Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Experimental Parasitology 110 (2005) 146–156

www.elsevier.com/locate/yexpr

Morphological characterization of the ovary and oocytes vitellogenesis of the tick Rhipicephalus sanguineus

(Latreille, 1806) (Acari: Ixodidae)

Patrícia Rosa de Oliveira a, Gervásio Henrique Bechara b, Sandra Eloisi Denardi a,Érika Takagi Nunes a, Maria Izabel Camargo Mathias a,¤

a Departamento de Biologia, Instituto de Biociências, UNESP, Av. 24 A, no. 1515, Cx. Postal 199, Rio Claro, SP 13506-900, Brazilb Departamento de Patologia Veterinária, FCAV, UNESP, Via de Acesso Prof. Paulo Castellane, s/n, Jaboticabal, SP 14884-900, Brazil

Received 18 October 2004; received in revised form 15 December 2004; accepted 16 December 2004Available online 16 March 2005

Abstract

This study presents the morphology of the ovary, as well as the process of the vitellogenesis in oocytes of the tick Rhipicephalussanguineus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ consists of a singletubular structure, continuous, and composed of a wall formed by small epithelial cells with rounded nuclei which delimit thelumen. The oocytes in the diVerent developmental stages in this tick species were classiWed into Wve stages (I–V). They remainattached to the ovary during vitellogenesis by a cellular pedicel and afterwards the mature oocytes (stage V) are released into theovary lumen. 2005 Elsevier Inc. All rights reserved.

Keywords: Rhipicephalus sanguineus; Tick; Ovary; Vitellogenesis; Histology; Ultrastructure

1. Introduction

Ticks are ectoparasites of great medical-veterinarianimportance because besides causing huge blood loss inthe infested animals, they can act as vectors for proto-zoa, virus, and bacteria and other pathogens (Flecht-mann, 1973).

Numerous studies are currently under way to Wnd aneVective control strategy that would minimize the dam-age caused by these parasites. A new tick controlapproach is an immunological one, consisting in theidentiWcation, isolation, and synthesis of proteins thatprotect tissues and organs of the tick, mainly those of thereproductive system, with the objective of developing avaccine (Tellam et al., 1992; Willadsen, 1997).

¤ Corresponding author. Fax: +55 19 3526 4136.E-mail address: [email protected] (M.I. Camargo Mathias).

0014-4894/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2004.12.016

Because few studies have focussed on the internalorgans of the tick Rhipicephalus sanguineus, the presentwork has aimed to contribute new data concerning mor-phological, histological, and ultrastructural aspects of thefemale reproductive system of R. sanguineus. In addition,it attempts to establish the dynamics of the vitellogenesisprocess in this species to provide information that couldcontribute to the future control of this parasite.

2. Materials and methods

Semi-feeding females of R. sanguineus ticks used inthis study were from the tick colony maintained undercontrolled (28 °C, 80% humidity, and 12 h photoperiod)at the Department of Animal Pathology, VeterinaryCollege, UNESP—Jaboticabal, SP, Brazil. Equipmentfrom the Histology and Electron Microscopy

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Laboratories of the Biology Department at the Biosci-ences Institute, UNESP—Rio Claro, SP, Brazil, wereutilized throughout the study.

Twenty Wve specimens maintained in the refrigeratorfor thermal shock anesthesia were dissected in salinesolution (NaCl 7.5 g/L, Na2HPO4 2.38 g/L and KH2PO42.72 g/L). The ovaries were removed, sectioned and theoocytes were drawn using a camera lucida coupled to aZeiss stereomicroscope.

2.1. Scanning electron microscopy

The ovaries removed were Wxed in Karnovisky for24 h, and dehydrated in a graded 70–100% ethanol andacetone series. The material were processed by criticalpoint drying, sputtered with gold, and examined andphotographed in a PHILLIPS 505 scanning electronmicroscopy (SEM).

2.2. Histology

Ovaries were Wxed in 4% paraformaldehyde. Thematerial was dehydrated in ethanol, embedded in JB4resin for 24 h at 4 °C and transferred to plastic mouldspreviously Wlled with resin containing a catalyzer. Afterresin polymerization, the material was sectioned using aSorvall JB4 microtome (Bio-Red) and stained withhematoxylin and eosin, following routine histologicalprocedures.

2.3. Histochemistry

Histochemical tests were applied in order to detectthe presence of the following compounds: proteins (Bro-mophenol Blue according to Pearse, 1985); lipids (NileBlue according to Lison, 1985); polysaccharides (PASaccording to Junqueira and Junqueira, 1983).

2.4. Transmission electron microscopy

The material was Wxed in 2.5% glutaraldehyde, post-Wxed in 1% OsO4, and embedded in Epon Araldite.Ultrathin sections were contrasted with acetate andlead citrate. The material was then embedded in pureEpon resin and polymerized at 60 °C for 72 h. After-wards, screens containing ultrathin sections of thematerial were observed and photographed in a Phillips100 transmission electron microscopy (TEM).

3. Results

3.1. Ultramorphology

The scanning electron microscopy allowed the obser-vation of a unique and tubular ovary in females of R.

sanguineus arranged in the posterior third part of thehorseshoe shaped body where a large number of oocytesof several sizes are Wxed and developed simultaneouslyand yet asynchronically (Figs. 1A and B). The oocytesadhered to the ovary are distributed in two distinctregions: the oocytes in early developmental stages arefound in the distal region and larger and mature oocytesin later developmental stages are found in the proximalregion (Figs. 1A and B).

3.2. Histology

Histologically, the ovary of the tick R. sanguineus wasclassiWed as panoistic, without specialized nurse cells.The ovary of R. sanguineus possessed a narrow lumendelimited by a delicate wall of small epithelial cubic cellswith round nuclei (Fig. 2A). In the ovary wall, oocytesWxed by the pedicel are found in various developmentalstages, including some in reabsorption processes (Figs.2A, B, C, and E).

Fig. 1. (A,B) Scanning Electron Microscopy (SEM) of Rhipicephalussanguineus ovary showing oocytes in several phases of development. d,distal region; moo, mature oocyte; oo, young oocyte; ov, ovary; p,proximal region. Bars: A D 0.5 mm; B D 0.1 mm.

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3.3. Oocytes classiWcation

Because of the wide variation in morphological andhistological appearance of the oocytes, they were classi-Wed according developmental stage (Figs. 2 and 3).

Oocytes I or in early stages of development—con-sisted of a small elliptical cell, a clear germ vesicle with avery evident nucleolus, occupying great part of the cyto-

plasm, which had a homogeneous appearance, withoutgranulations. There was a thin plasmic membrane sur-rounding the oocyte (Figs. 2A, E, F, H, L, and 3A).

Oocytes II—were larger than oocyte I, had ellipticalmorphology and germ vesicles in the center. A thinhomogeneous granulation was observed in the cyto-plasm. Other histological characteristics were similar tothose found in stage I (Figs. 2A, E–H, M and 3B).

Fig. 2. Histological sections of Rhipicephalus sanguineus ovary. (A) Detail of oocytes I, II, and III stained by fucsine/toluidine blue. (B) Detail ofoocyte V stained by H-E. (C) Detail of oocyte in reabsorption process (ov) stained by fucsine/toluidine blue. (D) Oocytes V and oocyte in reabsorp-tion process (ov) stained by bromophenol blue. (E) Detail of oocytes I, II, III, and IV stained by fucsine/toluidine blue. (F) Oocytes I, II, III, and IVstained by bromophenol blue. (G–J) Oocytes I, II, III, IV, V, and oocyte in reabsorption process (ov) stained by PAS. (K–M) Oocytes I, II, III, IV, V,and oocyte in reabsorption process (ov) stained by Nile blue. ch, chorium; ep, ovary epithelium; gv, germ vesicle; nu, nucleolus; p, pedicel; pm, plas-mic membrane; yg, yolk granules. Bars: A,B D 0.05 mm; C D 0.01 mm; D–F D 0.05 mm; G D 0.01 mm; H–J D 0.02 mm; K–M D 0.05 mm.

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Oocytes III or in intermediate stage—were largerthan oocyte I and II, mostly rounded but also elliptical.The germ vesicle occupied the oocyte’s pole facing thepedicel. The plasmic membrane was thicker than in theother stages, with chorium characteristics. The cyto-plasm was full of yolk granules of various sizes: thesmaller grains occupy the central region and the largerones, the surroundings. The germ vesicle and the nucleo-lus were as in the previous stages (Figs. 2A, E–G, M and3C).

Oocytes IV–were larger than oocyte I, II, and III.They were round and the germ vesicle was no longerobserved due to the yolk granulation. The chorium wasalmost totally deposited. The cytoplasm possessedcountless yolk granules, in various sizes, randomly dis-tributed (Figs. 2E–G, K and 3D).

Oocytes V—In the Wnal stage of development, theywere the largest ones and round. The germ vesicle couldno longer observed due to a great yolk granulation.These oocytes presented a thick chorium, totally depos-ited (Figs. 2B, D, K, J and 3E).

3.4. Histochemistry

3.4.1. Bromophenol blue—protein detectionThis test showed that oocytes I and pedicel cells have

a small amount of elements composed of protein. Onlythe germ vesicle nucleolus showed moderate positivity(Fig. 2F).

In oocytes II a thin positive granulation was detectedand it was distributed uniformly in the cytoplasm. As faras plasmic and germ vesicle limits and pedicel cells wereconcerned a weak positivity was shown. The nucleoluspresented strong positivity (Fig. 2F).

The oocytes III had a cortex with small yolk granules.In the central region, smaller and strongly positive gran-ules were found, while in the surrounding area, largerones with strong and weak positivity appeared together.The germ vesicle, the chorium, the nuclear membraneand the pedicel cells reacted weakly. The nucleolusappeared strongly stained (Fig. 2F).

In oocytes IV, the cytoplasm showed large yolk gran-ules with strong positivity. However, weakly positive

Fig. 3. (A–E) Diagrammatic summary of vitellogenesis in ovarian cross-section of Rhipicephalus sanguineus. (A) Oocyte I. (B) Oocyte II. (C) OocyteIII. (D) Oocyte IV. (E) Oocyte V. gv, germ vesicle; ch, chorium; nu, nucleolus; yg, yolk granules.

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granules were also found in the surrounding area. Thechorium and the pedicel cells showed weak positivity(Fig. 2F).

In oocytes V, a large amount of large and stronglypositive granules was observed all over the cytoplasm. Inthe surroundings, weak positivity granules were present.The chorium was weakly positive (Fig. 2D).

The oocytes undergoing resorption showed corticalvacuoles with irregular morphology and weakly positivefor proteins. Smaller and positive yolk granules wereobserved occupying the oocyte’s center and larger posi-tive and negative granules in its surroundings. The cho-rium reacted weakly (Fig. 2D).

3.4.2. Nile blue—lipid detectionThis test revealed that all the oocytes present in the

ovary reacted positively, showing the presence of lipidsin these cells (Figs. 2K–M).

Oocytes I presented a homogeneous and strongly pos-itive cytoplasm (Fig. 2L).

Oocytes II, a thin positive granulation was observedand it was distributed homogeneously all over the cyto-plasm (Fig. 2M).

Oocytes III showed the cytoplasm with small yolkgranules, strongly positive, deposited in the center of thecell and some larger granules often located in the sur-roundings (Fig. 2M).

In some oocytes classiWed in the Wrst three stages, thepresence of the germ vesicle nucleolus was evident due tothe use of the fast stain with hematoxylin (Figs. 2L and M).

Oocytes IV showed the cytoplasm with many positivegranules that increase in size from the central to the sur-rounding regions. In the surroundings, the weakly posi-tive granules were found, which, in fact, could beforming complexes with other elements. The choriumshowed moderate positivity (Fig. 2K).

Oocytes V showed the cytoplasm full of yolk gran-ules, strongly positive. In the cortex, smaller weakly pos-itive granulations were observed (Fig. 2K).

Some oocytes with irregular outlines and apparentlyundergoing resorption were observed along the ovary,often in the proximal region. In these oocytes’ cyto-plasms negative and irregular vacuoles among the yolkgranules were detected and the chorium was weakly pos-itive (Fig. 2M).

A moderate positivity was detected in the cytoplasmas well as in the plasmic membrane of the ovary wallcells, including the pedicels (Figs. 2K–M).

3.4.3. PAS – polysaccharide detectionOocytes I had a weak cytoplasmatic positivity (Fig.

2H).Oocyte II showed the cytoplasm with a thin positive

and homogeneous granulation, evidencing the beginningof synthesis and/or incorporation of polysaccharides(Figs. 2G and H).

Oocytes III presented strongly positive cytoplasmaticgranules. The largest ones were located in the surroundingsand the smallest, in the oocyte’s central region (Fig. 2G).

Oocytes IV were strongly positive with yolk granulesin various sizes (Fig. 2G).

Oocytes V had strongly positive large granules, occu-pying all the cytoplasm (Fig. 2J).

The oocytes apparently undergoing resorption exhib-ited the cytoplasm with yolk granules moderatelystained, besides vacuoles in various sizes and irregularshape, weakly positive or negative to the test. These lastones occupied mainly the surroundings. The choriumdid not react to the test (Fig. 2I).

3.5. Ultrastructure

The ovary wall of the tick R. sanguineus was made upof small epithelial cells connected to each other by meansof interdigitations. The nuclei were large and irregular,and occupied the greater part of the cytoplasm that wasrestricted to a thin layer. In the proximal portion, theovary wall showed cells releasing secretion vesicles fromthe apical portions to the ovary lumen (Fig. 4A).

The pedicel cells, a structure that attaches the oocytesto the ovary wall, were cylindrical with a large irregularnucleus, heterochromatin, an evident nucleolus and vari-ous nuclear pores, suggesting the occurrence of cellularsynthesis processes (Figs. 4B and C). The most frequentorganelles were the round, elliptical, and halter shapemitochondria occupying all cytoplasm, concentrated inthe region next to the contact with the oocyte (Fig. 4B).Rough endoplasmic reticulum and many vacuoles dis-tributed in the cytoplasm were observed (Fig. 4C). Athick basal lamina supported the pedicel cells and envel-ops the oocyte, not penetrating in the interface of bothstructures (Fig. 4E). In this same region, a specializationof the oocyte and the pedicel cells membranes, similar tomicrovilli, was observed, (Fig. 4D). The cells that formeach pedicel presented interdigitations in their lateralmembranes (Fig. 4B).

Ultrastructurally, in oocytes I a homogeneous cortexwith some mitochondria in varied shapes was observedas well as large amounts of free ribosomes (Fig. 5A), inaddition to the round and central germ vesicle. The plas-mic membrane presented microvilli in direction to thebasal lamina (Fig. 5C), as well as vesicles containing elec-trondense material (Fig. 5C).

Oocytes II showed a large germ vesicle with dispersechromatin and many pores in their nuclear envelope(Fig. 5D). The presence of few and small lipidic granuleswas veriWed (Fig. 5B). Mitochondria in various shapes wereobserved, as well as numerous ribosomes, free or adhered tothe reticulum membrane (Fig. 5B). The plasmic membraneshowed specializations in longer and more frequent micro-villi in comparison to those found in oocytes I (Fig. 5B). Inthis region, the presence of vesicles was observed (Fig. 5B).

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Oocytes III showed numerous small granules withvaried morphology and content in the cytoplasm. Thelow electrondensity ones were most likely composed oflipid (Fig. 5F) and the others, of protein composed,appeared under two circumstances: (p1) less frequently,composed of more compact material with varied densi-ties or smaller, (p2) more frequently with a foam aspectand lower electrondensity (Fig. 5F). The larger yolkgranules with lipid or protein constitutions mostly tookup the surroundings of oocyte III and decreased gradu-ally in size in direction to the germ vesicle (Fig. 5E).Mitochondria with many shapes, free ribosomes andlamellar rough endoplasmic reticulum were observed(Fig. 5E), as well as numerous microvilli in the plasmicmembrane. Vesicles between these two regions, as in theprevious stage, were detected here (Fig. 5E).

As the oocytes progress in their development (stagesIV and V), the yolk granules also increased in amountsmaking the observation of the germ vesicle diYcult. Themicrovilli found in the plasmic membrane decreasedboth in size and in number (Wnal stage of vitellogenesis).

Oocytes IV showed a large quantity of granules ofprotein composed, in various sizes and electrondensity allover the cytoplasm (Figs. 5 and 6A) and were distin-guished according to their morphology: (p3) the oneswith homogeneous content and moderately electron-dense (Fig. 5G) and (p4) the ones with a more electron-dense central region, called dense bodies (Fig. 6A). Dropscomposed of lipid, smaller than granules of protein com-posed, were also detected all over the cytoplasm (Figs. 5and 6A), besides the presence of small mitochondria (Fig.6A). In this stage, the deposition of chorium was still

Fig. 4. (A–E) TEM of Rhipicephalus sanguineus ovary. (A) Detail of ovary epithelium. (B) General view of pedicel cells. (C) Detail of cytoplasmaround the pedicel cells nuclei. (D,E) Detail of contact between oocyte and pedicel cells. bl, basal lamina; ep, ovary ephitelium; lrer, lamellar roughendoplasmic reticulum; m, mitochondria; n, nuclei; ne, nuclear envelope; nu, nucleolus; o, oocyte; p, pedicel; po, nuclear pore; v, vacuoles; arrow, lat-eral membranes interdigitations; and head arrow, membrane invagination making the contact of pedicel/oocyte. Bars: A D 2.5 �m; B D 5 �m;C D 1 �m; D D 2 �m; E D 1 �m.

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happening. The transport of material to the surroundingsof the oocyte was detected (Fig. 6B). The content of theseexocytosis vesicles polymerized in the extra-cellular spacebetween the basal lamina and the plasmic membrane(Fig. 6B). The microvilli of the plasmic membranedecreased in number, due to the decrease in the incorpo-ration of material from the haemolymph (Fig. 6B).

The chorium of oocytes V was completely depositedand was constituted of two layers: the outer, thinner andmoderately electrondense, denominated exochorium;and the inner, thicker, and weakly electrondense, denom-inated endochorium, which establishes direct contactwith the oocyte (Figs. 6C and E). Numerous and smallgranules composed of lipid were found in the cytoplasm,as well as large granules of protein composed, of homo-geneous material and electrondense medium also presentin oocyte IV (Fig. 6C).

In some oocytes the presence of myelinic bodiesand vacuoles, structures related to degeneration orundergoing resorption, was detected (Figs. 6D and F).

In these oocytes, the chorium was thin and Wbrillar(Figs. 6D and F).

4. Discussion

The morphological analysis of the ovaries of adultticks R. sanguineus revealed that it is a single continuoustubular structure, similar to a horseshoe, located in theposterior third part of the body. This information wassimilar to the data obtained by Till (1961) in R. appendi-culatus, by Sonenshine (1991) in Dermatocentor ander-soni and D. variabilis and by Denardi et al. (2004) inAmblyomma cajennense.

It was possible to classify the ovary of the tick R. san-guineus as of panoistic type, since all cells present corre-spond to oogonia or future oocytes. In this type ofovary, nurse cells are not found. This classiWcation wasadopted by Denardi et al. (2004) for the tick A. cajenn-ense.

Fig. 5. (A–G) Transmission electron microcopy (TEM) of Rhipicephalus sanguineus oocytes in several phases of development. (A) Detail of the cyto-plasm of oocytes I. (B) Peripheric region of oocytes I. (C) Detail of the peripheric cytoplasm of oocytes II. (D) Perinuclear region of oocytes II. (E,F)Detail of the cytoplasm of oocytes III. (G) Detail of the peripheric cytoplasm of oocytes IV. bl, basal lamina; gv, germ vesicle, l, lipid granule; m,mitochondria; mv, microvilli; ne, nuclear envelope; pm, plasmic membrane; p1, protein granule 1; p2, protein granule 2; p3, protein granule 3; andarrow, secretion vesicles. Bars: A D 1�m; B D 0.25 �m; C, D D 1 �m; E D 5 �m; F D 1 �m; G D 2 �m.

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Morphologically, the ovary of the tick R. sanguineuswas not segmented in distinct regions of developmentsimilar to that found in Diplopoda. Newport (1841)described the ovary of the diplopod Iulus terrestris, as along simple bag, covered by a great number of oocytes.According to this author and also to Denardi et al.(2004) for A. cajennense, most oocytes did not reachmaturity, since they had been retarded by the earlierdevelopment of other adjacent and more proximal ones.In females of R. sanguineus the same characteristics havebeen veriWed, as well as the presence of oocytes indiVerent stages of development, characterizing an asyn-

chrony in their development along the ovary of theseindividuals.

According to Wagner-Jevseenko (1958) and Denardiet al. (2004), in tick ovaries and Fontanetti and Cunha(1993), in diplopod ovaries, the oogenesis was processeddistal-proximally. The present study has demonstratedthe same disposition of oocytes along the ovary of R.sanguineus.

The ovary wall of R. sanguineus possessed oocytes indiVerent stages of development as observed by Till(1961) in R. appendiculatus, by Balashov (1983) in Hya-lomma asiaticum.

Fig. 6. (A–F) Transmission electron microcopy (TEM) of Rhipicephalus sanguineus oocytes in several phases of development. (A) Detail of centralregion of oocytes IV. (B) Peripheric region of oocytes IV. (C,E) Peripheric region of oocytes V. (D,F) Detail of the cytoplasm of oocytes in reabsorp-tion process near of the peripheric region. bl, basal lamina; ch, chorium; enc, endochorium; exc, exochorium; l, lipid granule; m, mitochondria; mb,myelinic bodies; pm, plasmic membrane; p3, protein granule 3; p4, protein granule 4 or proteic bodies; and v, vacuoles. Bars: A D 2 �m; B D 1 �m;C D 5 �m; D D 2 �m; E D 1 �m; F D 1 �m.

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Through histological and histochemical techniquesthe oocytes of R. sanguineus were classiWed in stages thatvaried from I to V based in that proposal by Denardiet al. (2004) for A. cajennense ticks.

The ultrastructural analysis revealed that oocytes Iand II possessed the plasmic membrane with microvilliand vesicles between the membrane and the basal lam-ina, which indicates that a yolk exogenous incorporationhas already started in these stages.

In oocytes II, the transfer of material from thenucleus to the cytoplasm by nuclear pores has beenobserved with consequent increase in the quantity ofribosomes, as well as the deposition of small electron-dense granules in this region. These structural character-istics can indicate the beginning of the occurrence ofendogenous yolk synthesis processes in oocytes II,besides the exogenous production of yolk. The transportof material through the nuclear pores, probably RNArmaterial, has also been observed in oocytes from theAtya scabra by Cruz-Landim (1997).

A variety of techniques have shown that oocytes I upto stage IV were attached to the ovarian epithelium by astructure denominated pedicel, as observed also by Till(1961) in R. appendiculatus, by Balashov (1983) in H. asi-aticum and by Denardi et al. (2004) in A. cajennense.

According to Said (1992) and Denardi et al. (2004), inA. cajennense, the pedicel would release the most devel-oped oocytes to the interior of the ovary’s lumen, due toa continuous growing pressure against the ovary wall.

According to these studies, the pedicel’s function,besides Wxing the oocyte to the ovary wall, was still ques-tionable. Some authors believe that its cells could also beinvolved in yolk synthesis and supply to aid oocytegrowth; others, believe they only function to Wx the oocyteto the ovary wall (Brinton and Oliver, 1971; Diehl, 1970).

The pedicel’s cells in direct contact with the oocyte inR. sanguineus, possessed numerous microvilli in theirmembrane, which interdigitate with the ones located inthe oocyte’s surface. Near the oocyte/pedicel contact amore electrondense material found in vesicles as well asmany mitochondria was detected. These characteristicscan indicate that in this tick species a transport of sub-stances from pedicel cells to the interior of the oocytemight be happening, establishing thus the helping func-tion in the oocyte’s development.

According to the histochemical data in R. sanguineus,the yolk granulation from oocytes III presented variedaYnity to dyes speciWc for proteins, lipids, and polysac-charides. King (1960) in Drosophila (Diptera) and Cruz-Landim and Caetano (1981) in Atta (Hymenoptera) reg-istered that this characteristic could be related to a varia-tion in the quantity of these elements in the granules.These granules were located mainly in the central regionof oocytes III, suggesting that in these oocytes, anendogenous yolk production might be happening, andthe smaller granules would gradually fuse with larger

ones while migrating towards the periphery. These datawere also registered by Fontanetti and Cunha (1993) inoocytes from the diplopod Rhinocricus padbergi.

Mitochondria have been found all over the cytoplasmof oocytes, pointing out to a preparation of these cellsfor the period of exogenous incorporation of the vitello-genesis, in which these organelles become prerequisitefor the transportation of material coming from the hae-molymph (Balashov, 1972).

Camargo-Mathias and Caetano (1998) in studies withthe ant Neoponera villosa, suggested that the origin ofthe lipid in the oocytes of these insects would be themitochondria conWrming the data from other authors(Bonhag, 1958; Wigglesworth, 1964). Ranade (1933) hadpreviously stated that the mitochondria would be trans-formed in lipid yolk bodies and suggested that the lipidmaterial in the oocytes would come from breaking anddestruction of the mitochondrial crests.

In the cytoplasm of oocytes III of R. sanguineus thepresence of lipids and a low amount of smooth endo-plasmic reticulum, the organelle responsible for lipidsynthesis, were detected. This suggests that lipid compo-nents could have the mitochondria as their source, aswell as exogenous sources.

Numerous microvilli were found in the plasmic mem-brane of oocyte III and between this membrane and thebasal lamina, vesicles, as in oocyte II. Possibly, a highincorporation rate of exogenous elements for the compo-sition of yolk might be happening in these cells and theywould have been synthesized by other tissues, transportedby the haemolymph to the ovary and absorbed by oocytesthrough pinocytic vesicles, thus characterizing the exis-tence of an extra-ovarian source of yolk. Similar observa-tions have been made by Anderson (1970), in oocytes ofother animals, Balashov (1983) in H. asiaticum andDenardi et al. (2004), in oocytes of A. cajennense.

According to Balashov (1983), the exogenous sourcesof yolk production in ticks would be located in theirintestine cells. However, Sonenshine (1991) has sug-gested the participation of fat body in tick vitellogenesis,in which the cells would secrete great amounts of thevitellogenic protein in the haemolymph, which would, inturn, be taken up by developing oocytes via endocytosis;this could also occur in R. sanguineus.

Consequently, there are three probable sources of ele-ments for the oocytes’ growth in R. sanguineus: anendogenous one, through synthesis processes inside theoocyte, and two exogenous ones: the haemolymph andthe pedicel cells.

Balashov (1983) suggested that in H. asiaticum ticks,the yolk has a dual origin (endogenous and exogenous).However, the author states that the endogenous produc-tion occurred before the exogenous production. This con-tradicts our results since oocytes II possessed morphologicevidences that conWrmed the transport of elements com-ing from the exterior to the interior of the oocyte.

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According to Balashov (1983), the vitellogenesis inticks would only produce granules constituted of pro-teins and lipids. Ramamurty (1968) stated that, ininsects, the yolk is constituted by lipids, proteins andpolysaccharides, deposited in this sequence, and thatcould be free or chemically bound to other elements. Thepresent study with R. sanguineus females has shown datathat disagree with Balashov (1983), since the use of his-tochemical techniques revealed positivity for the threeelements, indicating that the yolk, in this species, has aglycolipoproteic character.

Regarding the depositing sequence of yolk in R. san-guineus oocytes, the results obtained here conWrm thesequence suggested by Ramamurty (1968), since inoocytes I, there was a signiWcant amount of lipids and lit-tle or no proteic and polysaccharidic content; in oocytesII, the amount of protein was higher than that of polysac-charides, which were only found on oocytes III to V.

In oocytes IV, chorium deposition is already takingplace and the microvilli were still detected, since theincorporation process of haemolymph material was stillgoing on. Charniaux-Cotton and Payen (1985) registeredin Panulirus that the retraction of the plasmic membraneand consequent disappearance of the specializationswould occur at the end of the vitellogenesis. Talbot(1981) related this event to the proximity of the ovula-tion process.

In oocytes V, the chorium is completely deposited.Chorium elaboration and deposition, responsible forpreserving the oocyte’s structure, comes to an end (Hin-ton, 1982; King, 1960).

The oocyte’s chorium in this tick species was subdividedin two layers: the endochorium (more internal and elec-trondense) and the exochorium (more external and lesselectrondense). This has also been observed by Camargo-Mathias and Caetano (1998) in Neoponera villosa ants.

Along the ovary, mostly in the proximal region,oocytes of irregular shape and showing cellular disorga-nization, autophagic vacuoles, and myelinic bodies intheir cytoplasm were often noticed. These structures sug-gest that these oocytes are probably undergoing resorp-tion. In insects, this process was frequently described andoccurs due to a series of physiologic, ecologic, andbehavioral order factors, such as food shortage, inter-ruption of yolk deposition, and lysis of the yolk granules(Bell and Bohm, 1975; Camargo-Mathias and Caetano,1998; Lusis, 1963). Denardi et al. (2004) observed thesame process occurring in oocytes of A. cajennense, how-ever, the causes are still not known.

Acknowledgments

We thank Dr. Alberto A. Guglielmone (INTA, Rafa-ela, Santa Fé, Argentina) for tick supplying of Argentin-ean origin, to Miss Monika Iamonte, Mr. Gerson de

Melo Souza, Mr. Antônio Teruyoshi Yabuki, Mr.Ronaldo Del Vecchio and Miss Cristiane Marcia Mileofor their technical support and FAPESP (Fundação deAmparo à Pesquisa do Estado de São Paulo) for Wnancialsupport (Grant No. 02/01180-8). Part of this work hasbeen facilitated through the International Consortium ofTicks and Tick-Borne Diseases (ICTTD-2) supported bythe INCO-DEV Program of the European Union undercontract number ICA4-CT-2000-30006.

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