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Veterinary Parasitology 130 (2005) 131–140

Biological and DNA evidence of two dissimilar populations

of the Rhipicephalus sanguineus tick group

(Acari: Ixodidae) in South America§

Matias P.J. Szabo a,c,*, Atilio J. Mangold b, Carolina F. Joao c,Gervasio H. Bechara c, Alberto A. Guglielmone b

a Faculdade de Medicina Veterinaria, Universidade Federal de Uberlandia, Av. Para 1720/Campus Umuarama,

Bloco 2T, CEP 38400-902 Uberlandia, MG, Brazilb Instituto Nacional de Tecnologıa Agropecuaria, Estacion Experimental Agropecuaria Rafaela,

CC 22, CP 2300, Rafaela (Santa Fe), Argentinac Universidade Estadual Paulista, Jaboticabal, SP, Brazil

Accepted 8 March 2005

Abstract

In this work, the biology, mitochondrial DNA and fertility of hybrids from two strains of Rhipicephalus sanguineus, from

Brazil and Argentina, were compared. Engorged larvae, nymphs and adults from Argentina weighed more and the engorgement

period of adult females was significantly longer than those of their Brazilian counterparts, whereas adult female tick yield rate

was higher for the Brazilian strain. High intraspecific divergence of mitochondrial DNA was detected between R. sanguineus

from Brazil and Argentina. On the other hand, a strong genetic relationship was detected between European and Argentinean R.

sanguineus populations while the Brazilian population appeared to be related to the African Rhipicephalus turanicus. Adult

hybrid females laid eggs, which were mostly unviable, whereas a mean of more than 1400 larvae hatched per egg mass from pure

Brazilian and Argentinean strains. These results showed that differences between these strains are greater than previously

assumed and that the biosystematic status of R. sanguineus ticks from South America should be re-evaluated. Wide variations,

such as these might account for the reported worldwide differences in biology and vector capacity of this species.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Rhipicephalus sanguineus; Argentina; Brazil; Biology; DNA; Hybrid

§ Note: Nucleotide sequence data reported in this paper are

available in the GenBankTM, EMBL and DDBJ databases under

the accession number(s).

* Corresponding author. Tel.: +55 34 3218 2228;

fax: +55 34 3218 2521.

E-mail address: szabo@famev.ufu.br (Matias P.J. Szabo).

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

doi:10.1016/j.vetpar.2005.03.008

1. Introduction

The ixodid tick Rhipicephalus sanguineus, com-

monly referred to as the ‘‘kennel tick’’ or ‘‘brown dog

tick’’, is widely distributed in the America, Europe,

Africa, Asia and Australia and is probably the most

.

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140132

prevalent of all ixodid species (Pegram et al., 1987a,b).

It has been linked to tick-borne diseases, such as spotted

and boutonneuse fever and ehrlichioses in man and

babesiosis and ehrlichioses in dogs (Walker et al.,

2000). It is an introduced tick species in the South

American region. Different routes were probably

involved in colonization of the subcontinent. For

instance, its presence in Brazil was recognized early in

the XX century by Rohr (1909), while it was first found

in Argentina (city of Buenos Aires) in 1938 (Roveda,

1954), and the first Chilean finding of R. sanguineus

occurred in Santiago in 1974 (Tagle, 1976). It is

generally believed that R. sanguineus sensu stricto is

the only representative of the genus in South America.

Because of the importance of R. sanguineus as a

parasite of dogs and its vector capacity, it has been a

frequent subject of research. But, even though most

observations published throughout the world broadly

match, a few important regional differences seem to

exist. R. sanguineus is the principal vector of

Rickettsia conorii, the agent of Boutonneuse fever,

to humans in the countries around the Mediterranean

coast (Estrada-Pena and Jongejan, 1999). There are

several records of humans bitten by R. sanguineus in

the Neotropical countries, namely Argentina, Brazil,

Chile, Mexico (southern region), Panama, Peru,

Puerto Rico, Venezuela and Uruguay (Guglielmone

et al., 2003). Schenone (1996) reported that 2.2% of

cases of arthropod bites in humans in Chile were due

to R. sanguineus, which causes a pruriritic dermatitis

that is sometimes very intense in children. Ehrilichia

canis, or a closely related species, has been isolated

from man in Venezuela (Perez et al., 1996), and

evidence suggests that R. sanguineus is the vector

(Unver et al., 2001). In USA, human cases of tick-bite

vary according to the region studied (Estrada-Pena and

Jongejan, 1999). In Africa, R. sanguineus is pre-

dominantly a parasite domestic dog but may also

infest man and, probably rarely, his domestic livestock

(Walker et al., 2000). Elsewhere, it is confined almost

entirely to domestic dogs (Pegram et al., 1987b).

R. sanguineus also seems to display a variability in

size in different regions. A comparison of published

data showed that engorged R. sanguineus females

from Brazil (Bechara et al., 1995; Szabo et al., 1995)

may weigh 50% less than those from the USA

(Jittapalapong et al., 2000) and Japan (Inokuma et al.,

1997).

Another variable observation regards the capacity

of dogs to develop resistance to R. sanguineus

following several infestations. In a few instances, it

was observed that dogs are unable to acquire

resistance even after several infestations (Chabaud,

1950; Theis and Budwiser, 1974; Szabo et al., 1995),

whereas other authors detected some degree of

resistance following reinfestation (Inokuma et al.,

1997; Jittapalapong et al., 2000).

In view of the above differences among R.

sanguineus ticks, it would seem desirable to more

precisely evaluate these variations between popula-

tions. Such knowledge may contribute an under-

standing of regional differences in the epidemiology

of diseases transmitted by R. sanguineus as well as

possible variations in susceptibility to acaricides. The

objective of this work was to compare the biology and

mitochondrial DNA and fertility of hybrids from two

strains of R. sanguineus within South America, one

from Jaboticabal city, Sao Paulo, Brazil and the other

from Rafaela, Santa Fe, Argentina.

2. Material and methods

2.1. Ticks

R. sanguineus colonies were established from

engorged females detached from healthy dogs in

Jaboticabal, SP, Brazil and from Rafaela, Santa Fe,

Argentina. Jaboticabal is located approximately

600 m above sea level in the Southeast of Brazil

(2181502200S; 4881805800W), has a subtropical climate

with a dry winter and a hot and humid summer.

Rafaela (318150S; 618290W) is located in central

Argentina at an altitude of 63 m; the climate is

continental with a hot summer and cold winter.

The Brazilian strain colony has now been main-

tained for over 10 years (Szabo et al., 1995) but is

periodically renewed with the inclusion of local tick

samples. The colony with the Argentinean strain was

set up specifically for this work from 10 engorged

females collected from 3 healthy dogs.

Tick colonies of both R. sanguineus strains were

reared in the laboratory as previously described

(Bechara et al., 1995). Unless otherwise stated, ticks

were kept under a constant temperature of 27 8C, a

relative humidity of 80 � 5% and a 12-h light:12-h

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140 133

dark photoperiod. A continuous tick supply was

maintained by feeding adults, nymphs and larvae on

tick-bite naive New Zealand rabbits. One of the authors

(AAG) and Ivan G. Horak, at the Faculty of Veterinary

Science, University of Pretoria, South Africa confirmed

the identity of ticks from both colonies.

2.2. Hosts

Male and female domestic mongrel dogs or New

Zealand white rabbits were used as hosts. Every

animal was tick-bite naive at the beginning of the

experiments. Dogs were born and raised in a tick-free

kennel at the Pathology Department of the School of

Veterinary Science of Jaboticabal, Sao Paulo State

University-UNESP. Rabbits were obtained from the

Sao Paulo State University’s Bioterium (Botucatu-SP,

Brazil). Water and commercial food were available

‘‘ad libitum’’ to animals.

2.2.1. Comparison of feeding and reproductive

parameters of R. sanguineus ticks from pure

Argentinean and Brazilian strains

Tick-bite naıve hosts were infested with both the

Rafaela R. sanguineus strain from Argentina and the

Jaboticabal R. sanguineus strain from Brazil as

described elsewhere (Szabo et al., 1995). Briefly,

ticks from each strain were confined in a feeding

chamber on the host’s dorsum. Two feeding chambers

were glued to each host for simultaneous separate

infestation with both strains. Fifty nymphs and 10

adult tick pairs from the same strain were fed together

in each chamber on each dog (n = 7). Larvae were fed

separately on rabbits (n = 7), since simultaneous

infestation of dogs with all instars proved to be a

very difficult procedure. Two dogs were infested with

larvae for comparative purposes. Every larval infesta-

tion consisted of 300 specimens from each strain.

Feeding chambers were examined every day and

engorged detached ticks weighed individually

(adults) or in daily batches (larvae and nymphs) and

kept under constant temperature and relative humidity

as described above. Tick yield, engorged female

and egg mass weights, engorging, pre-oviposition

and incubation periods, larval-hatchability rates and

efficiency rate of female ticks in converting their food

reservoir to eggs were used for a comparison of

strains. In this initial experiment, larval-hatchability

rates were assessed by visual inspection as described

elsewhere (Szabo et al., 1995).

2.2.2. Comparison of mitochondrial 12S rDNA

sequences of tick strains from Argentina and Brazil

Comparison of tick DNA from both strains was

performed as described elsewhere (Mangold et al.,

1998). Briefly, living ticks from the Argentinean and

Brazilian strains were immersed in 70% alcohol and

kept at �20 8C until processing. DNA was extracted

from a single unfed male tick and PCR amplification

and sequencing was carried out to obtain a � 400 bp

fragment of the 30 half of the mitochondrial 12S rDNA

sequences. PCR conditions included an initial

denaturation step at 94 8C for 2 min followed by 35

cycles for 45 s at 94 8C, 45 s for primer annealing at

55 8C and 45 s for primer extension at 72 8C. A final

extension step was carried out for 7 min at 72 8C. The

primers used for the amplification and sequencing of

the 400 bp fragment (part of the II and to the III

domain, Hickson et al., 1996) were: forward, 50-AAA

CTA GGA TTA GAT ACC CTA TTA TTT TAG-30;reverse, 50-CTA TGT AAC GAC TTA TCT TAA TAA

AGA GTG-30. All PCR reactions were performed in a

100 ml volume. Negative controls (no template) were

always run simultaneously. A 10 ml volume of the

reaction mixture was examined by 1% agarose-gel

electrophoresis followed by staining with ethidium

bromide. The amplified DNA was purified using

Concert1 Rapid PCR Purification System (Invitrogen

Corp.) according to the manufacturer’s protocol.

Purified PCR products were sequenced using the

ABI PRISM Dye Terminator Cycle Sequencing Ready

Reaction kit and an Applied Biosystems 373A gene

sequencer.

Ten-mitochondrial 12S rDNA sequences of R.

sanguineus and R. turanicus available in the GenBank

were also used for comparative sequence analysis.

R. sanguineus (U95915) and R. turanicus (U95916)

from Israel reported by Norris et al. (1999), R.

sanguineus (AF133056) from Egypt, R. sanguineus

(AF150020) from France, R. turanicus (AF133057)

from Turkey, R. turanicus (AF150018) from France,

R. turanicus (AF150017) from Zimbabwe, R. tur-

anicus (AF150015) strain 1 from Israel, R. turanicus

(AF150014) strain 35 from Israel and R. turanicus

(AF150013) strain 63 from Israel reported by Beati

and Keirans (2001).

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140134

Aligned sequences were examined using the

computer program MEGA Version 2.1 (Kumar

et al., 2001). A similarity matrix was constructed

and a neighbor-joining tree was generated from

Kimura two-parameter distance measure.

2.2.3. Cross-mating of R. sanguineus ticks from

Argentina and Brazil to detect fertility of hybrid

offspring

Naive rabbits, each fitted with three feeding

chambers, were used for the cross-mating of ticks.

Six adult female R. sanguineus from the Brazilian

strain were mated with six males from Argentina in

the first chamber on each rabbit, and six females

from Argentina and six males from Brazil in the

second chamber. For control purposes, six couples of

either the Brazilian or Argentinean strain were

released in the third chamber. Later batches of 300

hybrid larvae obtained from females cross-mated on

different hosts were fed on tick-bite naive rabbits to

obtain hybrid nymphs and sequentially hybrid adults.

Engorged ticks were weighed and kept as previously

described.

2.2.4. Comparison of feeding and reproductive

parameters of adult hybrid R. sanguineus and pure

Argentinean and Brazilian strains

Six tick-bite naive rabbits each fitted with three

feeding chambers were used for comparisons. Six

couples of ticks were released in each chamber.

Hybrid adults from each origin (offspring of Brazilian

females mated with Argentinean males or vice versa)

were each released into five chambers and, for control

purposes, either pure Argentinean or pure Brazilian

ticks were each released into four chambers. Care

was taken to infest the three chambers on each rabbit

with ticks from different experimental group. Biolo-

gical parameters were determined as described above

but larval-hatching rates were obtained by counting

Table 1

Means, standard deviation and statistical analysis of the biological param

strains fed on tick-bite naive rabbits

Larvae strain/host Weight (mg) Engorging period (days)

Argentina/rabbit (n = 7) 0.47 a � 0.04 3.38 a � 0.54

Brazil/rabbit (n = 7) 0.29 b � 0.03 3.12 a � 0.33

Number of hosts used for feeding is in brackets. Means in a column foll

unhatched eggs and larvae from each female under a

stereomicroscope.

2.3. Statistical analysis

Data on the biological parameters of ticks in each

experiment were compared using one-way analysis

of variance and Tukey’s Multiple Comparison Test

( p > 0.05).

3. Results

3.1. Comparison of feeding and reproductive

parameters of R. sanguineus from pure Argentinean

and Brazilian strains

Feeding and reproductive parameters of R. sangui-

neus strains from Argentina and Brazil, where

compared on tick-bite naive rabbits (larvae) and dogs

(nymphs and adults). Engorged larvae, nymphs and

adults from Argentina weighed 62, 43 and 49%

more than their Brazilian counterparts ( p < 0.001)

(Tables 1–3). The engorgement period of adult female

ticks from Argentina was significantly longer than

those from Brazil ( p < 0.01), whereas adult female

tick yield rate was higher for the Brazilian strain

( p < 0.05) (Table 3). The low yield of engorged adult

females from Argentina prompted us to test the

maintenance of ticks from this strain at a constant

temperature of 20 8C, a relative humidity of 80 � 5%

and a 14-h light:10-h dark photoperiod for 2 months

before infestations. Two of the seven dogs were

infested with ticks kept under these conditions, and

tick yield of engorged females increased to 100%.

These data were pooled with the other results, since

other biological did not differ significantly (Table 3).

Unfed adult ticks from Argentina were, on visual

inspection darker, and when engorged, the females

eters of R. sanguineus tick larvae from Brazilian and Argentinean

Moulting period (days) Moulting rate (%) Tick yield (%)

5.01 a � 0.96 91.01 a � 9.04 58.61 a � 22.39

5.34 a � 0.72 95.31 a � 5.52 48.38 a � 20.55

owed by the same letter do not differ significantly ( p < 0.05).

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140 135

Table 2

Means, standard deviation and statistical analysis of the biological parameters of R. sanguineus tick nymphs from Brazilian and Argentinean

strains fed on tick-bite naive dogs

Nymphs strain/host Weight (mg) Engorging period (days) Moulting period (days) Moulting rate (%) Tick yield (%)

Argentina/dog (n = 7) 5.84 a � 0.95 5.19 a � 0.30 11.09 a � 1.28 96.67 a � 8.82 72.0 a � 25.59

Brazil/dog (n = 7) 4.08 b � 0.43 5.09 a � 0.48 10.81 a � 0.58 92.44 a � 12.91 71.43 a � 18.39

Number of hosts used for feeding is in brackets. Means in a column followed by the same letter do not differ significantly ( p < 0.05).

had a greenish coloration as opposed to a greyer

pattern of engorged females of the Brazilian strain.

3.2. Comparison of mitochondrial 12S rDNA

sequences of tick strains from Argentina and Brazil

The lengths of the 12S rRNA gene fragment for

R. sanguineus from Argentina (GenBank accesion

number AY559841) was 355 bp and for R. sanguineus

from Brazil (GenBank accesion number AY559842)

was 356 bp. The absolute nucleotide differences

between R. sanguineus sequences were 27 (8%) and

the alignment between them is shown in Fig. 1.

The sequence variations among R. sanguineus and

R. turanicus populations range between 0 and 8.3%

(Table 4). The lowest intraspecific variations were

observed between R. sanguineus from Argentina,

France and Egypt (range, 0–0.6%) and between

R. turanicus from Israel (strain 1) and Turkey (0.3%).

On the other hand, the highest intraspecific divergence

was detected between R. sanguineus from Brazil and

Israel (8.3%). However, the sequence variations

observed between R. sanguineus from Brazil and

R. turanicus from Zimbabwe was only 2.4%. The

neighbor-joining analysis yielded the tree shown in

Fig. 2. The results provided a strong support (99%) for

Table 3

Means, standard deviation and statistical analysis of the biological par

Argentinean strains fed on tick-bite naive dogs

Adult females

strain/host

FW

(mg)

EMW

(mg)

EP

(days)

POP

(days)

Argentina/dog*

(n = 7)

231.06 a � 22.14 97.79 a � 69.22 11.88 a � 3.15 3.29 a �

Brazil/dog

(n = 7)

154.77 b � 26.07 87.59 a � 18.77 7.04 b � 0.46 4.09 a �

FW, engorged female weight; EMW, egg mass weight; EP, engorging period; PO

rate; ERCE, efficiency rates of female ticks in converting their food reservoir

significantly ( p < 0.05). Number of hosts used for feeding is in brackets.* Pooled data from dogs infested with unfed adult ticks kept at 20 8C and a 1

dark photoperiod (n = 5).

the cluster containing the Argentinean R. sanguineus

and others Mediterranean R. sanguineus. A high

bootstrap value (100%) supported the close relation-

ship between R. sanguineus from Brazil and R.

turanicus from Zimbabwe (AF150017). Furthermore,

there are two clusters formed by four different

populations of R. turanicus from Israel and one from

Turkey.

3.3. Cross-mating of R. sanguineus ticks from

Argentina and Brazil to detect fertility of offspring

Female ticks mated with males from the other

strain attached readily, engorged fully and laid viable

egg masses. Adult females, which mated with males of

the other strain, tended to display feeding and

reproductive characteristics similar to those of their

original strain, although females from Argentina

attached and engorged more rapidly than when mated

with males from the same strain (data not shown).

Hybrid larvae and nymphs exhibited development

patterns comparable to those of the pure strains but

engorged weights were higher and lower when

compared to pure Brazilian and pure Argentinean

strains, respectively. Representative data are shown in

Table 5.

ameters of adult R. sanguineus female ticks from Brazilian and

IP

(days)

LH

(%)

ERCE

(%)

Tick yield

(%)

0.49 17.22 a � 0.60 95.94 a � 5.15 46.70 a � 31.19 38.57 a � 45.62

0.68 18.52 a � 1.30 94.45 a � 5.62 58.29 a � 5.17 87.14 b � 17.99

P, pre-oviposition period; IP, incubation period; LH, larval-hatchability

to eggs. Means in a column followed by the same letter do not differ

4-h light:10-h dark photoperiod (n = 2) or at 27 8C and a 12-h light:12-h

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140136

Fig. 1. Alignment of nucleotide sequences (50–30) of the 12S rRNA gene of R. sanguineus from Argentina and Brazil.

Table 4

Matrix of sequence divergence and absolute nucleotide differences on pair wise comparisons of the 12S mitochondrial rRNA gene for 12 R.

sanguineus and R. turanicus populations

OTUs 1 2 3 4 5 6 7 8 9 10 11 12

1 Rt Israel35 – 1.5 1.3 4.1 5 5.9 7.1 6.6 6.6 6.4 5.7 6.8

2 Rt Israel01 5 – 0.3 3.5 4.8 6.2 6.8 5.9 5.9 5.8 5.1 6.2

3 Rt Turkey 4 1 – 2.9 4.2 5.4 6.0 4.8 4.8 5.5 4.5 5.1

4 Rt Israel63 14 12 9 – 1.2 5.3 5.9 5.9 5.9 6.1 5.9 6.2

5 Rt Israel 17 16 13 4 – 6.2 6.8 7.4 7.4 7.7 7.4 6.8

6 Rt Zimbabwe 20 21 17 18 21 – 2.4 8.3 8.3 8.3 8 7.7

7 Rs Brazil 24 23 19 20 23 8 – 8 8 8 7.7 8.3

8 Rs France 22 20 15 20 25 28 27 – 0 0.6 2.4 3.9

9 Rs Argentina 22 20 15 20 25 28 27 0 – 0.6 2.4 3.9

10 Rs Egypt 20 18 17 19 24 26 25 2 2 – 2.6 4.2

11 Rt France 19 17 14 20 25 27 26 8 8 8 – 5.0

12 Rs Israel 23 21 16 21 23 26 28 13 13 13 17 –

Proportion of nucleotide differences (102) are shown in the upper right matrix and nucleotide differences, in the lower left matrix. OTUs,

operational taxonomic units; Rt, R. turanicus; Rs, R. sanguineus.

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140 137

Fig. 2. Neighbor-joining tree (unrooted) using Kimura two-parameter distance. Numbers represent the percentages of bootstrap support.

3.4. Comparison of feeding and reproductive

parameters of adult hybrid R. sanguineus

and pure Argentinean and Brazilian strains

Feeding and reproductive data were available from

five of seven rabbits. Pre-oviposition periods, efficiency

rates of female ticks in converting their food reservoir to

eggs and total numbers of eggs did not differ

significantly among the ticks with different genetic

backgrounds, although Brazilian ticks laid fewer eggs

than the others (Table 6). Females from Argentina were

significantly heavier and had significantly heavier egg

masses than their Brazilian counterparts. Hybrid

females, originating from Argentinean females mated

with Brazilian males (Haf) and Brazilian females mated

Table 5

Means and standard deviation of the biological parameters of R. sanguineu

other strain (Argentinean or Brazilian) and fed on tick-bite naive rabbits

Nymph strain Weight (mg) Engorging

period (days)

F.arg/M.bra (n = 6) 5.69 � 0.48 4.90 � 0.56

F.bra/M.arg (n = 8) 6.22 � 0.43 4.91 � 0.74

Pure Argentinean (n = 4) 6.57 � 0.65 4.93 � 0.77

Pure Brazilian (n = 4) 4.11 � 0.56 5.34 � 0.58

F, female; M, male. Number of hosts used for feeding is in brackets.

with Argentinean males (Hbf) had intermediate

engorged female and egg mass weights. From these,

the engorged Haf weight was significantly greater than

that from pure Brazilian strains (Table 6). Ticks of the

Argentinean strain, displayed the lowest tick yield and

the longest engorging period, differing significantly

from all other tick groups (Table 6).

Very few larvae hatched from egg masses from both

hybrid tick groups (Haf, 0.06%; Hbf, 0.045%),

whereas a mean of almost 1400 and 4000 larvae

hatched per egg mass from pure Brazilian and pure

Argentinean strains, respectively (Table 7 and Fig. 3).

The incubation period the few hybrid eggs that

hatched were almost double in relation to the

incubation period of the pure strains (Table 6).

s tick nymphs, offspring of female ticks mated with males from the

Moulting

period (days)

Moulting

rate (%)

Tick yield (%)

10.90 � 0.93 100.0 � 0.0 69.35 � 21.29

11.18 � 0.56 100.0 � 0.0 65.94 � 34.26

11.78 � 0.54 98.18 � 3.65 77.95 � 18.11

9.73 � 0.63 100.0 � 0.0 72.67 � 21.01

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140138

Table 6

Means and standard deviation and statistical analysis of the biological parameters of hybrid and pure strain R. sanguineus adult female ticks fed

on tick-bite naive rabbits

Adult females FW (mg) EMW (mg) EP (days) POP (days) IP (days) ERCE (%) Tick yield (%)

Haf (n = 4) 271.6 a � 33.3 162.2 ab � 15.0 11.8 a � 1.7 2.6 a � 0.1 29.4 a � 5.7 58.9 a � 3.7 87.5 a � 15.9

Hbf (n = 5) 252.9 ab � 62.3 139.7 ab � 46.5 14.98 a � 3.6 2.2 a � 0.4 33 a � 1.4 49.6 a � 5.9 73.3 a � 30.3

Argentinean (n = 3) 309.8 a � 23.5 198.6 a � 23 25.3 b � 8.1 2.5 a � 0.7 16 b � 0.0 61.5 a � 6.0 16.6 b � 16.5

Brazilian (n = 3) 173.4 b � 33.7 103.9 b � 20 8.8 a � 0.3 2.5 a � 0.3 19 b � 0.5 59.9 a � 2.5 100 a � 0.0

Haf, hybrid ticks offspring of Argentinean females mated with Brazilian males; Hbf, hybrid ticks descendent from Brazilian females mated with

Argentinean males; FW, engorged female weight; EMW, egg mass weight; EP, engorging period; POP, pre-oviposition period; IP, incubation

period; ERCE, efficiency rates of female ticks in converting their food reservoir to eggs. Means in a column followed by the same letter do not

differ significantly ( p < 0.05). Number of hosts used for feeding is displayed in brackets.

Table 7

Means and standard deviation and statistical analysis of the number of eggs, larvae and hatching rate in the egg mass of hybrid and pure strain R.

sanguineus adult female ticks fed on tick-bite naive rabbits

Strain (number of hosts/ticks) Number of

emerging larvae

Number of

unhatched eggs

Number of eggs

per egg mass

Eclosion rate (%)

Haf (n = 4/21) 2.0 a � 1.3 3820.1 a � 782.2 3822.0 a � 782.9 0.06 a � 0.04

Hbf (n = 5/22) 0.1 a � 0.2 3262.1 a � 958.6 3262.2 a � 958.4 0.045 a � 0.1

Argentina (n = 3/3) 3977.5 a � 886 374 a � 256 4351.5 a � 630 91.8 a � 5.9

Brazil (n = 3/18) 1396.6 a � 263.4 926.8 a � 456.8 2323.4 a � 352.2 58.8 a � 13.2

Haf, hybrid ticks offspring of Argentinean females mated with Brazilian males; Hbf, hybrid ticks descendent from Brazilian females mated with

Argentinean males. Means in a column followed by the same letter do not differ significantly ( p < 0.05). Number of hosts used for feeding and

total number of female ticks recovered is in brackets.

4. Discussion

Several dissimilarities were observed between the

feeding and reproductive parameters of R. sanguineus

Fig. 3. Larval-hatch rate of R. sanguineus hybrid females, offspring

of Argentinean females mated with Brazilian males (HybridArgF)

and Brazilian females mated with Argentinean males (HybridBraF)

and pure strains from Argentina and Brazil.

ticks from Argentina and Brazil. Engorged ticks from

Argentina were approximately 50% heavier, had a

longer engorging period and displayed a lower female

tick yield rate. Size and morphological variations

among R. sanguineus strains are not an unexpected

feature in such a widely distributed tick species. For

example, morphological variations, both intra- and

interpopulations, were detected in the adanal plates,

genital aperture, spiracular plates, hypostomal denti-

tion and palpi in R. sanguineus ticks from eight states

of Brazil (Ribeiro et al., 1996). At the same time, the

lower tick yield of the strain from Argentina was

associated with an unwillingness of the adult ticks to

attach to the hosts. Exposure of this tick to an

increased daily light regimen before infestation took

place enhanced attachment of the parasite. This

behaviour indicates a strong influence of season on

ticks from Argentina, possibly an adaptation to local

environment.

To further evaluate differences between the two

strains, mitochondrial 12S rDNA sequences of tick

from Argentina and Brazil were compared. Results

showed that differences between both strains are wider

M.P.J. Szabo et al. / Veterinary Parasitology 130 (2005) 131–140 139

than supposed before. Moreover, cross-mating of both

tick strains produced practically non-fertile females.

The strong genetic relationship between European and

Argentinean populations may indicate a common

European origin, while the Brazilian population

appears related to the African R. turanicus. Differ-

ences in the colonization of these countries might have

lead to the introduction of ticks of the R. sanguineus

group from different origins. Alternatively, climate

and environmental conditions of each country might

have favoured survival of ticks from different

geographical origins. Last but not the least, South

America may be favourable for speciation with a

divergence over time in the tick populations intro-

duced with colonization.

It is true that there is no a simple definition of species

(Kunz, 2002). Nevertheless, several authors consider

production of viable progeny as a strong indication for

species definition (Guglielmone et al., 2003). Our

results show that crossing alleged R. sanguineus from

Brazil and Argentina produce sterile hybrids; there are

also noteworthy size and biological differences

between them. These facts added to an ample

divergence in 12S rDNA sequences indicate that they

may belong to different species of Rhipicephalus.

Unfortunately R. sanguineus belongs to a group

difficult to deal with in this context. Rhipicephalus

species do not provide a wide array of discriminator

characters (Beati and Keirans, 2001). In fact, this R.

sanguineus complex comprises several tick species and

the biosystematic status of the majority of them has

been confused; consequently, they have often been

misidentified (Pegram et al., 1987a,b). In addition, since

it is believed that R. sanguineus is the only

representative of this genus in South America, there

was, until now, little incentive to seek for other species.

It is, thus, important to more carefully look at

R. sanguineus ticks of different origin within South

America. In this regard, it would be specially

interesting to compare populations all along from

Santa Fe (Argentina) to Jaboticabal (Brazil) to

determine whether there are populations with inter-

mediate features or to determine whether populations

are allopatrically separated or, eventually, if there is

sympatry between them.

Differences in so-called R. sanguineus and R.

turanicus from various parts of the world indicate that

considerable confusion exists for a precise definition of

either species. The task seems to be further complicated

as the 12S rDNA sequences of specimens morpholo-

gically identified as R. turanicus are characterized by a

high level of variability, indicating that R. turanicus-

like morphology may cover a spectrum of distinct

species (Beati and Keirans, 2001). Moreover, sympatry

of R. sanguineus and R. turanicus is likely to occur as

has been recently described from Southern Switzerland

(Bernasconi et al., 2002). These authors also observed

a close relationship between the sequence of a

tick collected on a dog from Costa Rica (classified as

R. turanicus) with the corresponding sequence of

R. turanicus from Zimbabwe also used in the present

study (AF150017). In any situation it seems that there

are at least two species of the R. sanguineus complex in

South America but it is premature to name the species

we worked with until an exhaustive worldwide revision

of R. sanguineus and R. turanicus and other related

species is performed.

Whatever the real biosystematic status of

R. sanguineus in South America is different popula-

tions of R. sanguineus might be associated with

differences in tick behaviour, biology and vectoring

capacity. Awareness of these differences is important,

as such knowledge might be needed for the control of

ticks and tick-borne diseases in different locations.

Acknowledgements

We would like to acknowledge CNPq for financial

support, FAPESP for scholarship (Carolina F. Joao).

We are also grateful to INTA and Fundacion Argeninta

for the support to AJM and AAG. We are indebted to

Professor Ivan G. Horak for confirming tick identi-

fications and reviewing the manuscript, for Karina,

Viviane, Marcos and Augusto for the counts of tick

larvae and eggs. Part of this work has been facilitated

through the International Consortium on Ticks and

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

INCO-DEV program of the European Union under

Contract number ICA4-CT-2000-30006.

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