Life cycle of tortoise tick Hyalomma aegyptiumunder laboratory conditions

Pavel Siroky • Jan Erhart • Klara J. Petrzelkova • Martin Kamler

Received: 7 February 2011 / Accepted: 5 March 2011 / Published online: 24 March 2011� Springer Science+Business Media B.V. 2011

Abstract The tortoise tick Hyalomma aegyptium has a typical three-host life-cycle.

Whereas its larvae and nymphs are less host-specific feeding on a variety of tetrapods,

tortoises of the genus Testudo are principal hosts of adults. Ticks retained this trait also in

our study under laboratory conditions, while adults were reluctant to feed on mammalian

hosts. Combination of feeding larvae and nymphs on guinea pigs and feeding of adults on

Testudo marginata tortoises provided the best results. Feeding period of females was on

average 25 days (range 17–44), whereas males remain after female engorgement on tor-

toise host. Female pre-oviposition period was 14 days (3–31), followed by 24 days of

oviposition (18–29). Pre-eclosion and eclosion, both together, takes 31 days (21–43).

Larvae fed 5 days (3–9), then molted to nymphs after 17 days (12–23). Feeding period of

nymphs lasted 7 days (5–10), engorged nymphs molted to adults after 24 days (19–26).

Sex ratio of laboratory hatched H. aegyptium was nearly equal (1:1.09). The average

P. Siroky (&)Department of Biology and Wildlife Diseases, Faculty of Veterinary Hygiene and Ecology, Universityof Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republice-mail: sirokyp@vfu.cz

J. ErhartInstitute of Parasitology, Biology Center, Academy of Sciences of the Czech Republic, Branisovska31, 370 05 Ceske Budejovice, Czech Republic

K. J. PetrzelkovaInstitute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Kvetna 8, 603 65 Brno,Czech Republic

K. J. PetrzelkovaLiberec Zoo, Masarykova 1347/31, 460 01 Liberec, Czech Republic

M. KamlerDepartment of Parasitology, Faculty of Veterinary Medicine, University of Veterinary andPharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic

Present Address:M. KamlerBee Research Institute Dol, Dol 94, 252 66 Libcice nad Vltavou, Czech Republic

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Exp Appl Acarol (2011) 54:277–284DOI 10.1007/s10493-011-9442-8

weight of engorged female was 0.95 (0.72–1.12) g. The average number of laid eggs was

6,900 (6,524–7,532) per female, it was significantly correlated with weight of engorged

female. Only 2.8% of engorged larvae and 1.8% of engorged nymphs remained un-molted

and died. Despite the use of natural host species, feeding success of females reached only

45%. The whole life-cycle was completed within 147 days (98–215).

Keywords Hyalomma aegyptium � Testudo � Life-cycle � Laboratory rearing

Introduction

Availability of pathogen-free ticks in sufficient numbers is inevitable condition for any

experimental study with ticks and tick-borne agents. Laboratory rearing methods were

developed for many tick species in the past, particularly for model species used in studies

of tick biology and epidemiology of tick-borne diseases (i.e. Chen et al. 2009; Ghosh and

Azhahianambi 2007; Krober and Guerin 2007; Liu et al. 2005; Rechav and Fielden 1997;

Simo et al. 2004; Slovak et al. 2002; Srivastava and Varma 1964; Yeruham et al. 2000). On

the other hand, little attention was given to tick species being assumed to have lower

economic importance.

Hyalomma aegyptium (Linnaeus, 1758) distributed in Mediterranean area from Atlantic

coastland of Morocco through Northern Africa, Balkan countries, Middle East, and Cau-

casus region to Central Asia, Afghanistan, and Pakistan (Kolonin 1983), belongs to such

understudied species. H. aegyptium is dominant species among ticks parasitizing tortoises

in western Palaearct (Apanaskevich 2003; Robbins et al. 1998; Siroky et al. 2006;

Sweatman 1968), possessing typical three-host life cycle. Larvae and nymphs are less host-

specific infesting tortoises, lizards, birds, small mammals and even men (Apanaskevich

2004; Kolonin 2004; Vatansever et al. 2008). Nevertheless, tortoises of the genus Testudoare principal hosts of adult ticks. Other hosts (e.g. hares and hedgehogs) are for adult ticks

reported rarely (Hoogstraal 1956; Hoogstraal and Kaiser 1960).

Hyalomma aegyptium is known as a vector and definitive host of tortoise-specific

apicomplexan blood parasite Hemolivia mauritanica (Sergent et Sergent, 1904). In a frame

of our studies on vectorial capability of H. aegyptium we have got high requirement of

pathogen-free ticks (Siroky et al. 2004, 2007, 2010). The laboratory rearing of thousands of

H. aegyptium ticks provided controlled conditions to collect information about basic traits

of its life-cycle, feeding, and reproduction. These data are summarized in the presented

paper.

Materials and methods

Origin and keeping of ticks

Tick laboratory breeding colony was established by five consecutively imported engorged

females of H. aegyptium. Two females were collected in July 2001 from tortoises Testudomarginata Schoepff, 1792 at locality Volos, Eastern Greece (39�2002700N, 22�5404900E).

Third female was collected in June 2004 from hedgehog Erinaceus concolor Martin, 1838

near Areopoli, South of Peloponnesus peninsula, Greece (36�4001000N, 22�2205800E). Last

two engorged females were collected in April 2005 from tortoises Testudo graeca Lin-

naeus, 1758 at locality Qualat Samaan, NW Syria (36�1905800N, 36�5004900E). The ticks

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were kept in cylindrical glass tubes (23 mm in diameter, 70 mm height) filled with strip of

filter paper, closed with cotton wool pads, and stored in shaded box under 22–25�C, and

relative humidity (RH) 60–85%.

Host species

Unsexed outbred guinea pigs having weight 300–500 g and originating from a breeding

facility of Institute of Parasitology Academy of Sciences of the Czech Republic, Ceske

Budejovice were used as host species for feeding of larvae and nymphs. Adult male and

5 year old captive bred juvenile tortoises Testudo marginata originating from private

breeding stock of the first author were used as natural host species for adult ticks. We also

tried to feed immature tick stages on tortoises and vice versa adult H. aegyptium on guinea

pigs, but without significant success. Adult ticks were unwilling to feed on guinea pigs. On

the other hand, it was difficult to safely manage and control feeding of small immature

stages of ticks on tortoise body.

Technique of ticks feeding

One plastic feeding chamber was glued to clipped back of each guinea pig. Afterwards,

ticks were introduced into this chamber, which was immediately closed with dense nylon

cloth. Guinea pigs were kept in open enclosure 110 9 85 9 38 cm (length 9 width 9 -

height) under temperature 22–24�C, and RH 50–70%, fluctuating slightly according to

season, and controlled daily.

Adult ticks (5 males ? 5 females) were put together with host tortoise into twill sack,

which was totally closed for 48 h. Then, the sack was opened and position and attachment

of ticks was controlled. Host tortoises were kept in closed vivarium 100 9 50 9 45 cm

(l 9 w 9 h) under 18–28�C and RH 35–55%. Position and feeding state of ticks were

controlled at least once a day.

Collection of data on life cycle

We recorded duration of feeding periods of H. aegyptium larvae, nymphs, and females,

defined as interval between insertion of ticks into feeding chamber (for premature stages)

or into twill sack with tortoise (for tick females). Lasting of molting period represents time

between spontaneous detachments of engorged larvae and nymphs, respectively, and their

molting to forthcoming life stage. Weight of ten selected engorged females was recorded

immediately after detachment from host on laboratory scales RADWAG WAS 220/C/2

(Radwag, Radom, Poland) and rounded with accuracy 10 mg. Period between their

detachment and appearance of first eggs represents pre-oviposition period. Lasting of

oviposition and number of eggs laid was recorded for the same ten females. The eggs were

removed daily from these females. Eggs from the other females were removed in 3–5 days’

intervals to avoid their repeated disturbing. Behavior of ticks and their movement on hosts

was also registered daily.

Data analysis

To reveal the relationship among duration of feeding periods, weight of engorged females,

lasting of oviposition and number of eggs laid we performed several Spearman’s

Exp Appl Acarol (2011) 54:277–284 279

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correlations. Bonferoni corrections (with added mean correlation between variables as a

parameter) were used for P values (Sankoh et al. 1997). The analyses were performed

using the STATISTICA software (version 8.0, StatSoft, 2008).

Results

The duration of H. aegyptium life cycle under laboratory conditions divided into particular

life stages is given in Table 1.

Feeding of adult ticks

Feeding females (N = 30) remained usually after attachment on the same place over all

feeding period. They changed feeding place exceptionally, usually when firstly attached to

carapace. Females preferred for feeding the inguinal area and places around hind limbs of

tortoises (66.7%). Four females (13.3%) engorged in area around forelimbs, other one

(3.3%) on the neck, five females (16.7%) engorged successfully on carapace in seams

between carapace scutes. Six females (20%) originally attached to carapace seams changed

place to inguinal and tight area, and then engorged. Comparing to females, males were

observed to change places on tortoise body more frequently. Feeding period of females was

24.87 ± 5.18 (N = 30; range 17–44) days. Engorged females weighted immediately after

spontaneous detachment 0.95 ± 0.14 (N = 10; range 0.72–1.12) g. Feeding success of

females reached 45% (N = 75), forty females (53.3%) did not attached or died during

feeding, and five females (6.7%) were rubbed by tortoise movement. Males remained

attached on tortoise host after females’ detachment until their removal or death.

The pre-oviposition and oviposition

Interval between detaching of engorged female and the appearance of the first eggs was

14.3 ± 5.94 (N = 30; range 3–31) days. Oviposition period of selected females lasted

24 ± 3.21 (N = 10; range 18–29) days. Number of eggs laid per one female was

6,900 ± 294 (N = 10; range 6,524–7,532).

Table 1 Duration of respectivelife stages in life cycle of tickHyalomma aegyptium

Stage Duration (days)

Minimum Maximum Mean

Engorgement of females 17 44 24.9

Pre-oviposition 3 31 14.3

Oviposition 18 29 24

Pre-eclosion and eclosion 21 43 31

Feeding of larvae 3 9 5.1

Pre-molting and molting to nymphs 12 23 16.6

Feeding of nymphs 5 10 6.9

Pre-molting and molting to adults 19 26 23.8

Total 98 215 *147

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There were no significant relationships among feeding periods, weight of engorged

females, lasting of oviposition and number of eggs laid with exception of correlation

between weight of engorged females and number of eggs (rS = 0.83; P \ 0.01).

The pre-eclosion and eclosion

First larvae appeared after 21–35 days. Later, they formed clusters on the wall of glass

tubes or on cotton wool pads. Forming of this clusters were connected with finishing of

eclosion. Clusters were formed 31 ± 5.62 (N = 20; range 21–43) days after oviposition.

Feeding period of larvae and nymphs

Pre-feeding period of larvae and nymphs was not tested. Larvae of H. aegyptium fed

5.13 ± 1.45 days (N = 2,004; range 3–9) till complete engorgement on guinea pigs.

Nymphs need 6.9 ± 1.2 days (N = 1,600; range 5–10) to complete feeding on guinea

pigs. Engorged larvae and nymphs, respectively, were seen freely moving in feeding

chamber, including nylon cloth. Since number of specimens of both life-stages—larvae and

nymphs, given to feeding chamber was only roughly estimated, we have no data on

percentage of feeding success.

The pre-molting period, molting, sex ratio, and longevity

Engorged larvae molted to nymphs 16.6 ± 2.95 (N = 500; range 12–23) days after their

detachment from host. Pre-molting period of engorged nymphs was 23.8 ± 1.92

(N = 500; range 19–26) days. Fifty-six (2.8%) of all engorged larvae and 28 of all

engorged nymphs (1.8%) remained unmolted and died. From 737 engorged nymphs 347

and 378 molted to males and to females, respectively (12 died unmolted; sex ratio 1:1.09).

Under presented laboratory conditions, unfed H. aegyptium larvae and nymphs both sur-

vived for approximately 4–5 months. Longevity of adults is fairly over 1 year (see also

Siroky et al. 2010).

Discussion

We have discovered that feeding period of adult H. aegyptium on its natural host species is

rather variable under laboratory conditions. We suppose that its duration depends on

attachment site on tortoise body, respectively on availability of capillary blood at this

place. Generally, observed feeding period of females is longer than is usual in other ixodid

tick species (e.g. Chen et al. 2009; Hadani et al. 1969; Liu et al. 2005; Slovak et al. 2002;

Yeruham et al. 2000). Srivastava and Varma (1964) described similar phenomenon of

prolonged feeding in unfertilized females of Rhipicephalus sanguineus. We have kept

males and females separately from their hatching, which is why it could be the case. We

have never tested to feed separately females only. On the other hand, we have observed

such a prolonged feeding particularly in females attached to carapace. Lower density of

blood vessels expected at that places could explain these observations.

Hadani et al. (1969) successfully, despite with some reluctance, fed adult H. aegyptiumon rabbits. Comparing to that study we have bad experience with feeding of adult H.aegyptium on laboratory mammals. We have tried to use mice, guinea pigs, and rabbits, as

Exp Appl Acarol (2011) 54:277–284 281

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hosts, but we did not achieve any success. Ticks remained usually unattached in feeding

chambers, or if exceptionally attached; they displayed no interest to feed.

Immature stages of H. aegyptium fed readily on guinea pigs; hence, we did not test other

host species. Feeding period of larvae and nymphs fit well that feeding periods as presented

previously for H. aegyptium by Hadani et al. (1969). Feeding periods recorded in this study

for pre-adult stages of H. aegyptium are fairly comparable to those periods observed in

many other ixodid tick species, including members of the genus Hyalomma (i.e. Chen et al.

2009; Hadani et al. 1969; Magano et al. 2000; Rechav and Fielden 1997; Slovak et al.

2002; Srivastava and Varma 1964).

Observed duration of both, pre-oviposition as well as oviposition in this study fall into

intervals reported for these stages also in other studied ixodid ticks. Hadani et al. (1969)

observed pre-oviposition interval overlapping with that obtained in our study. Species

having more pronounced seasonality could possess much longer pre-oviposition period, for

example Dermacentor reticulatus up to 113 days (Slovak et al. 2002), but see Liu et al.

(2005) for data dealing with seasonality of Dermacentor silvarum.

Comparing parameters of reproduction, only significant relationship was found between

weight of engorged female and number of laid eggs (similarly e.g. Chen et al. 2009; Liu

et al. 2005). Yeruham et al. (2000) reported for Rhipicephalus bursa other significant

positive correlation between weight of engorged female and duration of oviposition.

Process of oviposition in H. aegyptium was thoroughly described by Sweatman (1968).

This author worked with engorged H. aegyptium females collected in nature. He reports

that engorged tick females were collected from tortoises Testudo kleinmanni in Lebanon.

Since this host species does not occur in Lebanon, the tortoises were certainly T. graeca(Fritz and Havas 2007). Tick females collected by Sweatman (1968) weighted up to

1,462.8 mg and subsequently laid up to tremendous clutch of 16,427 eggs. Both weight as

well as clutch size in his study exceeded remarkably our observations.

Optimistic scenario counting with the shortest periods for each stage makes possible

obtain three generations of H. aegyptium per year under presented laboratory conditions.

Nevertheless, both, tick as well as host tortoises display in the nature clear seasonality

disabling such a fast development. Average duration of respective life stages recorded

within our study is similar as those observed in other ixodid ticks (i.e. Hadani et al. 1969;

Pospelova-Shtrom and Petrova-Piontkovskaya 1949; Slovak et al. 2002; Yeruham et al.

2000).

Nearly equal sex ratio of laboratory hatched H. aegyptium is in contrast to our previous

field observations, where males clearly dominate on tortoises (Siroky et al. 2006). This trait

could be caused by mating habits with long lasting host attachment of males observed also

in lab. Tick females drop off after engorgement, whereas males remain attached on tortoise

body much longer. Thus, cumulative effect of long-term remaining tick males could

explain their virtual dominance on tortoises under field conditions (Siroky et al. 2006).

Other possible explanation—the higher mortality of females, was not recorded in this

study.

Despite they are generally undervalued; reptiles serve as reservoirs of numerous

important pathogens (e.g. Bodetti et al. 2002; Stenos et al. 2003; Yadav and Sethi 1979).

Particularly long-living tortoises could have potential in long-term maintenance of natural

foci of infectious diseases and their ticks can serve as vectors (Burridge and Simmons

2003; Peter et al. 2000). Ticks of tortoises (including H. aegyptium) as blood sucking

arthropods have indisputable potential to play a role in transmission of pathogenic agents

(Blanc 1961; Siroky et al. 2010). Therefore, knowledge of biology of such host-specific

ticks should not be overlooked.

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Acknowledgments Eva Praskova, Dana Travnıckova, and Michaela Zapletalova helped with laboratoryprocedures.

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