Food and Chemical Toxicology 47 (2009) 1255–1264

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Food and Chemical Toxicology

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Action of the chemical agent fipronil on the reproductive processof semi-engorged females of the tick Rhipicephalus sanguineus (Latreille, 1806)(Acari: Ixodidae). Ultrastructural evaluation of ovary cells

Patrícia Rosa de Oliveira a, Gervásio Henrique Bechara b, Maria Aparecida Marin Morales a,Maria Izabel Camargo Mathias a,*

a Departamento de Biologia, I.B., UNESP, Avenida 24 A 1515, Cx. Potal 199, CEP 13506-900, Rio Claro, SP, Brazilb Departamento de Patologia Veterinária, FCAV, UNESP, Via de Acesso Prof. Paulo Castellane s/n, CEP 14884-900, Jaboticabal, SP, Brazil

a r t i c l e i n f o

Article history:Received 28 August 2008Accepted 18 February 2009

Keywords:Rhipicephalus sanguineusFipronilOvaryAcaricideUltrastructureReproduction

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.02.019

* Corresponding author. Tel.: +55 19 3526 4135; faE-mail address: micm@rc.unesp.br (M.I. Camargo-

a b s t r a c t

The ovary of the tick Rhipicephalus sanguineus consists of a wall of epithelial cells and a large number ofoocytes in five different developmental stages (I–V), which are attached to the wall by a pedicel. The pres-ent study provides ultrastructural information on the effects (dose–response) of the acaricide fipronil(Frontline�) on ovaries of semi-engorged females of R. sanguineus, as well as it demonstrates some pos-sible defense mechanisms used by oocytes to protect themselves against this chemical agent. Individualswere divided into four groups. Group I was used as control while groups II, III and IV were treated withfipronil at the concentrations of 1, 5 and 10 ppm, respectively. Fipronil at the concentration of 10 ppmhad the strongest effect on the development of oocytes. At this concentration, even oocytes that reachedthe final developmental stage exhibited damaged cell structures. Moreover, the observation in fipronil-treated R. sanguineus ticks of damaged cellular components such as plasmic membrane, mitochondriaand protein granules (due to alteration in the protein synthesis), and cellular defense mechanisms suchas increase in the amount of cytoplasmic microtubules and large amounts of digestive vacuoles and mye-lin figures, were only possible by means of ultrastructure.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Ticks, one of the most important groups among arthropods, areectoparasites of vertebrates and thus may transmit several patho-gens to their hosts (Sonenshine, 1991).

The voracity of these ectoparasites to consume blood may resultin serious damage for their hosts, especially when several ticks in-fest the same individual (Sonenshine, 1991).

In the case of the tick Rhipicephalus sanguineus, dogs are its mostcommon host, although it may also be found in cats and rabbits.Studies have also reported this species infesting buffalos, camels,cattle, goats, horses, sheep, bats, reptiles, in addition to severalbirds and even humans (Walker, 1994).

The ovary, organ responsible for the reproduction of R. sanguin-eus, is panoistic, and therefore lacks nurse cells or follicular cells,confirming the data obtained for Amblyomma cajennense (Denardiet al., 2004), R. (Boophilus) microplus (Saito et al., 2005), and A. triste(Oliveira et al., 2006) ticks. It consists of a wall of epithelial cellsthat in certain regions proliferate and form pedicels. The latterare structures that attach oocytes (in different developmental

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x: +55 19 3534 0009.Mathias).

stages) to the ovary and synthesize and provide them with yolkprecursors (Till, 1961; Denardi et al., 2004; Oliveira et al., 2005,2006; Saito et al., 2005).

According to the literature, chemical control with acaricides isthe most efficient method against ticks since immunological andbiological controls still play a supplemental role.

Currently, new compounds are being released into the marketsuch as fipronil (active ingredient of Frontline�) (Taylor, 2001).Fipronil is an extremely active pesticide and is highly toxic to birds,reptiles and fish (Tingle et al., 2003). According to Tingle et al.(2003), in laboratory animals, fipronil cause abnormal gait andposture, ruffled fur, lethargy, tremors and convulsions.

Fipronil degrades slowly on vegetation and relatively slowly insoil and in water. One of its main degradation products, fipronildesulfinyl, is generally more toxic than the parent compound andis very persistent (Tingle et al., 2003).

As this drug is relatively new in the market, all the implicationsof its use are not fully understood (Sabatini et al., 2001; Oliveiraet al., 2008).

Thus, the present study analyzed the action (dose–response) offipronil at sub-lethal concentrations (1 ppm, 5 ppm and 10 ppm)on ovary cells of R. sanguineus semi-engorged females, aiming toassess which cells’ components would be damaged by the acaricide

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as well as to understand what are the defense mechanisms used bygerm cells (oocytes) in an attempt to defend themselves and tominimize damage.

2. Material and methods

2.1. R. sanguineus ticks

In the present study, a total of 60 partially fed females of R. sanguineus tickswere used throughout the experiment. They were supplied by the tick colony main-tained at the São Paulo State University-UNESP, campus of Jaboticabal-SP, Brazil,under controlled conditions (29 �C, 80% humidity and 12 h photoperiod) in a BOD(Biological Oxygen Demand) incubator and fed on white New Zealand rabbits.

2.2. Analysis with fipronil (n� CAS: 120068-37-3)

The fipronil was obtained from the so-called Regent� 800 WG, the commercialproduct made by BASF and its degree of purity is of 80%.

The concentrations of fipronil, (RS)-5-amino-1-(2,6-dichloro-a,a,a-trifluoro-p-tolyl)-4-trifluoro methylsulfinylpyrazole-3-carbonitrile, used were based on LC50

of 10 ppm determined previously on a pilot test by the authors. The concentrationscorrespond to 10% of the LC50 (1 ppm), 50% of the LC50 (5 ppm) and the normal LC50

(10 ppm). The control group was exposed only to the placebo (distilled water).The 60 females of R. sanguineus, after being washed in a sieve with tap water,

were dried on soft absorbent paper. After that, 45 females were divided into threegroups of 15 females each and immersed in Petri dishes containing the above dif-ferent concentrations of fipronil, for 2 min. The control group was also composedof 15 females that had been immersed in distilled water for the same period. Tickswere then dried in absorbent paper and placed in an incubator for 7 days.

2.3. Transmission electron microscopy (TEM)

All the ticks maintained in the refrigerator for thermal shock anesthesia weredissected in a phosphate buffered saline-PBS solution (NaCl 7.5 g/L, Na2HPO4

2.38 g/L and KH2PO4 2.72 g/L).The ovaries were fixed in 2.5% glutaraldehyde for 2 h, postfixed in 1% OsO4 for

2 h, contrasted in 2% uranyl acetate and dehydrated in a graded acetone series (50%,70%, 90% and twice in 100%). Next the material was submerged in a mixture of ace-tone and resin (1:1) for 12 h, embedded in Epon Araldite for 12 h and then includedin pure Epon resin and polymerized at 60 �C for 72 h. The material was sectionedand ultrathin sections were contrasted with uranyl acetate and lead citrate during25 and 10 min, respectively.

Afterwards, screens containing ultrathin sections of the material were exam-ined and photographed in a Phillips 100 Transmission Electron Microscope fromthe Biosciences Institute, UNESP, campus of Rio Claro-SP, Brazil. Then, the figureswere built: each page contains all the obtained results of a specific group(Fig. 1 = group I; Fig. 2 = group II; Fig. 3 = group III; Fig. 4 = group IV) and each linecorresponds to developmental stages of oocytes (I–V).

3. Results

3.1. Females of group I (control)

The morphohistological description of the ovaries and the vitel-logenesis dynamics of R. sanguineus has been reported by Oliveiraet al. (2005). The obtained results in females of group I correspondto those of the control group and the histological data were com-plemented by ultrastructural techniques.

R. sanguineus females of group I (control) present ovaries con-sisted of oocytes in five developmental stages (I–V), which are at-tached to the ovary wall by a pedicel.

Ooctyes I present a round-shaped germ vesicle, with little het-erochromatin and very prominent nucleolus, that occupies a largeregion in the center of the cytoplasm of the cell (Fig. 1A).

The cytoplasm is homogeneous, with large quantities of freeribosomes and polyribosomes, as well as mitochondria of shapesranging from round to elliptical, or even dumbbell-like, locatedmainly in the periphery of the oocyte (Fig. 1A).

In the plasmic membrane surrounding the oocyte, specializa-tions are present as small microvilli facing the basal lamina(Fig. 1B). In the region between the basal lamina and the mem-brane, some vesicles with electron-dense material are observed(Fig. 1B).

Oocytes II exhibit germ vesicle with disperse chromatin as wellas few and small electron-lucent lipid droplets in the periphery ofthe cell (Fig. 1C and D).

The most common organelles still are very electron-dense mito-chondria of various shapes and sizes that occupy the peripheralregion of oocytes II (Fig. 1C). Numerous ribosomes are alsoobserved, free or forming polyribosomes attached to the mem-brane of little developed lamellar rough endoplasmic reticulum(Fig. 1C and D).

Enclosing the oocyte, the plasmic membrane (with microvilli)lies on a basal lamina differentiated in two regions: an innermostlayer, thicker and in direct contact with microvilli; and outermostone, thinner and fibrillar (Fig. 1D). Microvilli present in this oocyteare longer and more abundant than those present in oocyte I. In theregion between the plasmic membrane and basal lamina, vesiclesare still observed (Fig. 1D).

Oocytes III exhibit several, although small, secretion granuleswith various contents. Those containing lipids are electron-lucent(Fig. 1E) and those with proteins, more electron-dense (Fig. 1Eand F).

Larger granules, containing proteins and lipid droplets, arefound mainly in the periphery of oocytes III, while smaller granulesare located in the central region (Fig. 1E and F).

Regarding organelles, mitochondria of various shapes and withcristae arranged in parallel arrays are observed throughout thecytoplasm (Fig. 1E), as well as numerous ribosomes and lamellarrough endoplasmic reticulum (Fig. 1E and F).

Oocytes III also exhibit numerous microvilli in the plasmicmembrane and a thick and continuous basal lamina subdividedinto two regions (Fig. 1F). Vesicles between these two regions, asin the previous stage, are still observed (Fig. 1F).

As the development of oocytes progresses (oocytes IV and V),the number and size of yolk granules decreases, as well as thenumber of microvilli in the plasmic membrane. These characteris-tics correspond to the final stage of vitellogenesis.

In oocytes IV, a large amount of protein granules of various sizesand electron densities (Fig. 1G and H) is observed. Lipid droplets,on the other hand, are electron-lucent, smaller than protein gran-ules, and distributed throughout the cytoplasm of the oocyte(Fig. 1G). Few small mitochondria among yolk granules are also ob-served (Fig. 1H).

In the peripheral region of oocytes IV, chorium deposition be-gins, as exocytic vesicles polymerized in the extracellular space be-tween the basal lamina and the plasmic membrane (Fig. 1H).

Ultrastructurally, oocytes V exhibit cytoplasm filled with largeand electron-dense yolk granules, probably consisting of proteins(Fig. 1J), as well as small and electron-lucent lipid droplets locatedin between large protein granules and in the periphery of the oo-cyte, near the plasmic membrane (Fig. 1I and J).

Surrounding the oocyte V, the chorium is completely depositedand consists of two layers: the exochorium, an outermost, thinnerand moderate electron-dense layer; and the endochorium, aninnermost, thicker and little electron-dense layer in direct contactwith the oocyte (Fig. 1J).

3.2. Females of group II (1 ppm)

Individuals treated with fipronil at this concentration presentoocytes characterized by different levels of cytoplasmic vacuola-tion, in addition to other ultrastructural alterations, whencompared to those of the control group. However, most character-istics of the cells remain unaffected.

Oocytes I exhibit early stages of cytoplasmic vacuolation mainlynear the peripheral region (Fig. 2B). In the plasmic membrane, thenumber and size of microvilli decreased compared to oocytes I ofthe control group (Fig. 2B and C). On the other hand, the basal

Fig. 1. (A)–(J). Ultrastructure of Rhipicephalus sanguineus ovary of the group I (control). (A) Detail of the cytoplasm near the germ vesicle of oocytes I. (B) Peripheral region ofoocytes I. (C) Detail of the peripheral cytoplasm of oocytes II. (D) Peripheral region of oocytes II. (E) Detail of the cytoplasm of oocytes III. (F) Peripheral region of oocytes III.(G) Detail of the cytoplasm of oocytes IV. (H) Peripheral region of oocytes IV. (I) Detail of the cytoplasm of oocytes V. (J) Peripheral region of oocytes V. bl = basal lamina;ch = chorium; gv = germ vesicle; l = lipid granule; lrer = lamellar rough endoplasmic reticulum; m = mitochondria; mv = microvilli; ne = nuclear envelope; p = proteic yolkgranules, pm = plasmic membrane. Bars: A = 1 lm; B = 1 lm; C = 1 lm; D = 1 lm; E = 2 lm; F = 1 lm; G = 4 lm; H = 2 lm; I = 4 lm; J = 5 lm.

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lamina, in addition to presenting a clear subdividion, is thicker(Fig. 2B and C). The germ vesicle remains unchanged (Fig. 2A).

Oocytes II exhibit irregular-shaped germ vesicle and evagina-tion on its sheath (Fig. 2D). Several vacuoles occupy the periphery

as well as the central region of the cytoplasm of the cell (Fig. 2D–G). The peripheral cytoplasm also exhibits some autophagicvacuoles (Fig. 2G), while the plasmic membrane present fewermicrovilli compared to that of oocytes II of the control group

Fig. 2. (A)–(T). Ultrastructure of Rhipicephalus sanguineus ovary of the group II (1 ppm). (A) Detail of the germ vesicle of oocytes I. (B) Peripheral cytoplasm of oocytes I. (C)Peripheral region of oocytes I. (D) Detail of the cytoplasm near the germ vesicle of oocytes II. (E) Cytoplasm of oocytes II. (F) Detail of the cytoplasm of oocytes II. (G) Peripheralregion of oocytes II. (H) Detail of the germ vesicle of oocytes III. (I) Detail of the cytoplasm near the germ vesicle of oocytes III. (J) Cytoplasm near the germ vesicle of oocytesIII. (K) Detail of the cytoplasm of oocytes III. (L) Peripheral region of oocytes III. (M) Detail of the cytoplasm near the germ vesicle of oocytes IV. (N) Detail of the cytoplasm ofoocytes IV. (O) Detail of the peripheral cytoplasm of oocytes IV. (P) Peripheral region of oocytes IV. (Q) Peripheral cytoplasm of oocytes V. (R) Detail of the cytoplasm ofoocytes V. (S) Detail of the peripheral cytoplasm of oocytes V. (T) Peripheral region of oocytes V. a = disorganized areas; av = autophagic vacuoles; bl = basal lamina;ch = chorium; gv = germ vesicle; l = lipid granule; lrer = lamellar rough endoplasmic reticulum; m = mitochondria; mb= myelinic bodies; mv = microvilli; ne = nuclearenvelope; nu = nucleolus; p = proteic yolk granules; pm = plasmic membrane; v = vacuoles; arrow = infoldings in nuclear envelope. Bars: A = 4 lm; B = 5 lm; C = 1 lm;D = 2 lm; E = 2 lm; F = 2 lm; G = 2 lm; H = 5 lm; I = 10 lm; J = 4 lm; K = 2 lm; L = 0.5 lm; M = 5 lm; N = 2 lm; O = 2 lm; P = 1 lm; Q = 1 lm; R = 0.5 lm; S = 2 lm;T = 2 lm.

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Fig. 3. (A)–(X). Ultrastructure of Rhipicephalus sanguineus ovary of the group III (5 ppm). (A) Detail of the germ vesicle of oocytes I. (B) Detail of the cytoplasm of oocytes I. (C)Cytoplasm of oocytes I. (D) Peripheral region of oocytes I. (E) Detail of the cytoplasm near the germ vesicle of oocytes II. (F) Cytoplasm of oocytes II. (G) Detail of the cytoplasmof oocytes II. (H) Detail of the peripheral cytoplasm of oocytes II. (I) Detail of the peripheral region of oocytes II. (J) Peripheral region of oocytes II. (K) Detail of the cytoplasmnear the germ vesicle of oocytes III. (L) Detail of the cytoplasm of oocytes III. (M) Detail of the filaments of oocytes III. (N) Peripheral region of oocytes III. (O) Detail of the germvesicle of oocytes IV. (P) Detail of the cytoplasm near the germ vesicle of oocytes IV. (Q) Detail of the peripheral cytoplasm of oocytes IV. (R) Peripheral region of oocytes IV. (S)Detail of the cytoplasm of oocytes V (second type). (T) Peripheral cytoplasm of oocytes V (second type). (U) Detail of the peripheral region of oocytes V (second type). (V)Peripheral region of oocytes V (second type). (W) Detail of the peripheral cytoplasm of oocytes V (first type). (X) Detail of the cytoplasm of oocytes V (first type).av = autophagic vacuoles; bl = basal lamina; ch = chorium; f = cytoskeleton elements; gv = germ vesicle; l = lipid granule; lrer = lamellar rough endoplasmic reticulum;m = mitochondria; mb = myelinic bodies; mv = microvilli; ne = nuclear envelope; nu = nucleolus; p = proteic yolk granules; pm = plasmic membrane; v = vacuoles;vrer = vesicular rough endoplasmic reticulum; arrow = infoldings in nuclear envelope. Bars: A = 10 lm; B = 2 lm; C = 2 lm; D = 1 lm; E = 5 lm; F = 2 lm; G = 1 lm;H = 2 lm; I = 1 lm; J = 1 lm; K = 4 lm; L = 2 lm; M = 0.25 lm; N = 0.5 lm; O = 5 lm; P = 2 lm; Q = 2 lm; R = 1 lm; S = 2 lm; T = 4 lm; U = 1 lm; V = 2 lm; W = 4 lm;X = 2 lm.

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Fig. 4. (A)–(P). Ultrastructure of Rhipicephalus sanguineus ovary of the group IV (10 ppm). (A) General view of oocyte II. (B) Detail of the cytoplasm of oocytes II. (C) Detail ofthe peripheral cytoplasm of oocytes II. (D) Peripheral region of oocytes II. (E) General view of oocyte III. (F) Cytoplasm of oocytes III. (G) Detail of the cytoplasm of oocytes III.(H) Peripheral region of oocytes III. (I) Detail of the cytoplasm of oocytes IV. (J) Peripheral region of oocytes IV. (K) Detail of the peripheral region of oocytes IV. (L) Generalview of oocyte V. (M) Cytoplasm of oocytes V. (N) Detail of the cytoplasm of oocytes V. (O) Detail of the peripheral cytoplasm of oocytes V. (P) Peripheral region of oocytes V.av = autophagic vacuoles; bl = basal lamina; ch = chorium; f = cytoskeleton elements; l = lipid granule; m = mitochondria; mb = myelinic bodies; mv = microvilli; p = proteicyolk granules; pm = plasmic membrane; v = vacuoles; vrer = vesicular rough endoplasmic reticulum, * = electrondense material around the oocyte. Bars: A = 10 lm; B = 2 lm;C = 2 lm; D = 1 lm; E = 10 lm; F = 1 lm; G = 0.25 lm; H = 2 lm; I = 1 lm; J = 2 lm; K = 2 lm; L = 10 lm; M = 4 lm; N = 2 lm; O = 5 lm; P = 2 lm.

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(Fig. 2G). In the region between the basal lamina and the plasmicmembrane, in addition to few microvilli and vesicles, some myelinfigures are observed (Fig. 2G).

Oocytes III present deep infoldings in the nuclear sheath, result-ing in a very irregular-shaped germ vesicle (Fig. 2H–J). In the cyto-

plasmic region near the germ vesicle, disorganized areas areobserved and protein granules and lipid droplets of yolk are no long-er visible (Fig. 2I). Organelles, such as mitochondria and lamellarrough endoplasmic reticulum, are still observed. However a moder-ate quantity of vacuoles is also observed, including autophagic ones

P.R. de Oliveira et al. / Food and Chemical Toxicology 47 (2009) 1255–1264 1261

throughout the cell (Fig. 2J and K). In the peripheral region, betweenthe basal lamina and the plasmic membrane, myelin figures arepresent (Fig. 2L).

Oocytes IV exhibit small vacuoles around large protein gran-ules and lipid droplets of yolk (Fig. 2M–O) in the cytoplasm. Inthe peripheral region of the cells, the chorium, although present,is thinner and more electron-dense than those observed inoocytes IV of the control group (Fig. 2O and P). The remainingcharacteristics are similar to those of oocytes IV of the controlgroup.

In oocytes V, instead of high quantities of large protein granulesof yolk observed in the control group in this stage, many cytoplas-mic vacuoles are present in the periphery and in the central regionof the cell (Fig. 2Q–T), as well as remaining large protein granules(Fig. 2Q). The chorium is no longer observed and a thin membraneis present instead (Fig. 2Q–T).

3.3. Females of group III (5 ppm)

Individuals of group III present the highest number of modifiedoocytes, in addition to large altered cytoplamic areas when com-pared to the previous group.

Oocytes I exhibit distinct germ vesicle with infoldings in itssheath, one main nucleolus, large and prominent, and smaller ones,which were not previously observed (Fig. 3A). In the cytoplasm,larger quantities of vacuoles than that observed in oocytes I ofthe group II (Fig. 3B and C) fill the cytoplasm as well as the regionadjacent to the plasmic membrane. In the latter, microvilli are nolonger observed (Fig. 3D). Some vacuoles near the membrane areruptured, releasing their contents into the region between theplasmic membrane and the basal lamina (Fig. 3D).

Oocytes II exhibit more cytoplasmic vacuolation when com-pared to oocytes II of group II (Fig. 3E–G), including vacuolation be-tween the basal lamina and the plasmic membrane (Fig. 3H and I).In the cytoplasm, myelin figures are also observed, as well as auto-phagic vacuoles and lamellar and vesicular rough endoplasmicreticulum (Fig. 3E–H). In the peripheral region, also between theplasmic membrane and the basal lamina, larger quantities of mye-lin figures are observed (Fig. 3J).

Oocytes III exhibit irregular germ vesicle as a result of the sev-eral infoldings in its sheath (Fig. 3K). In the cytoplasm, very irreg-ular protein granules are observed with several areas undergoingdegeneration while the number of lipid droplets of yolk decreases(Fig. 3K and L). Cytoskeleton filaments and an increase in cytoplas-mic vacuolated areas, mainly near the germ vesicle (Fig. 3K–M), arealso observed. The organelles usually found in oocytes III are few-er; only rough endoplasmic reticulum is observed, while mito-chondria become gradually scarcer (Fig. 3L). In the periphery ofthe oocyte, the basal lamina underwent changes characterized bythe outermost layer separated from the innermost one, with irreg-ularities as if it lacked lamina (Fig. 3N).

Oocytes IV exhibit smaller germ vesicle, with altered shape andheterochromatin adhered to the internal region of the nuclearsheath (Fig. 3O). In the cytoplasm, the number of lipid droplets de-creases (Fig. 3Q). Protein granules and mitochondria remain un-changed compared to those of the control group (Fig. 3P–R).Intense vacuolation is also observed around large yolk granulesas well as in regions near the germ vesicle and between the chori-um and the basal lamina (Fig. 3O–R). In oocytes in this stage, thechorium is thinner than that of oocytes IV of the control groupand similar to that of oocytes of group II in the same stage(Fig. 3Q and R).

Oocytes V exhibit large protein granules of yolk with modifica-tions and various electron densities (Fig. 3S). Lipid droplets are nolonger observed. Myelin figures are present as well as many vacu-

oles of various sizes in the cytoplasm (Fig. 3S and T). In theperiphery, the chorium is thin and accompany the projections ofthe plasmic membrane of the oocyte. Also, the chorium is not sub-divided (endo and exo) and is ruptured in some regions, releasingportions of the cytoplasm into the extracellular space (Fig. 3T–V).The basal lamina does not surround the cell entirely, as it is alsoruptured (Fig. 3V).

Oocytes V with other characteristics are also observed, althoughless frequently (Fig. 3W and X). These oocytes still have intact yolkgranules (Fig. 3W and X), cytoplasm with numerous vacuoles ofseveral sizes (Fig. 3W and X), unaltered basal lamina, and choriumwithout subdivisions, thicker than that of the oocyte V describedpreviously, although thinner than that of oocyte V of the controlgroup (Fig. 3W).

3.4. Females of group IV (10 ppm)

Ovaries of individuals treated with fipronil at a concentration of10 ppm present many oocytes with extensive altered and vacuo-lated areas, indicating a process of cell death.

Oocytes I can no longer be identified due to advanced degener-ation stages.

Oocytes II exhibit intense vacuolation, occupying more thanhalf of the cytoplasmic region (Fig. 4A–D), including the region be-tween the basal lamina and the plasmic membrane (Fig. 4A, C andD). Many myelin figures and autophagic vacuoles are observed inthe cytoplasm, in addition to few lipid droplets, mitochondria,and vesicular rough endoplasmic reticulum (Fig. 4B and C). The ba-sal lamina is present only in some regions where the plasmic mem-brane exhibits few microvilli (Fig. 4A, C and D).

Oocytes III exhibit extensive vacuolated areas throughout thecell, including inside of protein granules of yolk (Fig. 4E–H) thatstill exhibit irregular shape and damaged areas (Fig. 4E–G), similarto those observed in oocytes III of group III. Lipid droplets are nolonger present. Regarding organelles, the rough endoplasmic retic-ulum is vesiculated and mitochondrial cristae are disorganized(Fig. 4F). In the central cytoplasm, many autophagic vacuoles, mye-lin figures, and cytoskeleton elements are present (Fig. 4F and G).In the peripheral cytoplasm, deposition of electron-dense materialis observed forming a strip surrounding oocytes III (Fig. 4H). Theplasmic membrane no longer has microvilli and is in direct contactwith the basal lamina (Fig. 4H).

Oocytes IV exhibit few infoldings in the membrane, alteringthe round shape observed in previous groups (Fig. 4J and K). Inthese oocytes, intense vacuolation around and inside large proteingranules of yolk, as well as in the peripheral region near the ped-icel is observed (Fig. 4I–K). These protein granules have lost theiroriginal shape and became irregular with damaged areas (Fig. 4Iand J). Lipid droplets are not longer observed. The cytoplasm stillexhibits mitochondria with altered cristae, cytoskeleton elements,and myelin figures (Fig. 4I–K). The chorium presents pores thatrelease portions of cytoplasm filled with vacuoles and myelin fig-ures into the region between the plasmic membrane and the ba-sal lamina (Fig. 4J and K). In addition, the chorium of oocytes IVof group III is thinner than that of oocytes IV of previous groups(Fig. 4J and K).

Oocytes V exhibit many infoldings in the plasmic membrane(Fig. 4L and O) and several vacuoles filling the cytoplasm, even in-side larger protein granules of yolk (Fig. 4L–N and P). Some of theseprotein granules, located in the periphery, are undergoing frag-mentation (Fig. 4L and O). Lipid droplets are no longer observed,unlike in oocytes V of other treatment groups (II and III). In theperipheral region, the basal lamina is preserved and the choriumis thinner than that of oocytes V of the control group and withoutsubdivisions (endo and exo) (Fig. 4L, O and P).

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4. Discussion

Seeking to better understand the dynamics of tick reproduction,several studies have been conducted (Denardi et al., 2004; Saitoet al., 2005; Oliveira et al., 2006), including on R. sanguineus(Oliveira et al., 2005). In the present study, the ultrastructure ofthe ovaries of semi-engorged females of R. sanguineus treated withfipronil in various concentrations was described (dose–response),aiming at assessing the damage caused to germ cells, as well asunderstanding what are the mechanisms used by these animalcells to protect themselves against this chemical agent. Theseultrastructure techniques allow the observation of small regionsdue to their great magnification, making so hard to get imagesshowing the cell in its totality, mainly in the case of the largestcells oocytes. For the visualization of total cells, we have actuallyused histological techniques and light microscopy as shown byOliveira et al. (2008).

According to Oliveira et al. (2005), the ticks’ ovary of the Ixod-idae family (genus Rhipicephalus, Amblyomma, etc.) consists of awall of epithelial cells and a large number of oocytes which are at-tached to the ovary wall by a cell pedicel. Because of the wide var-iation in morphological and histological appearance of the oocytes,they are classified according to developmental stage: I–V. Similarresults were obtained by Till (1961) in R. appendiculatus, Balashovand John (1983) in Hyalomma asiaticum, Denardi et al. (2004) inA. cajennense and Oliveira et al. (2006) in A. triste.

In the individuals of the control group, oocytes of all develop-mental stages exhibited typical morphological characteristics al-ready described for other ticks (Denardi et al., 2004; Saito et al.,2005; Oliveira et al., 2006). Thus, in the present study the ultra-structural characteristics of oocytes of the control group will notbe discussed, only the changes caused by the application of fipro-nil. The main criteria used to recognize cell injuries caused bychemical agents were: the presence of digestive vacuoles and mye-lin figures, as well as disorganized membranes, mitochondria andendoplasmic reticulum.

In oocytes I of R. sanguineus females treated with fipronil at1 ppm (group II), changes such as few vacuoles in the cytoplasm,fewer and smaller microvilli in the plasmic membrane, and basallamina already subdivided in two regions, suggest that fipronil isalready affecting these cells, by modifying their structure.

In the females treated with fipronil at 5 ppm (group III), thegerm vesicle of oocytes I exhibited infoldings in its sheath andthe cytoplasm presented large amounts of vacuoles, includingsome ruptured ones, near a plasmic membrane with no micro-villi. The absence of these membrane specializations (or eventhe reduced number) in these oocytes might be an attempt tominimize internal cell damage, by decreasing the surface areawith the hemolymph, consequently decreasing the uptake ofthe acaricide.

In individuals of group IV, treated with fipronil at 10 ppm, oo-cytes I were no longer observed, suggesting that fipronil in thisconcentration caused the total destruction of cells leaving onlyan amorphous mass. The vulnerability of these cells in this devel-opmental stage might be due to the absence of the chorium, theprotective membrane of eggs with special characteristics thatmight decrease the rate of acaricide uptake in the cell, as therewould be one more barrier to be overcome.

Thus, oocytes I of individuals subjected to the treatments II–IVsustained progressive damage caused by fipronil, which at 10 ppm,culminated in cell death.

According to Oliveira et al. (2005) and the observations in thecontrol group (group I), in oocytes II of R. sanguineus, vitellogenesisbegins with the synthesis of vitellogenic precursors by the cell(endogenous synthesis) as well as the uptake of elements produced

by other tissues (exogenous or extraovarian uptake of yolk) thatare later absorbed from the hemolymph by the oocyte.

Oocytes II of individuals treated with fipronil at 1 ppm (groupII), exhibited infoldings in the sheath of the germ vesicle, myelinfigures and vacuoles, including autophagic ones, in the cytoplasm,in addition to fewer microvilli in the plasmic membrane comparedto those of the control group. These changes suggest that in thisconcentration, damaged cell organelles in oocytes II might be con-tained and lysed in vacuoles.

According to Junqueira and Carneiro (2006) and Carvalho andPimentel (2007), autophagic vacuoles are structures found mainlyin cells where degradation and recycling of damaged portions andcytoplasmic organelles are taking place, which would explain thepresence of these organelles in fipronil-treated oocytes II.

Individuals exposed to 5 (group III) and 10 ppm (group IV) offipronil, exhibited oocytes II with even more vacuolation, severalmyelin figures; although, lamellar and vesicular rough endoplas-mic reticulum were also observed.

Specifically in individuals exposed to fipronil at 10 ppm, oocytesII exhibited further damage, including a decrease in exogenous up-take of yolk precursors, as microvilli in the plasmic membranewere reduced to even lower numbers. Regarding the rough endo-plasmic reticulum, its presence also suggests that despite the inter-ruption of exogenous uptake of yolk and the damage sustained bythe cells, they might still be trying to maintain protein synthesis(endogenous synthesis), although in lower scale and thus preserv-ing and continuing their development.

On the other hand, it is necessary to take in to account thatvesiculation of the rough endoplasmic reticulum of oocytes II trea-ted with fipronil might also be an indication of early stages of celldeath, as this is one of the most affected organelles. This character-istic was also described by Silva de Moraes and Bowen (2000)studying this process in insects.

Oocytes III of individuals treated with fipronil at 1 ppm under-went changes and exhibited, in addition to vacuolation, the pres-ence of myelin figures located between the basal lamina and theplasmic membrane, probably a result of the action of the fipronilin cell structures, which might have caused the cell to eliminateremnants to the extracellular space.

Oocytes III of individuals treated with fipronil at 5 and 10 ppmshowed large vacuolated areas and the presence of cytoskeletonelements in the cytoplasm, in addition to changes in the proteingranules, which became irregular shaped and exhibited disorga-nized areas.

Chapman (1998), studying degenerating oocytes of insects, re-ported that they exhibit disorganized yolk, where the protein vitel-logenin is released from granules by autophagic digestion. Thisvitellogenin, however, is not disposed, but returns to the hemo-lymph, as the permeability of the plasmic membrane increased.This process probably occurred in the oocytes III of our study, asa long strip of electron-dense material was observed underneaththe plasmic membrane, suggesting that these are the same pro-teins from ruptured granules of yolk protein, as a result of the ef-fects of fipronil.

On the other hand, the appearance of cytoskeleton elements inthe cytoplasm of oocytes III of the groups treated with fipronil at 5and 10 ppm of fipronil might be associated with the need of thecell to isolate its cytoplasm (that might still be functional) fromdamaged areas, preventing further damage and still attemptingself-preservation. This role of cytoskeleton elements in oocyteshas been previously suggested by Jedrzejowska and Kubrakiewicz(2007).

Specifically oocytes III of individuals treated with fipronil at10 ppm lacked lamellar rough endoplasmic reticulum. Hacker(2000) reported that this might be due to the dilation and

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fragmentation of these organelles, which might give rise to vesicu-lar rough endoplasmic reticulum. These data might explain thelarge quantity of vesicular rough endoplasmic reticulum observedin oocytes III of R. sanguineus of this treatment group.

In addition, in oocytes III of group IV, mitochondria were swol-len and cristae were disorganized and/or absent. According toProskuryakov (2002), these changes might be due to the rapid in-crease in the quantity of calcium ions or cell stress, factors thatcould alter the structure of the internal membrane of mitochondriathus making them permeable. This process might also result inmitochondrial death, directly affecting ATP levels in the cell andconsequently, the respiratory metabolism, which in turn could leadto cell death.

In addition, in oocytes III of group IV, the uptake of yolk com-pounds from the hemolymph was halted and a complete retractionof microvilli of the plasmic membrane was observed.

In oocytes IV of individuals exposed to 1 ppm, 5 ppm and10 ppm of fipronil, vacuolated areas were observed, as well asthe presence of a thinner chorium than the control group. The pro-gressive increase in cytoplasmic changes and cell vacuolationwhen comparing oocytes IV of groups II–IV, suggest that muchmore intense autophagic processes are taking place in these oo-cytes of individuals treated with higher concentrations of fipronil,probably an attempt of the cell to eliminate larger quantities ofdamaged elements.

Oocytes V, according to Oliveira et al. (2005) and the observedin R. sanguineus females of the control group, are characterizedby having the largest protein granules of yolk, few and small lipiddroplets in their cytoplasm, as well as chorium completely depos-ited and subdivided into endo and exochorium. This, however, wasnot observed in oocytes V of individuals exposed to fipronil at1 ppm, where yolk granules were replaced by large vacuolatedareas and the oocyte membrane did not exhibit the typical charac-teristics of chorium, as it was very thin.

In individuals exposed to 5 ppm, two types of oocytes V wereobserved with distinct morphology. One type was characterizedby yolk granules of various electron densities, myelin figures, cyto-plasmic vacuoles, and thin chorium lying on a ruptured basal lam-ina. The second type exhibited intact yolk granules, severalvacuoles, and a thicker chorium than that of the first type lyingon an intact basal lamina. These results clearly show that oocytesV may react differently to the action of fipronil. Mature oocytes,presenting a thicker chorium with all layers, might have lower per-meability to external agents, although it does not form a total bar-rier against the acaricide, as the cytoplasm of these cells exhibitdisorganization clearly caused by the fipronil action.

This stronger or weaker reaction to the effects of this chemicalagent also explains the presence of normal oocytes near oocyteswith prominent morphological changes, confirming the resultsobtained by Schwartz and Osborne (1995) and also shows that celldeath does not occur simultaneously throughout the ovary ofR. sanguineus.

In individuals exposed to 10 ppm, oocytes V with prominentchanges were observed, confirming that this concentration offipronil causes the most damage in germ cells of R. sanguineus.

According to the literature, the reproductive system is con-trolled by complex interactions involving the central nervous andreproductive systems. Fipronil is a chemical agent that affects spe-cifically the central nervous systems, preventing gamma-aminobu-tyric acid (GABA) from binding to its receptors (Sattelle, 1990; Coleet al., 1993) and consequently, the flux of chloride ions into the cell(Sattelle, 1990; Cole et al., 1993). Recent studies conducted byMcCarthy (1995), Colborn (1998), and Davis et al. (2000) also re-ported that fipronil might also be an hormonal disruptor, blockingthe action of other hormones and/or controlling them, via mecha-nisms triggered by the central nervous system (Gray, 1998;

Sonnenschein, Soto, 1998; Baker, 2001). Thus, the present studyshowed that oocytes of R. sanguineus might react against the actionof fipronil, by presenting a series of morphological changes thatmay result in cell death.

Friesen et al. (2003), in studies with the tick A. hebraeum re-ported that the chemical agent avermectin induced the interrup-tion and degeneration of yolk present in oocytes of specimens,confirming the data obtained by the present study.

Friesen and Kaufman (2003), however, detected the inhibitionof the uptake of vitellogenin, the main protein of the yolk, by oo-cytes of the tick A. hebraeum exposed to the insecticide cypermeth-rin, which probably also occur in the ovary of R. sanguineusexposed to fipronil, due to the decrease in the number of microvilliin the plasmic membrane of oocytes.

Thus, our findings showed that females of the tick R. sanguineus,when exposed to fipronil, exhibit oocytes with different responsesto the drug. Thus, in the same ovary treated with a certain concen-tration of fipronil, several oocytes with morphological changeswere observed as well as interruption of vitellogenesis that re-sulted in cell death, and others that, although not completely, werestill able to maintain part of their cells structures intact. These datademonstrated that fipronil is an efficient agent to reduce the fertil-ity of R. sanguineus females.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We would like to thank Mr. Marcos Aparecido Pizano and Mr.Ronaldo Del Vecchio for their technical support, to FAPESP (Grantno. 06/52599-0) and CNPQ (Grant no. 308733/2006-1) for financialsupport. Part of this work has been facilitated through the Interna-tional Consortium on Ticks and Tick-borne Diseases (ICTTD-3)Coordination Action financed by the INCO Program of the EuropeanCommission Project No. 510561.

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