Life cycle of tortoise tick Hyalomma aegyptium under laboratory conditions

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Life cycle of tortoise tick Hyalomma aegyptiumunder laboratory conditionsPavel Siroky Jan Erhart Klara J. Petrzelkova Martin KamlerReceived: 7 February 2011 / Accepted: 5 March 2011 / Published online: 24 March 2011 Springer Science+Business Media B.V. 2011Abstract 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 mammalianhosts. Combination of feeding larvae and nymphs on guinea pigs and feeding of adults onTestudo 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 ofoviposition (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 ofnymphs 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 averageP. 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.czJ. ErhartInstitute of Parasitology, Biology Center, Academy of Sciences of the Czech Republic, Branisovska31, 370 05 Ceske Budejovice, Czech RepublicK. J. PetrzelkovaInstitute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Kvetna 8, 603 65 Brno,Czech RepublicK. J. PetrzelkovaLiberec Zoo, Masarykova 1347/31, 460 01 Liberec, Czech RepublicM. KamlerDepartment of Parasitology, Faculty of Veterinary Medicine, University of Veterinary andPharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech RepublicPresent Address:M. KamlerBee Research Institute Dol, Dol 94, 252 66 Libcice nad Vltavou, Czech Republic123Exp Appl Acarol (2011) 54:277284DOI 10.1007/s10493-011-9442-8weight of engorged female was 0.95 (0.721.12) g. The average number of laid eggs was6,900 (6,5247,532) per female, it was significantly correlated with weight of engorgedfemale. Only 2.8% of engorged larvae and 1.8% of engorged nymphs remained un-moltedand died. Despite the use of natural host species, feeding success of females reached only45%. The whole life-cycle was completed within 147 days (98215).Keywords Hyalomma aegyptium Testudo Life-cycle Laboratory rearingIntroductionAvailability of pathogen-free ticks in sufficient numbers is inevitable condition for anyexperimental study with ticks and tick-borne agents. Laboratory rearing methods weredeveloped for many tick species in the past, particularly for model species used in studiesof tick biology and epidemiology of tick-borne diseases (i.e. Chen et al. 2009; Ghosh andAzhahianambi 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). Onthe other hand, little attention was given to tick species being assumed to have lowereconomic importance.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 suchunderstudied 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 (Apanaskevich2004; 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 ticksreported 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 ofH. aegyptium ticks provided controlled conditions to collect information about basic traitsof its life-cycle, feeding, and reproduction. These data are summarized in the presentedpaper.Materials and methodsOrigin and keeping of ticksTick laboratory breeding colony was established by five consecutively imported engorgedfemales 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 ticks278 Exp Appl Acarol (2011) 54:277284123were kept in cylindrical glass tubes (23 mm in diameter, 70 mm height) filled with strip offilter paper, closed with cotton wool pads, and stored in shaded box under 2225C, andrelative humidity (RH) 6085%.Host speciesUnsexed outbred guinea pigs having weight 300500 g and originating from a breedingfacility of Institute of Parasitology Academy of Sciences of the Czech Republic, CeskeBudejovice were used as host species for feeding of larvae and nymphs. Adult male and5 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 alsotried 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. Onthe other hand, it was difficult to safely manage and control feeding of small immaturestages of ticks on tortoise body.Technique of ticks feedingOne 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 nyloncloth. 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 attachmentof 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 cycleWe 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 timebetween spontaneous detachments of engorged larvae and nymphs, respectively, and theirmolting to forthcoming life stage. Weight of ten selected engorged females was recordedimmediately after detachment from host on laboratory scales RADWAG WAS 220/C/2(Radwag, Radom, Poland) and rounded with accuracy 10 mg. Period between theirdetachment and appearance of first eggs represents pre-oviposition period. Lasting ofoviposition and number of eggs laid was recorded for the same ten females. The eggs wereremoved daily from these females. Eggs from the other females were removed in 35 daysintervals to avoid their repeated disturbing. Behavior of ticks and their movement on hostswas also registered daily.Data analysisTo reveal the relationship among duration of feeding periods, weight of engorged females,lasting of oviposition and number of eggs laid we performed several SpearmansExp Appl Acarol (2011) 54:277284 279123correlations. Bonferoni corrections (with added mean correlation between variables as aparameter) were used for P values (Sankoh et al. 1997). The analyses were performedusing the STATISTICA software (version 8.0, StatSoft, 2008).ResultsThe duration of H. aegyptium life cycle under laboratory conditions divided into particularlife stages is given in Table 1.Feeding of adult ticksFeeding females (N = 30) remained usually after attachment on the same place over allfeeding period. They changed feeding place exceptionally, usually when firstly attached tocarapace. Females preferred for feeding the inguinal area and places around hind limbs oftortoises (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 seamsbetween carapace scutes. Six females (20%) originally attached to carapace seams changedplace to inguinal and tight area, and then engorged. Comparing to females, males wereobserved to change places on tortoise body more frequently. Feeding period of females was24.87 5.18 (N = 30; range 1744) days. Engorged females weighted immediately afterspontaneous detachment 0.95 0.14 (N = 10; range 0.721.12) g. Feeding success offemales reached 45% (N = 75), forty females (53.3%) did not attached or died duringfeeding, and five females (6.7%) were rubbed by tortoise movement. Males remainedattached on tortoise host after females detachment until their removal or death.The pre-oviposition and ovipositionInterval between detaching of engorged female and the appearance of the first eggs was14.3 5.94 (N = 30; range 331) days. Oviposition period of selected females lasted24 3.21 (N = 10; range 1829) days. Number of eggs laid per one female was6,900 294 (N = 10; range 6,5247,532).Table 1 Duration of respectivelife stages in life cycle of tickHyalomma aegyptiumStage Duration (days)Minimum Maximum MeanEngorgement of females 17 44 24.9Pre-oviposition 3 31 14.3Oviposition 18 29 24Pre-eclosion and eclosion 21 43 31Feeding of larvae 3 9 5.1Pre-molting and molting to nymphs 12 23 16.6Feeding of nymphs 5 10 6.9Pre-molting and molting to adults 19 26 23.8Total 98 215 *147280 Exp Appl Acarol (2011) 54:277284123There were no significant relationships among feeding periods, weight of engorgedfemales, lasting of oviposition and number of eggs laid with exception of correlationbetween weight of engorged females and number of eggs (rS = 0.83; P \ 0.01).The pre-eclosion and eclosionFirst larvae appeared after 2135 days. Later, they formed clusters on the wall of glasstubes or on cotton wool pads. Forming of this clusters were connected with finishing ofeclosion. Clusters were formed 31 5.62 (N = 20; range 2143) days after oviposition.Feeding period of larvae and nymphsPre-feeding period of larvae and nymphs was not tested. Larvae of H. aegyptium fed5.13 1.45 days (N = 2,004; range 39) till complete engorgement on guinea pigs.Nymphs need 6.9 1.2 days (N = 1,600; range 510) to complete feeding on guineapigs. Engorged larvae and nymphs, respectively, were seen freely moving in feedingchamber, including nylon cloth. Since number of specimens of both life-stageslarvae andnymphs, given to feeding chamber was only roughly estimated, we have no data onpercentage of feeding success.The pre-molting period, molting, sex ratio, and longevityEngorged larvae molted to nymphs 16.6 2.95 (N = 500; range 1223) days after theirdetachment from host. Pre-molting period of engorged nymphs was 23.8 1.92(N = 500; range 1926) days. Fifty-six (2.8%) of all engorged larvae and 28 of allengorged nymphs (1.8%) remained unmolted and died. From 737 engorged nymphs 347and 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 45 months. Longevity of adults is fairly over 1 year (see alsoSiroky et al. 2010).DiscussionWe have discovered that feeding period of adult H. aegyptium on its natural host species israther variable under laboratory conditions. We suppose that its duration depends onattachment site on tortoise body, respectively on availability of capillary blood at thisplace. Generally, observed feeding period of females is longer than is usual in other ixodidtick 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 ofprolonged feeding in unfertilized females of Rhipicephalus sanguineus. We have keptmales and females separately from their hatching, which is why it could be the case. Wehave never tested to feed separately females only. On the other hand, we have observedsuch a prolonged feeding particularly in females attached to carapace. Lower density ofblood 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, asExp Appl Acarol (2011) 54:277284 281123hosts, but we did not achieve any success. Ticks remained usually unattached in feedingchambers, 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 otherhost species. Feeding period of larvae and nymphs fit well that feeding periods as presentedpreviously for H. aegyptium by Hadani et al. (1969). Feeding periods recorded in this studyfor pre-adult stages of H. aegyptium are fairly comparable to those periods observed inmany 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 intointervals 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. Specieshaving more pronounced seasonality could possess much longer pre-oviposition period, forexample 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 betweenweight of engorged female and number of laid eggs (similarly e.g. Chen et al. 2009; Liuet al. 2005). Yeruham et al. (2000) reported for Rhipicephalus bursa other significantpositive 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 reportsthat 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 to1,462.8 mg and subsequently laid up to tremendous clutch of 16,427 eggs. Both weight aswell as clutch size in his study exceeded remarkably our observations.Optimistic scenario counting with the shortest periods for each stage makes possibleobtain three generations of H. aegyptium per year under presented laboratory conditions.Nevertheless, both, tick as well as host tortoises display in the nature clear seasonalitydisabling such a fast development. Average duration of respective life stages recordedwithin 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 previousfield observations, where males clearly dominate on tortoises (Siroky et al. 2006). This traitcould be caused by mating habits with long lasting host attachment of males observed alsoin lab. Tick females drop off after engorgement, whereas males remain attached on tortoisebody much longer. Thus, cumulative effect of long-term remaining tick males couldexplain their virtual dominance on tortoises under field conditions (Siroky et al. 2006).Other possible explanationthe higher mortality of females, was not recorded in thisstudy.Despite they are generally undervalued; reptiles serve as reservoirs of numerousimportant 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 naturalfoci of infectious diseases and their ticks can serve as vectors (Burridge and Simmons2003; Peter et al. 2000). Ticks of tortoises (including H. aegyptium) as blood suckingarthropods 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-specificticks should not be overlooked.282 Exp Appl Acarol (2011) 54:277284123Acknowledgments Eva Praskova, Dana Travnckova, and Michaela Zapletalova helped with laboratoryprocedures.ReferencesApanaskevich DA (2003) K diagnostike vida Hyalomma (Hyalomma) aegyptium (Acari, Ixodidae) [Todiagnostics of Hyalomma (Hyalomma) aegyptium (Acari: Ixodidae)]. Parazitologija 37:4759 (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]. 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Vet Parasitol 89:109116284 Exp Appl Acarol (2011) 54:277284123Life cycle of tortoise tick Hyalomma aegyptium under laboratory conditionsAbstractIntroductionMaterials and methodsOrigin and keeping of ticksHost speciesTechnique of ticks feedingCollection of data on life cycleData analysisResultsFeeding of adult ticksThe pre-oviposition and ovipositionThe pre-eclosion and eclosionFeeding period of larvae and nymphsThe pre-molting period, molting, sex ratio, and longevityDiscussionAcknowledgmentsReferences /ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 149 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 599 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /DocumentCMYK /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /UseDocumentProfile /UseDocumentBleed false >> ]>> setdistillerparams> setpagedevice

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