insecticidal and genotoxic potential of two semi-synthetic derivatives of dillapiole for the control...

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ARTICLE IN PRESSG ModelUTGEN 402518 1–13

Mutation Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology andEnvironmental Mutagenesis

jo ur nal home page: www.elsev ier .com/ locate /gentoxComm uni t y ad dress : www.elsev ier .com/ locate /mutres

nsecticidal and genotoxic potential of two semi-synthetic derivativesf dillapiole for the control of Aedes (Stegomyia) aegypti (Diptera:ulicidae)

edro Rauel Cândido Domingosa, Ana Cristina da Silva Pintob,oselita Maria Mendes dos Santosb, Míriam Silva Rafaelb,∗

Post-Graduation Program in Genetics, Conservation and Evolutionary Biology/Instituto Nacional de Pesquisas da Amazônia – INPA, BrazilLaboratory of Vectors of Malaria and Dengue/CPCS/INPA, Manaus, AM, Brazil

r t i c l e i n f o

rticle history:eceived 19 November 2013eceived in revised form 21 July 2014ccepted 23 July 2014vailable online xxx

a b s t r a c t

The effects of two semi-synthetic dillapiole derivatives, ethyl-ether dillapiole and n-butyl ether dillapiole,on eggs and larvae of Aedes aegypti were studied in view of the need for expansion and renovation ofstrategic action to control this mosquito – the vector of Dengue virus –, which currently shows a highresistance to chemical insecticides. Eggs and third-instar larvae of A. aegypti that had been exposed todifferent concentrations of these two compounds showed toxicity and susceptibility, with 100% mortality.

eywords:ioassayio-insecticideengue virusssential oil

Classical cytogenetic assays showed genotoxicity caused by the two compounds in A. aegypti from thecumulative effect of nuclear abnormalities, indicating that these derivatives may be potential alternativesto control A. aegypti.

© 2014 Published by Elsevier B.V.

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iological control

. Introduction

Dengue is a major arbovirus that infects about 50 million peo-le per year worldwide [1]. Its main vector is the mosquito AedesStegomyia) aegypti (Linnaeus, 1762). Because a vaccine againstifferent types of viruses is lacking, and in view of the occur-ence of several cases of resistance to chemical insecticides, asell as the increase in registered cases during periods of epi-emic dengue in the world [2–10], new alternative methods areecessary to effectively control this mosquito. Bio-insecticides orlant extracts as repellent substances have been a viable and effec-ive alternative for biological control of insect pests and/or vectors2,11–14].

Piper aduncum (family, Piperaceae) has been used to con-

Please cite this article in press as: P.R.C. Domingos, et al., Insecticiddillapiole for the control of Aedes (Stegomyia) aegypti (Diptera: Culhttp://dx.doi.org/10.1016/j.mrgentox.2014.07.008

rol larvae of A. aegypti in Argentina, Bolivia, Peru and Brazil15–17]. The essential oil extracted from this plant has bio-nsecticidal effects [18]. It contains dillapiole [18,19], with potential

∗ Corresponding author at: Instituto Nacional de Pesquisas da Amazônia, Labora-ory of Vectors of Malaria and Dengue/CPCS, Av. André Araújo, 2.936 Petrópolis, CEP9067-375, Manaus, AM, Brazil. Tel.: +55 92 3643 3066.

E-mail addresses: [email protected] (P.R.C. Domingos),[email protected] (A.C. da Silva Pinto), [email protected] (J.M.M. dos Santos),[email protected] (M.S. Rafael).

ttp://dx.doi.org/10.1016/j.mrgentox.2014.07.008383-5718/© 2014 Published by Elsevier B.V.

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application to control A. aegypti [12,18,20–23]. Dillapiole hasa highly stable chemical structure, which offers potential forproduction of semi-synthetic derivatives as alternative sourcesto enhance the action and the physicochemical properties ofagents with confirmed pharmacological or bio-insecticidal activ-ity [24–27]. Semi-synthetic derivatives synthesized from dillapiolewere most effective as insecticide (adulticide) in Aedes atropal-pus, when compared with the parent compound (dillapiole),according Belzile et al. [28], and in adults of A. aegypti accord-ing Pinto et al. [25]. Moreover, the small structural variationsthat occur among the monoterpenes R-carvone, S-carvone, R,S-carvone, R-limonene, S-limonene, R,S-menthol, �-terpinene and3-carene, culminate in a large variation in toxicity of these com-pounds to larvae of A. aegypti [29]. This indicates the potentialof manipulation of natural substances with high stability, suchas dillapiole, for the synthesis of new compounds with improvedactivity.

Bioassays for evaluating the genotoxicity and mortality of dil-lapiole in A. aegypti were successfully performed by Rafael et al.[23], showing genotoxic effects of the insecticide in samples of A.

al and genotoxic potential of two semi-synthetic derivatives oficidae), Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014),

aegypti. We evaluated the insecticidal and genotoxic activity of twosemi-synthetic derivatives of dillapiole against larvae of A. aegypti,in order to understand the toxic and genotoxic activity of thesesubstances in this mosquito.

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dx.doi.org/10.1016/j.mrgentox.2014.07.008


http://www.sciencedirect.com/science/journal/13835718

http://www.elsevier.com/locate/gentox

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ARTICLEUTGEN 402518 1–13

P.R.C. Domingos et al. / Muta

. Materials and methods

.1. Material used

Specimens of A. aegypti were collected in the neighborhood of Puraquequara03◦03′06.95′′ S and 59◦52′31.38′′ W), municipality of Manaus, Amazonas, Brazil.he samples were used to form colonies (generations F1, F2, F3 and F4) in thensectary of the Coordenac ão de Sociedade, Ambiente e Saúde (Coordination of Soci-ty, Environment and Health) – CSAS, Instituto Nacional de Pesquisas da AmazôniaNational Institute for Amazonian Research) – INPA/Ministério da Ciência e Tecnolo-ia (Ministry of Science and Technology) – MCT. Ethyl-ether dillapiole (1KL39-B)nd n-butyl-ether dillapiole (1KL43-C) compounds (Fig. 1) were assayed against. aegypti individuals in our biological tests. After isomerization, oxy-mercuration,poxidation, bis-hydroxylation (oxidation) reactions with dillapiole, Pinto et al. [25]roduced a series of derivatives, among which 1KL39-B and 1KL43-C. Dimethylulfoxide (DMSO) at concentrations of 2% was used as a solvent to solubilize theerivatives in water.

.2. Bioassays of toxicity and genotoxicity

Two independent bioassays with the F1 generation of A. aegypti were conducted.n Bioassay I we established two lethal concentrations (LC) 50% (LC50) and 90% (LC90)or both compounds 1KL39-B and 1KL43-C, causing mortality of eggs and third-nstar larvae exposed for 24 h. In Bioassay II we evaluated acute genotoxicity (larvaleuroblasts) and residual genotoxicity (oocytes of adults emerged from these larvae)fter exposure of the third-instar larvae to the compounds for 4 h in the aquatichase.

Bioassay I: We used the compounds 1KL39-B at concentrations of 40, 50, 60, 70nd 80 �g/mL, and 1KL43-C at concentrations of 12.5, 20, 25, 30 and 40 �g/mL toreat eggs and larvae, respectively. A total of 200 eggs and 200 larvae were parti-ioned into five replicates (n = 20) for each concentration. Exposure was continuedor 24 h. After this time, eggs and larvae were transferred to vessels containing tapater. After evaluation of the mortality caused by each concentration in the samples

f eggs and larvae, the final mortality curve and the values of LC50 and LC90 werestablished.

Bioassay II: We selected the concentrations of 70 and 80 �g/mL for 1KL39-B and0 and 30 �g/mL for 1KL43-C, which were higher than the LC50 value established inioassay I. A total of 200 third-instar larvae, divided into five replicates (n = 20) for


ach concentration were transferred to vessels containing tap water and exposed tooth semi-synthetics for 4 h. Then, 30 larvae were used to prepare cytological slidesnd the remaining larvae were transferred to cups with tap water, where they wereaintained until adulthood. Upon reaching the adult phase, 30 female mosquitoesere used to prepare cytological slides and the remaining larvae were used for

ig. 1. (A) Ethyl-ether dilapiole. (B) n-Butyl ether dilapiole (Pinto, personal commu-ication).

ig. 2. Probit analysis to determine the lethal concentration (LC) against larvae of A. aegs. mortality after treatment with 1KL39-B; (B) Concentration vs. mortality after treatme

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PRESSesearch xxx (2014) xxx–xxx

formation of couples (n = 30 males, 30 females), and crossed in order to obtain thenext generation (F2). The steps developed in this bioassay were repeated for gener-ations F3 and F4, the next generation emerging from the eggs laid by couples of theprevious generation. At the end of each generation the average oviposition for eachtreatment was evaluated.

2.3. Genotoxic activity at the cellular level

Genotoxic effects of the compounds 1KL39-B and 1KL43-C were evaluated byclassical cytogenetics. The cytological preparations were prepared with the spread-ing method [30], with cerebral ganglia of the third-instar larvae and ovaries of adultfemales emerged from larvae exposed to the semi-synthetics in Bioassay II. TheF1 progeny obtained from crosses of individuals exposed to two concentrations of1KL39-B and 1KL43-C, received the same treatments to obtain the F2 and subse-quent generations (F3 and F4). The exposure of A. aegypti to these products for 4 hin four successive generations aimed to highlight the potentially deleterious effectsin different and successive generations of A. aegypti.

Neuroblasts (n = 10,000) and oocytes from ovaries (n = 10,000) in interphasenuclei were analyzed for normality or occurrence of structural and morphologicalabnormalities (buds, micronuclei, poly-nuclei and other malformations). Moreover,300 neuroblasts and 300 oocytes were analyzed for normality or the occurrenceof aberrant structures and morphological abnormalities (chromosomal breaks,fragmentation or chromosomal bridges, micronuclei and other chromosomal mal-formations). Analysis of the interphase nuclei and of the nuclei in division across thefour generations tested (F1, F2, F3 and F4), was performed to investigate differencesbetween them.

2.4. Statistical analysis

The lethal concentrations (LC50 and LC90) of the compounds 1KL39-B and 1KL43-C on the samples of A. aegypti exposed for 24 h (Bioassay I) were determined from theProbit analysis [31], by use of StatPlus® portable v5.8.4. The differences in oviposi-tion average of individuals, and in the average frequency of abnormal nuclei betweenthe test concentrations and between the generations of each treatment (Bioassay II)were evaluated for statistical significance by means of two-way analysis of variance(two-way ANOVA, P < 0.05) and Tukey’s test (P < 0.05), both performed with the aidof GraphPad Prism 6.0.

3. Results

The mortality of eggs laid by A. aegypti exposed to the semi-synthetics 1KL39-B and1KL43-C was 100%, after a 24-h exposure tothe lowest concentrations of both compounds (40 �g/mL of 1KL39-B and 12.5 �g/mL of 1KL43-C). The LC50 and LC90 observed from themortality curves plotted for exposure of third-instar larvae of A.aegypti for 24 h were 61.8 ± 1.9 and 89.0 ± 1.7 �g/mL for the treat-ment with 1KL39-B, respectively, while for treatment with 1KL43-C


the LC50 and LC90 values were 18.6 ± 1.9 and 27.1 ± 2.3 �g/mL,respectively (Fig. 2).

In Bioassay II we used 1KL39-B at concentrations of 70 and80 �g/mL and 1KL43-C at 20 and 30 �g/mL. Both tests were

ypti exposed for 24 h to semi-synthetic derivatives of dillapiole. (A) Concentrationnt with 1KL43-C.

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ARTICLE IN PRESSG ModelMUTGEN 402518 1–13

P.R.C. Domingos et al. / Mutation Research xxx (2014) xxx–xxx 3

Fig. 3. Frequency of nuclear abnormalities (%) observed in interphase nuclei (A and C) and division nuclei (B and D) in generations F1 to F4. (A) Anomalies in interphaseneuroblasts and oocytes treated with 1KL39-B. (B) Anomalies in cell division of neuroblasts and oocytes treated with 1KL39-B. (C) Anomalies in interphase neuroblasts ando ytes ta

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ocytes treated with 1KL43-C. (D) Anomalies in cell division of neuroblasts and oocre 20 and 30 �g/mL, respectively.

stablished after determining the LC50 of each compound. Theortality, according to ANOVA analysis was not significantly dif-

erent between the samples exposed to derivatives 1KL39-B andKL43-C (1.25–3.75%) compared with the control (1.25%), after 4 hf exposure. However, with respect to nuclear abnormalities innterphase (neuroblasts and oocytes) and cell division (neurob-asts and oocytes), there were differences between the treatments

ith 1KL39-B at 70 �g/mL and 80 �g/mL, and between 1KL43-C at0 �g/mL and 30 �g/mL (Fig. 3. The average frequency of nuclearbnormalities varied significantly between both treatments andhe control, as well as between the lowest and highest test con-entration of each compound, according to Tukey’s test (Annex C).bnormalities in the F2, F3 and F4 generations, respectively, werelso recorded (Fig. 3). Despite the fact that ANOVA indicates vari-tion between treatments and generations (Annex B), the resultsbtained with Tukey’s test showed no significant variation in theumber of nuclear abnormalities observed between the F4 and F3enerations (Annex C), except in neuroblasts in division (treatmentith 80 �g/mL of 1KL39-B) and in interphase neuroblasts (treat-ent with 20 and 30 �g/mL of 1KL43-C). Nonetheless, the values

emained much higher than those observed in the earlier genera-ions.

There was a significant reduction in average number of eggs laid


y exposed A. aegypti (Annex B), and there is a gradual reductionrom the F1 to the F4 generation when compared with the con-rols, according to Tukey’s test (Annex C). A significant decreaselso occurred with increasing concentration of each semi-synthetic

reated with 1KL43-C. B80 and B70 are 70 and 80 �g/mL, respectively. C20 and C30

agent (Table 1). The difference was significant between the 70and 80 �g/mL concentrations of 1KL39-B and between the 20 and30 �g/mL concentrations of 1KL43-C, as well as between both treat-ments and the control. This variation was significant since the F1generation after exposure of A. aegypti to these concentrations for4 h (Bioassay II), showed a mean of 37.6 ± 4.4 (at 80 �g/mL for1KL39-B) and 27.0 ± 5.6 (at 30 �g/mL for 1KL43-C) eggs laid inthe F4 generation, while the control value was 99.6 ± 9.9 eggs laid(Table 1).

The cytotoxicity and genotoxicity analyses by classical cyto-genetics showed formation of morphological abnormalities ininterphase nuclei (Fig. 4b–f), both in neuroblasts of larvae andin oocytes of adult females, after treatment with both semi-synthetic derivatives. Chromosomal alterations were also evidentin nuclei (Fig. 5b–f) observed both in neuroblasts and oocytes. Theseresults were evident in the four generations exposed to the semi-synthetics, with the frequency of alterations increasing in the lastgenerations (Fig. 3. The difference between individuals in the con-trols and in the treatment groups increased in the later generations,according to Tukey’s test (Annex C), as can be seen in Fig. 3.

4. Discussion


The larvicidal activity of semi-synthetic derivatives of natu-ral compounds in mosquitoes is scarcely documented. However,results with essential oils and their derivatives have demonstratedlarvicidal, ovicidal and repellent activities in A. aegypti [25,32].

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Table 1Average rate of oviposition and standard deviations of adult females of Aedes aegypti exposed to 1KL39-B and 1KL43-C, and untreated controls, along different generations(F1, F2, F3 and F4). The concentrations correspond to the quantity of this product in tap water. The control contains only tap water and DMSO (2%).

Compound semi-synthetic Concentration (�g/mL) Generation

F1 F2 F3 F4

1KL39-B 70 64.40 ± 7.76 53.40 ± 6.23 41.40 ± 5.03 38.60 ± 3.5180 56.80 ± 6.61 54.40 ± 6.69 38.20 ± 3.96 37.60 ± 4.39

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1KL43-C 20 98.80 ±30 51.40 ±

Control 0 100.40 ±

hese compounds are volatile, such as monoterpenes, terpenes,romatic compounds derived from phenol and aliphatic com-ounds with biological activities suitable for controlling A. aegypti11,14,25,29,32–34].

Essential oils containing aromatic compounds as main compo-ent had their larvicidal activity tested successfully in experimentso control larvae of A. aegypti [11,23,33,34]. Maia et al. [18], Costa24] and Pinto et al. [25] reported chemical conversion of dillapi-le as a tool for synthesis of a great variety of possible derivativesith improved pharmacological or insecticidal properties. In our

tudy, the compounds derived from dillapiole, ethyl-ether dillapi-le (1KL39-B) and n-butyl-ether dillapiole (1KL43-C) were toxic to. aegypti, causing 100% mortality of eggs and larvae after 24 h ofxposure (Fig. 2. In addition to the high mortality, both compoundsaused a reduction in the average number of eggs laid (Table 1),aused a delay in larval development, and induced genotoxic dam-


ge in neuroblasts of the third-instar larvae and oocytes of adultemales (Fig. 3. In mammalian cells, Burkey et al. [35] observedenotoxic effects of the aromatic compounds safrole and eugenol,

ig. 4. Interphase nuclei from neuroblasts (A, D, E and F) and oocytes (B and C) of A. aegypcontrol); (B) Nuclear bridge between nuclei after treatment with 70 �g/mL of 1KL39-B;treated with 20 �g/mL of 1KL43-C); (E) Micronucleus (arrow) in interphase cell after trafter treatment with 20 �g/mL of 1KL43-C). Magnification, 600–1000×.

73.00 ± 5.70 65.80 ± 6.10 52.20 ± 8.76 45.00 ± 8.15 32.80 ± 4.32 27.00 ± 5.612 100.00 ± 11.85 99.20 ± 12.21 99.60 ± 9.91

with the same basic structure of dillapiole and also with provenpotential larvicidal activity. However, similar data on insects arescarce and these do not include the assessment of genotoxicity [23].

Our results in A. aegypti showed a decrease in the numberof eggs of adult females after the crossing of each couple fromthe first generation (F1) previously treated, at the larval stage,with ethyl-ether dillapiole (1KL39-B) and n-butyl-ether dillapiole(1KL43-C). This level of reduction in the number of eggs graduallyincreased with increasing concentrations of the two compounds inthe F2 and F3, and reached a maximum in the last generation (F4)of A. aegypti, which showed intolerance to the cumulative toxiceffect of these derivatives. Rafael et al. [23] also showed a reductionin the average number of eggs per couple in adult females of A.aegypti at the stage when the larvae were treated with dillapiole(200 and 400 �g/mL), and this reduction was associated with anincrease in the concentration of dillapiole and its cumulative effect


on generations tested. The literature does not provide data onexposure of A. aegypti to essential oils or their derivatives, or onreduced oviposition per adult female of A. aegypti, but only on the

ti (F4 generation) stained with Giemsa 8% phosphate buffer. (A) Interphase nucleus (C) Bi-nucleate cell treated with 80 �g/mL of 1KL39-B; (D) Nuclear segmentationeatment with 30 �g/mL of 1KL43-C); (F) Nuclear segmentation (arrow) and buds

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Fig. 5. Dividing nuclei from neuroblasts (A, B, and F) and oocytes (C, D and E) of A. aegypti (generation F4) stained with Giemsa 8% phosphate buffer. (A) Metaphasen 30 �gc tment( �g/m

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ucleus (control); (B and C) Breakage (arrows) in metaphase (after treatment withhromosomes, respectively; (D) Micronucleus (arrow) in metaphase cell (after treaafter treatment with 70 �g/mL of 1KL39-B) and telophase (after treatment with 30

epulsive character of these oils when they were applied in reser-oirs, which reduced the number of eggs laid [36]. This repellentctivity of essential oils and their derivatives is another potentialpplication for such compounds in controlling A. aegypti [13,32].owever, semi-synthetic derivatives to control mosquitoes ofedical importance are still scarce. A semi-synthetic derivative

f dillapiole for use against A. atropalpus, as a synergistic agent ofhe insecticide phototoxin alpha-terthienyl, was the first examplef insecticidal action from chemically modified compounds onhe basis of dillapiole [28]. In A. aegypti, larvicidal effects of

onoterpenes and their semi-synthetic derivatives were reporteds suitable alternatives to control this mosquito [29].

This study represents the first on A. aegypti, a mosquito ofedical importance, which was exposed to 1KL39-B and 1KL43-C

emi-synthetics, with toxic effects and mortality on eggs andarvae (100%), respectively, after 24 h of exposure. It also is therst report on genotoxicity in larval neuroblasts and oocytesf adult females of A. aegypti treated with both compounds. Atudy in larval neuroblasts and oocytes of the pupae of A. aegyptihowed toxic and genotoxic effects of dillapiole, in addition toeducing to oviposition of adult females treated at the larval stageith dillapiole at 200 and 400 �g/mL [23]. In the present study,

hose characteristics were also observed in A. aegypti, treatedith 1KL39-B and 1KL43-C. However, the mortality data of the

hird-instar larvae (100%) exposed to concentrations of 80 �g/mLf 1KL39-B and 30 �g/mL of 1KL43-C for 24 h were more significantn comparison with the mortality of third-instar larvae exposed to


illapiole (67% mortality at 400 �g/mL, 53% at 200 �g/mL) for 72 h23]. This study aimed to evaluate the genotoxic effects in larvaend adults of A. aegypti, but the exposure and the determinationf lethal concentrations (LC50) were performed in the larval stage,

/mL of 1KL43-C) and pro-metaphase (after treatment with 70 �g/mL of 1KL39-B) with 80 �g/mL of 1KL39-B); (E and F) Chromosomal bridges (arrows) in anaphaseL of 1KL43-C) nuclei, respectively. Magnification, 1000×.

only. The different concentrations for both compounds used in thisassay aimed to monitor the appearance of chromosomal damage inthird-instar larvae and adult females from F1 to the F4 generation.

Dillapiole derivatives with variations in the positions of the radi-cals and degrees of oxygenation have been used against third-instarlarvae of A. aegypti to ascertain their potential larvicidal activity,with dillapiole as a control compound (Quadros, personal commu-nication). Compounds with a higher degree of oxygenation in theirradical species in comparison to dillapiole induced high mortalityamong larvae. It confirms our results of greater acute toxicity of thesemi-synthetics 1KL39-B and 1KL43-C, which seems to have con-tributed to the strongly reduced number of eggs laid, the delayedlarval development, and the genotoxic damage in neuroblasts ofthe third-instar larvae and in oocytes of adult females.

Besides the higher degree of oxygenation of the semi-syntheticderivatives 1KL39-B and 1KL43-C in relation to dillapiole, previ-ously related to increased toxicity of these derivatives (Quadros,personal communication), a higher degree of lipophilicity mayalso be related to increased toxicity of compounds as potentialinsecticide [28,29], which may explain the differences foundbetween the LC50 of 1KL39-B and 1KL43-C. Belzile et al. [28]observed a high level of toxicity of semi-synthetic derivativesof dillapiole with higher lipophilicity, when these agents wereused as a synergistic agent with the insecticide phototoxin alpha-terthienyl against A. atropalpus. Semi-synthetic derivatives ofnatural monoterpenes, which have a high degree of lipophilicity,also showed higher toxicity against third-instar larvae of A. aegypti


[29]. This may explain the difference observed between the toxicityof the derivatives 1KL39-B and 1KL43-C in the present study, inwhich the 1KL43-C showed greater toxicity due to its greaterlipophilicity compared with 1KL39-B, when comparing the LC50

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alues obtained from the mortality curve (18.6 ± 1.9 �g/mL forKL43-C and 61.8 ± 1.9 �g/mL for 1KL39-B).

Increasing concentrations of the semi-synthetic derivativesKL39-B and 1KL43-C increased the frequency of abnormalitiesbserved in interphase nuclei and in other phases of cell division ineuroblasts and oocytes. In our results, a high potential for cumula-ive effects of the semi-synthetics was observed in larvae exposedor 4 h, when compared with the genotoxic potential of dillapiole inarvae exposed for 72 h [23]. These authors report also micronucleind chromosomal aberrations after treatment of the third-instararvae with dillapiole. However, the results obtained with semi-ynthetics 1KL39-B and 1KL43-C against A. aegypti exposed for fourours show nuclear malformations, buds, poly-nuclear cells andicronuclei in higher frequencies than in A. aegypti exposed to dil-

apiole for 72 h. The occurrence of nuclear buds also showed anncrease in frequency after treatment of larvae of A. aegypti with theemi-synthetic derivatives, as was also observed in larvae exposedo dillapiole [23].

Studies on dillapiole in higher organisms and mammalian cellseported mutations, chromosomal aberrations and aneuploidy37]. In A. aegypti, the increase in the frequency of poly-nuclearells or nuclear fragmentation observed in this study may be dueo defective cell division in anaphase. Anaphase bridges are featuresisible by non-disjunction of chromosomes and/or chromatids dur-ng anaphase [38,39]. The anaphase bridges observed in neuroblastsf third-instar larvae and oocytes of adult females after treatmentf A. aegypti with 1KL39-B and 1KL43-C may be related to defec-ive separation of sister chromatids, but also to inhibition of mitoticpindle formation. Micronuclei were frequent in neuroblasts (lar-ae) and oocytes (adult females) of A. aegypti treated with 1KL39-Bnd1KL43-C, as well as after treatment with dillapiole [23], and maye associated with breakage occurring in the despiralization pro-ess of the DNA molecule, when this is more vulnerable to external


gents [23]. In the present study, micronuclei were seen in inter-hase, pro-metaphase and metaphase nuclei, and they occurred ineuroblasts and oocytes. Micronuclei in pro-metaphase may disap-ear in the process of metaphase condensation [40]. Chromosome

Cell type/generation Interphase nuclei

F1 F2 F3

1KL39-B (�g/mL)70 Neuroblasts 13.52 ± 0.57 12.22 ± 1.23 31.50 ± 0.43

80 15.34 ± 1.15 17.18 ± 0.84 37.34 ± 2.20

0 (control) 4.64 ± 0.48 5.04 ± 0.15 5.86 ± 0.61

70 Oocytes 16.06 ± 0.64 25.40 ± 4.34 37.28 ± 0.90

80 7.62 ± 1.24 28.74 ± 1.34 39.64 ± 1.25

0 (control) 8.48 ± 1.23 8.64 ± 1.23 8.97 ± 0.97

1KL43-C (�g/mL)20 Neuroblasts 9.06 ± 1.02 17.20 ± 1.27 21.92 ± 1.10

30 12.68 ± 0.69 29.78 ± 2.77 27.48 ± 4.45

0 (control) 4.64 ± 0.48 5.04 ± 0.15 5.86 ± 0.61

20 Oocytes 11.70 ± 0.50 18.86 ± 1.97 33.86 ± 0.88

30 15.46 ± 1.52 30.66 ± 1.87 49.96 ± 0.82

0 (control) 8.48 ± 1.23 8.64 ± 1.23 8.97 ± 0.97

359

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PRESSesearch xxx (2014) xxx–xxx

breakage in the pro-metaphase and metaphase was observed aftertreatment with 1KL39-B and 1KL43-C. These breakages are relatedto the most fragile regions of the chromosome [23,41,42], suchas the secondary constrictions observed on chromosome 3 of A.aegypti [23].

5. Conclusion

The semi-synthetic derivatives of dillapiole (1KL39-B and1KL43-C) may be suitable alternatives to control A. aegypti, becausethey show highly toxic and genotoxic effects. Studies in the futureshould increase our knowledge about mechanisms involved in thisprocess, and should indicate how to apply these semi-synthetics inpreventing natural and artificial breeding of A. aegypti.

Funding source

None.

Conflict of interest

None.

Acknowledgements

We are grateful to the Fundac ão de Amparo à Pesquisa do

Estado do Amazonas – FAPEAM (Universal Project – Process no.1036/2011), Conselho Nacional de Desenvolvimento Científico eTecnológico – CNPq (Universal project – Process no. 480926/2011-5), and Ministério da Ciência, Tecnologia e Inovac ão – MCTI (projectno. 06.178/2012–2013).

Annex A. Average frequency and standard deviation (%) ofnuclear abnormalities in cells (neuroblasts and oocytes) ofA. aegypti treated with 1KL39-B or 1KL43-C. F1, F2, F3 and F4are the experimental generations.

Nuclei in division

F4 F1 F2 F3 F4

31.82 ± 2.14 6.82 ± 2.81 16.22 ± 2.11 42.61 ± 5.08 43.79 ± 7.6535.68 ± 2.94 13.36 ± 1.51 27.93 ± 1.91 47.40 ± 4.73 53.15 ± 2.94

6.10 ± 0.57 3.23 ± 1.31 4.56 ± 1.38 3.33 ± 0.78 4.99 ± 1.2539.24 ± 2.14 15.96 ± 1.34 28.06 ± 2.83 46.90 ± 4.86 47.34 ± 4.0542.24 ± 1.81 16.61 ± 3.19 34.74 ± 7.87 49.79 ± 2.38 47.74 ± 2.14

7.99 ± 0.79 6.62 ± 2.96 4.92 ± 1.64 4.06 ± 1.24 5.61 ± 1.74

25.24 ± 1.47 8.73 ± 1.26 15.93 ± 4.69 40.34 ± 3.65 41.35 ± 4.95


37.38 ± 1.81 24.21 ± 2.65 34.59 ± 2.68 47.56 ± 2.04 46.30 ± 4.656.10 ± 0.57 3.23 ± 1.31 4.56 ± 1.38 3.33 ± 0.78 4.99 ± 1.25

33.94 ± 0.77 15.12 ± 1.67 24.56 ± 5.59 46.19 ± 2.56 40.32 ± 2.0548.74 ± 3.05 22.13 ± 3.96 38.77 ± 4.69 49.84 ± 7.60 50.04 ± 3.22

7.99 ± 0.79 6.62 ± 2.96 4.92 ± 1.64 4.06 ± 1.24 5.61 ± 1.74


G ModelM

AQ3(

364

365

366

ARTICLE IN PRESSUTGEN 402518 1–13


nnex B. Analysis of variance by two-way ANOVA of average frequency of oviposition and nuclear abnormalities in cellsneuroblasts and oocytes) of A. aegypti treated with 1KL39-B or 1KL43-C. F1, F2, F3 and F4 are the experimental generations.

Oviposition – 1KL39-B

Two-way ANOVA OrdinaryAlpha 0.05

Source of variation % of total variation P value P value summary Significant?

Interaction 2.812 0.0132 * YesRow factor 5.87 <0.0001 **** YesColumn factor 83.93 <0.0001 **** Yes

ANOVA table SS DF MS F (DFn, DFd) P value

Interaction 1196 6 199.4 F (6, 48) = 3.046 0.0132Row factor 2497 3 832.5 F (3, 48) = 12.72 <0.0001Column factor 35711 2 17856 F (2, 48) = 272.8 <0.0001Residual 3142 48 65.46

No of missing values 0

Oviposition – 1KL43-C



Interaction 6.006 <0.0001 **** YesRow factor 9.774 <0.0001 **** YesColumn factor 76.79 <0.0001 **** Yes


Interaction 2896 6 482.7 F (6, 48) = 6.466 <0.0001Row factor 4713 3 1571 F (3, 48) = 21.04 <0.0001Column factor 37028 2 18514 F (2, 48) = 248.0 <0.0001Residual 3584 48 74.66


Nuclei abnormalities (neuroblasts interphasic) – 1KL39-B





Interaction 0.115 6 0.01917 F (6, 48) = 99.77 <0.0001Row factor 0.494 2 0.247 F (2, 48) = 1286 <0.0001Column factor 0.2692 3 0.08975 F (3, 48) = 467.1 <0.0001Residual 0.009222 48 0.0001921


Nuclei abnormalities (oocytes interphasic) – 1KL39-B





Interaction 0.1173 6 0.01955 F (6, 48) = 62.17 <0.0001Row factor 0.6527 2 0.3264 F (2, 48) = 1038 <0.0001


Column factor 0.2495 3

Residual 0.01509 48



0.08318 F (3, 48) = 264.5 <0.00010.0003144


ING ModelM

8 tion Research xxx (2014) xxx–xxx

C

TA

P value P value summary Significant?

<0.0001 **** Yes<0.0001 **** Yes<0.0001 **** Yes

MS F (DFn, DFd) P value

0.05572 F (6, 48) = 47.98 <0.00010.5327 F (2, 48) = 458.7 <0.00010.2305 F (3, 48) = 198.4 <0.00010.001161


<0.0001 **** Yes<0.0001 **** Yes<0.0001 **** Yes


0.04386 F (6, 48) = 35.51 <0.00010.6275 F (2, 48) = 508.0 <0.00010.1467 F (3, 48) = 118.8 <0.00010.001235


<0.0001 **** Yes<0.0001 **** Yes<0.0001 **** Yes


0.01308 F (6, 48) = 41.07 <0.00010.2327 F (2, 48) = 730.5 <0.00010.05215 F (3, 48) = 163.7 <0.00010.0003186


<0.0001 **** Yes<0.0001 **** Yes<0.0001 **** Yes


0.03423 F (6, 48) = 156.4 <0.00010.3795 F (2, 48) = 1734 <0.00010.1282 F (3, 48) = 585.9 <0.00010.0002188

367



hromosomal alterations (neuroblasts in nuclear division) – 1KL39-B

wo-way ANOVA Ordinarylpha 0.05

Source of variation % of total variation

Interaction 15.57

Row factor 49.63

Column factor 32.2

ANOVA table SS DF

Interaction 0.3343 6

Row factor 1.065 2


Residual 0.05574 48


Chromosomal alterations (oocytes in nuclear division) – 1KL39-B



Interaction 13.04Row factor 62.2

Column factor 21.82

ANOVA table SS DF


Row factor 1.255 2


Residual 0.05929 48


Nuclei abnormalities (neuroblasts interphasic) – 1KL43-C



Interaction 10.97

Row factor 65.04

Column factor 21.86

ANOVA table SS DF


Row factor 0.4655 2


Residual 0.01529 48


Nuclei abnormalities (oocytes interphasic) – 1KL43-C



Interaction 15.11

Row factor 55.83

Column factor 28.29

ANOVA table SS DF


Row factor 0.7589 2


Residual 0.0105 48



Chromosomal alterations (neuroblasts in nuclear division) – 1KL43-C


PRESS



G ModelM

AQ4o8Q5

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ARTICLE IN PRESSUTGEN 402518 1–13





Interaction 0.2146 6 0.03576 F (6, 48) = 40.02 <0.0001Row factor 1.206 2 0.6029 F (2, 48) = 674.7 <0.0001Column factor 0.3875 3 0.1292 F (3, 48) = 144.6 <0.0001Residual 0.04289 48 0.0008936


Chromosomal alterations (oocytes in nuclear division) – 1KL43-C





Interaction 0.2213 6 0.03689 F (6, 48) = 26.56 <0.0001Row factor 1.321 2 0.6604 F (2, 48) = 475.5 <0.0001Column factor 0.3445 3 0.1148 F (3, 48) = 82.69 <0.0001Residual 0.06667 48 0.001389


nnex C. Comparative average frequency by Tukey’s test of oviposition and nuclear abnormalities in cells (neuroblasts andocytes) of A. aegypti treated with 1KL39-B or 1KL43-C. F1, F2, F3 and F4 are the experimental generations. B70 and B80: 70 and0 �g/mL of 1KL39-B, respectively. C20 and C30: 20 and 30 �g/mL of 1KL43-C, respectively.

Tukey’s multiple comparisons test Mean diff 95% CI of diff Significant? Summary

Oviposition – 1KL39-BF1

Control vs. 70 mg/mL 36 23.62 to 48.38 Yes ****Control vs. 80 mg/mL 43.6 31.22 to 55.98 Yes ****70 mg/mL vs. 80 mg/mL 7.6 −4.775 to 19.98 No ns

F2Control vs. 70 mg/mL 46.6 34.22 to 58.98 Yes ****Control vs. 80 mg/mL 45.6 33.22 to 57.98 Yes ****70 mg/mL vs. 80 mg/mL −1 −13.38 to 11.38 No ns

F3Control vs. 70 mg/mL 57.8 45.42 to 70.18 Yes ****Control vs. 80 mg/mL 61 48.62 to 73.38 Yes ****70 mg/mL vs. 80 mg/mL 3.2 −9.175 to 15.58 No ns

F4Control vs. 70 mg/mL 61 48.62 to 73.38 Yes ****Control vs. 80 mg/mL 62 49.62 to 74.38 Yes ****70 mg/mL vs. 80 mg/mL 1 −11.38 to 13.38 No ns

Oviposition – 1KL43-CF1

Control vs. 20 mg/mL 1.6 −11.62 to 14.82 No nsControl vs. 30 mg/mL 49 35.78 to 62.22 Yes ****20 mg/mL vs. 30 mg/mL 47.4 34.18 to 60.62 Yes ****

F2Control vs. 20 mg/mL 27 13.78 to 40.22 Yes ****Control vs. 30 mg/mL 55 41.78 to 68.22 Yes ****


20 mg/mL vs. 30 mg/mL 28

F3Control vs. 20 mg/mL 33.4

Control vs. 30 mg/mL 66.4

20 mg/mL vs. 30 mg/mL 33

F4Control vs. 20 mg/mL 47.4

Control vs. 30 mg/mL 72.6

20 mg/mL vs. 30 mg/mL 25.2


14.78 to 41.22 Yes ****

20.18 to 46.62 Yes ****53.18 to 79.62 Yes ****19.78 to 46.22 Yes ****

34.18 to 60.62 Yes ****59.38 to 85.82 Yes ****11.98 to 38.42 Yes ****


ING ModelM


95% CI of diff Significant? Summary

−0.02733 to 0.01933 No ns−0.03553 to 0.01113 No ns−0.03793 to 0.008731 No ns−0.03153 to 0.01513 No ns−0.03393 to 0.01273 No ns−0.02573 to 0.02093 No ns

−0.01033 to 0.03633 No ns−0.2031 to −0.1565 Yes ****−0.2063 to −0.1597 Yes ****−0.2161 to −0.1695 Yes ****−0.2193 to −0.1727 Yes ****−0.02653 to 0.02013 No ns

−0.04173 to 0.004931 No ns−0.2433 to −0.1967 Yes ****−0.2267 to −0.1801 Yes ****−0.2249 to −0.1783 Yes ****−0.2083 to −0.1617 Yes ****−0.006731 to 0.03993 No ns


−0.1232 to −0.06355 Yes ****−0.2420 to −0.1824 Yes ****−0.2616 to −0.2020 Yes ****−0.1486 to −0.08895 Yes ****−0.1682 to −0.1086 Yes ****−0.04945 to 0.01025 No ns



−0.1513 to −0.03660 Yes ***−0.4152 to −0.3005 Yes ****−0.4270 to −0.3123 Yes ****−0.3212 to −0.2065 Yes ****−0.3331 to −0.2184 Yes ****−0.06920 to 0.04552 No ns

−0.2030 to −0.08830 Yes ****−0.3977 to −0.2830 Yes ****−0.4553 to −0.3406 Yes ****−0.2521 to −0.1374 Yes ****−0.3096 to −0.1949 Yes ****−0.1149 to −0.0001991 Yes *

−0.04216 to 0.07616 No ns

373


0 P.R.C. Domingos et al. / Muta

Tukey’s multiple comparisons test Mean diff

Nuclei abnormalities (neuroblasts interphasic) – 1KL39-BControl

Generation F1 vs. generation F2 −0.004




Generation F2 vs. generation F4 −0.0106Generation F3 vs. generation F4 −0.0024

B70Generation F1 vs. generation F2 0.013



Generation F2 vs. generation F3 −0.1928Generation F2 vs. generation F4 −0.196Generation F3 vs. generation F4 −0.0032

B80Generation F1 vs. generation F2 −0.0184





Generation F3 vs. generation F4 0.0166

Nuclei abnormalities (oocytes interphasic) – 1KL39-BControl





Generation F2 vs. generation F4 −0.00324Generation F3 vs. generation F4 0












Chromosomal alterations (neuroblasts in nuclear division) – 1KL39-BControl



















Chromosomal alterations (oocytes in nuclear division) – 1KL39-BControl



Generation F1 vs. generation F3 0.02556 −0Generation F1 vs. generation F4 0.01006 −0Generation F2 vs. generation F3 0.00856 −0Generation F2 vs. generation F4 −0.00694 −0Generation F3 vs. generation F4 −0.0155 −0

PRESS


.03360 to 0.08472 No ns

.04910 to 0.06922 No ns

.05060 to 0.06772 No ns

.06610 to 0.05222 No ns

.07466 to 0.04366 No ns


ING ModelM

tion Research xxx (2014) xxx–xxx 11





−0.1114 to −0.05136 Yes ****−0.1586 to −0.09856 Yes ****−0.1918 to −0.1318 Yes ****−0.07724 to −0.01716 Yes ***−0.1104 to −0.05036 Yes ****−0.06324 to −0.003157 Yes *

−0.2010 to −0.1410 Yes ****−0.1780 to −0.1180 Yes ****−0.2770 to −0.2170 Yes ****−0.007043 to 0.05304 No ns−0.1060 to −0.04596 Yes ****−0.1290 to −0.06896 Yes ****





−0.1224 to −0.02172 Yes **−0.3665 to −0.2658 Yes ****−0.3766 to −0.2759 Yes ****−0.2944 to −0.1938 Yes ****

374









Generation F3 vs. generation F4 −0.00444B80





Generation F2 vs. generation F4 −0.1301Generation F3 vs. generation F4 0.0205

Nuclei abnormalities (neuroblasts interphasic) – 1KL43-CControl






Generation F3 vs. generation F4 −0.0024C20







C30Generation F1 vs. generation F2 −0.171



Generation F2 vs. generation F3 0.023Generation F2 vs. generation F4 −0.076


Nuclei abnormalities (oocytes interphasic) 1KL43-CControl





Generation F3 vs. generation F4 0












Chromosomal alterations (neuroblasts in nuclear division) – 1KL43-CControl







C20Generation F1 vs. generation F2 −0.07204Generation F1 vs. generation F3 −0.3161




Generation F2 vs. generation F4 −0.2542 −0Generation F3 vs. generation F4 −0.0101 −0

C30Generation F1 vs. generation F2 −0.1038 −0Generation F1 vs. generation F3 −0.2336 −0

PRESS


.3045 to −0.2039 Yes ****

.06042 to 0.04022 No ns

.1541 to −0.05344 Yes ****

.2839 to −0.1832 Yes ****


IN PRESSG ModelM



−0.2713 to −0.1706 Yes ****−0.1801 to −0.07948 Yes ****−0.1675 to −0.06688 Yes ****−0.03772 to 0.06292 No ns


−0.1571 to −0.03163 Yes **−0.3734 to −0.2479 Yes ****−0.3147 to −0.1892 Yes ****−0.2790 to −0.1536 Yes ****−0.2203 to −0.09487 Yes ****−0.004049 to 0.1214 No ns

−0.2291 to −0.1036 Yes ****−0.3398 to −0.2144 Yes ****−0.3418 to −0.2164 Yes ****−0.1735 to −0.04801 Yes ***−−

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Generation F3 vs. generation F4 −0.0155C20












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[

[

0.1755 to −0.05001 Yes ****0.06473 to 0.06073 No ns

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insecticidal and genotoxic potential of two semi-synthetic derivatives of dillapiole for the control...

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