Morphological, histological, and ultrastructural characterization of degenerating salivary glands in...

0

d

www.elsevier.com/locate/vetpar

Veterinary Parasitology 129 (2005) 299–311

Morphological, histological, and ultrastructural studies

of the ovary of the cattle-tick Boophilus microplus

(Canestrini, 1887) (Acari: Ixodidae)

Kelly Cristina Saito a,*, Gervasio Henrique Bechara b, Erika Takagi Nunes a,Patricia Rosa de Oliveira a, Sandra Eloisi Denardi a,

Maria Izabel Camargo Mathias a

a Departamento de Biologia, I.B., Universidade Estadual Paulista Julio de Mesquita Filho, Av. 24 A, No. 1515 Cx,

Postal 199, CEP 13506-900, Rio Claro, SP, Brasilb Departamento de Patologia Veterinaria, FCAV, UNESP, Jaboticabal, SP, Brasil

Accepted 4 September 2004

Abstract

This study presents the morphology of the ovary, as well as the dynamics of the vitellogenesis process in oocytes of the cattle-

tick Boophilus microplus. The ovary of these individuals is of the panoistic type; therefore, it lacks nurse cells. This organ

consists of a single tubular structure, continuous, and composed of a lumen delimitated by a wall of small epithelial cells with

rounded nuclei. In this tick species, the oocytes were classified into six stages varying from I to VI and according to: cytoplasm

appearance and presence of the germ vesicle, yolk granules, and chorion. Oocytes of various sizes and at different developmental

stages remain attached to the ovary through a cellular pedicel until completing stage V. Afterwards, they are liberated into the

lumen and from there to the exterior. Some oocytes (classified as type VI) showed an atypical appearance indicating that some of

the cellular components would be undergoing a degenerative process and/or reabsorption.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Boophilus microplus; Cattle-tick; Ovary; Vitellogenesis; Histology; Ultrastructure

1. Introduction

The economic importance of ticks is widely

acknowledge and is related to their feeding habits.

When feeding, many species of ticks transmit diseases

to man and other animals. The diseases transmitted by

* Corresponding author. Tel.: +55 19 35340009.

304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved

oi:10.1016/j.vetpar.2004.09.020

ticks are caused by protozoa, viruses, Rickettsias, and

spirochetes and cause the appearance of dermatosis

and other more severe infections (Rey, 1973). Among

the species belonging to the family Ixodidae is the tick

Boophilus microplus, known as the southern cattle-

tick, which is of great veterinary importance for

transmitting the disease known as cattle babesiosis or

cattle fever. This disease causes severe economical

.

K.C. Saito et al. / Veterinary Parasitology 129 (2005) 299–311300

losses to the cattle industry in many countries (Arthur,

1996).

Numerous studies are currently under way aiming

at finding an efficient control strategy that would

minimize the damages caused by these parasites. A

new control perspective uses immunological methods

consisting of the identification, isolation, and synth-

esis of proteins that protect the tissues and organs of

the tick, mainly those of the reproductive system,

aiming to obtain an anti-cattle-tick vaccine (Tellam

et al., 1992; Willadsen, 1997).

According to Sonenshine (1994), the female

reproductive system of ticks generally consists of a

large U-shaped ovary located at the posterior region of

the body, with a pair of oviducts, an uterus, a muscular

connection tube, vagina, and genital opening. After

fertilization, the ovary increases in size considerably

exhibiting eggs at all developmental stages (Rey,

1973). The ovary is an organ with a lumen delimitated

by a wall of small epithelial cells of rounded nuclei, in

which the oocytes at the different developmental

stages are attached by means of a pedicel (Sonenshine,

1994). Denardi et al. (2004) studied the ovaries of the

lone star tick Amblyomma cajennense and suggested

that the oocytes are developed simultaneously but

asynchronously; therefore, it is possible to observe

throughout the ovaries of these individuals oocytes

from the initial developmental stages to the most

mature ones.

In view of the data mentioned above and due to the

existence of few and out-of-date studies focusing on the

internal organs of the cattle-tick B. microplus, the

present work was aimed at collecting new data on the

morphological, histological, and ultrastructural features

of the female reproductive system of B. microplus, as

well as to attempt to establish the dynamics of the

vitellogenesis process in these individuals. The final aim

is to provide information that would contribute to the

future control of this parasite.

2. Material and methods

About 20 females of the cattle-tick B. microplus

were collected from tick colonies maintained in

controlled conditions (28 8C, 80% humidity and 12 h

photoperiod) at the Department of Animal Pathology,

Veterinary College, UNESP, Jaboticabal, SP, Brazil.

Equipment from the Histology and Electron Micro-

scopy Laboratories of the Biology Department at the

Biosciences Institute, UNESP, Rio Claro, SP, Brazil,

was utilized throughout the study.

Individuals were maintained in the refrigerator

(4 8C) for thermal shock anesthesia and dissected in

saline solution.

2.1. Morphology

Individuals were anesthetized and the ovaries were

removed and schematically drawn with the aid of

camera lucida coupled to a Zeiss stereomicroscope.

2.2. Scanning Electron Microscopy (SEM)

The ovaries were removed, fixed in Karnovsky for

24 h, and dehydrated in a graded 70–100% ethanol

and acetone series. The material was processed by

cryptal point drying, sputtered with gold, and

examined and photographed under a SEM Phillips

505.

2.3. Histology

The ovaries were fixed in 4% paraformaldehyde.

The material was then dehydrated in ethanol,

embedded in JB4 resin during 24 h at 4 8C and then

transferred to plastic moulds previously filled with

resin containing a catalyzer. After resin polymeriza-

tion, the material was sectioned using a Sorvall JB4

microtome (Bio Rad) and stained with hematoxiline

and eosin, following routine histological procedures.

2.4. Histochemistry

Histochemical tests were applied in order to detect

the presence of the following compounds: proteins

(Bromophenol Blue); lipids (Nile Blue); polysacchar-

ides (simultaneous staining with PAS/ Alcian Blue);

acids (Toluidine Blue); and calcium (Von Kossa).

2.5. Transmission Electron Microscopy (TEM)

The material was fixed in 2.5% glutaraldehyde,

postfixed in 1% OsO4, and embedded in Epon

Araldite. The material was then included in pure

Epon resin and polymerized at 60 8C for 72 h.

https://www.researchgate.net/publication/8233213_Morphological_characterization_of_the_ovary_and_vitellogenesis_dynamics_in_the_Amblyomma_cajennense_Acari_Ixodidae?el=1_x_8&enrichId=rgreq-752e12b6-3906-4f95-b327-29bec0f9999f&enrichSource=Y292ZXJQYWdlOzc4OTI4Njc7QVM6OTkxMzUwMDQ4NzI3MjNAMTQwMDY0NzAzNzE2OQ==

K.C. Saito et al. / Veterinary Parasitology 129 (2005) 299–311 301

Ultrathin sections were contrasted with acetate and

lead citrate. Afterwards, screens containing ultrathin

sections of the material were examined and photo-

graphed in a TEM Phillips MC 100.

3. Results

3.1. Ultramorphology

The application of scanning electron microscopy

techniques (SEM) allowed us to verify that the ovary

of the southern cattle tick consists of a single tubular

organ, in the shape of a horseshoe, to which numerous

rounded oocytes of various sizes and at various

developmental stages are attached (Fig. 1). This

morphological variation of the oocytes is related to the

fact that their development does not proceed

uniformly, in other words, the different developmental

stages are processed simultaneously but asynchro-

nously throughout the ovary (Fig. 1).

3.2. Histology

The ovary of the tick B. microplus is composed of a

lumen delimitated by a wall of small epithelial cells

with rounded nuclei, to which the oocytes in the

different developmental stages are attached through a

pedicel (Figs. 2 and 3A,B). Once mature, the oocytes

are liberated inside the lumen and from there to the

exterior (Fig. 2).

The pedicels, which attach the oocytes to the

ovarian wall, are composed of cells that resemble

those that compose the epithelial wall (Fig. 3A,C).

Since the differences found in the oocytes were

significant, we classified them following the criteria

previously established for the oocytes of A. cajennense

by Denardi et al. (2004). The classification suggested

for the oocytes found in the ovary of B. microplus is as

follows.

3.3. Oocyte I or primary oocyte

Characterized by its small size and frequently

rounded shape. The plasmic membrane is relatively

thin and delimitates a homogeneous cytoplasmic

content containing a germ vesicle with an evident

nucleolus (Figs. 2, 3B, 4A,D and 5A).

3.3.1. Oocyte II or pre-vitellogenic oocyte

It is larger when compared to the previous stage. A

fine granulation is observed dispersed throughout the

cytoplasm and the germ vesicle with a nucleolus still

evident (Figs. 2, 3A,D, 4A and 5A,B).

3.3.2. Oocyte III or vitellogenic oocyte

Its volume is considerably increased in relation to

the previous stages. The cytoplasm contains a coarser

granulation and the germ vesicle appears located at the

pole, next to the ovarian wall. Surrounding the oocyte,

at the external surface of the plasmic membrane, starts

the deposition of the chorion (Figs. 2, 3A,E, 4C,E and

5B).

3.3.3. Oocyte IV

Large yolk granules appear at the central region of

the cytoplasm, arising from smaller granules at the

peripheral region. The presence of a germ vesicle is

still evidenced. The thick chorion appears completely

deposited (Figs. 2; 3 C; 4C, E).

3.3.4. Oocyte V

This type of oocyte is well developed and

surrounded by a thick chorion. The cytoplasm

appears completely filled by numerous and large yolk

granules; the germ vesicle is rarely observed (Figs. 2,

3A and 4C,F).

3.3.5. Oocyte VI

Possesses an abnormal morpho-histological aspect

characterized by a cytoplasmic disorganization and

numerous folds at the chorion, rendering it an irregular

contour (Figs. 1, 3A, 4B,E and 5C). They were

observed in all individuals examinated.

4. Histochemistry

4.1. Bromophenol Blue test for the detection of

proteins (according to Junqueira and Junqueira, 1983).

This technique revealed the presence of proteins in

the plasmic membrane and inside all the oocytes.

Nevertheless, the distribution of this material through-

out the cytoplasm was not uniform: in stages I and II,

the proteins appeared dispersed homogeneously; in

stage III the proteins appeared as a fine granulation


Fig. 1. Scanning electron micrographs (SEM) of the ovary of Boophilus microplus. (A) General view of the ovary with inumerous rounded

oocytes. (B) Detail of external oocytes surface. oo: young oocyte; or: oocyte in reabsorption process; ov: ovary. Bars: A, B = 0.1 mm.

(Fig. 3E); in stage IV, the granules were larger; and in

stages V and VI, the proteins appeared as yolk

granules. The chorion was evident in stages III–VI.

4.2. Simultaneous staining PAS/Alcian Blue for

the detection of polysaccharides (according to

Junqueira and Junqueira, 1983).

The results suggest that the concentration of

polysaccharides gradually increases as the oocytes

develop (Fig. 4A–C).

In the cytoplasm of the oocytes I–III were found

polysaccharides forming a coarse granulation

(Fig. 4A,C). In the more developed stages (IV–VI)

the polysaccharides are contained inside large yolk

granules, probably forming complexes with lipids and

proteins (Fig. 4B,C).

In stage IV, the larger yolk granules are located at

the central region while the smaller ones appear at

the periphery of the oocytes. Large yolk granules

occupy the whole cytoplasm of oocytes in stage V

(Fig. 4C).


Fig. 2. Diagrammatic summary of oogenesis in ovarian cross-section of Boophilus microplus. I: oocyte I; II: oocyte II; III: oocyte III; IV: oocyte

IV; V: oocyte V; ec: ovary ephitelium; lu: lumen; ovd: oviduct; gv: germinal vesicle.

4.3. Nile Blue test for the detection of lipids

(according to Junqueira and Junqueira, 1983).

This histochemical test revealed the presence of

lipids in the plasmic and nuclear membranes and also

inside the oocytes (Fig. 4D–G). The concentration of

this material stored in the cytoplasm of the oocytes

varied according to the various developmental

stages.

The presence of a high concentration of lipids

in the cytoplasm of all oocytes was verified.

However, in the stages III–VI, lipids were only

present at the spaces between the yolk granules

(Fig. 4D–G).

The chorion reacted positively to this test, thus

indicating the presence of lipids in its composition

(Fig. 4E,G).

4.4. Von Kossa test for the detection of calcium

(according to Junqueira and Junqueira, 1983).

The results revealed that calcium is present in the

oocytes III–VI, distributed throughout their cytoplasm

(Fig. 5A–C).


Fig. 3. (A and B) Histological sections of Boophilus microplus ovary stained by H–E. (C and D) Detail of oocytes stained by Toluidine Blue. (E)

Detail of oocyte III by Bromophenol Blue. I: oocyte I; II: oocyte II; III: oocyte III; IV: oocyte IV; V: oocyte V; VI: oocyte VI or in reabsorption

process; ch: chorium; ec: ovary ephitelium; gv: germ vesicle; lu: lumen; nu: nucleolus; n: nucleus; pe: pedicel; pm: plasm membrane; yg: yolk

granules. Bars: A, B = 0.2 mm; C = 0.05 mm; D, E = 0.01 mm.

5. Ultrastructure

The ultrastructural analysis of the oocytes of B.

microplus revealed details that could not be observed

through conventional histological and histochemical

techniques.

Small epithelial cells with rounded nuclei

compose the ovarian wall of the southern cattle-tick.


Fig. 4. Histological sections of Boophilus microplus ovary. (A–C) Stained by PAS/Alcian Blue. (D–G) Stained by Nile Blue. I: oocyte I; II:

oocyte II; III: oocyte III; IV: oocyte IV; V: oocyte V; VI: oocyte VI; ch: chorium; ec: ovary ephitelium; gv: germ vesicle; lu: lumen; n: nucleolus;

pe: pedicel; yg: yolk granules. Bars: A, B, E, and G = 0.2 mm; C and F = 0.01 mm; D = 0.05 mm.

The oocytes at the different developmental stages are

attached to this wall through a cellular pedicel (Fig. 6J).

The cells that compose the wall and the pedicels

presented a varied morphology from rounded to oval

with a rounded nucleus surrounded by a fine layer of

cytoplasm, in which we noted the presence of vacuoles

and granular endoplasmic reticulum (Fig. 6J).

The basal lamina that appears in these cells was

differentiated into two layers: a more external and

thinner layer of fibrillar aspect and a more internal and

thicker layer (Fig. 6C). This basal lamina support the

more external cells that formed the pedicels and on the

oocytes, being absent at the point of contact between

the oocytes and the pedicel cells, in which we verified

a specialization of the membrane of these two cellular

types in the shape of interdigitations (Fig. 6J).

The cytoplasm of oocytes at the initial develop-

mental stages (I and II) shelters the germ vesicle and a


Fig. 5. Histological sections of Boophilus microplus ovary stained by Von Kossa. I: Oocyte I; II: oocyte II; III: oocyte III; VI: oocyte VI; ca:

calcium spheres; ec: ovary ephitelium; lu: lumen. Bars: A, B = 0.2 mm; C = 0.01 mm.


Fig. 6. Transmission electron micrographs of Boophilus microplus ovary. (A) Oocyte I. (B) The perinuclear region of oocyte II. (C) Peripheric

region of the oocyte III. (D) Chorium formation of oocyte IV. (E) The chorium of oocyte V completely deposited. (F) The cytoplasm of oocyte V

presented large yolk granules. (G and H) Myelinic bodies and autophagic vacuoles presents in oocyte VI. (I) Chorium of oocyte VI showing

myelinic bodies inside them. (J) Interface oocyte/pedicel region. av: autophagic vacuoles; bl: basal lamina; ch: chorium; g: glycogen; ger:

granular endoplasmic reticulum; gv: germ vesicle, l: lipid granule; m: mitochondria; mb: myelinic bodies; nu: nucleolus; o: oocyte, p: protein

granule; pe: pedicel; pm: plasmic membrane; sv: secretion vesicles; i: interdigitation between two pedicel cells; n: nucleus. Bars: A = 10 mm; B,

C and G = 2 mm; D, F, H and I = 1 mm; E and J = 5 mm.


small amount of material stored as a fine granulation

that renders a homogeneous appearance to the

cytoplasm (Fig. 6A,B).

During stage I, the chromatin of the germ vesicle is

poorly condensed and a large amount of granular

endoplasmic reticulum, glycogen, and ribosomes

appear free in the cytoplasm (Fig. 6A).

The oocytes in stage II, also known as pre-

vitellogenic oocytes, presented in their cytoplasm a

large amount of granular endoplasmic reticulum,

vesicles of varied morphologies, glycogen, and RNA

precursors that were found at the perinuclear region

together with numerous mitochondria (Fig. 6B).

In the cytoplasm of oocytes in stage III a larger

amount of glycogen, granular endoplasmic reticulum,

polyribosomes, and lipid droplets of varying shapes

and sizes were noted (Fig. 6C).

At the periphery of oocytes in stage III, in the

region next to the basal lamina, small secretion

vesicles being incorporated by the cell were observed

(Fig. 6C). The plasmic membrane presented numerous

infoldings towards the basal lamina and, in the spaces

formed between these structures, the export of vesicles

filled with an electrondense material was verified; this

material is deposited and then polymerizes for the

formation of the chorion.

As the oocytes progress in their development (stages

IVand V), the infoldings of the plasmic membrane tend

to decrease and a larger amount of yolk granules is

observed in the cytoplasm, resulting from the fusion of

small secretion vesicles (Fig. 6D–F).

In oocytes V, the chorion appeared completely

formed with micropores that probably facilitate the

oxygenation of the internal structures of the future egg

(Fig. 6E). During this same stage, the cytoplasm of the

oocyte presented large yolk granules (Fig. 6F).

The oocytes in stage VI presented in their

cytoplasm strong indications that some of their

components were undergoing degeneration and/or

reabsorption. We noted the presence of myelinic

bodies as well as mitochondria and endoplasmic

reticulum with altered morphology inside autophagic

vacuoles (Fig. 6G,H).

The chorion of the degenerating oocytes presented

characteristics that differed from the oocytes in

previous developmental stages, showing a few

myelinic bodies inside them (Fig. 6I) as well as a

disorganized cytoplasmic yolk.

6. Discussion

The ultramorphological analysis revealed that the

ovary of the cattle-tick consists of a single tubular

structure, continuous, and in the shape of a horseshoe,

located at the posterior third of the animal’s body. This

data corroborate the results obtained by Sonenshine

(1994) in the species Dermatocentor andersoni and D.

variabilis, by Denardi et al. (2004) in A. cajennense, and

by Oliveira et al. (2004) in Rhipicephalus sanguineus.

The application of histological techniques allowed

the classification of the ovary of B. microplus as

belonging to the panoistic type, in which all the cells

present, except the ones that compose the ovarian wall,

are oogonies that would give rise to the future oocytes.

Nurse cells are absent in this type of ovary. This

classification for B. microplus is the same described

for A. cajennense in the study made by Denardi et al.

(2004) and for R. sanguineus collected in Brazil and

Argentina (Oliveira et al., 2004).

Morphologically, the ovary of B. microplus is not

segmented into different developmental zones as

occur in the ovaries of most arthropods.

The morphological variation that exists between

the oocytes found in the ovary of this species was not

only restricted to the external aspects but also to their

internal composition. These differences were a

consequence of the different developmental stages

displayed by the oocytes, thus suggesting a simulta-

neous but asynchronous development of these cells,

which was also observed in the ovary of A. cajennense,

as reported by Denardi et al. (2004).

It was demonstrated that in the cattle-tick, the

oogenesis proceeds in a distal-proximal direction,

with the less developed oocytes occupying the

distal region while the more developed oocytes (or

vitellogenic) occupy the proximal region. This

configuration was also observed by Wagner-Jevseenko

(1958) in other species of ticks and by Fontanetti and

Cunha (1993) in millipedes.

The significant morphological and histochemical

differences related to the developmental stage and the

amount of yolk found in the oocytes of B. microplus

allowed the classification of the oocytes into stages

that varied from I to VI. This classification was based

on the one proposed by Denardi et al. (2004) for the

development of oocytes of the lone star tick A.

cajennense, in which the author described the


occurrence of oocytes in five different developmental

stages. We described an additional stage for the

oocytes of B. microplus, where were observed features

of degeneration. The main characteristics considered

for this classification were the aspect of the cytoplasm,

the presence of a germ vesicle, the presence and size of

yolk granules, and the presence of the chorion.

The histological study revealed that the oocytes in

stages I–IV, and rarely some of stage V are attached to

the ovarian wall through a pedicel. This structure

results from the proliferation of epithelial cells of the

ovarian wall during the development of the oocytes.

This phenomenon was described by Till (1961) and

Balashov (1983) for other species of ticks.

During the first studies performed with A.

cajennense, El Said (1992) suggested that the pedicel

releases the mature oocytes into the ovarian lumen due

to the continuous growth pressure they exert against

the ovarian wall. In addition to this function, Balashov

(1983) suggested that the pedicels would play the role

of the nurse cells, present in ovaries of the meroistic

type and thus absent in B. microplus, which aid in the

incorporation of proteins and lipids that would later be

incorporated into the oocytes.

The ultrastructural study revealed a specialization

of the plasmic membranes at the interface between

the pedicel cells and the oocytes, where they present

infoldings that interdigitate, thus increasing the con-

tact surface between these two types of cells. This

observation corroborates the idea that the pedicels

are actually aiding in the production of elements

that would later be incorporated into the oocytes of

B. microplus.

The basal lamina was observed leaning on the

plasmic membranes of the oocytes and pedicels, except

at the point of contact between these two cellular

types. The basal lamina was divided into two different

regions: a more external layer, thinner and with a

fibrillar aspect, and a more internal layer, thicker and of

granular appearance. This description of the basal

lamina coincides with the observations of Denardi et al.

(2004) in A. cajennense and of Silva (1999) in locust of

the genus Panulirus. In B. microplus, the basal lamina

does not penetrate the interface region between the

pedicel and the oocyte, the region of intense contact

between these two cellular types.

According to Reddy and Locke (1990), the basal

lamina’s main function would be to serve as a barrier

between the elements of the hemolymph and of the

oocytes, thus monitoring the traffic of macromole-

cules in both directions.

At the perinuclear region of the oocytes in the

developmental stages I and II, we observed an intense

traffic of material from the nucleus to the cytoplasm

through the nuclear pores, probably rRNA, with a

consequent increase in the amount of ribosomes as

well as the deposition of electrondense material at this

region.

The characteristics described above for oocytes I

and II were also described for A. cajennense by Denardi

et al. (2004) and for R. sanguineus by Oliveira et al.

(2004), thus suggesting the occurrence of an endogen-

ous synthetic activity of only structural proteins during

the first stage and later, during the second stage, a

production of vitellogenic proteins, including the

exogenous incorporation of yolk elements.

Oocytes at the end of stage II and at the beginning of

stage III presented a significant increase in the number

of organelles accumulated in the cytoplasm, which

might be related to the preparation of these cells for the

next stage, during which these organelles would be a

prerequisite for the endogenous production and/or the

exogenous incorporation of yolk (Balashov, 1972).

The abundance of rough endoplasmic reticulum in

oocytes III of B. microplus suggests that the presence

of this organelle indicates that endogenous synthesis

of yolk is occurring in the oocyte. In addition to

the endoplasmic reticulum, some authors relate the

participation of the Golgi complex in endogenous

vitellogenesis. Nevertheless, this organelle was not

observed in the oocytes of the cattle-tick, probably

indicating a small participation of these cells in the

vitellogenesis process. Norevang (1968), while

reviewing the ultrastructural aspects of oogenesis in

diverse animal groups, suggested that in most oocytes

the Golgi complex is not a prominent element and few

comments have been made with regard to their

function in oocyte physiology in general.

Consequently, we suggest that the vitellogenesis

process in B. microplus occurs through the endogen-

ous production of lipids and proteins until reaching

developmental stage III; afterwards, there are exo-

genous elements that are incorporated from the

hemolymph. The hypothesis of exogenous production

of yolk results from the observation of small yolk

vesicles located preferentially at the peripheral region


of the oocytes while larger yolk granules concentrate

at the central region, thus indicating that the material

might be incorporated through pinocytosis.

When the vitellogenesis process is completed

during stage V, the infoldings observed frequently

on the plasmic membrane of the oocytes of B.

microplus decrease in size and number until totally

disappearing, thus confirming that such specialization

would be related to the incorporation of materials from

an exogenous source in order to contribute to yolk

formation inside the oocytes.

Our results indicate that the yolk granules of

oocytes of the cattle-tick are only composed of

glycoproteic elements, with lipids being only present

in the cytoplasm between the granules. This observa-

tion disagrees with the results observed by Ramamurty

(1968) in insects and by Oliveira et al. (2004) in

R. sanguineus, species in which the yolk granules are

composed of lipids, proteins, and carbohydrates.

In oocytes at developmental stage V of B.

microplus, the large yolk granules are distributed

throughout the whole cytoplasm. Nevertheless, at the

peripheral region it is still possible to observe a thinner

granulation, which indicates the incorporation of

extra-ovarian materials via pinocytosis vesicles that

Denardi et al. (2004) described as originated from the

hemolymph in A. cajennense.

As the oocyte completes its development, it becomes

surrounded by the chorion, towhich numerous functions

are attributed, such as preservation of the egg with the

consequent preservation of the species (King, 1960);

protection against mechanical stimuli, desiccation, and

predation; and allowing gas exchange for the oxygena-

tion of the embryo (Hinton, 1982).

According to Camargo-Mathias (1993), in the ant

Neoponera villosa the chorion is structurally formed

by the endochorion (more internal and electrondense)

and by the exochorion (more external and less

electrondense), which are normally composed of

proteins, lipids, and carbohydrates synthesized by the

follicular cells in the case of meroistic polytrophic

ovaries (King, 1960).

Numerous authors believe that the chorion is

produced by the oocyte itself, originating from

vesicles of the endoplasmic reticulum and of the

Golgi, which fuse to the plasmic membrane of the

more developed oocytes and discharge their contents

to the extracellular space in which it polymerizes.

The ultrastructural study revealed that, in B.

microplus, the chorion is not composed of different

layers and, due to the absence of follicular cells, it is

composed of an electron-dense material produced by

the oocyte itself. This material later accumulates at the

space between the plasmic membrane and the basal

lamina; its composition in the cattle-tick consists

basically of compounds of a lipoproteic nature.

Inside the oocytes, mainly those in stages III–V, in

addition to the yolk granules we found calcium

spheres. This element was also observed by Denardi

et al. (2004) in the more developed oocytes of A.

cajennense and in millipedes by Kubrakiewicz (1989),

that suggested that this element originated from

systems of intracellular mineralization.

According to Camargo-Mathias et al. (1998), in

millipedes the accumulation of this mineral in the

shape of spherocrystals could represent a mechanism

of homeostasis or some sort of special reserve, which

would later be used in the calcification of the

exoskeleton of the millipede embryo (Pettit, 1970).

The actual function of this element present in the

cytoplasm of the oocytes of B. microplus cannot yet be

confirmed.

Throughout the ovary of B. microplus we observed a

few oocytes with an abnormal morpho-histological

appearance, characterized by a cytoplasmic disorgani-

zation and many folds on the chorion. The ultrastructure

of these oocytes revealed strong indications that some

of the cellular components would be undergoing a

degenerative process and/or reabsorption, since we

observed myelinic bodies as well as mitochondria and

endoplasmic reticulum inside autophagic vacuoles.

Thus, these oocytes appear to be undergoing

autolysis without being phagocytized, or without the

participation of hemolymph cells, phenomenon that was

also described by Crayon (1941) in his analyses with

oocytes of pagurid crabs undergoing reabsorption.

Oliveira et al. (2004) showed that in R. sanguineus

a process of reabsorption in some oocytes probably

occurs as a mechanism to recover certain nutrients.

Lusis (1963) even suggested that this process occurs

due to a series of physiological, ecological, and

behavioral factors, such as lack of food by a

determined period, which would interrupt the deposi-

tion of yolk and would promote the lysis of the

existing granules with a consequent rupture of the

cytoplasm (Bell and Bohm, 1975).


The causes for the appearance of oocytes undergoing

reabsorption in the tick B. microplus remain unknown.

The findings described here regarding the mor-

phological, histological, and ultrastructural aspects of

the female reproductive system of the cattle-tick

B. microplus may contribute to the future control of

this parasite, since the survival of the species depends

on this system.

Acknowledgments

This work has been supported by FAPESP

(Fundacao de Amparo a Pesquisa do Estado de Sao

Paulo). The authors wish to tank Ronaldo Del Vecchio,

Antonio Teruyoshi Yabuki, Monika Iamonte, Gerson

de Melo Souza and Cristiane Marcia Mileo for their

technical support. Part of this work has been facilitated

through the International Consortium of Ticks and

Tick-borne Diseases (ICTTD-2) supported by the

INCO-DEV program of the European Union under

contract number ICA4-CT-2000-30006.

References

Arthur, D.R., 1996. Ticks: a monograph of the Ixodoidea: on the

genera Dermatocentor, Anocentor, Cosmiomma. In: Boophilus e

Margaphorus, Cambridge University Press, London, p. 215.

Balashov, Y.S., 1972. A translation of bloodsucking ticks (Ixodidae)

vectors of diseases of man and animals. Misc. Publ. Entomol.

Soc. Am. 8 (5), 159–376.

Balashov, Yu.S., 1983. In: Raikhel A.S., Hoogstraal H. (Eds.), An

Atlas of Ixodid Tick Ultrastructure. Entomological Society of

America, 289 pp. (Special Publication).

Bell, W.J., Bohm, M.K., 1975. Oosorption in insects. Boil. Rev. 50,

373–396.

Camargo-Mathias, M.I., 1993. 157f. Histoquımica e ultra-estrutura

dos ovarios de operarias e rainhas de formigas Neoponera villosa

(Hymenoptera: Ponerinae). Dissertacao (Doutorado em Zoolo-

gia)- Instituto de Biociencias, Universidade Estadual Paulista,

Rio Claro.

Camargo-Mathias, M.I., Fontanetti, C.S., Mico-Balagues, E., 1998.

Histochemical studies of Rhinocricus padbergi Verhoeff ovaries

(Diplopoda, Spirobolida, Rhinocricidae). Cytobios, Cambridge

169–184.

Crayon, J., 1941. Morphologie et structure de I’appareil genital

femelle chez quelques pagures. Bull. Soc. Zool. France. LXVI,

95–122.

Denardi, S.E., Bechara, G.H., Oliveira, P.R., Nunes, E.T., Saito,

K.C., Camargo-Mathias, M.I., 2004. Morphological character-

ization of the ovary and vitellogenesis dynamics in the tick

Amblyomma cajennense (Acari: Ixodidae). Vet. Parasitol. 125,

379–395.

El Said, A., 1992. A contribution to the anatomy and histology of the

female reproductive system of Amblyomma cajennense (Acar-

ina: Ixodidae).. J. Egypt. Soc. Parasitol., Cairo. 22, 391–400.

Fontanetti, C.S., Cunha, M.A.S., 1993. Morfologia ovariana e

desenvolvimento dos ovocitos de Rhinocricus padberg Verhoeff

(Diplopoda, Spirobolida, Rhino cricidae). Ver. Brasil. Biol. 53

(1), 7–12.

Hinton, H.E., 1982. Biology of Insect Egg Shells. Pergamon,

Oxford.

Junqueira, L.C.U., Junqueira, L.M.M., 1983. Tecnicas basicas de

citologia e histologia. Livraria Editora Santos, 48–81.

King, R.C., 1960. Oogenesis in adult Drosophila melanogaster IX

Studies on the citochemistry and ultrastructure of developing

oocytes. Growth 24, 265–323.

Kubrakiewicz, J., 1989. Deposition of calcium salts in oocytes and

ovarian somatic tissue of millipedes. Tissue Cell 21, 443–

446.

Lusis, O., 1963. The histology and histochemistry of development

and resorption in the terminal oocytes of the desert locust

Schistocerca gregaria. Quart. J. Micro. Sci. 104, 57–68.

Norevang, A., 1968. Electron microscopic morphology of oogen-

esis. Int. Rev. Cytol. 23, 114–186.

Oliveira, P.R., Bechara, G.H., Denardi, S.E., Saito, K.C., Nunes,

E.T., Camargo-Mathias, M.I., 2004. Morphological character-

ization of the ovary and oocyte vitellogenesis of the tick

Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae).

Exp. Parasitol., in press.

Pettit, J., 1970. Sur la nature et l’accumulation de substances

minerals dans les oocytes des Polydesmus complanatus (Myr-

iapoda Diplopoda). Compte Rendu Hebd des Seances de l’.

Acad. Sci. 27, 2107–2110.

Ramamurty, P.S., 1968. Origin and distribution of glycogen during

vitellogenesis of the scorpion fly Panorpa communis. J. Insect.

Physiol. 14, 1325–1330.

Reddy, J.T., Locke, M., 1990. The size limited penetration of gold

particles through insect basal laminae. J. Insect Physiol. 36 (6),

397.

Rey, L., 1973. Parasitologia. Guanabara Koogan 633–641.

Silva, J.R.F., 1999. Estudo morfologico em ovarios de lagostas do

genero Panulirus. White 1847 (Crustacea: Decapoda: Palinur-

idae). Dissertacao (Doutor em Zoologia), Instituto de Biocien-

cias da Universidade de Sao Paulo, 187 pp.

Sonenshine, D.E., 1994. Biology of ticks V. 1. Ed.. Oxford Uni-

versity Press, New York, pp. 280–303.

Tellam, R.L., Smith, D., Kemp, D.H., 1992. Vaccination against

ticks. In: Yong, W.K. (Ed.), Animal Parasite Control Using

Biotechnology. CRC Press, Boca Raton, pp. 303–331.

Till, W.M., 1961. A contribution to the anatomy and histology of the

brown ear tick Rhipicephalus appendiculatus Neumann. Mem.

Entomol. Soc. South Africa 6, 1–124.

Wagner-Jevseenko, O., 1958. Fortpflanzung bei Ornithodorus mou-

bata und genitale Uebertragung von Borrelia duttoni. Acta

Trop., Basel. 15, 119–168.

Willadsen, P., 1997. Novel vaccines for ectoparasites. Vet. Parasit.

71, 209–222.

Morphological, histological, and ultrastructural characterization of degenerating salivary glands in...

Documents

Transcript of Morphological, histological, and ultrastructural characterization of degenerating salivary glands in...