Food and Chemical Toxicology 48 (2010) 825–830

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Food and Chemical Toxicology

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Permethrin-induced morphological changes in oocytes of Rhipicephalus sanguineus(Acari: Ixodidae) semi-engorged females

Gislaine Cristina Roma a, Karim Christina Scopinho Furquim b, Gervásio Henrique Bechara b,Maria Izabel Camargo-Mathias a,*

a Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista ‘‘Júlio de Mesquita Filho”, UNESP, Avenida 24 A, 1515, 13506-900, Rio Claro, SP, CP 199, Brazilb Departamento de Patologia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso Prof. Paulo Castellane, s/n, 14884-900, Jaboticabal, SP, Brazil

a r t i c l e i n f o

Article history:Received 12 August 2009Accepted 17 December 2009

Keywords:Rhipicephalus sanguineusPermethrinAcaricideOocytesCytotoxicityReproduction

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.12.016

* Corresponding author. Tel.: +55 19 35264135; faxE-mail address: micm@rc.unesp.br (M.I. Camargo-

a b s t r a c t

The permethrin, active ingredient of the Advantage� Max3 – Bayer, has been widely used in the chemicalcontrol of ticks. These ectoparasites are one of the most important animal groups that cause seriousdamage to their hosts. This study evaluated the toxic effects of permethrin in oocytes of Rhipicephalussanguineus semi-engorged females subjected to four treatments: group I (control – distilled water), groupII (206 ppm of permethrin), group III (1031 ppm of permethrin) and group IV (2062 ppm of permethrin).Results demonstrated that permethrin is a potent chemical agent causing major structural changes inoocytes, such as emergence of large vacuolated cytoplasm regions, reducing the amount of yolk granulesand decreasing the size of oocytes, culminating with cell death. As reported in the literature, theseoocytes changes, besides affect the tick nervous system, also drastically reduce or prevent the reproduc-tion process in females of R. sanguineus ticks subjected to this compound.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The economic importance of ticks is widely acknowledged.When feeding, many species of ticks transmit diseases caused byprotozoa, virus, bacteria (rickettsias and spirochetes) to man andother animals, as well as produce dermatosis and other more se-vere infections (Rey, 1973).

Among the species belonging to the Ixodidae family is the tickRhipicephalus sanguineus, an ectoparasite of dogs of great medicaland veterinary importance that produces debilitating effects dueto blood losses in affected animals and transmission of severalpathogens (Borges et al., 2007).

The female reproductive system of Ixodidae ticks is constitutedby an ovary with a pair of oviducts, uterus, a muscular connectiontube, vagina and genital opening (Sonenshine, 1991; Coons andAlberti, 1999). According to Denardi et al. (2004) in Amblyommacajennense, Oliveira et al. (2005, 2008, 2009) in R. sanguineus, Saitoet al. (2005) in Boophilus microplus and Oliveira et al. (2006) inA. triste, the ovary is a single and tubular continuous structure witha horseshoe shape and connected to a genital opening through oneor more oviducts.

The use of chemical acaricide is still the most efficient methodof control against ticks. Among the chemical compounds is the

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: +55 19 35340009.Mathias).

permethrin, active ingredient of the Advantage� Max3 – Bayer, asynthetic pyrethroid that works by quickly paralyzing the nervoussystem of the ticks (Mencke et al., 2003).

In this way, due to the wide use of the permethrin in chemicalcontrol of the tick and the scarce data in the literature about theaction of this acaricide in the reproductive system of the females’ticks, the present study aimed to investigate, through morpho-histological techniques, the behavior of oocytes of semi-engorgedfemales of the dog tick R. sanguineus exposed to different concen-trations of permethrin, attempting to detect possible changes infemale germinative cells of this tick species.

2. Material and methods

2.1. Rhipicephalus sanguineus ticks

A total of 60 R. sanguineus semi-engorged tick females weighing 27 mg in aver-age were used throughout the experiment. The specimens were supplied by the tickcolony maintained at the Brazilian Central of Studies on Ticks Morphology (BCSTM)at the São Paulo State University, Rio Claro, SP, Brazil, under controlled conditions(28 ± 1 �C, 80% humidity and 12 h photoperiod) in an Eletrolab EL 202 BOD (Biolog-ical Oxygen Demand) incubator and fed on New Zealand White rabbits (Approvedby Comitê de Ética em Pesquisa e Mérito Científico – UNIARARAS, Protocol No.011/2009).

The feeding laboratorial conditions of R. sanguineus ticks in the hosts were asstated elsewhere (Bechara et al., 1995). In summary, ticks were placed inside a feed-ing chamber consisting of a plastic tube (2.5 cm of diameter and 3 cm of height)glued, on the previous day, with an atoxic and non-lesive preparation (BritanniaAdhesives-Unit 4, UK) to the shaved back of the hosts.

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2.2. Dilution assays of permethrin (CAS No.: 52645-53-1)

The permethrin used in this study was purchased from Fersol Indústria e Comé-rcio S/A (Mairinque, SP, Brazil) and was designated by the manufacturers as 38.4%pure (384 g/L). The concentration of permethrin (3-phenoxybenzyl (1RS, 3RS, 1RS,3SR)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) used in thiswork was based on LC50 of 2062 ppm determined previously on a pilot test by Romaet al. (2009). The doses correspond to 10% of the LC50 (206 ppm), 50% of the LC50

(1031 ppm) and the normal LC50 (2062 ppm). The control group was exposed onlyto the placebo (distilled water).

The R. sanguineus females, after being washed in a sieve with tap water, weredried on soft absorbent paper. Afterwards, 60 females were divided into fourgroups of 15 specimens each and immersed in Petri dishes containing eitherthe above different concentrations of permethrin or distilled water, for 5 min.Ticks were then dried on absorbent paper and placed in an Eletrolab EL 202BOD incubator (28 ± 1 �C, 80% humidity and 12 h photoperiod) for 7 days. The

Fig. 1. Schematic drawing of the morphological changes observed in oocytes (I–V) of Rh(control), group II (206 ppm of permethrin), group III (1031 ppm of permethrin) and grstage III, IV = oocyte stage IV, V = oocyte stage V, cl = cell limit, ch = chorium, gv = germ

observation period was established like this because most often the effect ofthe acaricide is not immediate as it acts slowly on the physiology of the specimenanalyzed.

2.3. Histology

The R. sanguineus females were dissected on Petri dishes containing phosphatebuffered saline-PBS solution (NaCl 0.13 M, Na2HPO4 0.017 M, KH2PO4 0.02 M, pH7.2).

The ovaries were removed, fixed in 4% paraformaldehyde and 0.9% NaCl in 10%phosphate buffer (0.1 M – pH 7.5), dehydrated in an alcoholic series (70%, 80%, 90%and 95%) at 15 min intervals. Infiltration was made with Leica resin at 4 �C and thematerial embedded in plastic moulds at 4 �C to delay premature polymerization.The moulds with material were filled and covered with Leica resin and the polymer-ization completed at room temperature (about 37 �C).

ipicephalus sanguineus treated with permethrin in different concentrations: group Ioup IV (2062 ppm of permethrin). I = oocyte stage I, II = oocyte stage II, III = oocytevesicle, v = vacuoles, yg = yolk granules.

G.C. Roma et al. / Food and Chemical Toxicology 48 (2010) 825–830 827

Sections with 4 lm thickness were mounted on glass slides, stained with hema-toxylin–eosin (HE) and examined and photographed in a Motic BA 300 photomicro-scope. This device and other equipments were from the Histology Laboratory of theBiology Department at the Bioscience Institute, São Paulo State University, RioClaro, SP, Brazil.

3. Results

3.1. Ticks of group I (control)

The description of the female reproductive system of R. sanguin-eus was already reported by Oliveira et al. (2005). In the presentwork were obtained the same results related by these authors tothe control group (Figs. 1 and 2A–E).

Fig. 2. Histological sections of the Rhipicephalus sanguineus oocytes stained with hematoOocytes (I–V) of the group II (206 ppm of permethrin). (L–R) Detail of the oocytes (I–(2062 ppm of permethrin). I = oocyte stage I, II = oocyte stage II, III = oocyte stage III, IVnu = nucleolus, v = vacuoles, p = pedicel.

3.2. Ticks of group II (treated with 206 ppm of permethrin)

Females of R. sanguineus exposed to 206 ppm of permethrinpresented few oocytes with morphological changes when com-pared with the control group.

The oocytes I becomes more elongated and the germ vesicleshifts to the pole of the oocyte, toward the pedicel. In the cyto-plasm, the presence of weakly stained large vacuoles is observed(Figs. 1 and 2F).

In oocytes II, vacuolation can be observed around the germ ves-icle, and the weakly stained larger vacuoles can also be seen dis-tributed preferentially at the pole opposite the attachment of theoocytes to the pedicel. There is also a slight decrease in the largeyolk granules (Figs. 1 and 2G).

xylin and eosin (HE). (A–E) Detail of the oocytes I–V of the group I (control). (F–K)V) of the group III (1031 ppm of permethrin). (S–X) Oocytes II–V of the group IV= oocyte stage IV, V = oocyte stage V, cl = cell limit, ch = chorium, gv = germ vesicle,

Table 1Reduction percentage in the size of the oocytes IV and V subjected to the differentpermethrin concentrations in comparison with control group.

Permethrin concentrations

Oocytes 206 ppm 1031 ppm 2062 ppm

IV 3% 6% 11%V 2% 5.5% 9%

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The oocytes III present more vacuolated regions mainly nearthe germ vesicle and the periphery of the oocyte towards the ped-icel. Moreover, it is observed that the cytoplasm contains feweryolk granules (Figs. 1 and 2H).

The oocytes IV are characterized by the appearance of vacuolessurrounding the germ vesicle and the large granules of yolk (Figs. 1and 2I).

The oocytes V exhibit vacuoles around large yolk granules thatlose their original rounded shape and become irregular. Somegranules seem broken and releasing part of their contents intothe cytoplasm. At this stage the shape of the oocytes has an irreg-ular appearance with folds in their protective membranes (Figs. 1and 2J–K).

3.3. Ticks of group III (treated with 1031 ppm of permethrin)

Females of R. sanguineus subjected to this concentration of per-methrin present higher number of oocytes with morphologicalchanges when compared to the group II. Besides this, the oocytesexhibit more significant changes.

The oocytes I completely lose their original form and take on apear shape with large vacuoles occupying most of the cytoplasm.Other smaller vacuoles are found around the germ vesicle that islocated at the pole of the oocyte towards the pedicel (Figs. 1 and2L).

In the oocytes II the appearance of the yolk granules is similarto the previous group, but an intense vacuolation occurs in theperiphery of the oocyte immediately adjacent to the cell limit (Figs.1 and 2M).

The oocytes III present vacuolated regions, mainly around thegerm vesicle and the periphery of the oocyte near the pedicel.There are even fewer yolk granules when compared to the previousgroup (Figs. 1 and 2N).

The oocytes IV show many vacuoles between and around thelarge yolk granules located mainly at the cell periphery. They canalso be rounded or have already shown signs of disorganizationby the presence of folds in their protective membranes (Figs. 1and 2O–P).

The oocytes V have the same characteristics found to this stagein the group II; however, there is an increase in the cytoplasm vac-uolation (Figs. 1 and 2Q–R).

3.4. Ticks of group IV (treated with 2062 ppm of permethrin)

The ovaries of R. sanguineus subjected to this permethrin con-centration have many oocytes with greater morphological changeswhen compared to the other treatment groups.

The oocytes I suffer extreme changes, can not be found and arereplaced by amorphous masses.

The oocytes II exhibit extreme vacuolation, restricting the areaoccupied by yolk granules to the opposite side of their connectionwith the pedicel. The surroundings of the germ vesicle are mark-edly vacuolated (Figs. 1 and 2S).

The oocytes III exhibit vacuolation mainly in the cell peripheryand around the germ vesicle (Figs. 1 and 2T).

The oocytes IV suffer great cytoplasm vacuolation mainlyaround the large granules of yolk and in the cytoplasm regions fac-ing the pedicellar pole (Figs. 1 and 2U).

The oocytes V exhibit cytoplasm vacuolation around the yolkgranules, which are less evident when compared to the other treat-ment groups, and show large empty spaces in the regions near thepedicel also. Some oocytes have completely altered their originalform, displaying irregular contours and constriction in the medianregion. At this stage there is probably fusion of the yolk granules,since they are almost twice as big in size when compared withthose of the control group (Figs. 1 and 2V–X).

In general, in the treatment groups II, III and IV, there is aprogressive decrease in size of oocytes IV and V as the permethrinconcentration increases (Figs. 1 and 2I–K, O–R, U–X) (Table 1).

4. Discussion

Data on the direct influence of acaricides in other systems of thetick with the exception of the nervous system are scarce. Fewworks, as those performed by Davey et al. (1998), Friesen et al.(2003), Friesen and Kaufman (2003) and Oliveira et al. (2008,2009) describe that some acaricides can act as inhibitors of thedevelopment and the reproduction of ticks. Thus, due to the wide-spread use of permethrin and the scarce data in the literatureabout its action on the female reproductive system of ticks, thisstudy provides information on changes that occur in germinativecells of semi-engorged females of R. sanguineus exposed to differentconcentrations of this chemical acaricide.

Females of R. sanguineus subjected to 206 ppm, 1031 ppm and2062 ppm of permethrin showed changes in oocytes induced bythis chemical agent when compared with those of the controlgroup. These changes concern mainly the presence, size and num-ber of vacuoles in the oocytes cytoplasm, number of granules ofyolk stored and number and size of oocytes modified.

The morphology and dynamics of the vitellogenesis in R. san-guineus has been previously described by Oliveira et al. (2005),which showed that the ovary is of panoistic type where whole ger-minative cells correspond to oogonia or future oocytes, not pos-sessing specialized nurse and follicular cells.

The ovary of ticks is constituted by a wall formed by epithelialcells with spherical nuclei, which delimitates a lumen and where alarge number of oocytes are attached by the pedicel cells. Thisstructure, which previously was just considered by its function ofattaching the oocytes to the ovary wall (Diehl, 1970; Brinton andOliver, 1971), nowadays is known for its relationship with thevitellogenesis (Oliveira et al., 2007).

The development of the oocytes of R. sanguineus is asynchro-nous, since the oogenesis occurs in the distal-proximal directionalong the ovary. Thus, the oocytes in early developmental stagesare found in the distal region and larger and mature oocytes in la-ter developmental stages are found in the proximal region. Thesecharacteristics were also observed in other species of ticks (Denar-di et al., 2004; Oliveira et al., 2005; Saito et al., 2005).

The R. sanguineus oocytes were classified into five stages ofdevelopment in accordance with the cytoplasm aspect; locationof the germ vesicle; presence, amount and composition of the yolkgranules, and the presence of chorium (Oliveira et al., 2005).

This study showed that some oocytes in the early stages ofdevelopment become more elongated and with some vacuoles inthe cytoplasm, which moved the germ vesicle to the cell pole nearthe pedicel, in females of R. sanguineus exposed to 206 ppm of per-methrin. These data show that the acaricide, even at low concen-trations, would be able to cause changes in the germinative cellsin this tick species.

In specimens subjected to the concentration of 1031 ppm ofpermethrin the number of oocytes modified was higher in relationto those subjected to the previous concentration. Moreover, theoocytes I assumed an irregular pear shape and presented larger

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and more numerous vacuoles in the cytoplasm, suggesting thatthis concentration of permethrin caused more damage in the tickreproductive system.

In the group subjected to the 2062 ppm of permethrin oocytes Iwere no longer observed, but only a cytoplasm mass indicatingthat as higher concentration of this pyrethroid, major changeswould be sustained by germinative cells, which lead to autophagiccell death. These data, when analyzed under the point of view ofthe control of ectoparasites, become important seen that blockingthe reproduction of ticks would also be reducing its biological suc-cess. These data corroborate those found by Oliveira et al. (2008)for this same tick species subjected to fipronil, the active ingredi-ent of Frontline� acaricide.

The presence of vacuoles in oocytes I of R. sanguineus suggestthat they would be autophagic and responsible for the degradationof cell organelles that were damaged by permethrin. These datajustified the increase in vacuoles in these oocytes as the concentra-tion of the acaricide is high, and consequently causes more damageto the cell.

The oocytes II and III of R. sanguineus semi-engorged female ex-posed to 1031 and 2062 ppm of permethrin showed more signifi-cant cell changes, such as extensive cytoplasm vacuolation andreduction in number of yolk granules in relation to the concentra-tion containing 206 ppm of this acaricide, suggesting that theaction of permethrin is intensified in higher concentrations.Oliveira et al. (2008) also reported an increase in cell changes inthe oocytes of R. sanguineus exposed to higher concentrations offipronil.

Friesen et al. (2003) and Friesen and Kaufman (2003) analyzingthe ovaries of Amblyomma hebraeum tick females exposed to aver-mectin and cypermethrin acaricides, respectively, observed thatthese compounds cause a decrease in production of vitellin ele-ments in the oocytes, and reduction in the synthesis of ecdister-oids, which reduces the production and release of vitellogenins(the main yolk protein) to the hemolymph and its subsequent cap-ture by oocytes. In this study were observed that the yolk granulesof oocytes II and III became less evident at higher concentrations ofpermethrin, especially at concentration of 2062 ppm, suggestingthat the changes reported by the authors mentioned above couldalso be occurring here.

This work also showed that the oocytes IV and V of R. sanguineussubjected to permethrin present morphological changes as theconcentration of acaricide increases. In fact, very few altered oo-cytes IV and V were found in the concentration of 206 ppm (pres-ence of vacuoles in the cytoplasm). However, at permethrinconcentrations of 1031 ppm and 2062 ppm this number was muchhigher, especially in the latter concentration, where more than 80%of oocytes had modified their original morphology. Similarly, thenumber and size of vacuoles, probably autophagic, between andaround the granules of the yolk was intensified when exposed tohigher concentrations of permethrin, signaling the need to recycleportions of the cell cytoplasm damaged by the action of permeth-rin (as damaged organelles) or to degrade the whole cell.

The results of this study showed that the increase in the con-centration of permethrin caused a gradual decrease in the amountof yolk granules in the oocytes IV and V in relation to the controlgroup, suggesting that they broken and released their contents,which corroborate the data of Oliveira et al. (2008) in studies withfemales of R. sanguineus exposed to the fipronil chemical agent. Inoocytes V, in addition to the breakdown of granules, they may alsohave been fusing, because they have become twice larger in size incomparison to the control group. These structural changes alsosuggested that even if the oocytes get to reach the stage III unal-tered, they could still suffer the harmful action of permethrin instages IV and V of development (Oliveira et al., 2008).

The oocytes V subjected to 206 ppm, 1031 ppm and 2062 ppmof permethrin also showed irregular contours, suggesting that thechorium, the membrane highly resistant and responsible for pro-tecting the eggs (Hinton, 1982), could also have been damaged.Thus, the chorium would lose its original protective function,allowing the endocytosis of acaricide on the oocytes and theirconsequent detrimental action in these cells. Moreover, at theconcentration of 2062 ppm, many oocytes V showed a constric-tion in their median region, as well as the reduction of yolkgranules.

The effect of permethrin on the development of the ovary of R.sanguineus would also reflect changes in the diameter of the oo-cytes. In this sense, the oocytes I, II and III did not show differencesin size in different treatment groups, while oocytes IV and V wereprogressively smaller to the concentrations of 206 ppm, 1031 ppmand 2062 ppm, respectively. This is probably due to the fact thatpermethrin reduces the synthesis (endogenous) and incorporation(exogenous) of vitellin elements by oocytes, and causes the break-down and/or the fusion of the few existing granules. Thus, smalleraccumulation of yolk would lower the diameter of the oocyte,affecting its structural organization, making it impossible to gener-ate a new individual. Davey et al. (1998), Friesen and Kaufman(2003) and Oliveira et al. (2008) also reported reduction in the sizeof ticks oocytes exposed to different acaricides.

Thus, based on the results obtained here, were observed that fe-males of R. sanguineus showed changes in oocytes I, II, III, IV and Vin all treatment groups with permethrin, especially in the concen-tration of 2062 ppm, which would suggest that exposure to thisconcentration would cause major changes in the structure of theoocytes. Moreover, data obtained here showed that permethrineven when applied at concentrations much lower than those foundin the products sold commercially – acaricides currently availablein the market have permethrin in concentrations greater than300,000 ppm – would be effective for compromising the entirereproductive development of R. sanguineus female ticks, reducingor eliminating their viability. In addition, based on data obtainedin this study; it was shown that even if there was oviposition,the eggs probably would not be viable, due to major structuralchanges caused by the action of permethrin when they were inthe stage of oocytes V.

Oliveira et al. (2008, 2009), studying the action of fipronil, ahighly toxic chemical compound, reported that this would be apowerful agent in reducing the fertility of R. sanguineus females.Similarly, in this study it was found that permethrin, although clas-sified as medium toxic (Garcia-Garcia et al., 2005), would also beable of causing damage that leads to the loss of reproductive effi-ciency of ectoparasites.

This study also showed another very important aspect aboutthe reproduction of ticks, i.e., confirmed that the cells of the pedicelfixing the oocytes to the ovary wall are, in addition to being amechanical function, a real function in the capture (hemolymph)and production of elements of the yolk. In fact, the greater changesin the cytoplasm of oocytes of individuals exposed to permethrin,such as vacuolation, always occurred with greater frequency andintensity in the pole of the oocyte closer to the pedicel. This indi-cates that many of the substances taken from the hemolymphand transferred into the oocyte (like the yolk elements) have themeans to access the cells of the pedicel, which, in this case, is alsothe route of permethrin.

Based on the information described above it is concluded thatpermethrin is a powerful chemical agent that acts by reducingand/or preventing the reproduction of females of R. sanguineusticks, since many of the oocytes from individuals subjected to dif-ferent treatments with such acaricide exhibited major changes inthe germinative cells in relation to the control group.

830 G.C. Roma et al. / Food and Chemical Toxicology 48 (2010) 825–830

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Authors are grateful to the Fundação de Amparo à Pesquisa doEstado de São Paulo – FAPESP (Grant Nos. 07/57809-5 and 07/59020-0) and to the Conselho Nacional de Desenvolvimento Cientí-fico e Tecnológico – CNPq (Grant No. 308733/2006-1 and G.H. Bec-hara and M.I. Camargo-Mathias academic carrier researchfellowships) for the financial support and to Mr. Gérson de MelloSousa for technical support.

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