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: [email protected]
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
278 Exp Appl Acarol (2011) 54:277–284
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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
280 Exp Appl Acarol (2011) 54:277–284
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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.
References
Apanaskevich DA (2003) K diagnostike vida Hyalomma (Hyalomma) aegyptium (Acari, Ixodidae) [Todiagnostics of Hyalomma (Hyalomma) aegyptium (Acari: Ixodidae)]. Parazitologija 37:47–59 (inRussian)
Apanaskevich DA (2004) Parazito-khozjainye svjazi vidov roda Hyalomma Koch, 1844 (Acari, Ixodidae) iikh svjaz s mikroevoljucionnym processom [Host-parasite relationships of the genus Hyalomma Koch,1844 (Acari, Ixodidae) and their conection with microevolutionary process]. Parazitologija 38:515–523(in Russian)
Blanc G (1961) Comportement de Rickettsia burneti Derrick chez la tique Hyalomma aegyptium (LIN) et latortue terrestre Testudo graeca LIN. Path Microbiol 24(Suppl):21–26
Bodetti TJ, Jacobson ER, Wan C, Hafner L, Pospischil A, Rose K, Timms P (2002) Molecular evidence tosupport the expansion of the hostrange of Chlamydophila pneumoniae to include reptiles as well ashumans, horses, koalas and amphibians. Syst Appl Microbiol 25:146–152
Burridge MJ, Simmons LA (2003) Exotic ticks introduced into the United States on imported reptiles from1962 to 2001 and their potential roles in international dissemination of diseases. Vet Parasitol113:289–320
Chen Z, Yu Z, Yang X, Zheng H, Liu J (2009) The life cycle of Hyalomma asiaticum kozlovi Olenev, 1931(Acari: Ixodidae) under laboratory conditions. Vet Parasitol 160:134–137
Fritz U, Havas P (2007) Checklist of chelonians of the world. Vertebr Zool 57:149–368Ghosh S, Azhahianambi P (2007) Laboratory rearing of Theileria annulata-free Hyalomma anatolicum
anatolicum ticks. Exp Appl Acarol 43:137–146Hadani A, Cwilich R, Rechav Y, Dinur Y (1969) Some methods for the breeding of ticks in the laboratory.
Refuah Veterinarith 26(3):87–100Hoogstraal H (1956) African Ixodoidea. I. Ticks of the Sudan. Department of the navy, bureau of medicine
and surgery. Washington, DC, USAHoogstraal H, Kaiser MN (1960) Some host relationships of the tortoise tick. Hyalomma (Hyalommasta)
aegyptium (L.) (Ixodoidea, Ixodidae) in Turkey. Ann Entomol Soc Am 53:457–458Kolonin GV (1983) Mirovoe rasprostranenie iksodovykh kleshchey. Rody Hyalomma, Aponomma,
Amblyomma [World distribution of ixodid ticks. Genera Hyalomma, Aponomma, Amblyomma]. Nauka,Moskva, SSSR (in Russian)
Kolonin GV (2004) Reptiles as hosts of ticks. Russ J Herp 11:177–180Krober T, Guerin PM (2007) In vitro feeding assays for hard ticks. Trends Parasitol 23:445–449Liu J, Liu Z, Zhang Y, Yang X, Gao Z (2005) Biology of Dermacentor silvarum (Acari: Ixodidae) under
laboratory conditions. Exp Appl Acarol 36:131–138Magano SR, Els DA, Chown SL (2000) Feeding patterns of immature stages of Hyalomma truncatum and
Hyalomma marginatum rufipes on different hosts. Exp Appl Acarol 24:301–313Peter TF, Burridge MJ, Mahan SM (2000) Competence of the African tortoise tick, Amblyomma marmoreum
(Acari: Ixodidae), as a vector of the agent of heartwater (Cowdria ruminantium). J Parasitol86:438–441
Pospelova-Shtrom MV, Petrova-Piontkovskaya SP (1949) K biologii Hyalomma marginatum, H. detritum,H asiaticum v laboratornykh usloviyakh [To the biology of Hyalomma marginatum, H. detritum, H.asiaticum under laboratory conditions]. Issledovaniya po kraevoi, eksperimentalnoi i opisatelnoiparazitologii 6: 87–97 (in Russian)
Rechav Y, Fielden LJ (1997) The effect of various host species on the feeding performance of immaturestages of the tick Hyalomma truncatum (Acari: Ixodidae). Exp Appl Acarol 21:551–559
Robbins RG, Karesh WB, Calle PP, Leontyeva OA, Pereshkolnik SL, Rosenberg S (1998) First records ofHyalomma aegyptium (Acari: Ixodida: Ixodidae) from the Russian spur-thighed tortoise, Testudograeca nikolskii, with an analysis of tick population dynamics. J Parasitol 84:1303–1305
Sankoh AJ, Huque MF, Dubey SD (1997) Some comments on frequently used multiple endpoint adjust-ments methods in clinical trials. Stat Med 16:2529–2542
Simo L, Kocakova P, Slavikova M, Kubes M, Hajnicka V, Vancova I, Slovak M (2004) Dermacentorreticulatus (Acari, Ixodidae) female feeding in laboratory. Biologia, Bratislava 59:655–660
Siroky P, Kamler M, Modry D (2004) Long-term occurrence of Hemolivia cf. mauritanica (Apicomplexa:Adeleina: Haemogregarinidae) in captive Testudo marginata (Reptilia: Testudinidae): Evidence forcyclic merogony? J Parasitol 90:1391–1393
Exp Appl Acarol (2011) 54:277–284 283
123
Siroky P, Petrzelkova KJ, Kamler M, Mihalca AD, Modry D (2006) Hyalomma aegyptium as dominant tickin tortoises of the genus Testudo in Balkan countries, with notes on its host preferences. Exp ApplAcarol 40:279–290
Siroky P, Kamler M, Frye FL, Fictum P, Modry D (2007) Endogenous development of Hemolivia mauri-tanica (Apicomplexa: Adeleina: Haemogregarinidae) in the marginated tortoise Testudo marginata(Reptilia: Testudinidae): evidence from experimental infection. Folia Parasitol 54:13–18
Siroky P, Kubelova M, Modry D, Erhart J, Literak I, Spitalska E, Kocianova E (2010) Tortoise tickHyalomma aegyptium as long term carrier of Q fever agent Coxiella burnetii–evidence from experi-mental infection. Parasitol Res 107:1515–1520
Slovak M, Labuda M, Marley SE (2002) Mass laboratory rearing of Dermacentor reticulatus ticks (Acarina,Ixodidae). Biologia, Bratislava 57:261–266
Srivastava SC, Varma MGR (1964) The culture of the tick Rhipicephalus sanguineus (Latreille) (Ixodidae)in the laboratory. J Med Entomol 1:154–157
Stenos J, Graves S, Popov VL, Walker DH (2003) Aponomma hydrosauri, the reptile-associated tickreservoir of Rickettsia honei on Flinders island, Australia. Am J Trop Med Hyg 69:314–317
Sweatman GK (1968) Temperature and humidity effects on the oviposition of Hyalomma aegyptium ticks ofdifferent engorgement weights. J Med Entomol 5:429–439
Vatansever Z, Gargili A, Aysul NS, Sengoz G, Estrada-Pena A (2008) Ticks biting humans in the urban areaof Istanbul. Parasitol Res 102:551–553
Yadav MP, Sethi MS (1979) Poikilotherms as reservoirs of Q-fever (Coxiella burnetii) in Uttar Pradesh.J Wildl Dis 15:15–17
Yeruham I, Hadani A, Galker F (2000) The life cycle of Rhipicephalus bursa Canestrini and Fanzago, 1877(Acarina: Ixodidae) under laboratory conditions. Vet Parasitol 89:109–116
284 Exp Appl Acarol (2011) 54:277–284
123