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Life cycle of Ornithodoros mimon (Acari: Argasidae)under laboratory conditions
Gabriel Alves Landulfo • Luisa Vianna Pevidor • Janio dos Santos Sampaio •
Hermes Ribeiro Luz • Valeria Castilho Onofrio • Joao Luiz Horacio Faccini •
Darci Moraes Barros-Battesti
Received: 30 December 2011 / Accepted: 17 April 2012 / Published online: 9 May 2012� Springer Science+Business Media B.V. 2012
Abstract Ornithodoros mimon Kohls et al. is an argasid tick, originally described from
larvae collected on bats from Bolivia and Uruguay. In Brazil the species is aggressive to
humans and animals. Nymphs and adults of O. mimon were collected from the roof of a
residence in Araraquara, Sao Paulo, Brazil, whose residents were bitten by ticks. Once in
the laboratory, they were fed on rabbits and maintained in biological oxygen demand
incubator at 27 ± 1 �C and 90 ± 10 % relative humidity. The females, after mating, laid
eggs that resulted in larvae that were identified by the original description and also by the
paratypes examination (RML 50271-50274) deposited at the United State National Tick
Collection, Georgia, GA, USA. The life cycle of this species was obtained through the
acquisition of two generations of ticks (F1 and F2) in the laboratory using rodents and
rabbits as hosts. The biological parameters of larva, nymph and adult stages of both
generations were recorded from infestations of the laboratory hosts. Larvae showed a
profile of feeding for days on the host, whereas the nymphs and adults fed only for few
minutes. First nymphal instar (N1) molted to second nymphal instar (N2) without blood
meal. The species life cycle was elucidated for the first time.
Keywords Ornithodoros mimon � Argasidae � Life cycle � Laboratory
G. A. Landulfo (&) � J. dos Santos Sampaio � H. R. Luz � J. L. H. FacciniDepartamento de Parasitologia Animal (DPA), Instituto de Veterinaria (IV),Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropedica, RJ 23890-000, Brazile-mail: [email protected]
L. V. Pevidor � V. C. OnofrioLaboratorio de Parasitologia, Instituto Butantan, Av. Vital Brasil 1500, Sao Paulo,SP 05503-900, Brazil
D. M. Barros-BattestiLaboratorio Especial de Colecoes Zoologicas, Instituto Butantan, Av. Vital Brasil 1500,Sao Paulo, SP 05503-900, Brazile-mail: [email protected]
123
Exp Appl Acarol (2012) 58:69–80DOI 10.1007/s10493-012-9567-4
Introduction
The life cycle of argasid ticks includes eggs, larval stage, two to nine nymphal instars and
the adult stage (Vial 2009). In general, the nymphs and adults feed rapidly on the host,
around 20–40 min, while the larvae remain fixed to the host for 7–10 days, though in rare
cases for only a few minutes (Barros-Battesti et al. 2012). Females of argasids lay multiple
batches of eggs (gonotrophic cycles), each batch normally after a blood meal and some-
times new mating (Hoogstraal 1985; Vial 2009).
According to the most recent list of ticks in the world, the Argasidae family includes
195 species (Guglielmone et al. 2010; Nava et al. 2010; Dantas-Torres et al. 2012),
distributed in five genera: Antricola Cooley & Kohls, Argas Latreille, NothoaspisKeirans & Clifford, Otobius Banks, and Ornithodoros Koch. The genus Ornithodoroshas the largest number of species, with 113 having been described, of which 52 occur in
the neotropical region and 15 of them occur in Brazil: O. rudis, O. talaje, O. capensis,
O. rostratus, O. brasiliensis, O. nattereri, O. hasei, O. jul, O. stageri, O. marinkellei,O. mimon, O. setosus, O. rondoniensis, O. fonsecai (Dantas-Torres et al. 2009;
Guglielmone et al. 2010) and O. cavernicolous (Dantas-Torres et al. 2012). Some of
these species, such as O. talaje, O. capensis, O. brasiliensis, and O. rostratus, are
of veterinary and medical importance because they are vectors of pathogenic agents or
infest humans (Brumpt 1915; Estrada-Pena and Jongejan 1999; Labruna and Venzal
2009; Martins et al. 2011).
The species O. mimon is a soft tick that parasitizes bats very aggressive to human. It
was originally described from larvae collected on two species of bats from Bolivia and
Uruguay (Kohls et al. 1969). Later the species was found infesting bats from Argentina
and Brazil (Venzal et al. 2004; Graciolli et al. 2008). In the latter country, there are
reports of parasitism of O. mimon in humans (Barros-Battesti et al. 2011). Although
larva and adult stage have been recently redescribed and described (Barros-Battesti et al.
2011), respectively, there are no published reports yet of the life cycle of this species.
Herein we have studied the life cycle of this tick species for two generations in the
laboratory, from specimens collected in a residence in the municipality of Araraquara,
Sao Paulo state, Brazil, where the residents were bitten by the ticks (Barros-Battesti
et al. 2011). Thus, the findings obtained on the life cycle of O. mimon will contribute
with information for better knowledge of the species, and provides subsidies for
researchers maintain colonies in laboratories, a fundamental step in research involving
ticks.
Materials and methods
Origin of the ticks
The colony of O. mimon was started from nymphs and adults collected from roof of a
residence in the municipality of Araraquara, Sao Paulo (218470S, 488100W). In the lab,
these ticks were fed on rabbits (New Zealand breed) without previous contact with ticks or
acaricide products. After feeding the ticks were kept in a biological oxygen demand (BOD)
incubator at 27 �C ± 1 and 90 ± 10 % relative humidity (RH). The females mated and
laid eggs, from which the larvae hatched. These were identified as O. mimon based on the
original description of Kohls et al. (1969), and later the larvae were compared with the
paratypes deposited in the tick collection of the University of Georgia, United States
70 Exp Appl Acarol (2012) 58:69–80
123
(USNTC, United States National Tick Collection) under the numbers RML 50271-50274
by one of us (VC Onofrio). The life cycle of O. mimon in laboratory conditions was
observed for two generations (larvae to larvae), using gerbils and rabbits as hosts.
Tick infestation
In a total, six rodents of the species Meriones unguiculatus (gerbil) and 10 rabbits of the
New Zealand breed were used as hosts. The animals were provided by the Central Bio-
terium of the Butantan Institute. The use of these animals was approved by the Butantan
Institute’s animal Ethics Committee (protocol 739/10).
Tick stages of O. mimon were infested with age around 20–40 days old (larvae with
30–40 days old) because preliminary tests have indicated this interval is good to the
success of engorgement. Specimens with 15 days old can feed on the hosts but the
recovery rate is around 20 % as well as specimens with age above than 40 days.
Gerbils were used as hosts for the larval stage and first nymphal instar (N1) of O.mimon. In order to minimize the number of host for larvae infestation, a total three rodents
were used. For the generation F1 a host was infested and for the generation F2 two hosts
were infested. Rabbits of the New Zealand breed were used as hosts for all the biological
stages of O. mimon. All instars of nymphs and adults from two generations were fed on this
host; however, only larvae of the first generation were fed on it.
During the feeding of the larvae of the first and second generation, the gerbils were
anesthetized by intramuscular injection of 0.07 ml of Ketamine Chlorhydrate (Vetan-
arcol�). After being anesthetized, the animals were placed in PVC tubes (4 cm in
diameter and 11 cm long), closed at the ends with wire screen, for infestation. These
tubes were necessary to limit their movement in the first 18 h, so as to maximize the
attachment of the larvae. Subsequently, the animals were removed from the tubes and
placed in the polypropylene boxes. Double-sided tape was affixed to the boxes’ edges to
prevent the ticks from escaping. The boxes were lined with wood shavings for collection
of the engorged larvae after natural detachment from the host. One gerbil received 100
larvae of the F1 generation and the other two each received 170 larvae of the F2
generation. The larvae selected for infestation had between 30 and 40 days old. The
boxes were inspected daily to find engorged larvae and the feeding period (from the
start of infestation to the natural detachment) was recorded. Those recovered were
individually placed in labeled flasks, kept in the BOD incubator at the same conditions
above mentioned, and daily examined to record the molting period (interval between
retrieval of the engorged larva and its molting to the next stage or instar). Three
different gerbils were used as hosts for the nymphs of the first instar (N1) of the first
generation. The animals were also anesthetized and prepared as described above. Each
host was infested with groups of 8–9 nymphs (with 20–40 days old), for a total of 26
N1 ticks. The feeding time (time between natural attachment and detachment from the
host) was recorded. After feeding, the N1 ticks were also individualized and placed
under the same conditions before mentioned to obtain the parameter of the non-parasitic
phase.
To feed the larvae on the rabbit, a containment chamber of cotton was fixed with non-
toxic glue on the host’s dorsum, which had been previously trichotomized. This chamber
was necessary to prevent the escape of ticks and also to observe of the biological
parameters. Additionally, a plastic collar was placed around the neck of the animal to
limit its movement and also to prevent removal of the chamber. One host was just
infested with 270 larvae of generation F1 (with 30–40 days old). The chamber was sealed
Exp Appl Acarol (2012) 58:69–80 71
123
with adhesive tape and the rabbit was placed in an individual cage at room temperature.
It was daily observed to collect engorged larvae that had detached naturally. The feeding
period was registered and the engorged larvae were kept under the same conditions
already described. Nymphs of each instar with 20–40 days old were fed on new
trichotomized rabbits. Each host was infested with groups of 5–15 nymphs. No chamber
was used in this case because the nymphs of all instars feed for few minutes. Again the
feeding time was recorded. The nymphs that failed to attach to the host within 60 min
were removed and placed in the BOD incubator. After engorgement, the nymphs of all
instars were individualized and placed under the same conditions above described. They
were daily examined to record the molting period. In order to observe molting without
blood meal, some nymphs of first and second instars were kept in starvation. The
emerged adult ticks (with 20–40 days old) were fed on new rabbits under the same
procedure and conditions as the nymphs. After feeding, couples were separated in Petri
dishes to record mating. After copulation, each female was isolated for observation and
recording of the number of fertile egg batches, in each gonotrophic cycle. The biological
parameters of the fertilized females, such as preoviposition and oviposition periods,
number of eggs per batch and egg incubation period, were daily observed. The females
were weighed before and after the blood meal and again after oviposition, as well as the
egg batch, with an electronic balance with precision of 0.0001 g. The egg production
index (EPI) and nutritional index (NI) were calculated according to Bennett (1974):
EPI = (weight of eggs/initial weight of engorged female) 9 100, NI = (weight of eggs/
initial weight of engorged female - female residual weight) 9 100.
Statistical analysis
The comparative biological parameters were statistically analyzed for normality, and the
data were submitted to the parametric t test. The nonparametric Mann–Whitney test was
used for data not normally distributed.
Results
Larvae
Of the 100 larvae of the F1 generation that infested the gerbil and of the 270 that infested
the rabbits, a total of 60 (60 %) and 102 (38 %) engorged larvae were recovered,
respectively. On the gerbil, the feeding period ranged from 5 to 8 days (5.4 ± 0.7 days), as
shown in Table 1. The highest percentage (73.3 %) of engorged larvae that detached from
the gerbil host was recorded on the fifth day after infestation. The larvae fed on the rabbit
detached between 5 and 9 days (5.2 ± 0.7 days) after infestation (Table 1). Similarly,
nearly 80 % of them dropped off on the fifth day. The average molting period for the larvae
fed on the gerbil was 6.7 ± 0.6 days, while the respective period for those fed on the rabbit
was 6.8 ± 0.5 days (Table 1). The majority of the larvae fed on both host species molted
to the first nymphal stage (N1). There were no significant differences (p [ 0.05) between
the biological parameters of the larvae of the F1 generation fed on the two host species
(Table 1).
The larvae of generation F2 were only fed on gerbils and their feeding period spent from
4 to 8 days (4.8 ± 0.9 days), with a recovery percentage of 45.6 % (155/340) (Table 1).
Most of the larvae detached on the fourth day (N = 72). The molt period ranged from 4 to
72 Exp Appl Acarol (2012) 58:69–80
123
Ta
ble
1B
iolo
gic
alp
aram
eter
so
fth
est
ages
of
Orn
ith
od
oro
sm
imo
no
fth
eg
ener
atio
ns
F1
and
F2
fed
inth
ela
bora
tory
ho
sts
Sta
ge
Bio
logic
alP
aram
eter
s1st
Gen
erat
ion
2nd
Gen
erat
ion
1st
Gen
erat
ion
2nd
Gen
erat
ion
Ho
stR
abb
itR
abbit
Rod
ent
Ro
den
t
Lar
vae
Fee
din
gp
erio
d(d
ays)
5.2
a±
0.7
(5–
9)
–5
.4aB
±0
.7(5
–8
)4
.8A
±0
.9(4
–8
)
Mo
ltin
gp
erio
d(d
ays)
6.8
a±
0.5
(5–
8)
–6
.7aB
±0
.6(5
–7
)6
.25
A±
0.8
(4–
7)
Per
cen
tag
eo
fm
olt
ing
(%)
93
.13
–9
3.3
39
5.5
N1
Fee
din
gti
me
(min
.)2
7.3
8aA
±1
0.4
(10
–6
0)
33
.8B
±8
.05
(15
–55
)2
7.1
a±
5.6
(20
–42
)–
Mo
ltin
gp
erio
d(d
ays)
11
.5a*A
±1
.6(9
–1
4)
12
.3B
±1
.4(1
0–
14
)1
3.4
b*
±3
.1(9
–1
9)
–
Per
cen
tag
eo
fm
olt
ing
(%)
10
01
00
10
0–
N2
Fee
din
gti
me
(min
.)2
6.1
A±
8.3
(13–
42
)3
1.4
B±
9.6
(15–
50
)–
–
Mo
ltin
gp
erio
d—
N3
(day
s)1
1.2
A±
1.5
(10
–14
)1
5.7
B±
2(1
2–
18
)–
–
Mo
ltin
gp
erio
d—
#(d
ays)
14
.3A
±3
.2(1
2–
18
)1
5.5
B±
2.2
(12
–18
)–
–
Mo
ltin
gp
erio
d—
$(d
ays)
11
.5A
±2
.1(1
0–
13
)1
5.6
B±
2(1
2–
18
)–
–
Per
cen
tag
eo
fm
olt
ing
(%)
10
01
00
––
N3
Fee
din
gti
me
(min
.)3
7.6
A±
7(2
7–4
8)
31
.6A
±8
.7(2
3–
44
)–
–
Mo
ltin
gp
erio
d—
#(d
ays)
15
±1
.7(1
4–
17
)–
––
Mo
ltin
gp
erio
d—
$(d
ays)
15
.33
A*
±3
(12
–19
)1
9.6
A*
±5
(16
–30
)–
–
Per
cen
tag
eo
fm
olt
ing
(%)
10
08
7.5
––
Mal
eF
eed
ing
tim
e(m
in.)
26
.2a
±9
(15
–40
)1
6.9
a±
4.1
(10
–23
)–
–
Fem
ale
Fee
din
gti
me
(min
.)2
8.4
a±
8.4
(17–
40
)2
7.6
b±
5.3
(21
–38
)–
–
The
val
ues
pre
sente
din
the
table
are
mea
n±
stan
dar
dd
evia
tio
n(r
ange
inp
aren
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es)
a*
val
ues
inth
esa
me
lin
efo
llo
wed
by
dif
fere
nt
low
erca
sele
tter
s(c
om
par
iso
no
fro
den
tan
dra
bb
it)
are
sig
nifi
can
tly
dif
fere
nt
by
Ma
nn
-Wh
itn
eyte
st:
p\0
.05
A*
val
ues
inth
esa
me
lin
efo
llo
wed
by
dif
fere
nt
cap
ital
lett
ers
(co
mpar
iso
nb
etw
een
F1
and
F2
inro
den
ts)
are
sign
ifica
ntl
yd
iffe
ren
tb
yth
eM
an
n-W
hit
ney
test
:p\
0.0
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val
ues
inth
esa
me
lin
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llo
wed
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fere
nt
low
erca
sele
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s(c
om
par
iso
no
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den
tan
dra
bb
it)
are
sig
nifi
can
tly
dif
fere
nt
by
tte
st:
p\
0.0
5A
val
ues
inth
esa
me
lin
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llo
wed
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dif
fere
nt
cap
ital
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ers
are
sign
ifica
ntl
yd
iffe
ren
tb
yt
test
:p\
0.0
5a
val
ues
inth
esa
me
colu
mn
foll
ow
edb
yd
iffe
ren
tlo
wer
case
lett
ers
un
der
lin
ed(c
om
par
iso
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etw
een
mal
ean
dfe
mal
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gn
ifica
ntl
yd
iffe
ren
tb
yt
test
:p\
0.0
5
Exp Appl Acarol (2012) 58:69–80 73
123
7 days (6.25 ± 0.8 days), and the highest number of molts occurred on the seventh day
after feeding. Of the 155 engorged larvae, 148 (95.5 %) molted to first nymphal instar
(N1). Comparing the two generations, the biological parameters of generation F1 fed on
the gerbils were numerically higher than those of the larvae of F2 (p \ 0.05) (Table 1).
Nymphs (N1, N2 and N3)
The N1 ticks of generation F1 were fed on both host species. Of the 56 nymphs emerged
from larvae fed on gerbils, 26 N1 were fed on a new gerbil and the feeding times ranging
from 20 to 42 min (27.1 ± 5.6 min) (Table 1). The remaining of the N1 (N = 30) were
kept without meal. Of the 95 N1 of generation F1 that emerged from larvae fed on rabbits,
fifty of them were selected randomly to infest one rabbit. Again the remaining of the N1
(N = 45) were kept in starvation. The engorged nymphs took between 2 and 23 min to fix
on the host and the feeding time ranging 10–60 min (27.38 ± 10.4 min) (Table 1). All of
N1 fed on both host species molted and became second-instar nymphs (N2). It was
observed that 4 N1 molted to N2 between 14 and 28 days, without blood meal. Although
the average feeding time of the N1 of generation F1 fed on gerbils was shorter than those
fed on rabbits, the difference was not statistically significant (p [ 0.05). However, there
was a significant difference in the molting period (Mann–Whitney: p \ 0.05), to the
nymphs fed on gerbils taking longer than those fed on rabbits. Considering that rabbits
were better hosts for nymphs than rodents, nymphs of the second and third (N3) instar of
generation F1, as well as all of generation F2, were only fed on rabbits. Of the total of 50
N2 emerged from N1 fed on rabbits, 17 of them became engorged, with the feeding time
varying between 13 and 42 min (26.1 ± 8.3 min) (Table 1). The remaining N2 (N = 33)
were kept in starvation. The engorged N2 took 10–18 days to molt, resulting in 3 males, 2
females and 12 nymphs of the third instar (N3). The sex ratio of the adults emerging from
N2 nymphs was 1.5#:1$. All N3 were fed and their biological parameters are shown in
Table 1. All of them molted in an interval from 11 to 19 days (15.25 ± 2.6 days), from
which 12 adults emerged, 9 females (75 %) and 3 males (25 %), for a sex ratio of 3$:1#.
Of the 148 N1 obtained from generation F2, 54 of them were fed on the rabbit hosts. The
N1 took between 4 and 60 min to fix on the rabbit and the feeding time ranged from 15 to
55 min (33.8 ± 8.05 min) (Table 1). The remaining of N1 (94) were kept without meal.
After the blood meal, the engorged N1 took between 10 and 14 days (12.3 ± 1.4 days) to
molt into second nymphs instar (N2). Of the 54 N2 obtained, 34 nymphs were fed and their
feeding time varied from 15 to 50 min (31.4 ± 9.6 min) (Table 1). Again the remaining
N1 (N = 20) were kept without meal blood. The molting period of the engorged N2
nymphs varied from 12 to 18 days. From these, 14 molted into males, 12 into females and
8 into N3. The sex ratio of the adults that emerged from the N2 was thus 1.16:1 (14#:12$).
The 8 N3 of generation F2 were fed and their biological parameters are shown in Table 1.
Of the 8 N3 fed, just 7 molted and became females. No molting was observed without a
blood meal in the generation F2. There was release of coxal fluid during the feeding
process by all the nymphal instars of O. mimon of both generations. There were significant
differences in the biological parameters of the N1 between generation F1 and F2,
(p \ 0.05), with the parameters for those of the first generation being numerically smaller.
The N2 of generation F1 also had smaller biological parameters than those of generation
F2 (p \ 0.05), as shown in Table 1. The biological parameters of the N3 of both gener-
ations were similar (p [ 0.05).
74 Exp Appl Acarol (2012) 58:69–80
123
Adults
The biological parameters of the adult stage of O. mimon were obtained from infestation
by 8 females and 6 males of generation F1 and 14 couples of generation F2. The
females of the first generation took 5–20 min to infest the rabbits and the feeding time
varied from 17 to 40 min (28.4 ± 8.4 min). The corresponding average time for the
males was 26.2 ± 9.0 min (Table 1). The parameter of the parasitic phase (feeding
time) of the first-generation adults did not statistically differ (p [ 0.05). In the second
generation, there was a significant difference between the sexes (p \ 0.05) for feeding
time, being longer for the females than the males (Table 1). Just as for the nymphs, the
adult ticks released coxal fluid while feeding. The weight of the engorged females of
both generations was 3–4 times greater than before feeding (Table 2). After feeding, the
adult ticks were placed in Petri dishes for mating. Some males of the first generation
were placed to copulate with more than one female, because there were fewer males
than females. The mating occurs very quickly. The male moves over the female until
reaching the ventral face. Upon reaching the female’s venter, the male deposits the
spermatophore in her genital opening and the content is absorbed in about 5 min,
fertilizing her. Mating can also occur on the host, as two males were observed mating
with females while the latter were feeding. The fertilized females of generation F1 took
an average of 12.2 ± 2.4 days to start laying the first eggs (preoviposition period) and
17.2 ± 5.0 days thereafter to complete laying all their eggs (oviposition period). These
observations refer to the females’ first gonotrophic cycle (Table 2). The oviposition of
some females was not continuous, because it was frequently interrupted for one to
3 days. In the second gonotrophic cycle, only six females were analyzed, because two
females died after engorgement. The average preoviposition period in the second
gonotrophic cycle was 10.3 ± 6.4 days and the oviposition period was 16.8 ± 7.3 days
(Table 2). The quantity of eggs varied between the two cycles, with a greater number
laid during the second one, but the difference was not significant (p [ 0.05). The EPI
and NI in the first gonotrophic cycle were smaller than those in the second cycle, but
again the differences were not statistically significant (p [ 0.05). The fertile eggs had a
mean incubation period of 13.4 ± 2.6 days in the first cycle and 12.0 ± 1.4 days in the
second cycle. The average hatching rate of larvae in the two cycles was above 70 %. In
the second generation, the fertilized females had in the first gonotrophic cycle an
average preoviposition period of 19.6 ± 8.4 days and oviposition period of
13.3 ± 6.2 days. In the second gonotrophic cycle, the mean preoviposition period
(12.5 ± 4.8 days) was significantly shorter (p \ 0.05) than in the first cycle, while the
mean oviposition period was similar, as shown in Table 2. The females laid an average
of 89.4 ± 42.6 eggs in the first cycle and 103.6 ± 35.13 eggs in the second cycle. The
average egg incubation periods in the first and second gonotrophic cycles were
11.9 ± 1.6 days and 13.09 ± 1.4 days, respectively. The average hatching rate of larvae
was above 80 %. The EPI and NI recorded for generation F2 were significantly higher
(p \ 0.05) in the second gonotrophic cycle (Table 2). Just as in the first generation,
three females of the second generation died after the second blood meal, resulting in the
observation of 11 females in the second cycle.
The life cycle from the larval stage of generation F1 to hatching of larvae of generation
F2 took 167 days with adults emerging from N2, e 175 days with females emerging from
N3. The life cycle (larva–larva) of generation F2 was completed in approximately
146 days.
Exp Appl Acarol (2012) 58:69–80 75
123
Ta
ble
2B
iolo
gic
alp
aram
eter
so
fth
en
on
-par
asit
icp
has
eo
fth
ead
ult
stag
eo
fO
rnit
hod
oru
sm
imo
n
Gen
erat
ion
F1
F2
Bio
log
ical
par
amet
ers—
go
no
tro
ph
iccy
cles
1�
2�
1�
2�
No
.o
ffe
mal
es8
61
41
1
Wei
gh
tb
efo
reth
eb
loo
dm
eal
(mg
)8
a±
3.5
(3–
14)
7.5
a±
2(4
.7–
10
.6)
4.2
a±
1.2
(3–
6.2
)5
.8b
±1
.2(3
.8–
7.8
)
Wei
gh
taf
ter
the
blo
od
mea
l(m
g)
22
.8a
±6
.5(1
7–
37
)2
1.6
a±
3.3
(16
–25
)1
9a
±5
.8(9
.7–
28
)1
8.1
a±
4.5
(12
.8–2
5.8
)
Pre
ovip
osi
tion
per
iod
(day
s)1
2.2
a±
2.4
(8–
15)
10
.3a
±6
.4(3
–2
2)
19
.6b
±8
.4(1
1–
39
)1
2.5
a±
4.8
(8–
25)
Ov
ipo
siti
on
per
iod
(day
s)1
7.2
a±
5(1
0–
22
)1
6.8
a±
7.3
(11
–30
)1
3.3
a±
6.2
(3–
30)
13
.6a
±3
(9–
19
)
Nu
mb
ero
feg
gs
13
7a
±5
0(8
4–
22
6)
14
1a
±2
2.5
(11
4–1
75
)8
9.4
a±
43
(30
–18
5)
10
3.6
a±
35
(59
–16
8)
Wei
gh
to
fn
um
ber
of
egg
s(m
g)
6.5
a±
1.7
(4.3
–8.7
)7
.3a
±1
.4(5
.2–
9.1
)4
.6a
±2
(1.6
–8
.9)
6.1
a±
2(3
.6–
8.6
)
Per
cen
tag
eo
fh
atch
ing
(%)
77
a±
11
.4(6
0–
92
)7
6a
±1
4(6
0.4
–8
8.6
)8
3.4
a±
10
.2(6
2.5
–1
00
)8
1.4
a±
10
.5(5
9.6
–1
00
)
EP
I(%
)2
9.6
a±
8.4
(21
–42
)3
3.6
a±
2(3
1.5
–3
6.2
)2
3.8
a±
7.4
(12
.8–3
4)
34
.4b
±9
.8(1
8.6
–5
0.8
)
NI
(%)
47
.8a
±1
2.6
(36
–7
4)
53
.2a
±2
(50
–55
)3
6.9
a±
15
.4(1
6–
76
)5
3.2
b±
15
.6(2
6.3
–8
5)
Incu
bat
ion
per
iod
of
egg
s(d
ays)
13
.4a
±2
.6(9
–1
8)
12
a±
1.4
(10
–14
)1
1.9
a±
1.6
(9–
14)
13
.09
a±
1.4
(10
–15
)
Val
ues
inth
eta
ble
sar
em
ean
±st
andar
dd
evia
tio
n(r
ange
inp
aren
thes
es)
av
alu
esin
the
sam
eli
ne
foll
ow
edb
yd
iffe
ren
tle
tter
sar
esi
gn
ifica
ntl
yd
iffe
ren
tb
yt
test
:p\
0.0
5
76 Exp Appl Acarol (2012) 58:69–80
123
Discussion
The biological cycle of O. mimon under laboratory conditions was observed for two
generations (F1 and F2) on laboratory animals, indicating that the species can be suc-
cessfully reared in the laboratory. We have established the best age of the specimens of O.mimon to infest hosts between 20 and 40 days, based on preliminary tests using rabbit as
host. It was observed that larvae with 15 or less days old can engorge as well as specimens
with age above than 40 days old, but, the recovery rate was less than 20 %. However there
are specific particularities for each species. Larvae of O. talaje with 5-10 days old were fed
on rats and on birds, but only 12.5 % of larvae were recovered from rat whereas 69 % were
recovered from birds (Schumaker and Barros 1995). Larvae of Ornithodoros amblusChamberlin, 1920 with the ages of 3–6 days old were fed on pigeon and the recovery rate
was 50 % (Khalil and Hoogstraal 1981).
In the first generation, the larvae fed on the gerbil had an engorgement success rate
higher than those fed on the rabbits (60–37.77 %). In the second generation, the
engorgement success rate of the larvae fed on gerbils was lower than in the first generation,
at 45 %. However, the success in recovering engorged larvae of both generations from
gerbils was greater than reported by Schumaker and Barros (1995) for the species O. talaje.
According to Hoogstraal (1985) and Sonenshine (1991), species of Ornithodoros that
parasitize bats and birds remain fixed to the host for several days, thus having feeding
habits similar to those of the Ixodidae. Sonenshine and Anastos (1960), Hoogstraal et al.
(1970), Khalil and Hoogstraal (1981) and Schumaker and Barros (1995) studied the
biology of ticks that infest bats and birds, respectively, Ornithodoros kelleyi Cooley and
Kohls 1941, Ornithodoros muesebecki Hoosgtraal 1969, O. amblus and O. talaje. All these
workers also observed that the larvae of these species feed slowly (many days) on the host.
The feeding profile of the larval stage of O. mimon, which is a bat parasite, observed in this
study was similar to these reports in the literature, because the species remained fixed for
days to both hosts, gerbils and rabbits. Besides this, the blood meal was essential for larval
ecdysis, since no larvae molted to the nymphal stage without feeding. This same pattern
has not been observed in some species of the Ornithodoros, such as Ornithodoros rostratusAragao 1911 and Ornithodoros turicata (Duges 1876), in which the larvae feeding for few
minutes (Clifford et al. 1964; Beck et al. 1986), and Ornithodoros savignyi (Audouin 1826)
(Khan and Srivastava 1988), Ornithodoros moubata (Murray, 1877) (Loomis, 1961) and
O. brasiliensis (Barros-Battesti et al. 2012), in which the larvae do not feed to molt to the
first nymphal instar.
The nymphal stage of O. mimon includes three instars (N1, N2 and N3). This finding
differs from those for other species of the Ornithodoros genus, which have between 4
and 6 instars such as O. kelleyi (Sonenshine and Anastos 1960), O. amblus (Khalil and
Hoogstraal 1981), O. muesebecki (Hoosgtraal et al. 1970), O. erraticus (Shoura 1987),
O. turricata (Beck et al. 1986), O. savignyi (Khan and Srivastava 1988) and O. talaje(Schumaker and Barros 1995). According to Hoogstraal (1985), nymphs can feed more
than once before the ecdysis process, because the first blood meal may have been
interrupted or insufficient for molting. This behavior was not observed in the present
study, because the nymphs of both generations molted with only one blood meal. Besides
this, the majority ([80 %) of the nymphs fed on both host species completed molting,
indicating that both rabbits and gerbils are adequate hosts for O. mimon nymphs.
Although there is information in the literature on species of the Ornithodoros genus that
can molt from N1 to N2 without feeding (Faccini and Barros-Battesti 2006), this is the
first observation of this fact for O. mimon, despite being observed for only a few
Exp Appl Acarol (2012) 58:69–80 77
123
specimens of generation F1. This phenomenon was observed by Hoogstraal et al. (1970)
for O. muesebecki, because the N1 of this species can molt to N2 (in two to 4 days)
without a blood meal.
The sexual maturity of O. mimon in both generations occurred after N2, demon-
strating a specific difference when compared to other Ornithodoros species, which reach
maturity only after the third or fourth nymphal instar (Hoogstraal et al. 1970; Khalil
and Hoogstraal 1981; Beck et al. 1986; Khan and Srivastava 1988; Schumaker and
Barros 1995). Besides this, the O. mimon males were more prevalent emerging from
N2, while most of the females emerged from N3. In fact, for species of the Orni-thodoros genus, the males are normally the first to reach sexual maturity (Schumaker
and Barros 1995).
In the present study the adult ticks had different feeding periods between the sexes, with
males feeding in less time than females, but both fed rapidly. This is characteristic of soft
ticks in both, nymphal and adult stages (Vial 2009). The mating observed for O. mimonwas similar to that described by Brumpt (1915) and Beck et al. (1986) for the species O.rostratus and O. turicata, respectively.
We have observed mating of O. mimon on the host, unlike reported elsewhere in the
literature (Hoogstraal 1985; Faccini and Barros-Batestti 2006). We also observed unfed
males copulating with engorged females in the Petri dishes, indicating that a blood meal
is not required for males to mate. This observation corroborates the findings of Shoura
(1987) for Ornithodoros erraticus (Lucas 1849), that has the same behavior. Irrespective
of whether mating occurred on the host or in the Petri dish, the fertilized females laid
fertile eggs in the two gonotrophic cycles. Although the preoviposition period has been
higher in the first gonotrophic cycle than the second gonotrophic cycle, the number of
eggs was smaller. This agrees with the findings of Khalil and Hoogstraal (1981) for O.amblus, of Shoura (1987) for O. erraticus and of Schumaker and Barros (1995) for O.talaje. These authors reported that this happens because the female of the first gono-
trophic cycle is still not fully mature, so a portion of the blood ingested in the first
feeding is used for development rather than egg production. The observation of inter-
ruption in the period of oviposition of some O. mimon females corroborates Khalil and
Hoogstraal (1981) for O. amblus, in which there also was an interval in the oviposition
period. The incubation period of eggs of O. mimon was similar to that noted by Schu-
maker and Barros (1995) for O. talaje, but shorter than that observed by Hoogstraal et al.
(1970) for O. musebecki. The high hatching rate ([70 %) demonstrates that the host
species utilized are suitable for this tick species and can be used to establish laboratory
colonies.
The EPI and NI levels obtained in the present study are similar to those reported by
Santos et al. (2011) for Argas miniatus Koch, 1844, although here we observed only a few
females of the two generations. The importance of these indices is that they indicate that
egg production efficiency is greater in the second gonotrophic cycle.
The life cycle was completed in a short period (146–175 days) to enable obtaining two
to three generations a year under laboratory conditions. Therefore, it can be concluded that
the soft tick O. mimon is easy to handle and colonize for use in other areas of research, such
as the transmission of pathogenic agents and tests of acaricides, among others.
Acknowledgments To Edson Maria Torres from City Hall of Araraquara who kindly helped us to collectthe ticks from the roof. This study was supported by Fundacao de Amparo a Pesquisa do Estado de SaoPaulo-FAPESP (grant 2007/57749-2 to DMBB) and by Conselho Nacional de Desenvolvimento Cientıfico eTecnologico-CNPq (grant 478950/2004-7 to DMBB).
78 Exp Appl Acarol (2012) 58:69–80
123
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