Amblyomma rotundatum (Koch, 1844) (Acari: Ixodidae) two-host life-cycle on Viperidae snakes.

Abstract

Amblyomma rotundatum is an ixodid tick that infests ectothermic animals and reproduces exclusively by parthenogenesis. This tick has been frequently reported to infest reptiles and amphibians, under natural conditions and sometimes in captivity. It was described in Brazil and several other countries of South, Central and North America. Although many studies have reported aspects of its biology, none of them has used regularly either ophidian as hosts, or controlled temperature, humidity and luminosity for parasitic stages. The objective of this experiment was to study the life cycle of A. rotundatum feeding on Viperidae snakes under room controlled conditions at 27 ± 1 °C temperature, 85 ± 10% relative humidity and 12:12 hours photoperiod for parasitic stages, and under B.O.D incubator conditions at 27 ± 1 °C temperature, 85 ± 10% relative humidity and scotophase for non-parasitic stages. The total duration of the life cycle ranged from 56 to 163 days (mean of 105 days). Two-host life cycle was observed for most of the ixodid population studied.

Full text

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www.cbpv.com.br/rbpv
Rev. Bras. Parasitol. Vet., Jaboticabal, v. 19, n. 3, p. 174-178, jul.-set. 2010
ISSN 0103-846X (impresso) / ISSN 1984-2961 (eletrônico)
*Corresponding author: Paulo Roberto de Oliveira
Professor Associado, Laboratório de Pesquisas em Endo e Ectoparasitoses,
Departamento de Medicina Veterinária Preventiva, Escola de Veterinária,
Universidade Federal de Minas Gerais – UFMG, Av. Antônio Carlos 6627,
CP 567, CEP 30123-970, Campus da UFMG, Belo Horizonte - MG, Brazil;
e-mail: pro@ufmg.br
Amblyomma rotundatum (Koch, 1844) (Acari: Ixodidae)
two-host life-cycle on Viperidae snakes
Ciclo dioxênico em Amblyomma rotundatum (Koch, 1844) (Acari: Ixodidae)
parasitando serpentes da família Viperidae
Daniel Sobreira Rodrigues1,2; Ricardo Maciel3; Lucas Maciel Cunha1;
Romário Cerqueira Leite4; Paulo Roberto de Oliveira4,5*
1Escola de Veterinária, Universidade Federal de Minas Gerais – UFMG
2Empresa de Pesquisa Agropecuária de Minas Gerais – EPAMIG
3Fundação Ezequiel Dias – FUNED
4Laboratório de Pesquisas em Endo e Ectoparasitoses, Departamento de Medicina Veterinária Preventiva, Escola de Veterinária,
Universidade Federal de Minas Gerais – UFMG
5Study Supervisor
Received May 7, 2010
Accepted July 12, 2010
Abstract
Amblyomma rotundatum is an ixodid tick that infests ectothermic animals and reproduces exclusively by
parthenogenesis. This tick has been frequently reported to infest reptiles and amphibians, under natural conditions and
sometimes in captivity. It was described in Brazil and several other countries of South, Central and North America.
Although many studies have reported aspects of its biology, none of them has used regularly either ophidian as hosts,
or controlled temperature, humidity and luminosity for parasitic stages. The objective of this experiment was to study
the life cycle of A. rotundatum feeding on Viperidae snakes under room controlled conditions at 27 ± 1 °C temperature,
85 ± 10% relative humidity and 12:12 hours photoperiod for parasitic stages, and under B.O.D incubator conditions
at 27 ± 1 °C temperature, 85 ± 10% relative humidity and scotophase for non-parasitic stages. The total duration of
the life cycle ranged from 56 to 163 days (mean of 105 days). Two-host life cycle was observed for most of the ixodid
population studied.
Keywords: Amblyomma rotundatum, two-host, life-cycle, Bothrops, Viperidae.
Resumo
Amblyomma rotundatum é um carrapato da família Ixodidae, parasito de animais pecilotérmicos, e que se reproduz
exclusivamente por partenogênese. Este carrapato é frequentemente relatado infestando répteis e anfíbios em condições
naturais e, às vezes, em animais de cativeiro. Ele já foi relatado no Brasil e em vários outros países das Américas do
Sul, Central e do Norte. Embora muitos estudos sobre sua biologia tenham sido publicados, nunca foram utilizados
ofídios como hospedeiros e, tão pouco, foram realizados ensaios com os estádios parasitários sob condições controladas
de temperatura, umidade e iluminação. O objetivo deste experimento foi estudar o ciclo biológico de A. rotundatum
se alimentando em serpentes da família Viperidae sob condições ambientais controladas a 27 ± 1 °C de temperatura,
85 ± 10% de umidade relativa do ar e 12:12 horas de fotoperíodo para estágios parasitários; assim como sob condições
iguais a 27 ± 1 °C de temperatura, 85 ± 10% de umidade relativa do ar e escotofase em estufas de germinação para
estádios não parasitários. A duração total do ciclo de vida variou de 56 a 163 dias (média de 105 dias). Observou-se
ciclo dioxênico para a a maior parte da população dos ixodídeos em estudo.
Palavras-chave: Amblyomma rotundatum, dioxênico, ciclo biológico, Bothrops, Viperidae.
Review ArticleFull Article

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v. 19, n. 3, jul.-set. 2010 Amblyomma rotundatum (Koch, 1844) (Acari: Ixodidae) two-host life-cycle on Viperidae snakes 175
Introduction
The tick A. rotundatum is a parasite that infests cold-blooded
animals, as some species of amphibians and reptiles, and was
first described in literature by Koch (1844 apud ROBINSON,
1926) from a female collected in the state of Pará, Brazil. The
origin and significance of males in the life cycle of A. rotundatum
remain unknown, since this ixodid reproduces exclusively by
parthenogenesis (LABRUNA et al., 2005). A. rotundatum is
believed to be one of the most ancient ticks of its genus. The small
number of laid eggs per gram, observed by Labruna et al. (1997),
is believed to be associated to the primitive ticks which evolved
along with amphibians and reptiles in the end of the Paleozoic
era and beginning of the Mesozoic era (HOOGSTRAL, 1985;
OLIVER Jr., 1989).
This parasite is geographically limited to the Americas, which
include at the moment the following countries: Brazil, Argentina,
Colombia, Bolivia, Peru, Venezuela, Dominican Republic,
Guatemala, Panama, Costa Rica, Jamaica, Granada, Martinique,
Trinidad and Tobago, Mexico and The United States of America
(ROBINSON, 1926; ARAGÃO, 1938; VOLGESANG, DIAS,
1953; JONES et al., 1972; OLIVER Jr. et al., 1993). With the
purpose of biological control of flies in sugar-cane plantations in the
state of Florida (USA), the introduction of Bufo marinus accidentally
also introduced the tick A. rotundatum, common parasite of this
toad specie (BECKLUND, 1968; OLIVER Jr. et al., 1993).
The infestation with these ticks is extremely pathogenic to hosts,
and even in small scales, it can lead them to death. Few studies
exist to confirm this hypothesis, but death has been attributed to
the inoculation of toxins (HANSON et al., 2007). It is known,
though, that the species transmits the hematozoon Hemolivia
stelatta in frogs, and that secondary infections and stress caused by
the parasitism may easily provoke septicaemia and lead the reptile
to death (MADER, 1996). These are the reasons why studies
about A. rotundatum are extremely important to zoos, bioteries,
centers for the extraction of poison and production of snake
poison antidotes, investigation of wild cycle diseases and reptile
and amphibian medicine. There are studies on the biology of this
tick, but none of them has either utilized ophidians as hosts in a
systematic form or controlled environmental conditions to assess its
parasitic stages, which is crucial, since the influence of temperature
on the duration of fixation periods is very much accentuated in
cold-blooded animals (BALASHOV, 1972). Thus, the goal of this
experiment is to observe the bionomic aspects of all the stages of
development of the species in question, using Viperidae Snakes
as hosts, under controlled condition of 27 ± 1 °C temperature,
75 to 95% relative humidity and photoperiod of 12 hours for
parasitic stages and scotophase for non-parasitic stages.
Material and Methods
The first step to set up a laboratory colony was to acquire
A. rotundatum engorged females. With this goal in mind, a
specimen of Bothropoides neuwiedi (Wagler, 1824) and another of
Rhinocerophis alternatus (Duméril, Bibron, Duméril, 1854), captured
in nature and taken to the center for the production of ophidian
poison antidote – FUNED (Ezequiel Dias Foundation) – Belo
Horizonte, Brazil, were placed in separate boxes to spontaneously
drop off engorged females. Six females were obtained, identified
according to Robinson (1926) and Aragão and Fonseca (1961),
individually accommodated in Petri dishes and incubated in a
BOD incubator at 27 °C temperature, 95% relative humidity
and scotophase. The laid eggs were placed in plastic syringes with
the anterior extremity clipped off and sealed with hydrophilic
cotton. The obtained larvae were used to produce ticks for this
experiment.
Seven Viperidae snakes were kept in captivity to carry out
experimental infestations and a boa snake (Boa constrictor) to produce
larvae and females. Sex and age were not taken into account, snakes
were identified (BÉRNILS, 2010), evaluated according to overall
heath and absence of ectoparasites, and weighted before being
submitted to experimentation. Three experimental infestations were
carried out: the first with 150 larvae per host, the second with five
females per host, and the third with 150 larvae and five females
per host. One R. alternatus and two Bothropoides jararaca (Wied,
1824) specimens were utilized in the first infestation. Conspecifics
adult ticks were utilized to infest one Bothrops moogeni (Hoge,
1966), one B. newiedi, and two B. jararaca specimens. The same
Viperidae host specimens of the second infestation were parasitized
at the third inoculation.
To carry out the experimental infestation with ticks, the restraint
of each host was necessary. This task was accomplished by making
the ophidian enter the first third of its body into a transparent
tube, which was then immobilized manually by holding both the
body of the animal and the tube (Figure 1). After this procedure,
the medium third of each serpent was wrapped with a flannel
strip of approximately 10 cm wide and 20 cm long and tied with
adhesive tape forming a cylinder. One of the ends of the cylinder
was closed with adhesive tape and the other kept open until the
ticks were introduced (Figure 2). It was then sealed to keep the
ticks from dispersing. The adult specimens, between 20 and 50 days
after molting from nymphs to females, were divided into groups
of five females on Petri dishes, 30 minutes before infestation. For
this purpose, the groups with best motility were chosen. Larvae
between 20 and 50 days after eclosion, accommodated in plastic
syringes, were inoculated by pressing the syringe embolus towards
the interior of the bag, leaving there as well the cotton placed in
the bottom of the syringe. After three days, all the hosts were
contained again to withdraw the flannel strips.
The hosts were kept in a room, accommodated in white or
transparent plastic boxes, with the superior edges taped up with
double-faced adhesive tape, numbered lids, potable water ad libitum
and with one rough stone to help in the physiological process of
skin change. Adult mice or little rats were made available every
15 to 20 days for feeding. The room with an area of 11.5 m2 and 3 m
high was equipped with an electric 12 watts heater-fan controlled
by an analogical thermostat with a 1.5 °C differential; a medium
size automatic-electric vaporizer, kept on 12 to 14 hours a day;
and an analogical timer set to turn on four fluorescent lights at
6:00 AM and turn off at 6:00 PM. Two digital thermo-hygrometers
placed at different points of the room, close to the hosts, also
monitored the temperature and relative humidity.

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176 Rodrigues, D.S. et al. Rev. Bras. Parasitol. Vet.
Observation took place daily as each serpent was transferred to
a handling box to inspect the container. The following bionomic
aspects were observed during the experiment: parasitic periods
and recovery rates of larvae, nymphs and female; premolt periods
and index of ecdysis of larvae and nymphs; weight of ovipositions
and engorged females; pre-oviposition, oviposition, and egg
incubation periods; larval hatchability; efficiency rates of female
ticks in converting their food reservoir to larvae and eggs.
The recovery rate was calculated by dividing the number of
specimens recovered by the total placed on the host. Although no
infestations with nymphs were carried out, the observed “larvae
to nymph parasitic periods” means the interval between larvae
infestation and nymph drop-off times. The differences between
the averages of larvae to nymph parasitic periods and the interval
containing larval parasitic and premolt larval periods were utilized
to estimate the nymph’s parasitic period.
The efficiency rates of female ticks in converting their food
reservoir to eggs (ERCE) and the efficiency rates of female ticks in
converting their food reservoir to larvae (ERCL) were calculated,
according to Szabó et al. (1995), by taking into account the weight
of egg mass (EW), weight of engorged females (FW) and larval
hatchability (LH), as follows (Equation 1):
EW × 100
ERCE = ERCL =
FW
Descriptive statistics was utilized in the evaluation of all
parameters observed (SAMPAIO, 2002).
EW × LH
FW
(1)
Results
The recovery rate of non-engorged larvae, engorged larvae,
non-engorged nymphs, engorged nymphs and engorged females
was of 20, 8, 6, 11 and 37% respectively. From the total of
recovered specimens after infestation with larvae, 45% did not do
or complete blood repast, 18% had the form of engorged larvae,
4% was non-engorged nymphs and 33% engorged nymphs.
Other observed parameters are shown in the Table 1 and the
Figure 3 shows the drop-off dynamics in the immature stages of
A. rotundatum, thus demonstrating the occurrence of the tick’s
two-host cycle. Seven nymphs at pre-molting period were detached
from a host which died during the experiment. Including it was
also possible to recover one non-engorged female from another
host. These facts indicates the possibility of A. rotundatum realize
one-host cycle either.
A 100% death rate of the Viperidae hosts was registered.
Clinical symptoms included anorexia, weakening, dysecdysis,
anemia, oral congestion, edema and caseous exudate, mucous
oral and nasal discharges, diarrhea, dermatitis and cutaneous
abscesses. Post-mortem alterations included diagnosis of stomachitis,
pneumonia, enteritis and septicaemia, the association of all them
being the most frequent cause. All animals presented stomachitis
symptoms. The B. constrictor utilized to maintain A. rotundatum
colony did not died possibly because its higher length and
weight than Viperidae snakes, as well the low number of ticks
inoculated.
Discussion
Values recorded in this study with regards to parasitic periods of
larvae, nymphs and females were higher than the values described
in literature thus far by Rohr (1909), Aragão (1912), Oba and
Shumaker (1983) and Amorim et al. (1996). This difference must
be related to the different environmental conditions during the
experiments. Oba and Shumaker (1983) observed full variation
of the cycle of 135 to 250 days under temperature of 27 °C and
80% RH for non-parasitic stages and environmental conditions for
parasitic stages. In this study, under controlled condition of 27 ± 1 °C
temperature and 75 to 95% relative humidity, it took 13 days for
females engorging period and a variation of 56 to 163 days for
the full biological cycle. The shortest periods observed in higher
room temperature confirmed the strong influence of temperature
on the periods of tick fixation in cold-blooded animals. Therefore,
recording the room temperature becomes crucial in the observation
and comparison of the fixation periods of this tick. Temperature
Figure 1. Ophidian’s manual immobilization by holding snake’s body
into a transparent tube.
Figure 2. A. rotundatum larvae infestation in a Viperidae snake
containing an alimentation chamber in its body. This apparatus were
constructed wrapping a flannel strip tied with adhesive tape.

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v. 19, n. 3, jul.-set. 2010 Amblyomma rotundatum (Koch, 1844) (Acari: Ixodidae) two-host life-cycle on Viperidae snakes 177
(1912) observed the occurrence of two-host cycle by employing
frogs to feed larvae, and noticing the same phenomenon also when
utilizing an ophidian and chelonian as hosts. From 1924 on, all
studies assessing the immature stages of this tick utilized frogs as
hosts and did not observe occurrence of ecdysis in the animal,
thereby characterizing it as three-host (OBA; SHUMAKER, 1983),
despite the observations Aragão (1912). Taking into account all of
these analyses, A. rotundatum tends to present three-host behavior
when fed on frogs, and when infesting ophidians and chelonians,
part of the population presents two-host behavior and the other
three-host. Even though no engorged female originated from
infestation with larvae was recuperated in this study, 7 nymphs
were collected molting on the host, plus a young female. This
detail suggests that, in a natural setting, a percentage of the
population must also undergo one-host life-cycle, since lower
temperatures are associated with higher ecdysis rates on the host
(BALASHOV, 1972).
In this experiment, the number of larvae and females inoculated
was determined by descriptions as done by Rohr (1909), Aragão
(1912) and Amorim et al. (1996), where the hosts frequently
died after infestation with this class of tick. Even then, despite
the small quantity of ticks inoculated per host, all of them
died. The symptoms observed are compatible with the available
information on the clinical aspects of ectoparasitism in reptiles.
Infestation by ectoparasites is commonly associated with loss of
appetite, depression, dysecdysis, dermatitis, ulcers and abscesses.
These conditions enough provoke immunosuppression and
occurrence of stomachitis, pneumonia, enteritis, septicaemia,
and animal death (MADER, 1996). However, one should not
discard the hypotheses of inoculation of toxins and pathogenic
microorganisms by the tick, since Hanson et al. (2007) describes
one case of tick paralysis caused by an A. rotundatum female in a
southern black racer, Coluber constrictor priapus, wild-caught in
the Florida Keys.
It is concluded that A. rotundatum presents a two-host life-cycle
when it obtain a blood meal in Viperidae Snakes, and more bioassays
are necessary to verify the hypothesis that this tick specie tends to
present a two-host life cycle when the reptiles are the hosts.
Acknowledgements
This study was sponsored by CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico), Brazil.
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Table 1. Bionomic parameters of Amblyomma rotundatum ticks
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Biological
parameter
Larval parasitic period (days)
Larval premolt period (days)
Nymph parasitic period (days)
Larval to nymph parasitic period (days)
Nymph premolt period (days)
Female engorging period (days)
Pre-oviposition period (days)
Oviposition period (days)
Egg incubation period (days)
Weight of engorged females (mg)
Weight of egg mass (mg)
Larval hatchability (%)
ERCE (%)a
ERCL (%)b
Total life cycle period (days)
aEfficiency rates of female ticks in converting their food reservoir to eggs.
b Efficiency rates of female ticks in converting their food reservoir to larvae.
Mean ± standard
deviation
15.58 ± 5.97
12.88 ± 1.96
10
38 ± 5.45
16.64 ± 0.86
12.60 ± 3.79
6.07 ± 1.94
18.36 ± 5.44
30.40 ± 7.57
1021 ± 255
488 ± 183
87 ± 6
47 ± 12
42 ± 12
105
Variation
width
9 to 31
10 to 17
-
25 to 57
15 to 19
6 to 21
4 to 10
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13 to 46
429 to 1338
143 to 762
75 to 95
28 to 59
25 to 54
56 to 163
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laboratory conditions (27 ± 1 °C, 85 ± 10% RH and scotophase).
0
5
10
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1 11 2131
Days
415161
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Engorged larvae
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