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 inour 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 onaverage 25 days (range 1744), whereas males remain after female engorgement on tor-
toise host. Female pre-oviposition period was 14 days (331), followed by 24 days of
oviposition (1829). Pre-eclosion and eclosion, both together, takes 31 days (2143).
Larvae fed 5 days (39), then molted to nymphs after 17 days (1223). Feeding period of
nymphs lasted 7 days (510), engorged nymphs molted to adults after 24 days (1926).
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: firstname.lastname@example.org
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
Exp Appl Acarol (2011) 54:277284DOI 10.1007/s10493-011-9442-8
weight of engorged female was 0.95 (0.721.12) g. The average number of laid eggs was
6,900 (6,5247,532) 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 (98215).
Keywords Hyalomma aegyptium Testudo Life-cycle Laboratory rearing
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
Hyalomma aegyptium (Linnaeus, 1758) distributed in Mediterranean area from Atlanticcoastland 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 tortoisesin 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-specificapicomplexan blood parasite Hemolivia mauritanica (Sergent et Sergent, 1904). In a frameof our studies on vectorial capability of H. aegyptium we have got high requirement ofpathogen-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 traitsof its life-cycle, feeding, and reproduction. These data are summarized in the presented
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 (392002700N, 225404900E).Third female was collected in June 2004 from hedgehog Erinaceus concolor Martin, 1838near Areopoli, South of Peloponnesus peninsula, Greece (364001000N, 222205800E). Lasttwo engorged females were collected in April 2005 from tortoises Testudo graeca Lin-naeus, 1758 at locality Qualat Samaan, NW Syria (361905800N, 365004900E). The ticks
278 Exp Appl Acarol (2011) 54:277284
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 2225C, andrelative humidity (RH) 6085%.
Unsexed outbred guinea pigs having weight 300500 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 privatebreeding 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 guineapigs, 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 2224C, and RH 5070%, fluctuating slightly according toseason, 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 1828C and RH 3555%. Position and feeding state of ticks werecontrolled 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 35 days
intervals to avoid their repeated disturbing. Behavior of ticks and their movement on hosts
was also registered daily.
To reveal the relationship among duration of feeding periods, weight of engorged females,
lasting of oviposition and number of eggs laid we performed several Spearmans
Exp Appl Acarol (2011) 54:277284 279
correlations. Bonferoni corrections (with added mean correlation between variables as a
parameter) were used for P values (Sankoh et al. 1997). The analyses were performedusing the STATISTICA software (version 8.0, StatSoft, 2008).
The duration of H. aegyptium life cycle under laboratory conditions divided into particularlife 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 female