cross groups under Dieldrin-pressure. Since no significant difference could befound between each of the two groups in theirsusceptibility to DDT or Dieldrin, only one of thegroup in each case was choosen to produce furtherback-cross generations. In the sixth back-crossthe colony DDT-R, selected for DDT, maintained71~ !tl M ~ ~ 34 ~-IITable 1. Dosage mortality response of parents, hybrids and offsprings from back-crossesafter selection of hybrids with DDT or Dieldrin.Generation Strain Selection DDT DieldrinPressure - LC50 R/N LC50 R7N-Parent Susceptible 0.32 0.031Parent Multi-R 170.00 531 2.55 82F1 ci"Rx,?S 51. 0 159 1.9 61!fRxci"S 44.0 137 1.82 581st Back Cross ci"(ci"RX,?S)X,?S DDT 36.0 112 0.58 18ci" (,?Rxci"S) x es DDT 31. 5 98 0.51 16ci" (ci"Rx !fS) X ,?S Dieldrin 19.9 62 0.75 24ci" (,?RXci"S) X ,?S Dieldrin 17.7 55 0.76 242nd Back Cross ci" (ci"RXci"S) x ss DDT 17.5 54 0.48 15ci" (ci"Rx ,?S) X ,?S Dieldrin 15.5 48 0.65 203rd Back Cross ci" (ci"Rx ,?S) X ,?S DDT 12.5 39 0.35 11ci" (ci"Rx !fS) X ,?S Dieldrin 11.9 37 0.45 144th Back Cross ci" (ci"Rx !fS) X ,?S DDT 26.5 82 0.31 10ci"(ci"RX,?S)X'?S Dieldrin 8.4 26 1.1 355th Back Cross ci" (ci"Rx ,?S) X ,?S DDT 62.5 195 0.21 6ci" (ci"Rx ,?S) x ss Dieldrin 5.6 17 1.5 486th Back Cross ci" (ci"Rx ,?S) X ,?S DDT 110.0 343 0.137 4ci"(ci"Rx,?S)x!fS Dieldrin 3.5 10 2.1 67100 FIG. I9080w(!) 70« 60I-Zw 50U 400:Wa. 302010.125 .2552 4 6 10CONCENTRATION20 40 60 10072Fig. 1. Changes in the dosage-mortality response against DDT of the progeny fromback crosses after selection of the hybrid with DDT.I\1j m n tr. m 34 1J-II1009080w 70C)60«...z 50w40Ua::W 300..2010.03125.0625 .125 .25 .5CONCENTRATION2 4Fig. 2. Changes in the dosage-mortality response against Dieldrin of the progeny fromback crosses after selection of the hybrids with DDT.1009080w 70C)« 60...Z 50WU 40a::w 300..201040 6020.5.125 .25 I 2 4 6 10CONCENTRATIONFig. 3. Changes in the dosage-mortality response against DDT of the progeny fromback crosses after selection of the hybrids with Dieldrin.73100 FIG.49080701LJ 60C>-c 50I-Z 401LJU 30a::1LJ 20a..10.03125.0625 .125 .25 .5CONCENTRATION2 4 6Fig. 4. Changes in the dosage mortality response against Dieldrin of the progenyfrom back crosses after selection of the hybrids with Dieldrin.Jr.~ /\ "1-\~, ~~+< \0,_- + / ~., -o~ ~., ......... lil' ~, e__ '"',+__ -;:-c'o OOT-R0-----0:::-::--./ 01.0 -80' - --e -__e_ _ ___ -.400 f1G.5375350325300275Q 250I-ri 225257550w 200U~ 175l-V) 150V)~ 125100P F1 IBC 2BC 3BC 4BC 5BC 6BCBACK CROSS GENERATIONFig. 5. Resistance pattern of the specific insecticide resistant strains and theircross-resistance characteristics.74JiJJ ill fl !'f. m 34 ~-IIits DDT-resistance in the strain; while therepeated back crossing with S-houseflies loweredDieldrin resistance practically to normal (Fig. 5).Similarly the colony Dld-R, selected for Dieldrin,developed resistance against Dieldrin and at thesame time its DDT-resistance (Fig. 5). Both DDT-Rand Dld-R strains have developed significantresistance and their response have been foundto be specific against those insecticides whichwere used for their selection. Any increase intheir cross tolerance against other hydrocarboninsecticides and cyclodiene group of compoundscould not be observed. After the selection pro-cedure described DDT-R and Dld-R strains weremaintained in isolation without further treatment.Inheritance of resistance:Summarized results of test on groups male andfemale in parental, F1 and Fr generations for DDTand Dieldrin resistance were given in Tables (2and 3) and illustrated in Figures (6,7,8 and 9).There is no evidence for a sex-linked factor ininheritance either in the F1 or the Fr progeniesof the crosses between male and female parentsof susceptible or resistant origin. In view of theclose agreement of the reciprocal crosses it isconcluded that resistance is autosomal, beingtransmitted to the offspring by either parents.Therefore, the results of one of the reciprocalcrosses have been selected and plotted for malesand females separately.Mortalities obtained with F1 houseflies in thecross in which DDT-R parent was used showthat resistance is partially dominant over sus-ceptibility as the houseflies were slightly lessresistant than the resistant parent and far moreresistant than the susceptible ones (Figs. 6 and 7).However, the higher mortality in F1 than theresistant parent indicated that the resistance isTable 2. Percentage mortalities of the parental and their reciprocal crosses.DDT(pg/f1y) IParent strain-Susceptible I DDT-Rt ~ t ~ Io"Sx ~DDT-R I ~SXo"DDT-RF1 hybrid I Frgeneration -F~hYbrid I F·r""""'ge-n....,e---:r-c"a;-iti-c"on.,--t ~ t ~ t ~ t ~0.03125 16.6 7.10.0625 26.6 14.10.125 43.7 24.50.25 49.1 39.9 5.2 2.3 4.8 3.50.5 73.3 51.7 3.2 2.4 6.8 4.5 15.1 10.1 7.2 5.2 14.5 12.21.0 81.6 81.3 7.1 5.3 9.5 8.0 23.5 21.3 10.1 9.2 24.4 24.52.0 91.5 89.6 9.3 6.7 19.1 10.3 52.4 40.5 18.2 11.5 45.6 39.44.0 100.0 96.8 12.5 9.7 22.5 18.5 56.1 45.5 21.5 19.4 57.2 42.66.0 16.9 13.5 27.9 23.2 63.5 47.2 28.5 24.2 64.5 50.0Table 3. Percentage mortalities of the parental and their reciprocal crosses.DId.IParent strain o"Sx~Dld-R I ~Sxo"Dld-R(pg/f1y) Susceptible I Dld-R F~l hybrid IFrgeneration -F1 hYbrid--1Frgenerationt ~ t ~ t ~ t ~ t ~ t ~0.015625 20.1 17.50.03125 59.8 41.3 5.2 6.2 4.5 7.10.0625 82.7 67.5 10.0 7.2 16.5 10.5 11.2 8.5 15.2 11.2O. 125 il00.0 89.6 3.1 2.3 29.9 25.7 31.2 32.5 26.8 24.1 30.5 28.40.25 I 100.0 6.2 4.6 42.5 34.4 61.5 40.5 45.1 36.4 55.6 41.20.5 I 15.9 9.1 70.1 60.1 90.0 78.2 68.5 58.2 88.1 75.41.0 I 33.7 24.3 90.1 71.2 96.2 85.3 85.3 75.4 95.4 84.2,2.0 55.4 41.6 96.2 79.3 100.0 89.1 92.4 80.5 100.0 90.14.0 i 85.3 59.5 100.0 90.4 90.3 100.0 91.3 95.575100908070I.LJ 60l:>4: 50I-Z 40I.LJU 30a:I.LJ 200-10FIG.6.125 .25 .5s2 4 6CONCENTRATIONMALE10 20 40Fig. 6. Dosage-mortality regression lines for male flies of the cross d'SX!f (DDT-R).100 FIG.7908070I.L/ 60o4: 50I-Z 40I.L/U 30a:I.L/ 200-10s FEMALE.125 .25 .5 2 4 6CONCENTRATION10 20 40 60Fig. 7. Dosage-mortality regression lines for female flies of the cross d'SX!f (DDT-R).not completely dominant. The test with the F2 isalmost intermediate between the susceptible andresistant parents. The pattern of the regressionlines for male and female parents, F1 and F2remained essentially the same, with slight inflationtowards greater susceptibility in the males76(Fig. 6). It is not clear whether this is due toreduced vigor in the male offspring or someinfluence of rearing media. The approximate1:2: 1 (1 susceptible homozygote, 2 heterozygote,1 resistant homozygote) segregation to be expectedin monofactorial inheritance is indicated by them ill H !'!- m 34 ~-II100908070w 60C><l: 50I-Z 40wU 300:wa..10DLD-R.015625.03125.0625 .125 .25 .5CONCENTRATION2 4 6Fig. 8. Dosage-mortality regression lines for male flies of the cross 0"S x !f (D1d-R).100908070w 60C><l: 50I-Z 40wU 300:Wa.. 2010FIG.9 's fEMALE4 62.015625.03125.0625 .125 .25 .5CONCENTRATIONFig. 9. Dosage-mortality regression lines for female flies of the cross o"Sx (-'i'- D1d-R).result. It is concluded, therefore, that the DDTresistance is in agreement with a single genehypothesis for inheritance of resistance.The regression lines obtained in the FJ generationof the cross in which Dld-R parent was usedshow a response intermediate to that of theparents (Figs. 8 and 9). It is indicated thatDieldrin resistance is neither dominant norrecessive. The dosage-mortality tests run in F 2generation and the figures obtained show thatin this case also the gene causes an intermediatedegree of resistance but at a slightly lower level77than the F J generation. There is no segregationin the F2 generation which indicates that theprogeny consists of varying genotypes. Thepresence of many genes is further proved by thewider variance of resistance to the insecticideswhich is reflected in F2 regression lines presentinggreater slopes as compared to the F J progeny.SummaryA multiple insecticide resistant strain, in thehousefly Musca domestica nebula, has been de-veloped by selection pressure of DDT and Dieldrinin alternate generations under laboratory conditionso as to provide material for isolation of thedifferent forms of resistance. Tests carried outby repeated back crossing combined with selectionpressure from DDT and Dieldrin produced specificDDT and Dieldrin resistant strains. Further,information on the mode of inheritance of DDTand Dieldrin resistance was obtained by deter-mining the relative toxicity of the insecticide tothe resistant and susceptible strains; the FJprogeny of the reciprocal crosses; F2 generationby inbreeding of the F J generation. The experimentwith DDT resistant houseflies demonstrated apopulation segregating in 1:2: 1 Mendalian ratiowhich indicated that resistance to DDT in thehousefly is inherited as major single factor. Whilethe experiment in which Dieldrin houseflies wereinvolved proved to be multifactorial inheritanceas the F J and F2 generations were both intermediatebetween their parents without showing anysegregation.Acknowledgement: The author is grateful toProf. S. M. Alam, Head, Department of Zoology,for providing facilities and encouragement duringthe course of the present study.ReferencesAbdullah, M.: Inheritance of Dieldrin resistancein the housefly. Jour. Hered, 52 (4), 179-182(1961) .Bruce, W. N., and G. C. Decker.: Housefly toler-ance for insecticides. Soap and SanitaryChemicals. 26(3), 122-125, 145-147 (1950).78Busvine, J. R.: The insecticide potency of BHCand chlorinated cyclodiene compounds andthe significance of resistance to them. Bull.Entomol. Research, 55, 271-288 (1964).Georghiou, G. P., March, R. B. and Printy, G. E.:A study of the genetics of Dieldrin resistancein the housefly, Musca domestica L. Bull.WId. Hlth. Org. 29, 155-165 (1963).Goodwin-Bailey, K. F., and M. Davies.: Occurrenceof Dieldrin resistance in wild Musca domesticaL. in England. Nature, 173, 216-217 (1954).Harison, C. M.: Inheritance of resistance to DDTin the housefly, Musca dcmesiica L. Nature,167, 855-856 (1951).Hess, A. D.: The significance of insecticide re-sistance in vector control programma, Am.J. Trop. Med. Hyg. 1, 371-388 (1952).Keiding, J.: Development of resistance in the fieldand studies of inheritance. Trans. 9th Intern.Congr, Entomol, 2, 340-345 (1953).Lanna, M. T.: The inheritance of insecticide re-sistance. Proceedings XI th Internat. Congressof Genetics. 1, 254 (1963).Lichtwardt, E. T.: Genetics of DDT-resistance ofan inbred line of housefly. Jour. Hered. 47(1), 11-16 (1956).Milani, R.: Genetic research on the resistance ofinsects to the action of toxic substances.Suppl. C, Oct. 1957. Inform. Circ, Probl.,WId. Hlth. Org. Div. Environm. Sanit. (1957).Milani, R.: The genetics of insect resistance toinsecticides. Proceedings Xltli Internat,Congress of Genetics. 1, 255 (1963).Oshima, C., and T. Hiroyoshi.: Genetic studiesof resistance to DDT and Nicotine sulphatein Drosophila virilis. Boiyu-Kagaku. (Bull.Inst. Insect. Control) 21, 65-70 (1956).Rahman, J. S., and N. H. Khan.: Inheritance ofDieldrin resistance in Musca domesiica nebula.Botyu-Kagaku. (Bull. Inst. Insect. Control)29, 19-21 (1964).Schoof, H. F., R. E. Siverly, and J. H. Coffey.:Dieldrin as a chemical control material oncommunity fly control programs. J.econ.Ent. 44, 803--807 (1951).

9.DDT, ディールドリン抵抗性イエバエの分離とその遺伝的性質

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9.DDT, ディールドリン抵抗性イエバエの分離とその遺伝的性質

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