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Veterinary Parasitology 129 (2005) 139–147

Comparison of the external morphology of Rhipicephalus

sanguineus (Latreille, 1806) (Acari: Ixodidae) ticks

from Brazil and Argentina

Patrıcia Rosa de Oliveira a, Gervasio Henrique Bechara b, Sandra Eloisi Denardi a,Kelly Cristina Saito a, Erika Takagi Nunes a, Matias Pablo Juan Szabo c,

Maria Izabel Camargo Mathias a,*

a Departamento de Biologia, I.B., Universidade Estadual Paulista Julio de Mesquita Filho, UNESP,

Av. 24A 1515, Cx. Postal 199, CEP 13506-900, Rio Claro, SP, Brazilb Departamento de Patologia Veterinaria, FCAV, UNESP, Via de acesso Prof. Paulo Castellane s/n,

CEP 14884-900, Jaboticaal, SP, Brazilc Faculdade de Medicina Veterinaria, Universidade Federal de Uberlandia, Av: Para, 1720/Campus Umuarama,

Bloco 2T, CEP: 38400-902, Uberlandia, MG, Brazil

Accepted 1 January 2005

Abstract

In the present study the external morphology of semi-engorged Rhipicephalus sanguineus females ticks from Brazil and

Argentina were compared by scanning electron microscopy. Many differences were detected among the R. sanguineus collected

at the two localities, such as body size, shape of the genital pore, and morphology of the sensory structures. All these

characteristics are fundamental for the diagnosis of species of the genus Rhipicephalus and thus indicate the need for further

comparisons and, the taxonomical revision of this species of tick in the Neotropics.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Rhipicephalus sanguineus; Ixodidae; External morphology; Brazil; Argentina

1. Introduction

All superior vertebrates are subject to attack by

ticks. Nevertheless, due to endothermy, mammals are

frequent hosts for this kind of parasite. Rhipicephalus

* Corresponding author. Tel.: +55 19 3526 4135;

fax: +55 19 3526 4136.

E-mail address: micm@rc.unesp.br (M.I.C. Mathias).

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetpar.2005.01.001

sanguineus, known as the brown dog tick is a

cosmopolitan species and presents a characteristic

reddish-brown coloration (Flechtmann, 1973).

Although dogs are the most common host of this

tick (Walker, 1994), it has also been recovered from

other animals, such as cats, rabbits, camels, bovines in

general, goats, horses, sheep, bats, reptiles, and ground

feeding birds (Flechtmann, 1973) and humans (Walker

et al., 2000).

.

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147140

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147 141

R. sanguineus is a species of both veterinary and

medical importance. It causes blood loss in the

host and transmits several pathogens such as

Babesia canis and Ehrlichia canis to dogs and

Rickettsia conori to humans, but regional differ-

ences seem to exist in vectoring capacity (Walker

et al., 2000).

Recent results by Mangold et al. (2004) and Szabo

et al. (2004) showed marked differences in mitochon-

drial DNA and biology of R. sanguineus collected in

Rafaela, Santa Fe, Argentina, and Jaboticabal, Brazil.

In addition, mating of hybrids of the tick R.

sanguineus from Rafaela, Santa Fe, Argentina, and

Jaboticabal, Brazil, resulted in non-viable mass of

eggs (Szabo et al., 2004).

In view of the existence of scarce literature on the

morphological study of ticks in general and consider-

ing the few studies concerning R. sanguineus in the

Neotropics and the differences already detected

between populations of this species from Brazil and

Argentina, the present study had as its objective

characterizing and comparing the external morphol-

ogy of females of the tick R. sanguineus collected in

Rafaela, Santa Fe, Argentina, and Jaboticabal, Sao

Paulo State, Brazil.

2. Materials and methods

2.1. Ticks

Semi-engorged female R. sanguineus ticks were

used for this study. Initial specimens were collected

from dogs in Jaboticabal, Brazil, and Rafaela, Santa

Fe, Argentina and kept in the laboratory under

controlled conditions (29 8C, 80% humidity, and a

12 h photoperiod) at the Department of Veterinary

Pathology, Jaboticabal, SP, Brazil. Semi-engorged

females measuring from 3 to 5 mm in diameter were

obtained by feeding unfed adults in feeding chambers

glued to the depilated dorsum of domestic dogs, as

described by Bechara et al. (1994).

Fig. 1. SEM of dorsal and ventral portions of females of R. sanguineus col

bar: 1 mm; (B) dorsal view of Brazilian tick, bar: 1 mm; (C) ventral view of

0.4 mm; (E) ventral portion of Argentinean tick, bar: 50 mm; (F) ventral por

bar: 50 mm; (H) anal plate of Brazilian tick, bar: 50 mm. id: idiosoma; ca:

scutum; ga: genital aperture; fc: deep fissure; ap: anal plate; v: anal valv

2.2. Scanning electron microscopy (SEM)

Ticks were fixed for 4 h in Karnovsky fixative

solution and then subjected to 10 min baths in an

increasing acetone series of 70, 80, 90, 95, and 100%

(twice in the last concentration). The material was

immediately transferred to a solution of 100% alcohol

and analytical grade acetone in the proportion of 1:1 for

15 min. Next, the material was subjected to a quick bath

in analytical grade acetone. Afterwards, the material

was critical-point dried, fixed to a metal stand with

double-faced adhesive tape, and sputtered with gold and

carbon. The prepared material was analyzed and

photographed under a PHILLIPS 505 scanning electron

microscope at the Electron Microscopy Laboratory of

the Biology Department at the Bioscience Institute—

UNESP, Rio Claro, SP, Brazil. External morphology of

the tick R. sanguineus females collected in Brazil and

Argentina were then compared.

3. Results

The results of morphological comparisons are

shown in Table 1.

4. Discussion

The ultramorphological data of R. sanguineus ticks

showed that females at the same feeding stage from

Argentina are larger than the ones from Brazil.

The palps of females of R. sanguineus from both

Argentina and Brazil were formed by four segments,

with the fourth being smaller than the others and

occupying a cavity at the ventral surface of the third

segment. This observation was also recorded by

Balashov (1983) while analyzing the external mor-

phology of Hyalomma asiaticum.

The same author verified that two groups of palpal

setae differed in shape. The first group showed a

needle shape, straight or slightly bent, pointed or

lected in Brazil and Argentina. (A) Dorsal view of Argentinean tick,

Argentinean tick, bar: 1 mm; (D) ventral view of Brazilian tick, bar:

tion of Brazilian tick, bar: 0.1 mm; (G) anal plate of Argentinean tick,

capitulum; g: groove genital; fe: festoons; a: sub-triangular area; sc:

e; ag: anal groove; se: smooth setae.

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147142

Fig. 2. SEM of dorsal and ventral portions of females of R. sanguineus collected in Brazil and Argentina. (A) Anterior dorsal portion of

Argentinean tick, bar: 0.1 mm; (B) anterior dorsal portion of Brazilian tick, bar: 0.1 mm; (C) chelicerae of Argentinean tick, bar: 10 mm; (D)

chelicerae of Brazilian tick, bar: 10 mm; (E) anterior ventral portion of Argentinean tick, bar: 0.1 mm; (F) anterior ventral portion of Brazilian

tick, bar: 0.04 mm; (G) ventral palps of Argentinean tick, bar: 10 mm; (H) ventral palps of Brazilian tick, bar: 10 mm. pa: palps; be: basis capituli;

c: chelicerae; p: porose area; eg: cervical groove; se: smooth setae; s: setae with a row of denticles; h: hypostome; sd: setae with several rows of

the denticles; I: first segment palps; II: second segment palps; III: third segment palps; IV: fourth segment palps.

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147 143

Table 1

Comparative external morphology of females of R. sanguineus tick females collected in Jaboticabal, Brazil, and Rafaela, Santa Fe, Argentina

Morphological character Argentina (n = 15) Brazil (n = 15)

Mean size of the tick 3.8 mm � 1.7 mm (Fig. 1A and C) 2.7 mm � 1.1 mm (Fig. 1B and D)

Genital aperture U-shaped groove delimitated by

a thick fold (Fig. 1E)

V-shaped groove delimitated by

a thick fold (Fig. 1F)

Anal aperture Anal plate with two valves and two grooves:

central and perianal (Fig. 1 G)

Anal plate with two valves and

two grooves: central and

perianal (Fig. 1H)

Presence and amount of setae

on the anal valves

Four pairs of setae (Fig. 1G) Three pairs plus one setae (Fig. 1H)

Mean size of the chelicerae 0.5 mm (Fig. 2A and C). 0.25 mm (Fig. 2B and D).

Presence of chitinous plates on

the chelicerae

Yes (Fig. 2C) Yes (Fig. 2D)

Size and distribution of

the chitinous plates

Chitinous plates of the same size distributed

equally throughout the length of the

chelicerae (Fig. 2A and C)

Higher concentration of the

smaller chitinous plates at the

distal region of the chelicerae

(Fig. 2B and D)

Palp morphology Constituted by four segments, with the

first three being larger than the fourth,

which occupies a cavity at the ventral

surface of the third segment (Fig. 2E)

Constituted by four segments,

with the first three being larger

than the fourth, which occupies

a cavity at the ventral surface

of the third segment (Fig. 2F)

Relation between the size of the

palps and the chelicerae

The palps are slightly longer than the

chelicerae (Fig. 2A and C)

The palps are much longer

than the chelicerae (Fig. 2B and D)

Presence and morphology of the

sensory structures

Sensilla and smooth setae or setae with one

or various rows of denticles along their

length (Figs. 2C and G, and 3A and C)

Sensilla and smooth setae

or setae with various rows of

denticles along their length

(Figs. 2D and H, and 3B and D)

Presence of area porose at the capitulum At the basis capituli (Fig. 2A) At the basis capituli (Fig. 2B)

Presence of fovea Next to the region of the scutum that is

in contact with the sub-triangular area

(Fig. 3A)

Next to the region of the

scutum that is in contact with

the sub-triangular area (Fig. 3B)

Presence of a groove on the coxae of

the first pair of legs

Yes (Fig. 3E) Yes (Fig. 1F)

Presence of festoons Yes (Fig. 3F and H) Yes (Fig. 3G and I)

Festoon morphology Delimitated only by deep lateral

grooves (Fig. 3F and H)

Delimitated by deep lateral

grooves and by the marginal

groove, forming well-defined

rectangular portions (Fig. 3G and I)

evenly thick, and with a smooth walls. The setae of the

second group appeared with thorns or denticles

throughout their surface. These denticles could vary

in size, from small to large, distributed in a row along

one side of the setae or spirally. In the females of R.

sanguineus collected in Argentina and Brazil, we

observed the presence of the first group of setae

described by Balashov (1983) (the needle-shaped

setae of smooth surface) on the second, third, and

fourth segments of the palps. The second group of

setae reported by Balashov (1983) was only detected

on the specimens from Brazil origin. Nevertheless, on

the internal margin of the first and second segments of

the palps of specimens from both locations, we noted

the presence of setae of a different morphology than

the ones described by Balashov (1983). These new

types of setae presented several rows of denticles of

the same size, surrounding mainly the median and

distal regions of the setae. None of these rows was

disposed in spiral, contrary to the morphology

described for other species of ticks (Balashov, 1983).

Setae of arthropods in general are structures of

sensory function that are necessary for monitoring and

recognizing the environment (Zacharuk, 1972). None-

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147144

Fig. 3. SEM of dorsal and ventral portions of females of R. sanguineus collected in Brazil and Argentina. (A) Anterior dorsal portion scutum

(sc) next to the point of contact with the sub-triangular area (a) of Argentinean tick, bar: 0.1 mm; (B) anterior dorsal portion scutum (sc) next

to the point of contact with the sub-triangular area (a) of Brazilian tick, bar: 0.1 mm; (C) legs of Argentinean tick, bar: 50 mm; (D) legs

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147 145

theless, some of them, mainly those on the internal

margin of certain segments of the palps of ticks, might

function as protective structures for the more delicate

mouthparts (Barnes and Ruppert, 1996). In the present

study we found setae that, due to their location, might

perform such protective functions. The setae that

possess denticles located on the internal margin of the

palps might play a role on the protection of the

hypostome, while the setae located on the body

surface might be related to the perception of

environmental changes.

The analysis of the ultramorphology of the ventral

hypostome, a structure that plays an important role in

the attachment of the tick to its host, showed that in

ticks of both origin this structure is covered by several

longitudinal rows of retrograde chitinous plates

throughout its surface, and, at its anterior end, by

small chitinous elevations that form the corona.

Similar data were reported by Till (1961) in

Rhipicephalus appendiculatus and by Balashov

(1983) in H. asiaticum.

Dorsally, the hypostome of specimens from both

localities appeared covered by the chelicerae, which

are encased by retrograde and overlaid chitinous

plates, thus corroborating the observations of Bala-

shov (1983) on other species of ticks. However, in the

specimens of R. sanguineus from Argentina, the

chelicerae were larger than the ones found in the

Brazilian specimens. In the Argentinean specimens,

the chitinous plates appeared distributed uniformly

throughout the chelicerae, while in the Brazilian

specimens we noted a higher concentration of these

structures of smaller size located at the distal region of

the chelicerae.

According to Walker et al. (2000), the comparison

of the size of palps and chelicerae provides an

important character used for the distinction between

males and females. It was possible in this work to

verify that the palps of female R. sanguineus collected

in Argentina were as long as the chelicerae while, in

the Brazilian specimens we detected a significant

difference in the size of these two structures. So, this

characteristic might be used to aid in the distinction of

R. sanguineus ticks from different origin.

of Brazilian tick, bar: 50 mm; (E) coxa of first pair of the legs of Argentinean t

bar: 0.1 mm; (G) posterior dorsal idiosoma of the Brazilian tick, bar: 0.1 mm

Brazilian tick, bar: 0.1 mm. se: smooth setae; si: sensilla; f: fovea; fc: deep

According to Balashov (1983), the end of the

chelicerae can move medio-laterally to cut the host

skin in this direction. Sonenshine (1991) suggested

that this same region of the chelicerae bear mechan-

osensory and chemosensory receptors providing

information about shear forces and chemical compo-

sition of host fluids, in addition to the possibility of

pheromone detection. In the present study we did not

find any sort of sensory structure on the ends of the

chelicerae of R. sanguineus from both localities.

At the basis capituli of specimens from both

localities we found setae and sensilla, in addition to a

pair of rounded depressions containing numerous

pores. Such depressions are known as porose area and

confirm the results reported by Balashov (1983) in

other species of ticks. The setae and sensilla might be

related to the reception of chemical or mechanic

stimulus. With regards to the porose area, these are

excretory apertures of dermal glands that secrete

substances for the lubrication of certain structures of

the gnathosoma (Balashov, 1983).

Dorsally, the females of R. sanguineus collected in

Argentina and Brazil presented an anterior region,

more resistant, which consists of sclerotized cuticle

and covers about half of the body. This region is

known as sub-triangular area. At this area we found

innumerous sensilla, which, due to their location,

might play a sensory role.

The present study confirmed that females of R.

sanguineus from both localities presented a great

capacity for body expansion, due to the presence of a

membranous, extensible cuticle at the posterior dorsal

face known as scutum. Balashov (1983) described that

such body expansion of the females is possible due to

the presence of a system of regular membranous

microfolds (grooves or striations) that run parallel to

the body of the animal and confer a great elasticity

during the feeding stage. At this same region of R.

sanguineus from Argentina and Brazil, we noted a

large number of setae, thus differing from the region

that comprehends the sub-triangular area.

According to Balashov (1983), in H. asiaticum,

anterior to scutum and next to the point of contact with

the sub-triangular area, there are two symmetrical

ick, bar: 0.1 mm; (F) posterior dorsal idiosoma of the Argentinean tick,

; (H) festoons of the Argentinean tick, bar: 0.1 mm; (I) festoons of the

fissure; sc: scutum; a: sub-triangular area; fe: festoons.

P.R. de Oliveira et al. / Veterinary Parasitology 129 (2005) 139–147146

round zones with numerous pores from which arise

several sensilla. In R. sanguineus collected in Brazil

and Argentina we detected the same structures,

enclosing about 15 sensilla in each zone. Sonenshine

(1991) suggested that these regions be thought to be

apertures of glands lying under the female cuticle and

to produce sexual pheromones.

The anal aperture of females of R. sanguineus from

both localities is situated on an anal plate at the

posterior region of the idiosoma ventral surface. The

anal plate of R. sanguineus possesses two valves and is

delimitated by a wide ring perianal fold, thus

corroborating the data obtained by Balashov (1983)

for other species of ticks. The same author reported

that both valves of this anal plate are movably

articulated with four setae arranged symmetrically on

each part. The present study demonstrated that in the

females of R. sanguineus collected in Argentina the

distribution of the setae is similar; however, in the

Brazilian specimens only three setae were distributed

in the same pattern reported by Balashov (1983).

According to Barnes and Ruppert (1996), the

number, shape, and location of the setae on the body of

ticks are constant for each developmental stage of any

given species, but they vary considerably from one

stage to another and also among different species, thus

being ideal characters for the identification and

classification of tick species.

The present study showed that the females of R.

sanguineus collected in Argentina presented rectan-

gular portions known as festoons, which are only

delimitated by deep lateral grooves. On the other hand,

in the Brazilian specimens the festoons were delimi-

tated by the lateral grooves and by the marginal groove.

The festoons were located on the posterior body margin.

Till (1961) also described this region in R. appendi-

culatus, with the same characteristics, locations, and

subdivided into 11 portions. In the present work we also

detected the same number of portions in the festoons of

R. sanguineus from both localities.

The genital aperture of females of R. sanguineus

collected in Argentina and Brazil was located on the

ventral surface of the body between the second pair of

legs, as reported previously for other tick species (Till,

1961). The genital aperture of R. sanguineus ticks

from Argentina appeared as a U-shaped groove, while

the Brazilian specimens presented a genital aperture of

broad V-shape. This observation is rather important as

genital aperture is considered an important feature to

distinguish the very similar species R. sanguineus and

Rhipicephalus turanicus. According to Walker et al.

(2000), genital aperture of R. sanguineus is U-shaped

while that of R. turanicus is broadly V-shaped.

Mitochondrial DNA analysis of R. sanguineus ticks

from Argentina and Brazil revealed that a strong

genetic relationship was detected between European

and Argentinean R. sanguineus populations while the

Brazilian population appeared to be related to the

African R. turanicus (Mangold et al., 2004). Moreover

crossmating ticks from both countries originated non-

fertile hybrids (Szabo et al., 2004). All these

observations together indicate that Rhipicephalus

ticks from Brazil are truly R. turanicus.

Unfortunately, the R. sanguineus group comprises

several tick species and the biosystematic status of the

majority has been confused (Walker et al., 2000). It is

however possible that more than one Rhipicephalus

species occurs in the Neotropics. Thus, a profound

taxonomical revision of this tick group is needed.

Acknowledgements

We thank Dr. Alberto A. Guglielmone (INTA,

Rafaela, Santa Fe, Argentina) for tick supplying of

Argentinean origin; to Miss Monika Iamonte, Mr.

Antonio Teruyoshi Yabuki and Miss Cristiane Marcia

Mileo for the technical support and FAPESP

( Fundacao de Amparo a Pesquisa do Estado de

Sao Paulo) for financial support (grant number 02/

01180-8). 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-2000-300069.

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