Acetamiprid, a member of the neonicotinoid insecticide family, is a new class of insecticide which has recently entered the market place. It is very selective and provides outstanding control of sucking pests such as aphids and whiteflies of major crops. In the present investigation, dominant lethal test (DLT) is adopted for the evaluation of the genotoxic effects of acetamiprid using Culex quinquefasciatus as an experimental model. In this experiment, the males hatched from larvae treated with LD40 were cross mated with normal females and the results were based on the number of hatched and unhatched eggs laid by these females. The statistical analysis of the results for LD40 treated groups gave the values of 37.526±3.886 as against 5.23±0.77 from the nontreated groups which indicated significant dominant lethality of p&lt;0.01. These results indicated that exposure of pesticides even at small dose level proved deleterious to the genome of mosquito and its subsequent generation

Bansal, Mamta

Chaudhry, Asha

Journal of Applied and Natural Science

171Journal of Applied and Natural Science 3 (2): 171-175 (2011)2008APPLIED    ANDNATURAL SCIENCEFOUNDATIONANSF JANSEvaluation of mutagenic potential of acetamiprid by dominant lethal test onCulex quinquefasciatusMamta Bansal1* and  Asha Chaudhry21Centre of Environment and Vocational Studies, Panjab University, Chandigarh-160014, INDIA2Mosquito Cytogenetics Unit, Department of Zoology, Panjab University, Chandigarh-160014, INDIA*Corresponding author. E-mail: mamta_peehu@yahoo.inAbstract: Acetamiprid, a member of the neonicotinoid insecticide family, is a new class of insecticide which hasrecently entered the market place. It is very selective and provides outstanding control of sucking pests such asaphids and whiteflies of major crops. In the present investigation, dominant lethal test (DLT) is adopted for theevaluation of the genotoxic effects of acetamiprid using Culex quinquefasciatus as an experimental model. In thisexperiment, the males hatched from larvae treated with LD40 were cross mated with normal females and the resultswere based on the number of hatched and unhatched eggs laid by these females. The statistical analysis of theresults for LD40 treated groups gave the values of  37.526±3.886 as against 5.23±0.77 from the nontreated groupswhich indicated significant dominant lethality of p<0.01. These results indicated that exposure of pesticides even atsmall dose level proved deleterious to the genome of mosquito and its subsequent generation.Keywords: Dominant lethality, Acetamiprid, Culex quinquefasciatusISSN : 0974-9411 (Print), 2231-5209 (Online)  All Rights Reserved © Applied and Natural Science Foundation   www.ansfoundation.orgINTRODUCTIONThe neonicotinoids are the newest major group ofinsecticides, which includes acetamiprid, imidacloprid,clothianidin, dinotefuran, nitenpyram, thiacloprid, andthiamethoxam (Tomizawa and Casida, 2005).Neonicotinoids have proved to be ideal alternatives toorganophosphates and carbamates (Elbert et al., 1998)with much lower rate of application as compared totraditionally used insecticides (Schmuck, 2001). In thepresent study, acetamiprid is selected which is usedagainst sucking insects, such as aphids and whiteflieson leafy vegetables, cole crops, citrus, cotton,ornamentals, and fruiting vegetables. Mammals andhumans are exposed to its residues during agriculturalpractice or when it enters the food chain. Once enteredin the body it attacks on the central nervous system ofinsect by binding of acetylcholine, the major excitatoryneurotransmitter in insects to the nAChRs, that furthercause excitation and paralysis, followed by the death ofthe insect. Acetamiprid has relatively low acute andchronic toxicity in mammals with no evidence ofcarcinogenicity, neurotoxicity, mutagenicity and/orendoctrine disruption (USEPA, 2002). Data related to itstoxicity is very scarce. But some of the recent studiesconducted by Kocaman and Topaktas (2007, 2009) onhuman peripheral lymphocytes in culture showed varioustypes of chromosomal aberrations and sister chromatidexchanges. In the last few years several toxicity testingprocedures have been designed, modified and improvedin such a way that they are acceptable to toxicologists(Lu, 1991; Anderson and Conning, 1993). In  recent years,a number of in vivo and in vitro protocols have beensuccessfully used to evaluate the genotoxic potentialof suspect environmental mutagens (Amer and Ali, 1968;Gaulden and Liang, 1982; Jain and Sarbhoy, 1988).Among them, dominant lethal test (DLT) is one such invivo procedure which is used for evaluating themutagenic potential of pesticides on the progenies ofthe treated parents. It is based on the frequency ofviable and nonviable embryos produced from crossesbetween treated males with untreated females in whichdominant lethal effect is manifested in the form ofembryonic deaths. Therefore, this test also helps todetermine the sensitivity of the  germ cells to thechemical mutagens (Manna and Sarkar, 1998).In the present investigations, a mosquito Culexquinquefasciatus was considered an ideal test systemas it has a high reproductive potential and only six asthe diploid number of chromosomes, wherebyabnormalities present in the germ cells can be easilydetected along with visible phenotypic changes in theadults. These mosquitoes  lay eggs in groups (egg rafts)in which it is convenient to observe all the eggs laid byan individual. In order to meet the present objectivesthe dominant lethality of acetamiprid was evaluated byat  LD40 dose level. Although, this  dose is consideredsublethal yet it prove  high enough to cause  detectableeffect.172 Mamta Bansal and  Asha Chaudhry  / J. Appl. & Nat. Sci. 3 (2): 171-175 (2011)MATERIALS AND METHODSAcetamiprid ((E)-N- [(6-chliro-3-pyridyl)]-N-cyano-N-methyl acetamidine) is commonly sold in the form ofwhite solid powder (Aventis Crop Sciences, U.S.A) underCAS no. 135410-20-7 and molecular formula C10H11ClN4(Fig. 1)  and molecular weight of 222.68. For the presentstudy, LD40 was calculated by probit analysis and werefound to be 8.9x10-4 µl/ml  (Finney, 1971, Fig.  2).The gravid females of Culex quinquefasciatus Say werecollected from village inhabitation of a rivulet, 20 kmsEast of Chandigarh. They were allowed to lay eggs inwater filled petridishes placed in the breeding cages. Theegg rafts obtained in this way were allowed to hatch andthe larvae were reared on a protein rich diet consisting ofa mixture of finely powdered dog biscuits and yeastpowder in the ratio of 6: 4 respectively. A colony wasraised under suitable conditions of temperature andhumidity in mosquito rearing laboratory (Krishnan, 1964;Singh et al., 1975). Fixed number of freshly hatchedhealthy fourth instar larvae were treated with LD40 doseof the pesticide by rearing them in acetamiprid containingrearing medium for 24 hours after which they weretransferred to pesticide free water and allowed to growupto adult stages. Similarly, parallel controls of larvaewere also reared upto adult stages and the freshly hatchedadults of both the sexes were fed on 10% sucrose/glucose solution. The treated males were crossmated withnontreated females after which the females were providedwith a blood meals by trapping a mice in a restrainer cagebefore keeping the same in the breeding cage (Muro andGoyer, 1969). After 4-5 days, females laid eggs which wereallowed to hatch and after one week all the eggs wereexamined under suitable magnification of a dissectingmicroscope. The eggs with open opercula wereconsidered as hatched while those with closed opercula(Figs. 3, 4) were taken as unhatched. The frequency ofunhatched egg was taken as the criterion to evaluate theeffects on the viability of embryos. Based on these figuresthe percentage frequency of induced lethality wascalculated by applying the following formula.Percentage frequency of unhatched eggs =No. of unhatched eggs in an egg raft X 100          Total no. of eggs in an egg raftStatistical analysis: The whole experiment was repeatedfive times and the statistical analysis was carried out byapplying Student t- test using significance level of 0.01.RESULTS AND DISCUSSIONAssessment of dominant lethal mutations throughcrossing experiments is a widely accepted parameter fordetermining the genotoxicity of environmental mutagens(Suter, 1975; Manna and Sarkar, 1998). In the present setof experiments, the percentage frequency of lethalmutations which produced nonviable eggs is presentedin Tables 1 and 2. Table 1 represents the percentagefrequency of unhatched eggs of nontreated individualsof Culex quinquefasciatus, whereas Table 2 representsthe percentage frequency of unhatched eggs of Culexquinquefasciatus treated with LD40 of pesticide.No. of  Egg  raft Total no. of eggs in an egg raft No. of eggs hatched No. of  eggs unhatched %  frequency of unhatched eggs 1 112 104 8 7.14 2 118 112 6 5.08 3 103 96 7 6.8 4 95 92 3 3.16 5 100 96 4 4 Table 1. Percentage frequency of unhatched eggs in controlstocks of Cx.  quinquefasciatus.No. of egg raft Total no. of  eggs in an egg raft No. of eggs hatched No. of  eggs unhatched % frequency of unhatched eggs 1 100 65 35 35.00 2 92 47 45 48.49 3 115 83 32 27.83 4 107 60 47 43.93 5 97 66 31 31.96 Table 2. Percentage frequency of unhatched eggs of Cx.quinquefasciatus  treated with LD40   of acetamiprid.Fig. 1. Chemical structure of acetamiprid.Culex quinquefaciatus Y=2.30+0.54x0123456781 2 3 4 5 6 7Log conc. (ul/ml)PROBIT OF KILLFig. 2. Relationship between the probit of kill and LD40 ofacetamiprid showing the regression line represented by theequation Y= a+ bx.173Fig. 6. Egg raft with open and closed opercula of treated Cx.quinquefasciatus.Accordingly, the average frequency of unhatched eggswas 37.44% as against 3.88% in the controls. Thepercentage frequency of dominant lethality was 37.52 ±3.88 as against 5.23 ± 0.77 in the controls. The t valuewas 7.50 at d.f. 4 which showed considerably significantvalue at p<0.01 (Table 3, Figs. 3, 4, 5, 6). Most of themutagens are known to have a damaging effect on theviability of the treated gametes and their chromosomeswhich ultimately reduces the normal production of viableembryos. This evidence was also provided by Brewen etal. (1975) who showed that the broken chromosomesdue to the mutagenic action of chemical mutagen wereeventually lost at anaphase, resulting in the death of thedeveloping embryo.  Bender et al. (1973, 1974) proposeda model by which chromosomal aberrations could beinduced by a mutagen in the germ cells. Their studiesstrongly supported the view that the dominant lethalitywas the outcome of the loss of chromosomal material byway of deletions in the chromosomes of gametes. Thiswas due to high frequency of damage to the male germcell chromosomes which ultimately resulted in earlyembryonic deaths. Sharma et al. (1989) performeddominant lethal tests using Cx. quinquefasciatus as anexperimental insect in which the extent of dominantlethality was assessed by measuring the percentagefrequency of hatched and unhatched eggs in both treatedType of larval stock No. of egg rafts counted %  frequency of unhatched eggs Standard deviation Standard error ‘t’ value Control 5 5.236 1.727 0.7724 7.505 Treated 5 37.526 8.689 3.886 d.f = 4 Table 3.  Statistical analysis of dominant lethality of Cx. quinquefasciatus treated with LD40   of acetamiprid.d.f. = degree of freedom, p<0.01Fig. 3. Egg raft with closed opercula of nontreated Cx.quinquefasciatus.Fig. 4. Egg raft with open opercula of nontreated Cx.quinquefasciatus.Fig. 5. Egg raft with closed opercula of  treated Cx.quinquefasciatus.Mamta Bansal and  Asha Chaudhry  / J. Appl. & Nat. Sci. 3 (2): 171-175 (2011)174and control stocks of mosquitoes. More recent,Chaudhry and her co- workers also performed similarexperiments with improved protocols of cytogenetics toevaluate the genotoxicity of some pesticides such aschloropyrifos, imidacloprid, cypermethrin, carbaryl andmonocrotophos F1 generations after mating treated maleswith different sets of virgin females. The results showedthe occurrence of significantly high level of dominantlethality (Chaudhry and Anand, 2004; Chaudhry et al.,2008; Chaudhry and Lovleen, 2008; Bhinder et al., 2009).Some of the recent reprints indicate that exposure toacetamiprid is related to induction of chromosomalaberrations and sister chromatid exchanges in humanperipheral lymphocytes at almost all the concentrationsand treatment times (Kocaman and Topaktas, 2007, 2009).To the contrary, the test carried out on lower animals liketermites showed that even at low dose of 4.8 ppm ofacetamiprid, more than 90% individuals died within 120hr of exposure  (Mo et al., 2005). Related to such studieson the toxicity of acetamiprid, the larvae of mosquitoCulex pipiens pallens were also exposed to pesticides,after which the results indicated that these larvae werehighly sensitive to acetamiprid as their mortality rate wassignificantly high as compared to the normal nontreatedstocks. The 1st instar larvae were the most susceptibleto acetamiprid as compared to  4th instar larvae whichwere found to be more tolerant to the action of acetamiprid(Mo et al., 2008). According to El Hassani et al., (2008)and  Yassine et al., (2009),  field studies showed thatacetamiprid was also found to affect the non-targetspecies of insects like honeybees (Apis mellifera). Whenthe toxicity of acetamiprid was compared withimidacloprid and thiamethoxam among the species oftermites it was found that acetamiprid was more toxicthan imidacloprid but less toxic than thiamethoxam (Rustand Saran, 2008).In summation it may be added that dominant lethal test isan ideal parameter for evaluating the genotoxic potentialof acetamiprid and other pesticides at different doseconcentrations which prove harmful to the genomiccontents of the test organism  mosquito. The presentstudy shows that genetic damage caused by such a lowerdose of LD40  is so high which further proves the risk ofacetamiprid at further higher doses. It also raises a pointof caution that, the exposure directly acting pesticidecould be deleterious to the genome of other living systemincluding man.ACKNOWLEDGEMENTSThe authors are grateful to Chairperson, Department ofZoology and Co-ordinator of Centre of Environment andVocational Studies, Panjab University, Chandigarh forproviding necessary facilities to carry out presentresearch work. The authors are also thankful to thephotographic section, Department of Biotechnology forproviding necessary facilities to present the photographs.REFERENCESAmer, S. M. and Ali,  E. M. (1968). Cytological effects ofpesticides III: Meiotic effects of some phenols.  Cytologia,33:  21-33.Anderson, D. and Conning, D. M. (1993).  Experimentaltoxicology: The basic issue. The Royal Society of Chemistry.Cambridage.  405-440.Bender, M. A., Bedford, J. S. and Mitchell, J. B. (1973).Mechanisms of chromosome aberration production II.Aberrations induced by 5- bromodeoxyuridine and visiblelight. Mutat. Res., 20: 403- 416.Bender, M. A.,  Griggs, H. G. and Bedford, J. S. (1974).Mechanisms of chromosome aberration production III.Chemicals and ionizing radiation. Mutat. Res., 23: 187- 212.Bhinder, P., Chaudhry, A. and Barna, B. (2009). rDNA ITS 2,polytene chromosomes and dominant lethal test basedgenotoxicity assessment oftwo pesticides using mosquitogenome, Proc. International Conference on Entomology,Punjabi University, Patiala, Punjab.Brewen, J. G., Payne, H. S., Jones, K. P. and Preston, R. J.(1975). Studies on chemically induced dominant lethality.The cytogenetic basis of MMS-induced dominant lethalityin post meiotic-male germ cells. Mutat. Res., 33: 239-250.Chaudhry, A. and Anand, P. K. (2004). Assessment ofdominant lethal effects of chlorpyrifos (CPF) usingmosquito genetics. Poll.  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Evaluation of mutagenic potential of acetamiprid by dominant lethal test on Culex quinquefasciatus

Acetamiprid, a member of the neonicotinoid insecticide family, is a new class of insecticide which has recently entered the market place. It is very selective and provides outstanding control of sucking pests such as aphids and whiteflies of major crops. In the present investigation, dominant lethal test (DLT) is adopted for the evaluation of the genotoxic effects of acetamiprid using Culex quinquefasciatus as an experimental model. In this experiment, the males hatched from larvae treated with LD40 were cross mated with normal females and the results were based on the number of hatched and unhatched eggs laid by these females. The statistical analysis of the results for LD40 treated groups gave the values of 37.526±3.886 as against 5.23±0.77 from the nontreated groups which indicated significant dominant lethality of p&lt;0.01. These results indicated that exposure of pesticides even at small dose level proved deleterious to the genome of mosquito and its subsequent generation.</jats:p

Mamta Bansal

Asha Chaudhry

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Evaluation of mutagenic potential of acetamiprid by dominant lethal test on Culex quinquefasciatus

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