RESEARCH
Recei ed: 11 Sep embe 2023 / Accep ed: 6 No embe 2023 / Published online: 16 No embe 2023
© The Au ho (s) 2023
Amalia Segu a and Ma a Ra ael con ibu ed equally o his wo k.
Ex ended au ho in o ma ion a ailable on he las page o he a icle
Tick in es a ion in spu - highed o oise popula ion: a
pilo s udy o un a eling epidemiological pa e ns and
demog aphic consequences
AmaliaSegu a1· Ma aRa ael2· Ri aVaz-Rod igues2· Osca Rod íguez1·
Ch is ianGo áza 2· Joséde la Fuen e2,3
Expe imen al and Applied Aca ology (2023) 91:661–679
h ps://doi.o g/10.1007/s10493-023-00863-7
Abs ac
Ec opa asi es, such as icks, modula e hos popula ion dynamics by impac ing demo-
g aphic ai s. They ansmi in ec ious agen s among hei hos s, posing a c i ical h ea
o animal and public heal h. This s udy aimed o cha ac e ize and analyze he Hyalomma
aegyp ium in es a ion on one o i s main hos s, he spu - highed o oise, i s e ec s on de-
mog aphic ai s, and o de e mine he di e si y o in ec ious agen s p esen in bo h icks
and o oises in he Maamo a o es (no hwes e n Mo occo). Ou esul s show ha 100%
o he o oises we e pa asi ized by adul icks in sp ing, an in es a ion in ensi y o 4 icks/
o oise (5.1 and 3.6 icks/ o oise in males and emales, espec i ely; 4.2 and 3.3 icks/
o oise in g a id and non-g a id emales, espec i ely) and an abundance anging om 1
o 12. Al hough wi hou signi ican di e ences, male o oises had highe ick abundances
han emales. The in e ac ion o o oise sex and body condi ion was signi ican ly ela ed
o ick abundance, male body condi ion dec eased wi h highe ick abundance in con as
o emales. Ne e heless, he in e ac ion o body condi ion and ep oduc i e s age o e-
males was no signi ican ly ela ed o ick abundance. G a id emales we e signi ican ly
associa ed wi h ick abundance, showing a sligh ly highe in es a ion han non-g a id
emales. Molecula analysis o pooled ick samples e ealed he p esence o Eh lichia
ewingii, Candida us Midichlo ia mi ochond ii, and Ricke sia a icae, wi h a minimum
in ec ion a e o 0.61 o 1.84%. Howe e , blood sample analysis o he o oises was in-
ec ious agen - ee, pinpoin ing a lack o signi ican heal h p oblems. Gi en he possible
e ec on he ansmission o zoono ic diseases by spu - highed o oises associa ed wi h
hei equen collec ion as pe s, i should be su eyed o con ol possible human heal h
p oblems. In conse a ion e ms, as a long-li ed species, he ole o ick in es a ion in
demog aphic ai s migh be included in he managemen and conse a ion p og ams o
spu - highed o oises.
Keywo ds Hyalomma aegyp ium · Su eillance · Tick-bo ne in ec ious agen s ·
To oise · Tes udo g aeca
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In oduc ion
Ec opa asi es may modula e hos popula ion dynamics by in luencing na u al selec ion
(Fi ze e al. 2004; Bull and Bu zaco 2006). Long in e ac ion be ween hos s and ec opa a-
si es impac s hos popula ion s uc u e and size, a ec ing de ence e ec i eness and esul -
ing in mos o he cases in adap a ion and co-e olu ion (Hwang and Kuang 2003; Esse e
al. 2019). To oises om he Tes udo genus ha e been deeply documen ed as hos s o ick
species o he Hyalomma genus, such as Hyalomma aegyp ium L. (Hoogs aal and Kaise
1960; Ši oký e al. 2006), a ec ing hose ec opa asi es hei li e-his o y ai s. Pa icula ly
high is he encoun e a e o H. aegyp ium wi h spu - highed o oise Tes udo g aeca in
Mo occo, Tunisia, Tu key and Alge ia (Gha bi e al. 2015; Tia e al. 2016; Segu a e al.
2019; Najja e al. 2020), and o a lesse ex end wi h Ma gina ed o oise Tes udo ma gina a
in G eece (Ši oký e al. 2006), Ho s ield´s o oise Tes udo ho s ieldii in I an (Ja anbakh e
al. 2015), and He mann´s o oise Tes udo he manni in Albania (Hoogs aal, 1956; Ši oký e
al. 2006; Bizhga e al. 2022). The endu ed con ac be ween H. aegyp ium and Tes udo may
depend on a complex in e play o ac o s in ol ing hos demog aphic ac o s such as sex,
ep oduc i e s age o popula ion densi y, hos -pa asi e ac o s including encoun e , compa -
ibili y and ecogni ion s a egies (Hobe g and B ooks 2008) and abio ic ac o s including
ele a ion, empe a u e, ain all and humidi y (Cumming 2002; Ja anbakh e al. 2015). In
pa icula , he e ec o ick pa asi ism is o en highe in male o oises han in emales
(Segu a e al. 2019; Laghzaoui e al. 2022; bu see Tia e al. 2016), ep esen ing ei he di -
e ences in exposu e o suscep ibili y o icks, such as male-speci ic beha iou in b eeding
ime by di e en ial habi a use (Robbins e al. 1998). Male pa asi ism migh esul in an
ex a biological cos i physiological aspec s such as body condi ion a e a ec ed (Segu a
e al. 2019). The e ec o pa asi ism in he ep oduc ion o o oise emales may in luence
esou ce alloca ion ade-o s, educing o inc easing ep oduc i e ou pu acco ding o di -
e en s a egies (e.g., Lockley e al. 2020). The e o e, emale ep oduc i e success migh be
comp omised as a di ec consequence o esou ce exploi a ion by pa asi es. Whe eas small
(young) in ec ed emales could use a be -hedging s a egy in a ou o li e ime ep oduc i e
success, olde in ec ed emales could adop a e minal in es men s a egy (e.g., Lockley e
al. 2020). Addi ional ex e nal ac o s, such as he limi a ion o ood esou ces, will a ou
esou ce alloca ion om cu en ep oduc ion o su i al (and u u e ep oduc ion) un il he
in ec ion has passed (e.g., Hu d 2001; Pollock e al. 2012).
Adul s o H. aegyp ium eed almos exclusi ely on o oises o he genus Tes udo. How-
e e , a e cases in o he hos s, such as ha es and hedgehogs, ha e been epo ed (Hoogs aal
and Kaise 1960; Gazyağci e al. 2010). La ae and nymphs a e less hos -speci ic and eed
on a a ie y o e eb a es (Es ada-Peña e al., 2017), including domes ic animals (dogs,
ca le, ho ses, o pigs; Aydin 2000), wild animals (liza ds, bi ds, hedgehogs, oden s, o
camels; Ka e al. 2011; Ši oký e al. 2011; Apanaske ich and Oli e 2014), and humans
(Va anse e e al. 2008; Bu sali e al., 2010). Ticks a e conside ed he second ec o o
human diseases and a e bo h ec o s and ese oi s o in ec ious agen s, ha bou ing bac-
e ial, i al, and p o ozoan mic oo ganisms (de la Fuen e e al. 2017). The mul i ude o
hos s a ec ed by H. aegyp ium poses a majo conce n as a ious dissemina ion scena ios
may occu , leading o epidemiological consequences. Indeed, se e al in ec ious agen s ha e
been de ec ed in H. aegyp ium collec ed om spu - highed o oise, such as Ricke sia spp.,
Eh lichia spp., Anaplasma spp., Coxiella bu ne ii, C imean-Congo haemo hagic e e
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i us (CCHFV) o Hemoli ia mau i anica (Tia e al. 2010; Bu sali e al., 2011; Paș iu e al.
2012; Kau man e al. 2016; Ba adas e al. 2019, 2020; Manoj e al. 2021; Mumcuoglu e al.
2022; Rjeibi e al. 2022). Pa icula ly in Mo occo, he p esence o H. mau i anica, Eh lichia
spp., Midichlo ia mi ochond ii, Wolbachia spp., elapsing e e bo eliae, F ancisella spp.,
and Ricke sia spp. has been epo ed om spu - highed o oise in es ed by H. aegyp ium
(e.g., Ha is e al. 2013; No e e al. 2021).
In ou s udy, we examined he p esence o in ec ious agen s in bo h H. aegyp ium icks
and spu - highed o oises and he ole o sex and emale ep oduc i e s age in o oises as
d i e s o ick in es a ion in he hos species. Spu - highed o oise ha e been ed-lis ed as
‘ ulne able’ by he In e na ional Union o Conse a ion o Na u e (IUCN 1996; Rhodin e
al. 2021) and one o hei main h ea s h ough hei dis ibu ion is he collec ion and ade as
pe s (Pé ez e al. 2004; Tia e al. 2019; Segu a e al. 2020). We selec ed a popula ion loca ed
in he Maamo a o es , a co k oak o es loca ed in no he n Mo occo ha is cha ac e ized
as highly humid, when compa ing wi h o he a eas o he o oise dis ibu ion ange, and
conside ed close o he op imum niche o he o oise dis ibu ion (Anadón e al. 2012). The
popula ion has been p e iously s udied in 2018 in a p i a e ese e whe e he e is no pe
ade and he unde g ow h is well p ese ed (Segu a e al. 2020). The s udy e ealed high
p e alence and mode a e in ensi y o ick pa asi ism, and he in luence o ick in es a ion
on o oise age, sex, body condi ion and popula ion densi y (Segu a e al. 2019). Indeed,
his spu - highed o oise popula ion has been ecognized as one o he denses documen ed
o da e (Segu a and Ace edo 2019). Howe e , he epidemiological s a us o he o oise
communi y p esen in he Maamo a o es is unknown, e en hough se e al demog aphic
s udies had discussed he di e en d i e s o his o oise popula ion (Segu a and Ace edo
2019; Segu a e al. 2019, 2021). The high collec ion and ade o he species in his o es
(Segu a e al. 2020) pinpoin o he po en ial ansmission o zoono ic pa hogen agen s. This
s udy aims o (i) de e mine adul pa asi e p e alence, in ensi y and abundance in o oises,
(ii) analyse he ole o o oise sex, o oise emale ep oduc ion s age and he in e ac ion
o bo h ac o s wi h he body condi ion as d i e s o ick pa asi ism in he species, and
(iii) iden i y and phylogene ically cha ac e ize ick-bo ne in ec ious agen s, including Ana-
plasma spp., Babesia spp., C. bu ne ii, Eh lichia spp., Hepa ozoon spp. / H. mau i anica,
Ricke sia spp., Bo elia spp., and CCHFV, in bo h H. aegyp ium icks and he spu - highed
o oise. This s udy will con ibu e o he design o app op ia e managemen and conse a-
ion plans and emphasizes he impo ance o su eillance and epidemiological p o iling o
bo h ec o s and hos s.
Ma e ials and me hods
S udy si e
The s udy was conduc ed in an a ea o low-ele a ion sandy soil (72–185 m abo e sea le el)
in he Maamo a o es (No hwes Mo occo; 34°02′54.19′′ N, 6°27′19.24′′ W, G ou-Bou e-
g eg basin). The s udy a ea was loca ed on he Medi e anean bioclima ic loo , wi h ho
and d y summe s, and he annual ange o a e age ain all was 300–500 mm and he mean
annual empe a u e 22º C. Maamo a o es is domina ed by co k oak ees Que cus sube ,
sca e ed endemic wild pea Py us mamo ensis, wild oli e Olea eu opaea, g een oli e Phyl-
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li ea la i olia, and mas ic Pis acia len iscus, and a spa se unde s o y o bush and sh ub
species such as Medi e anean b oom Genis a lini olia, Cy isus a bo eus, S au acan hus
genis oides, dwa palm Chamae ops humilis, F ench la ende La andula s oechas, sage-
lea ed ock ose Cis us sal ii olius, Halimium halimi olium, and Thymelaea ly h oides. The
s udy ook place on a p i a e ese e (3000 ha) cha ac e ized by well- ep esen ed unde -
g ow h (e.g., species ichness and co e ) when compa ed wi h o he unp o ec ed si es in
Maamo a (highly o e g azed by li es ock; Said e al. 2014).
Sampling
To oises we e cap u ed by hand be ween Ap il and May 2022 (Table S1) ollowing app o ed
e hical wildli e cap u e and managemen p o ocols. Each indi idual encoun e ed was sexed,
he body mass was de e mined using a p ecise balance (± 1 g), and he body size was mea-
su ed (± 1 mm) as he s aigh an e opos e io dis ance be ween he nuchal and sup acaudal
scu es using a callipe (ca apace leng h, CL). Collec ion and ick ex ac ion we e ca ied
ou wi hin a p i a e ini ia i e o he conse a ion o T. g aeca in Maamo a Fo es . All icks
a ached o he o oise body we e coun ed in he ield, and a ep esen a i e subsample was
collec ed o analysis o in ec ious agen s. The emo ed icks we e iden i ied a he species
le el wi h DNA ba coding o mi ochond ial genes. Blood was collec ed om he subca a-
pacial plexus using a 1-mL sy inge. Fo de e mining he emale ep oduc i e s age, emales
we e adiog aphed do so en ally wi h a po able X- ay a 60 kV (20 mA) a a dis ance o
1 m, acco ding o Gibbons and G eene (1979). The adiog aphy allowed he iden i ica ion
o g a id emales and assessed he clu ch size. Figu e 1 ep esen s he me hods employed
Fig. 1 Me hodological lowcha . Indi idual cha ac e is ics we e eco ded such as weigh , sex, body size
measu es and quan i ica ion o eggs. The ela ionship be ween he a iables was pe o med using gene al
linea models and linea models wi h he R so wa e. In addi ion, DNA was ex ac ed om icks and blood
samples collec ed om he o oises. Posi i e samples o he pa hogens analysed we e sequenced, and
phylogene ic ees we e gene a ed
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in his a icle. All o oises we e eleased immedia ely a e measu emen s and sample col-
lec ions a he cap u e si e.
Th ee pa asi ological indica o s we e calcula ed: (1) in es a ion p e alence, by di id-
ing he numbe o in es ed o oises by he numbe o examined o oises and mul iplying
i by 100, (2) mean in es a ion in ensi y, by di iding he numbe o icks by he numbe o
in es ed o oises, and (3) ick abundance, by di iding he numbe o icks by he numbe
o examined o oises. The o oise body condi ion (BC), which ep esen s he body mass
adjus ed o he body size (Nagy and Medica 1986), was de e mined by calcula ing esidual
alues h ough a linea eg ession analysis (all indi iduals pooled). In his analysis, he
na u al loga i hm (ln) o body mass was used as he dependen a iable, whe eas ln CL was
used as he independen a iable. The indi idual body-condi ion index measu es he ex en
o mass de ia ion compa ed o he expec ed alues based on he animal’s size, which can
change wi h age, s age o ep oduc ion, d ough and disease.
Bo h he icks and he blood o he o oises we e s o ed a -25 °C in ubes wi h RNAla e
and sodium hepa in, espec i ely, o u he analysis.
Tick DNA/RNA isola ion and PCR in ec ious agen s analysis
Nucleic acid ex ac ion was accomplished om indi idual ick samples and ick pools
(mean o 3,196 icks/pool, anging om 1 o 8 icks). The pools we e designed andomly,
acco ding o he numbe o icks collec ed in he ield. DNA and RNA we e ex ac ed om
he in e nal issues o icks, disca ding he ex e nal cu icle, and using TRI Reagen (Sigma-
Ald ich, S . Louis, USA), ollowing he manu ac u e ’s ins uc ions. The concen a ion
(ng/µL) and pu i y o samples we e e alua ed using a Nanod op One spec opho ome e
(The mo Scien i ic, Wal ham, USA), h ough he quan i ica ion o he nucleic acids a an
op ical densi y o 260 nm (OD260) and he a io o abso bance a 260/280 nm. The quali y
o he ex ac ion p o ocol and con i ma ion o ick species we e app aised by he ampli ica-
ion o he mi ochond ial 16S ibosomal DNA (16S DNA) gene and he cy och ome oxidase
subuni I (COI) gene o ou indi idual icks (Table 1). All samples we e es ed using con-
en ional polyme ase chain eac ion (PCR) aimed a de ec ing he p esence o Anaplasma
spp., Babesia spp., C. bu ne ii, Eh lichia spp., Hepa ozoon spp. / H. mau i anica, o Ricke -
sia spp., a nes ed PCR o he de ec ion o Bo elia spp., and a nes ed e e se ansc ip ion
(RT)-PCR o he iden i ica ion o he CCHFV. Table 1 p o ides in o ma ion on he speci ic
a ge ed egions o each PCR assay, he used p o ocol, and p ime s.
The PCR eac ions we e pe o med in a 25 µL olume, including 12.5 µL o PCR Mas e
Mix 2x (P omega, Madison, WI, USA), 1 µL o each p ime (10 µM wo king solu ion), 9
µL o RNase- ee wa e (The mo Scien i ic), and 1.5 µL o DNA sample. Fo he nes ed
RT-PCR assessmen o CCHFV, he comme cial ki Access RT-PCR Sys em (P omega,
Fi chbu g, WI, USA) was used acco ding o he manu ac u e ’s ins uc ions. The PCRs
we e conduc ed in a C1000 ouch PCR he mal cycle (Bio-Rad, He cules, CA, USA), wi h
he speci ic PCR agmen s isualized in 1.5% aga ose gel s ained wi h GelRed (Bio ium,
F emon , CA, USA) unde UV ansillumina ion.
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Sequencing and phylogene ic analysis
P esumed posi i e samples we e pu i ied and sequenced using he Sange me hod a Secu-
gen (Mad id, Spain). Sequences we e edi ed wi h he Ch omas so wa e .2.6.6., and
homology analysis was conduc ed using he Na ional Cen e o Bio echnology In o ma-
ion (NCBI) da abase, employing he Basic Local Alignmen Sea ch Tool (BLAST). The
Table 1 P ime s and PCR p o ocols acco ding o he pa hogen analysed
Pa hogen and
a ge gene
Sequence 5’-3’ (F: Fo wa d / R: Re e se) F ag-
men
(bp)
An-
neal-
ing
(ºC)
Re e ence
16S DNA F: CCGGTCTGAACTCAGATCAAGT
R: CTGCTCAATGATTTTTTAAATTGCTGTGG
460 48 Rod íguez e
al. 2022
COI F: GGTCAACAAATCATAAAGATATTGG
R: TAAACTTCAGGGTGACCAAAAATCA
650 50 Coimb a-
Do es e al.
2018
Anaplasma spp.
(16S RNA)
F: CAGAGTTTGATCCTGGCTCAGAACG
R: GAGTTTGCCGGGACTTCTTCTGTA
421 42 Mo aga
Fe nández e
al. 2022
Anaplasma spp.
(msp5)
F: GCATAGCCTCCGCGTCTTTC
R: TCCTCGCCTTGGCCCTCAGA
456 54 Mo aga
Fe nández e
al. 2022
Anaplasma spp.
(msp4)
F: CGGATCCTTAGCTGAACAGGAATCTTGC
R:
GGGAGCTCCTATGAATTACAGAGAATTGTTTAC
849 60 Mo aga
Fe nández e
al. 2022
Babesia spp.
(18S RNA)
F: AAT ACC CAA TCC TGA CAC AGG G
R: TTA AAT ACG AAT GCC CCC ACC
408 58 Ba adas e
al. 2020
Bo elia bu g-
do e i sensu
la o ( lagellin)
F1: GCATCACTTTCAGGGTCTCA
R1: TGGGGAACTTGATTAGCCTG
F2: CTTTAAGAGTTCATGTTGGAG
R2: TCATTGCCATTGCAGATTGT
390 55
and
58
No e e al.
2021
Coxiella bu -
ne ii (IS111a)
F: CAAGAATGATCGTAACGATGCGC
R: CTCGTAACACCAATCGCTTCG
349 63 Rjeibi e al.
2022
C imean-Congo
Haemo hagic
Fe e í us
(CCHFV S
segmen )
F1: TTGTGTTCCAGATGGCCAGC
R1: CTTAAGGCTGCCGTGTTTGC
F2: GAAGCAACCAARTTCTGTGC
R2: AAACCTATGTCCTTCCTCC
211 60
and
57
Mo aga-
Fe nández e
al. 2021
Eh lichia spp.
(16S RNA)
F: GGTACCYACAGAAGAAGTCC
R: TAGCACTCATCGTTTACAGC
345 54 Ba adas e
al. 2020; Gal
e al. 2008
Hepa ozoon
spp. / Hemoli ia
mau i anica
(18S RNA)
F: GTTTCTGACCTATCAGCTTTCGACG
R: CAAATCTAAGAATTTCACCTCTGAC
600 60 No e e al.
2021; Uj a i
e al. 2004
Ricke sia spp.
(16S RNA)
F: AGAGTTTGATCCTGGCTCAG
R: AACGTCATTATCTTCCTTGC
416 54 Rod íguez e
al. 2022
Ricke sia spp.
(ompA)
F: ATGGCGAATATTTCTCCAAAA
R: AGTGCAGCATTCGCTCCCCCT
630 54 Mo aga-
Fe nández e
al. 2019
Ricke sia spp.
(ompB)
F: GGGTGCTGCTACACAGCAGAA
R: CCGTCACCGATATTAATTGCC
618 53 Mo aga-
Fe nández e
al. 2019
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16S DNA sequence was deposi ed in GenBank unde he accession numbe OQ295899.
The COI pa ial sequences ob ained in his s udy we e a ibu ed he accession numbe s
OQ320497 and OQ556797. The 16S RNA pa ial sequences o Eh lichia iden i ied in
his s udy we e asc ibed he accession numbe s OQ9931657, OQ991496, OQ991497 and
OQ996270. The ou e memb ane p o ein A [ompA] pa ial sequence o Ricke sia was
submi ed o Genbank and assigned he accession numbe OR003919. Mul iple sequence
alignmen was ca ied ou using he Mul iple Sequence Compa ison by Log-Expec a ion
(MUSCLE) algo i hm. Phylogene ic analysis was pe o med in MEGA so wa e .11.0.13.
Co ec ed Akaike In o ma ion C i e ion (cAIC) was used o selec he bes - i model, and a
phylogene ic ee o posi i e in ec ious agen s was gene a ed using maximum likelihood
and Neighbo -Joining me hods. To ensu e he eliabili y o p oduced ees, 1000 boo s ap
eplica es we e implemen ed.
Blood nucleic acid isola ion and PCR in ec ious agen analysis
Blood DNA was ex ac ed om o oises wi h suspec ed in ec ious agen s p esen in ick
samples using he DNeasy Blood & Tissue Ki (Qiagen, Hilden, Ge many) and ollowing
he manu ac u e ’s ins uc ions. The samples we e es ed using con en ional PCR agains
Anaplasma spp., Eh lichia spp. and Ricke sia spp. (Table 1). The PCR p o ocol ollowed
he same indica ions as he one desc ibed o he ick in ec ious agen s esea ch.
S a is ical analysis
χ2 es s we e used o assess di e ences in in es a ion in ensi y be ween o oise sexes and
be ween g a id and non-g a id o oise emales. Two gene alized linea models (GLM)
wi h a Poisson dis ibu ion and loga i hmic link unc ion we e pe o med wi h he R .4.3.1
(2023) so wa e, o analyse he ela ionship be ween ick in es a ion a e ( ick abundance)
and (i) he o oise sex and he in e ac ion o body condi ion wi h sex, and (ii) emale ep o-
duc i e s age (g a id/non-g a id emales) and he in e ac ion o body condi ion wi h ep o-
duc i e s age. Fo all analyses, s a is ical signi icance was decla ed a α = 0.05 (con idence
le el o 95%).
Resul s
Tick in es a ion a e and o oise demog aphic ai s
In o al 520 icks (mos ly adul s wi h he excep ion o ou nymphs) we e coun ed on he
130 o oises cap u ed (98 emales, 32 males). O e all, he in es a ion p e alence was 100%
wi h all he o oises pa asi ized by icks, and he mean (± 95% con idence in e al) in es a-
ion in ensi y was 4 ± 0.42 icks/ o oise. Tick abundance anged om 1 o 12 icks/ o oise.
Males p esen ed highe in es a ion in ensi y (5.3 ± 1.11 icks/ o oise) han emales
(3.6 ± 0.40 icks/ o oise) bu he di e ences be ween hem we e no signi ican (χ2 = 2.4, d. .
= 1, P = 0.1). The model o de e mining he in es a ion a e e ec on sex and body condi ion
showed a signi ican ela ion o sex and a signi ican in e ac ion be ween body condi ion and
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sex. Males had highe ick abundances, and ick abundance dec eased in males in ela ion o
hei body condi ion (Table 2; Fig. 2).
G a id emales (34%; 1–5 eggs) p esen ed highe mean in es a ion in ensi y han non-
g a id emales (4.2 s. 3.3 icks/ o oise, n = 33 and 65, espec i ely) bu he di e ences
be ween hem we e no signi ican (χ2 = 1.08, d. . = 1, P = 0.29). The model showed a sig-
ni ican ela ion be ween ick abundance and he ep oduc i e s age o he emales, g a id
emales wi h highe ick abundance han non-g a id emales. In addi ion, i showed a lack
Table 2 S a is ical pa ame e s o he gene alized linea model (GLM) ca ied ou o de e mine ick abundance
a ia ion in ela ion o he o oise sex and he in e ac ion o body condi ion and sex in o oises
Model p edic o s Es ima e SE P
(In e cep ) 1.287e + 00 5.307e-02 24.252 < 0.01
Body condi ion -2.617e-05 4.945e-04 -0.053 0.96
Sex1
Males 2.620e-01 1.003e-01 2.613 < 0.01
Body condi ion × males -4.978e-03 1.083e-03 -4.597 < 0.01
1Class e e ence o he ca ego ical a iable sex is ‘ emale’
Fig. 2 Numbe o icks encoun e ed ca ego ized by sex and acco ding o body condi ion (BC). Female
da a a e ep esen ed in ed, male da a in blue
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o signi icance in he in e ac ion be ween body condi ion and he ep oduc i e s age o he
emales (Table 3).
PCR analysis
A sample o 163 icks (156 males, six emales, and one nymph) was used o DNA ex ac-
ion and analysis. All 163 icks we e con i med as H. aegyp ium by ba coding o 16S DNA
and COI genes. BLAST analysis e ealed 98.9–100% iden i y o wo icks, one iden i ied
o bo h genes, wi h H. aegyp ium (GenBank accession numbe s MG418679, AF132821
and KY548846). Phylogene ic analysis was pe o med o e alua e he gene ic associa ion
be ween he sequenced samples and o he Hyalomma species ob ained om he GenBank
da abase (Figs. 3 and 4). Bo h phylogene ic ees p esen clus e s o he geno ypes H. ma -
gina um, H. exca a um, H. aegyp ium and H. impel a um and an ou g oup o Ixodes icinus
(GenBank accession numbe MH645522 and MZ305543). The samples e ie ed in his
s udy clus e in he subg oup o H. aegyp ium, being agg ega ed wi h samples collec ed
om Tu key (KR870970) o Is ael (KU130407), in he case o 16S DNA sequences, and
om Is ael (KT989617), Mo occo (OL467652) o Alge ia (OL467646) in COI sequences.
Sequence and BLAST analysis o suspec ed posi i e samples e ealed ou ick pools
as posi i e o he Eh lichia 16S RNA gene (7.84%), and one (1.96%) as posi i e o he
Ricke sia ompA gene. BLAST analysis o he Eh lichia 16S RNA gene o H. aegyp ium
showed h ee samples sha ing 98–99% iden i y wi h Candida us M. mi ochond ii (Gen-
Bank accession numbe MG668797, OQ320500 o MK416236.1) and one wi h 99.6% iden-
i y o Eh lichia ewingii (GenBank accession numbe MW092750). One sample (isola e
12) posi i e o Eh lichia 16S RNA p esen ed a co-in ec ion wi h Ricke sia sha ing 99.7%
iden i y wi h Ricke sia a icae when a ge ing he ompA gene (GenBank accession numbe
MW874463).
Phylogene ic analysis o he Eh lichia 16S RNA (Fig. 5) con i med he classi ica ion
as Candida us M. mi ochond ii and E. ewingii. I shows a clus e be ween he isola es 44
(OQ996270), 20 (OQ991497) and 7 (OQ9931657) and Candida us M. mi ochond ii de ec ed
in H. ana olicum icks om China (MG668797), H. aegyp ium om Qa a (MW092748)
and Mo occo (MW293914), H. d omeda ii om Tunisia (MK416236) and H. u ipes om
Ghana (OQ320500). Conce ning isola e 12 (OQ991496), i clus e s wi h sequences iden i-
ied as E. ewingii collec ed om Haemaphysalis bandico a om Taiwan (OK345369) and
H. aegyp ium om Qa a (MW092750). In e ms o he Ricke sia ompA sequences, he
phylogene ic analysis con i ms he classi ica ion as R. a icae (Fig. 6). The posi i e sample
(isola e 12 - OR003919) clus e s wi h R. a icae sequences om Tu key (JQ691730) o
Table 3 S a is ical pa ame e s o he gene alized linea model (GLM) ca ied ou o de e mine ick abundance
a ia ion in ela ion o he ep oduc i e s age (g a id and non-g a id emales) and he in e ac ion o body
condi ion and ep oduc i e s age
Model p edic o s Es ima e SE P
(In e cep ) 1.448 0.084 17.108 < 0.01
Rep oduc i e s age
Non-g a id1-0.251 0.108 -2.316 < 0.01
Body condi ion -0.0004 0.0009 -0.478 0.63
Body condi ion × non-g a id 0.0004 0.001 0.449 0.65
1Class e e ence o he ca ego ical a iable sex is ‘g a id’
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Publishe ’s No e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
Au ho s and A ilia ions
AmaliaSegu a1· Ma aRa ael2· Ri aVaz-Rod igues2· Osca Rod íguez1·
Ch is ianGo áza 2· Joséde la Fuen e2,3
José de la Fuen e
[email p o ec ed]
1 BP 30, Sidi Allal el Bah aoui 15250, Mo occo
2 SaBio, Ins i u o de In es igación en Recu sos Cinegé icos (IREC), Consejo Supe io de
In es igaciones Cien í icas (CSIC), Uni e sidad de Cas illa-La Mancha (UCLM)-Jun a de
Comunidades de Cas illa-La Mancha (JCCM), Ronda de Toledo 12, Ciudad Real 13005, Spain
3 Cen e o Ve e ina y Heal h Sciences, Depa men o Ve e ina y Pa hobiology, Oklahoma
S a e Uni e si y, S illwa e , OK 74078, USA
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