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Iden i ying b eeding hos s o Ixodes icinus icks using s able iso ope analysis o hei
la ae : P oo o concep
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Sipa i, Saana; Kiljunen, Mikko; Nylund, Minna; Kallio, E a R.
Sipa i, S., Kiljunen, M., Nylund, M., & Kallio, E. R. (2024). Iden i ying b eeding hos s o Ixodes
icinus icks using s able iso ope analysis o hei la ae : P oo o concep . Ticks and Tick-bo ne
Diseases, 15(1), A icle 102252. h ps://doi.o g/10.1016/j. bdis.2023.102252
2024
Ticks and Tick-bo ne Diseases 15 (2024) 102252
A ailable online 21 Sep embe 2023
1877-959X/© 2023 The Au ho s. Published by Else ie GmbH. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
O iginal a icle
Iden i ying b eeding hos s o Ixodes icinus icks using s able iso ope
analysis o hei la ae – P oo o concep
Saana Sipa i
a
,
*
, Mikko Kiljunen
a
, Minna Nylund
b
, E a R. Kallio
a
a
Depa men o Biological and En i onmen al Sciences, Uni e si y o Jy ¨
askyl¨
a, Republic o Finland
b
Animal Heal h and Diagnos ic Uni , Finnish Food Sa e y Au ho i y, Republic o Finland
ARTICLE INFO
Keywo ds:
B eeding hos o icks
S able iso ope analyses
Tick la ae
T anso a ial aces o blood meal
ABSTRACT
Ticks a e impo an ec o s o zoono ic pa hogens. Ticks a e pa asi es ha a e dependen on hei hos s o blood
meal o de elop and ep oduce. The abundance o icks is dependen on he a ailabili y o sui able b eeding
hos s, o en medium- and la ge-sized mammals. So a he e has been a sho age o di ec me hods iden i ying he
b eeding hos s o he emale icks. In his s udy, we in oduce a s able iso ope analysis (SIA) me hod ha enables
us o iden i y he ophic g oup o he b eeding hos , i.e. he hos on which he ick mo he ed, by sampling
la al icks om he ield. We es ablished a e e ence da abase on he s able iso ope (SI) alues (δ
13
C and δ
15
N) o
he blood o po en ial ick hos species, and o la ae om Ixodes icinus emales, which ha e ed on known hos s.
By compa ing he SI alues om ield collec ed la al icks o ou e e ence da a, we can de e mine hei mos
likely hos species g oup. Ou esul s show ha he iso opic signa u es o I. icinus ick la ae e lec he die o
he b eeding hos o he mo he ick. SIA p o ed eliable in ca ego izing he b eeding hos s o I. icinus in o wo
dis inguishable ophic g oups; he bi o es and ca ni-omni o es. To ou knowledge, his is he i s ime ha
s able iso ope analyses ha e been applied o de ec anso a ial (i.e. o e -gene a ional) aces o a blood meal in
icks. The me hod p o ides an e icien , no el ool o di ec ly iden i ying ick b eeding hos s by sampling ield
collec ed la ae. Ixodes icinus is he mos impo an ec o o TBPs ( ick-bo ne pa hogens) in Eu ope, and o
p edic and mi iga e agains he u u e isks ha TBPs pose, i is c ucial o ha e de ailed knowledge on he hos s
ha suppo ick ep oduc ion in na u e.
1. In oduc ion
Mo e han hal o human pa hogens a e zoono ic, i.e. ansmi ed
om o he animal species o humans, and he majo i y o hese ha e
hei o igin in wildli e hos species (Jones e al., 2008). Inc easingly
ele an a e ec o -bo ne zoono ic in ec ions, o which he mos
impo an in he No he n hemisphe e a e caused by ick-bo ne pa ho-
gens (TBPs) (Jones e al., 2008; Rizzoli e al., 2011; Medlock e al., 2013;
Sykes and Makiello 2017; Pe ulionien˙
e e al., 2020). Ticks a e obliga e
pa asi es ha equi e blood meals om e eb a e hos s o de elop-
men and ep oduc ion (Randolph, 2004; Es ada-Pe˜
na and De La
Fuen e, 2014). Consequen ly, he hos s a ec he abundance o he icks
and con ibu e o TBPs ci cula ion, ul ima ely a ec ing he isks ha
TBPs pose o humans (Randolph, 2004).
The ick Ixodes icinus is he mos impo an ec o o TBPs in Eu ope
(Jo e e al., 2011; Jaenson e al., 2012; Medlock e al., 2013; Laaksonen
e al., 2017, 2018; Kjæ e al., 2019). Ixodes icinus ques s o hos s on he
ege a ion, wi h la ae, nymphs and adul emales eeding mos ly on
small-, medium- and la ge-sized animals, espec i ely (Randolph, 2004;
F ancische i e al., 2009; Mannelli e al., 2012; Es ada-Pe˜
na and De La
Fuen e, 2014). Females equi e su icien ly la ge blood meals, ypically
om a medium- o la ge-sized animal, o ep oduce. Hence, he a ail-
abili y o hese “b eeding hos s” is an impo an ac o de e mining ick
abundance. Howe e , i emains poo ly known which o he po en ial
hos s ac ually se e as b eeding hos s o he emales in di e en eco-
sys ems (Kilpa ick e al., 2017).
Iden i ying he hos s o he emale icks is challenging, because a e
eeding he ick de aches om he hos , p oduces eggs, and dies. So a ,
he b eeding hos po en ial o di e en e eb a e species has been
di ec ly examined based on ick in es a ion le els on wild-cap u ed o
hun ed indi iduals (T¨
alleklin and Jaenson, 1997). Al e na i ely, hos
compe ence has also been examined by holding wild-cap u ed animals
in cap i i y and measu ing he engo gemen and de achmen a es and
subsequen moul ing success o he icks (LoGiudice e al., 2003;
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (S. Sipa i).
Con en s lis s a ailable a ScienceDi ec
Ticks and Tick-bo ne Diseases
jou nal homepage: www.else ie .com/loca e/ bdis
h ps://doi.o g/10.1016/j. bdis.2023.102252
Recei ed 29 Ma ch 2023; Recei ed in e ised o m 28 Augus 2023; Accep ed 8 Sep embe 2023
Ticks and Tick-bo ne Diseases 15 (2024) 102252
2
Keesing e al., 2009; B unne e al., 2011). Due o high empo al and
indi idual-le el a ia ion in ick in es a ion a es (T¨
alleklin and Jaen-
son, 1997, Cayol e al., 2017), high numbe s o indi iduals pe hos
species need o be examined o ob ain accu a e es ima es. Consequen ly,
i is challenging o examine he ole o di e en e eb a e species as
hos s o icks. Al hough adi ional DNA based blood meal analysis can
de ec anss adial aces o hos s DNA om nymphs and adul s,
enabling he iden i ica ion o hos s on which he la ae and nymphs
we e eeding on (Humai e al., 2007; Mo a´nmo a´e al., 2007; Collini
e al., 2015; Kim e al., 2021; Goe he e al., 2021; So munen e al.,
2023), such me hods do no de ec anso a ial (i.e. o e -gene a ional)
aces o hos DNA (G´
omez-Díaz and Figue ola, 2010; Kim e al., 2021).
Thus, such blood meal analyses canno be used o iden i y b eeding hos s
o icks. Howe e , he iden i ica ion o b eeding hos s is pi o al in
es ima ing he ole o di e en hos species in d i ing ick ep oduc ion
and ick abundance in di e en ecosys ems.
In his s udy, we in oduce a me hod ha enables sampling la al
icks om he ield o iden i y he b eeding hos on a ophic le el (i.e.
he hos hei mo he ed on) by using s able iso ope analysis (SIA). As
‘you a e wha you ea ’ is he undamen al p inciple in SI ecology (F y,
2006), he hos animals a e expec ed o di e in hei SI alues e lec ing
hei die . SI alues o ni ogen (δ
15
N) and ca bon (δ
13
C) end o inc ease
in a p edic able manne in he ood web, and hence, can be used o
sepa a e consume s based on hei die sou ce (F y, 2006). Conse-
quen ly, he SI alue o ick la ae e lec s he SI alue o he b eeding
hos , which he emale, i.e. he mo he , ed on. Based on his p incipal,
we ha e es ablished a da abase, consis ing s able iso ope da a om
di e en po en ial hos species by collec ing blood samples om a a-
ie y o hos s, and la al samples om I. icinus emales ha ha e ed on
known hos s. By using he SI ma ke measu emen s om la ae
collec ed om ege a ion, we can de e mine he ophic g oup o he
mo he ’s hos species.
2. Ma e ial and me hods
2.1. S udy sys em and es ablishing e e ence da abase
In ou s udy sys em in Finland he expec ed b eeding hos s o
I. icinus a e he oe dee (Cap eolus cap eolus), which is impo an hos
o I. icinus in Eu ope (Vo e al., 2010; Medlock e al., 2013; Ho -
mees e e al., 2016; Mys e ud e al., 2021), in oduced whi e- ailed dee
(Odocoileus i ginianus), which is an impo an hos o Ixodes icks in
No h Ame ica (Eisen and S a o d, 2021), and moose (Alces alces)
(Q ille e al., 2013; Ho mees e e al., 2016; Fab i e al., 2021). In
addi ion, medium-sized animals, such as accoon dog (Nyc e eu es p o-
cyonoides), ed ox (Vulpes ulpes), badge (Meles meles), hedgehog (E i-
naceus eu opaeus), b own ha e (Lepus eu opaeus) and moun ain ha e
(Lepus imidus) a e wildli e hos s ha a e known o p o ide blood meals
o adul emale I. icinus (Jaenson & T¨
alleklin , 1996, T¨
alleklin and
Jaenson, 1997, Ho mees e e al., 2016, Wodecka e al., 2016, Jah a i
e al., 2017, Mys e ud e al., 2021) Also, dogs (Canis lupus amilia is) and
o he domes ic animals may suppo ick popula ions (Shaw e al., 2001;
Nijho e al., 2007; Jo e e al., 2014).
To c ea e a da abase o he s able iso ope alues (ni ogen and
ca bon), we u ilised blood samples om a a ie y o po en ial ick
b eeding hos species, including wild and domes ic mammals (Table 1).
All blood samples we e collec ed and p o ided by olun a y hun e s o
by he Finnish Food Au ho i y (go e nmen al au ho i y esponsible o
moni o ing he heal h and wellbeing o wild and domes ic animals in
Finland). Samples om hun ed indi iduals we e collec ed di ec ly a e
killing, while he samples om he Finnish Food Au ho i y we e
collec ed du ing he au opsy, a e unspeci ied ime a e he dea h.
Samples we e collec ed om se e al municipali ies ac oss Finland (see
deposi ed da a). The blood samples we e s o ed a −20 ◦C, un il he
p epa a ion o he samples o SIA.
To acqui e e e ence la ae, engo ged emales we e collec ed
di ec ly om hos s (Table 1), kep a oom empe a u e in a esealable
plas ic bag wi h a piece o mois pape issue o p e en d ying, un il he
emale ick laid eggs, and he eggs ha ched in o la ae. The engo ged
I. icinus emales we e p o ided by hun e s, pe owne s o we e collec ed
by esea che s ( icks om hedgehogs). The engo ged I. icinus emales
o igina ed om Cen al Finland (Jy ¨
askyl¨
a; n =11, and Luhanka; n =2)
and om an a chipelago in sou he n Finland (Po oo, n =19). Beside
wo samples (a accoon dog and a moun ain ha e), he blood and he
engo ged icks we e om di e en hos indi iduals.
A e he eggs had ha ched in o la ae, samples o he SIA analysis
we e collec ed (Fig. 1). Tick la ae samples we e collec ed by wiping he
inside o he plas ic bag wi h a whi e co on clo h, om which hey we e
collec ed wi h nano ape (Fig. 1a). The adhesion o nano ape is achie ed
wi hou chemical adhesi es, and i lea e no esidue on he subs a e.
Thus, he p o ocol is compa able wi h he me hod used o collec la al
icks om he ield wi h ab ic and ape (see “Collec ing la ae om he
ege a ion”). The ea e , he sample apes we e s o ed a −20 ◦C, un il
u he p ocedu es. To p epa e he samples o he SIA, he ick la ae
we e i s de ached om he ape by soaking he ape in a small amoun
o dis illed wa e , and hen sc aping he la ae in o he wa e wi h a help
o a small labo a o y spa ula (Fig. 1b). The la ae we e collec ed om
he wa e by using a plas ic loop (Fig. 1c), and placed in o a plas ic
cen i uge ube con aining 0.5 ml o dis illed wa e .
In o al, he da abase consis s o 103 blood samples, om 15 di e en
hos species and 32 ick la ae samples wi h known mo he s (Table 1).
Addi ionally, we included 4 samples o ick eggs ( om known mo he s),
ha ailed o ha ch, o he e e ence ick da a (Table 1). Se en een o he
la ges ick la ae samples we e di ided in o eplica e samples o he
SIA, and hei mean δ
13
C and δ
15
N alues we e used in he da a analyses.
2.2. E hical s a emen
All blood samples ha e been ob ained om dead indi iduals. Blood
samples we e p o ided by licenced hun e s, who ha e hun ed he ani-
mals ollowing species speci ic ules and egula ions in Finland. Dead
animals sen o Finnish Food Au ho i y we e sampled o blood du ing
Table 1
Lis o hos species included in he da abase, wi h numbe o samples pe species.
“Tick” e e s o Ixodes icinus la al ick samples and/o eggs, p oduced by he
engo ged I. icinus emales wi h a known hos . Tick samples om hedgehog
included bo h la ae and egg samples (sepa a ely). Samples om oe dee and
moose con ained only unha ched eggs. “The numbe o ick hos s” e e s o he
numbe o hos indi iduals he engo ged icks we e collec ed om. The e we e
se e al I. icinus emales pe one hos indi idual in hedgehog, accoon dog and
sheep. Excep o one accoon dog and a moun ain ha e, he blood samples and
icks we e collec ed om di e en hos indi iduals.
Hos species Sample ype Numbe o ick
hos s
Blood Tick
Badge (Meles meles) 4 0 0
B own bea (U sus a c os) 2 0 0
Ca (Felis ca us) 0 4 4
Dog (Canis lupus amilia is) 20 4 4
Fox (V. ulpes) 8 0 0
Hedgehog (E inaceus eu opaeus) 3 9 (2 eggs) 3
Lynx (Lynx lynx) 5 0 0
Mink (Neo ison ison) 4 0 0
Pine ma en (Ma es ma es) 2 0 0
Raccoon dog (Nyc e eu es
p ocyonoides)
14 5 1
Squi el (Sciu us ulga is) 1 0 0
Roe dee (Cap eolus cap eolus) 8 1 (eggs) 1
Eu opean ha e (Lepus eu opaeus) 2 0 0
Moun ain ha e (Lepus imidus) 3 1 1
Moose (Alces alces) 8 1 (eggs) 1
Whi e- ailed dee (Odocoileus
i ginianus)
19 1 1
Sheep (O is a ies) 0 6 3
S. Sipa i e al.
Ticks and Tick-bo ne Diseases 15 (2024) 102252
3
au opsy. The icks we e collec ed om he hun ed indi iduals o om
pe s wi h weeze s. Thus, no animal expe imen a ion pe missions ha e
been equi ed, excep o h ee hedgehogs ha we e caugh by hand and
om which icks we e emo ed wi h weeze s. Pe mission o cap u e,
handle and emo e icks om hedgehogs was p o ided by Cen al
Finland ELY cen e : KESELY/737/2021.
2.3. Collec ing la ae om he ege a ion
La al icks we e collec ed om he ege a ionusing a d agging
me hod: A co on lannel shee (1m
2
) was d agged along ege a ion and
checked e e y 10 m. Agg ega es o mo e han 20 la al icks, i loca ed
in a small, wi hin 15 ×15 cm a ea on he shee , we e sampled o SIA. A
leas 20 la ae pe agg ega e we e collec ed om he shee using nano
ape. I la a did no o m a clea agg ega e, bu we e sp ead ac oss
la ge a ea on he shee , a sample was no collec ed. The p o ocol aimed
o ensu e ha each sample o he ield collec ed la ae was o igina ed
om one emale ick. The shee was clea ed om all icks be o e
con inuing he d agging. The ape wi h he la ae was placed in a sealed
plas ic bag and s o ed a −20 ◦C un il u he p ocessing. La al ick
collec ions we e ca ied ou in an a chipelago in sou he n Finland
(Po oo, n =45 samples), and in u ban o es s in Cen al Finland
(Jy ¨
askyl¨
a, n =6 samples).
2.4. S able iso ope analysis
To emo e he wa e om he samples, bo h blood and ick la ae
samples we e eeze d ied (Ch is Alpha 1–2LD Plus, Ma in Ch is ,
Ge many). Fu he , 0.6 mg o each sample (in ick la ae 0.6 mg pe
sample, o he o al mass o he sample i <0.6 mg) we e measu ed in o a
in cup and used o he SIA. S able iso opes o ca bon and ni ogen we e
analyzed a he Uni e si y o Jy ¨
askyl¨
a, Finland, using a The mo Fin-
nigan DELTA
plus
Ad an age con inuous low s able iso ope- a io mass
spec ome y (CF-SIRMS) connec ed o a Ca lo E ba Flash EA1112
elemen al analyze . Resul s a e exp essed using he s anda d δ no a ion
as pa s pe housand (‰) di e ence om he in e na ional s anda ds.
The e e ence ma e ials used we e in e nal s anda ds o known ela ion
o he in e na ional s anda ds o Vienna Pee Dee belemni e ( o ca bon)
and a mosphe ic N
2
( o ni ogen). P ecision was always be e han
0.16‰ o ca bon and 0.21‰ o ni ogen based on he s anda d de i-
a ion o eplica es o he s anda ds un epea edly a e e e y 5 samples
in each sequence.
2.5. S a is ical analyses
Based on he δ
13
C and δ
15
N alues ecei ed om he s able iso ope
analyses, hos blood samples we e ca ego ized in o wo dis inguishable
g oups based on he die o he species: he bi o es and ca ni-omni o es
(Fig. 2). G oup “He bi o e” included samples om oe dee , Eu opean
ha e, moun ain ha e, moose, and whi e- ailed dee , and g oup “Ca ni-
omni o e” included samples om badge , bea , dog, ox, hedgehog,
lynx, mink, pine ma en, accoon dog and squi el (Fig. 2). Fo he die
g oups o he ick la ae hos (see below), sheep was included o he
”He bi o e” and ca o he “Ca ni-omni o e” g oup, as lis ed in Table 1.
These wo g oups we e used in he s a is ical da a analyses.
To es o he e ec o he hos species die on he δ
13
C and δ
15
N
alues (sepa a ely) in hei blood, and on he ick la ae (whose mo he
consumed he blood), we used Linea mixed-e ec s models (LMM) [R
package lme4 (Ba es e al., 2015)] wi h he iso ope δ- alues as esponse
a iables, die g oup (“He bi o e” s “Ca ni-omni o e”) as ixed ac o
and he hos species and o igin (O igin =municipali y he sample is
collec ed om) as andom ac o s. As he e was no in o ma ion o he
geog aphic o igin o he dog blood samples, all dog samples had he
same alue (RV) in he andom e ec .
2.5.1. MixSIAR analyses
The p opo ions o he blood sou ces (“He bi o e” hos s s “Ca ni-
omni o e” hos s) exploi ed by he icks ( e e ence icks and ield-
collec ed icks, sepa a ely) we e assessed using he Bayesian s able
iso ope mixing model MixSIAR e . 3.1.12 (S ock e al., 2018). Values
o δ
13
C and δ
15
N om he icks (consume da a), and om he blood
samples (sou ces da a) we e included in he model wi h a ophic
disc imina ion ac o (TDF, i.e., consume -sou ce di e ence, Δ). Fo he
e e ence da a, we an wo sepa a e models o es ima e which p o ides a
be e es ima e o he used hos g oups in compa ison o he known hos s
o he e e ence icks; model 1 was ca ied ou wi hou andom ac o
(wi h p ocess and esidual e o s uc u e), and in model 2 he ick
samples’ IDs ( unning numbe ) was added as andom ac o (p ocess
e o only). Adding ID as he andom ac o p oduces indi idual die
es ima ion o each sample, showing in which g oup (he bi o e o
ca ni-omni o e) he model ca ego ized each ick sample. Median alue
0.5 was used as he h eshold alue be ween he die g oups. The ield
collec ed ick da a was analyzed only wi h model wi hou andom ac-
o s (p ocess and esidual e o s uc u e), which pe o med be e wi h
he e e ence da a (see esul s). All models we e un wi h “long” Ma ko
Chain Mon e Ca lo (MCMC) op ion (Numbe o chains=3, Chain leng h
=300,000, Bu n-in=200,000, Thinning=100). Based on he
Gelman-Rubin and he Geweke diagnos ics all models con e ged
p ope ly.
TDF was calcula ed using he equa ion: Δ=δ
c
−δ
, whe e δ
c
e e s o
he δ
13
C and δ
15
N alues o he ick la ae (consume ) and δ
o he blood
( ood sou ce) (F y, 2006). As he blood samples and icks we e mos ly
collec ed om di e en hos indi iduals, we we e no able o calcula e
TDFs o indi idual ick-hos pai s. Ins ead, we calcula ed TDFs o he
Fig. 1. Sampling o he Ixodes icinus la al icks. The ick la ae we e sampled om lannel ab ic using nano ape (a), washing o he nano ape wi h dis illed wa e
o de ach he icks (b), and collec ing he la al icks om he wa e o a mic o ube wi h an inocula ion loop (c).
S. Sipa i e al.
Ticks and Tick-bo ne Diseases 15 (2024) 102252
4
species we had bo h blood and la al ick samples in ou da abase (e en
i om di e en hos indi idual) (Table 1). In case o mo e han one
sample pe hos species, we used hei a e age as a ep esen a i e alue
o ha pa icula species and issue (blood o ick la ae). Finally, he
TDFs used in ou s udy we e gene a ed by combining he species speci ic
TDFs and calcula ing hei mean and SD. Thus, he used TDF (±SD)
be ween ou ick la ae and blood samples was 1.14 (±0.84) o δ
13
C,
and 3.26 (±1.36) o δ
15
N (Table A1). All he da a analyses we e ca ied
ou wi h R 4.2.1. (R Co e Team, 2022).
3. Resul s
3.1. Iso opes o he e e ence blood and icks
The hos blood samples o med wo dis inguishable g oups based on
he δ
13
C and δ
15
N alues o he die o he species; he bi o es and ca ni-
omni o es (Fig. 2). Fu he , he ick la ae samples o known o igin
( e e ence icks) seemed o e lec he die o hei b eeding hos (Fig 2).
The die o he hos (he bi o e s ca ni-omni o e) had signi ican e ec
on he δ
13
C and δ
15
N alues in bo h, e e ence blood and e e ence ick
la ae (Table 2); in bo h sample ypes he he bi o es had lowe alues in
δ
13
C and δ
15
N han ca ni-omni o es.
3.2. Iso opes o e e ence and ield collec ed icks in ela ion o hos
g oups
The e e ence la ae and ield collec ed la al samples showed a
g ea a ia ion in he ca bon and ni ogen iso ope alues (Fig. 3). A e
co ec ing o TDF (Δ δ
13
C =1.14 (±0.84), Δ δ
15
N =3.26 (±1.36)),
e e ence ick iso ope alues ell isually in o he co ec hos ca ego ies,
as ca. 70% o e e ence icks’ mo he s we e collec ed om ca ni-
omni o es and ca. 30% om he bi o es (Fig. 3). Especially ca bon
iso ope alues seemed o sepa a e wo g oups o icks eeding ei he on
he bi o e o ca ni-omni o e hos g oups. Simila ly, ni ogen iso ope
alues in he e e ence la ae showed g ea a ia ion bu some la al
icks known o eed on he bi o ous hos s had o e lapping δ
15
N alues
wi h hose o ca ni-omni o ious hos s. Ca bon and ni ogen iso ope
alues o he ield collec ed ick la ae seemed o e lec mos ly he
alues o he bi o ous hos s, especially o ca bon iso opes, al hough
when obse ed isually, many indi iduals ell be ween wo hos g oups,
especially in ela ion o ni ogen iso ope alues (Fig. 3).
Fig. 2. The mean iso ope δ- alues (±SD) o hos blood (blue and g een ci cles) and e e ence Ixodes icinus ick la ae samples o known hos species (Red di-
amonds. The colo o he ame e e s o he ophic g oup o he o iginal hos ; g een =he bi o e, blue =ca ni-omni o e). The species names nex o he ick samples
e e o he hos species he ick mo he ed on.
Table 2
The e ec o he hos species die (he bi o e s ca ni-omni o e) on he alues o δ
13
C and δ
15
N in hei blood, and on he Ixodes icinus ick la ae (whose mo he
consumed he blood o a known hos ), es ed wi h LMM.
Tissue Response Fixed e ec Es ima e SE - alue d p- alue
Blood δ
13
C (‰) In e cep −24.92 0.25 −101.70 59 <0.001
T ophic g oup (he bi o e) −2.73 0.37 −7.33 13 <0.001
δ
15
N (‰) In e cep 7.40 0.30 24.33 59 <0.001
T ophic g oup (he bi o e) −3.54 0.50 −7.14 13 <0.001
Tick la ae δ
13
C (‰) In e cep −22.69 0.43 −53.08 20 <0.001
T ophic g oup (he bi o e) −4.05 0.68 −5.99 8 <0.001
δ
15
N (‰) In e cep 10.56 0.60 17.70 20 <0.001
T ophic g oup (he bi o e) −3.56 0.95 −3.74 8 0.006
S. Sipa i e al.

Ticks and Tick-bo ne Diseases 15 (2024) 102252
5
3.3. MixSIAR models o iden i y icks hos s
The obse ed p opo ions o he blood sou ces exploi ed by he
e e ence icks was 0.31 o he bi o e and 0.69 o ca ni-omni o e,
calcula ed di ec ly om he e e ence da a (Fig. 4a). Two MixSIAR
models we e used o es ima e he blood sou ces in he e e ence da a and
model 1 eached highe accu acy wi h lowe a iance compa ed o he
model 2 (Fig. 4a, pos e io densi y plo s in Appendix, Fig. A1 a and b).
Model 1 (model wi hou andom ac o s) p oduced es ima ion only o
he whole popula ion, while model 2 (indi idual samples as andom
ac o ) addi ionally p oduced indi idual die es ima ions o each sam-
ple. The indi idual es ima ions om model 2 co ec ly ca ego ized he
e e ence ick la ae samples in o he co ec die g oups, when median
h eshold alue 0.5 was applied (i.e. he model es ima ed he die o he
Fig. 3. MixSIAR isospace plo s o he Ixodes icinus ick la ae samples. The isospace plo s a e ep esen ing he e e ence ick la ae samples ( ed poin s), and he ield
cap u ed ick la ae samples (blue poin s) wi h he ophic g oups o he hos s (he bi o e and ca ni-omni o e) a e TDF (Δ δ
13
C =1.14 (±0.84), Δ δ
15
N =3.26
(±1.36)) ha e been applied o he hos blood samples (la ge illed ci cles wi h SD e o s).
Fig. 4. The MixSIAR model es ima ions o he p opo ions (median wi h uppe and lowe qua iles, wi h whiske s ep esen ing he 95% Bayesian c edible in e al)
o he blood sou ces exploi ed by he (a) e e ence and (b) ield collec ed Ixodes icinus ick la ae. “T ue alue” in panel (a) e e s o he obse ed alue, calcula ed
di ec ly om he e e ence da a.
S. Sipa i e al.
Ticks and Tick-bo ne Diseases 15 (2024) 102252
6
blood sou ce (hos ) o consis o e 50% o he bi o ous o ca ni-
omni o ous ood sou ce) (Table A2).
Fo he ield collec ed ick la ae, he model es ima ed ha a median
o 81% (95% C I 70–92%) o he la al samples we e om emale icks
ha had ed on he bi o es (Fig. 4b, MixSIAR pos e io densi y plo in
Appendix Fig. A1c).
4. Discussion
In his s udy, we show ha he iso opic signa u es o I. icinus ick
la ae e lec he die o he hos o he mo he ick. We es ablished a
e e ence da abase con aining in o ma ion on he s able iso ope alues
in he blood o 15 po en ial b eeding hos s species o I. icinus, and in ick
la ae om I. icinus emales ha ha e ed on known hos s. Wi h his
no el me hod we we e able o iden i y he ophic g oup o he b eeding
hos , i.e. he hos on which he ick mo he ed, by u ilizing ield
collec ed ick la ae. To ou knowledge, his is he i s analy ical
me hod enabling he iden i ica ion o ick b eeding hos s’ ophic g oup
h ough sampling ield collec ed la ae and i s s udy whe e s able
iso ope analyses ha e been applied o de ec anso a ial aces o a
blood meal in icks.
Ou analy ical me hod based on sc eening la al icks om he ield
p o ides a di ec and unbiased me hod o es ima e he ole o di e en
hos species o ick ep oduc ion in each ecosys em. Ea lie me hods
included co ela ion analyses be ween he abundances o hos s (Gilbe
e al., 2012; Ho mees e e al., 2017) o examina ion o ick in es a ion
on wild-cap u ed o hun ed indi iduals (T¨
alleklin and Jaenson, 1997;
LoGiudice e al., 2003; B unne e al., 2011). Howe e , co ela ions
be ween hos and ick abundance may no e eal causal ela ionship. In
addi ion, examining species o he ick in es a ion is e y labo ious and
may lead o biased conclusions o he ole o hos s species gi en insu -
icien numbe s o indi iduals a e examined. Fo ins ance, high ick
in es a ion load does no necessa ily mean ha he hos species is
impo an in suppo ing ick de elopmen o ep oduc ion, as he a e o
success ul eeding a ies subs an ially be ween hos species (LoGiudice
e al., 2003; Keesing e al., 2009; B unne e al., 2011). Mo eo e , he
ick in es a ion load on hos species may a y, o ins ance, depending
on hos densi y, o he species in he hos communi y composi ion, ick
abundance, habi a , ege a ion, and clima e, being po en ially e y
con ex dependen and equi ing ecosys em speci ic es ima ions on he
ole o any speci ic hos species o icks. Ou me hod p o ides an
excellen di ec , unbiased, low e o and ela i ely low-cos ool o
iden i y he hos , albei only on ophic le el, ha suppo he ep o-
duc ion o icks in di e en ecosys ems.
Despi e he di e ences in he s udy aims, wi h ou wo k ocusing on
anso a ial and ea lie s udies on anss adial ( om p e ious li e s age)
aces o blood meals, ou esul s a e in line wi h s udies showing SIA’s
applicabili y o iden i y he sou ce o a ick’s blood meal on a ophic
le el (LoGiudice e al., 2018; Kim e al., 2021). Fo ins ance, we we e
able o ca ego ize he b eeding hos s (based on he SIA alues in blood)
in o wo dis inguishable ophic g oups; he bi o es and
ca ni-omni o es. Simila ly, he Bayesian s able iso ope mixing model
MixSIAR (S ock e al., 2018), which was used o he e e ence icks, was
able o eliably sepa a e he he bi o ous and omni-ca ni o ous sou ces
o he blood meal wi h high accu acy. In addi ion, based on ou back-
g ound in o ma ion on he mammal communi y occupying he ield si es
he la ae samples we e collec ed om, he model’s es ima ion o he
ield collec ed ick da a is also plausible. Mo eo e , he TDF calcula ed
om ou e e ence da a (blood and icks) we e ela i ely close o he
alues ound om li e a u e on I. icinus icks, despi e he alues we
epo a e he i s ones p esen ing o e -gene a ional en ichmen ,
ins ead o di ec consume -sou ce ela ionship (Scmid e al., 2011;
Heylen e al., 2019.
To iden i y he blood meal hos o p e ious li e s age on a species
le el, he adi ional DNA based blood meal analysis has p o en o be
mo e p ecise compa ed o SIA (Kim e al., 2021). DNA ma ke s in
di e en species a e unique, while he die -de i ed SI ma ke s o en
o e lap wi h species om simila ecological niches (Bal enspe ge e al.,
2015; LoGiudice e al., 2018; Heylen e al., 2019; Goe he e al., 2021;
Kim e al., 2021; Goe he and Tel o d III, 2022), making species-le el
iden i ica ion di icul , o e en impossible. Fu he , he die o many
omni o ous hos species a ies be ween seasons. Fo example, b own
bea s ea mo e e eb a es and insec s in sp ing and summe , while
swi ching o mo e plan -based die du ing au umn (S ense e al., 2016).
Fo some species, he human ac i i y can s ongly a ec hei die , e.g.
ia eeding squi els and hedgehogs in u ban a eas. Howe e , he DNA
analysis has i s limi a ions oo; i is sensi i e o con amina ions, and
mos o all, i canno de ec anso a ial aces o blood meal because no
hos DNA is de ec able in he o sp ing (G´
omez-Díaz and Figue ola,
2010; Kim e al., 2021). Thus, despi e he lack o p ecision in SIA, i
p o ides a powe ul, complemen a y ool in ick hos iden i ica ion
( ophic g oup). When combined wi h backg ound in o ma ion on spe-
cies composi ion in a pa icula ecosys em, a he accu a e b eeding
hos es ima ions can be eached. Fo example, he ield collec ed ick
samples used in ou s udy we e mos ly om an a chipelago (Po oo,
Finland) wi h e y abundan ungula e communi y ( oe dee ,
whi e- ailed dee and moose), accompanied by a s able communi y o
mesop eda o s such as accoon dogs and oxes. The MixSIAR model
es ima ed ha ca. 81% o he b eeding hos s we e he bi o es, and 19%
ca ni-omni o es. The es ima ion e lec s well he obse ed species
composi ion in he a ea, as well as he p e ailing assump ion o dee
being one o he main b eeding hos s o Ixodes icks in Eu ope (Vo
e al., 2010; Medlock e al., 2013; Ho mees e e al., 2016; Mys e ud
e al., 2021).
While he impo ance o ungula es as b eeding hos s o icks seems
undispu ed, he e a e documen a ions o e.g. islands in Sweden wi h
es ablished ick popula ion, while comple ely lacking ungula es (Jaen-
son & T¨
alleklin , 1996). The e, he ick popula ion is main ained by
ha es. Fu he , he e is g owing e idence o many u ban si es, such as
pa ks and g eenspaces, becoming ho spo a eas o icks (Hans o d
e al., 2017, 2022; Cayol e al., 2018; Klemola e al., 2019; So munen
e al., 2020). The hos species d i ing ick ep oduc ion in many o hese
u ban si es is ye o be disco e ed, o which he SIA me hod demon-
s a ed he e p o ides an excellen ool. This in o ma ion is also essen ial
om he aspec o human heal h, as exposu e o icks, and hence, TBDs,
can be pa icula ly high in hese si es, compa ed o less densely popu-
la ed u al a eas.
The me hod in ou s udy was ca ied ou using only one species,
I. icinus, howe e , he me hod should be applicable o all pa asi ic ha d
ick species. Conside ing he global abundance and se e i y o ec o -
bo ne zoono ic in ec ions de i ed by a ick bi e, such as Lyme disease,
ick-bo ne encephali is (TBE) and anaplasmosis, an e icien and eliable
me hod o s udy he mechanisms behind he complex hos - ec o -
pa hogen in e ac ions d i ing icks and TBDs is o high ele ance.
Fu he , he SIAs abili y o de ec anso a ial aces o a blood meal, as
demons a ed he e, could po en ially be applied also on o he in e e-
b a e pa hogen ec o species, such as mosqui oes (Rasgon, 2008).
Fu he s udies a e wa an ed o imp o e he accu acy and p ecision,
bu also o make he me hod mo e use - iendly. Fo ins ance, wi h icks,
each sample should include 20 ick la ae a minimum. To inc ease he
likelihood o all he la ae o igina ing om he same emale, he ield
collec ed indi iduals need o be clus e ed as a clea agg ega e on he
shee o be accep ed as one sample. Thus, in he u u e, a s able iso ope
analysis op imized o smalle sample size would imp o e he p ac i-
cali y o he me hod (Langel and Dyckmans, 2014). To imp o e he
eliabili y, po en ial spa io empo al di e ences in he iso ope alues
p esen in he hos blood wi hin same species o species g oups need o
be conside ed and examined. Also measu emen s o accu a e ophic
en ichmen alues a e pi o al, as inco ec alues can signi ican ly
dis o he ou come (Bond and Diamond, 2011). Compound-speci ic
s able iso ope analysis (CSIA) o mac omolecules, such as a y acids
and amino acids, could p o ide means o dealing wi h a iabili y and
S. Sipa i e al.
Ticks and Tick-bo ne Diseases 15 (2024) 102252
7
o e lapping alues o hos species. Unlike in bulk iso ope analysis (as
used he e), speci ic mac omolecules may di e in syn hesis pa hway o
hei ou ing be ween ick hos species and hei die a y sou ces possibly
p o iding mo e dis inc iso ope a ios (Komonen e al., 2022). This could
allow dis inc ion o ick hos s in species o consume g oup le el. As a
collabo a i e e o , building up an ex ensi e, global and upda ing open
access da abase con aining in o ma ion on he blood SI alues and
species-speci ic ophic en ichmen ac o s o e a ious axa and spe-
cies, and o e di e en geog aphical loca ions as well as seasons should
be encou aged. This would p o ide a powe ul asse , bo h o ecological
and public heal h ela ed s udy ques ions.
CRediT au ho ship con ibu ion s a emen
Saana Sipa i: Me hodology, Da a cu a ion, Fo mal analysis, W i ing
– o iginal d a , W i ing – e iew & edi ing. Mikko Kiljunen: Concep-
ualiza ion, Da a cu a ion, Fo mal analysis, W i ing – e iew & edi ing.
Minna Nylund: Da a cu a ion, W i ing – e iew & edi ing. E a R.
Kallio: Concep ualiza ion, Me hodology, Da a cu a ion, W i ing – e-
iew & edi ing.
Decla a ion o Compe ing In e es
The au ho s con i m no con lic s o in e es s.
Da a a ailabili y
The aw da a and he codes a e p o ided as supplemen a y ma e ial.
Acknowledgmen s
We a e g a e ul o nume ous hun e s o p o iding blood samples
om hun ed indi iduals and o pe owne s o deli e ing icks o us. In
addi ion, we would like o hank P o . An i Oksanen and Ou i Simola
om Finnish Food Sa e y Au ho i y o hei e o o coo dina e samples
o us. We would like o hank also Saija Vuo enmaa and Nina Honkanen
o hei help wi h he sample p epa a ions o SIA. Funding om he
Academy o Finland (G an Nos 329332, 335651 and 329326 o E a R.
Kallio).
Supplemen a y ma e ials
Supplemen a y ma e ial associa ed wi h his a icle can be ound, in
he online e sion, a doi:10.1016/j. bdis.2023.102252.
Appendix
Appendix A: Fig. A1 p esen s he MixSIAR pos e io densi y plo s o
he e e ence da a models 1 and 2, and o he ield collec ed da a.
Table A1 p esen s he ophic disc imina ion ac o s o δ13C and δ15N
in la al Ixodes icks calcula ed om ou da a, and o e e ence, all he
TDF alues epo ed o Ixodes icks om ea lie li e a u e. In Table A2
we ha e lis ed all he indi idual die es ima ions (median wi h 95%
Bayesian c edible in e al) o each sample om he MixSIAR model 2.
Re e ences
Bal enspe ge , A.P., Hue mann, F., Hagelin, J.C., Welke , J.M., 2015. Quan i ying
ophic niche spaces o small mammals using s able iso opes (δ15N and δ13C) a wo
scales ac oss Alaska. Can. J. Zool. 93, 579–588.
Ba es, D., M¨
achle , M., Bolke , B.M., Walke , S.C., 2015. Fi ing linea mixed-e ec s
models using lme4. J. S a . So w. 67, 1–48.
Bond, A.L., Diamond, A.W., 2011. Recen Bayesian s able-iso ope mixing models a e
highly sensi i e o a ia ion in disc imina ion ac o s. Ecol. Appl. 21, 1017–1023.
B unne , J.L., Cheney, L., Keesing, F., Killilea, M., LoGiudice, K., P e i ali, A., Os eld, R.
S., 2011. Mol ing Success o Ixodes scapula is Va ies Among Indi idual Blood Meal
Hos s and Species. J. Med. En omol. 48, 860–866.
Cayol, C., J¨
a¨
askel¨
ainen, A., Koskela, E., Ky ¨
ol¨
ainen, S., Mappes, T., Siukkola, A., 2018.
Sympa ic Ixodes- ick species: pa e n o dis ibu ion and pa hogen ansmission
wi hin wild oden popula ions. Sci. Rep. 8, 16660.
Cayol, C., Koskela, E., Mappes, T., Siukkola, A., Kallio, E.R., 2017. Tempo al dynamics o
he ick Ixodes icinus in no he n Eu ope: epidemiological implica ions. Pa asi .
Vec o s 10, 1–11.
Collini, M., Albonico, F., Hau e, H.C., Mo a ino, M., 2015. Iden i ying he las
bloodmeal o ques ing sheep ick nymphs (Ixodes icinus L.) using high esolu ion
mel ing analysis. Ve . Pa asi ol. 210, 194–205.
Eisen, L., S a o d, K.C., 2021. Ba ie s o e ec i e ick managemen and ick-bi e
p e en ion in he Uni ed S a es (Aca i: ixodidae). J. Med. En omol. 58, 1588–1600.
Es ada-Pe˜
na, A., De La Fuen e, J., 2014. The ecology o icks and epidemiology o ick-
bo ne i al diseases. An i i . Res. 108, 104–128.
Fab i, N.D., Sp ong, H., Ho mees e , T.R., Hees e beek, H., Donna s, B.F., Widemo, F.,
Ecke, F., C omsig , J.P.G.M., 2021. Wild ungula e species di e in hei con ibu ion
o he ansmission o Ixodes icinus-bo ne pa hogens. Pa asi . Vec o s 14, 1–15.
F ancische i, I.M., Sa-Nunes, A., Mans, B.J., San os, I.M., Ribei o, J.M., 2009. The ole o
sali a in ick eeding. F on . Biosci. 14, 2051.
F y, B., 2006. S able iso ope ecology. Sp inge , New Yo k.
Gilbe , L., Ma ey, G.L., Ramsay, S.L., Hes e , A.J., 2012. The e ec o dee
managemen on he abundance o Ixodes icinus in Sco land. Ecol. Appl. 22,
658–667.
Goe he , H.K., Tel o d III, S.R., 2022. Limi ed capaci y o dee o se e as
zoop ophylac ic hos s o Bo elia bu gdo e i in he No heas e n Uni ed S a es.
Appl. En i on. Mic obiol. 88, e00042. -22.
Goe he , H.K., Ma he , T.N., Buch hal, J., Tel o d III, S.R., 2021. Re o ansposon-based
blood meal analysis o nymphal dee icks demons a es spa io empo al di e si y o
Bo elia bu gdo e i and Babesia mic o i ese oi s. Appl. En i on. Mic obiol. 87,
e02370. -20.
G´
omez-Díaz, E., Figue ola, J., 2010. New pe spec i es in acing ec o -bo ne in e ac ion
ne wo ks. T ends Pa asi ol. 26, 470–476.
Hans o d, K.M., Fon ille, M., Gillingham, E.L., Coipan, E.C., Pie zsch, M.E., K awczyk, A.
I., Vaux, A.G.C., Cull, B., Sp ong, H., Medlock, J.M., 2017. Ticks and Bo elia in
u ban and pe i-u ban g een space habi a s in a ci y in sou he n England. Ticks Tick
Bo ne Dis. 8, 353–361.
Hans o d, K.M., Wheele , B.W., Tschi en, B., Medlock, J.M., 2022. Ques ing Ixodes
icinus icks and Bo elia spp. in u ban g een space ac oss Eu ope: a e iew.
Zoonoses Public Heal h 69, 153–166.
Heylen, D., Schmid , O., Dau el, H., Ge n, L., Kampen, H., New on, J., G ay, J., 2019.
Hos iden i ica ion in un ed icks om s able iso ope composi ions (δ13C and δ15N).
Med. Ve . En omol. 33, 360–366.
Ho mees e , T.R., Coipan, E.C., Van Wie en, S.E., P ins, H.H.T., Takken, W., Sp ong, H.,
2016. Few e eb a e species domina e he Bo elia bu gdo e i s.l. li e cycle.
En i on. Res. Le . 11, 043001.
Ho mees e , T.R., Sp ong, H., Jansen, P.A., P ins, H.H.T., an Wi en, S.E., 2017. Dee
p esence a he han abundance de e mines he popula ion densi y o sheep ick,
Ixodes icinus, in Du ch o es s. Pa asi . Vec o s 10, 1–8.
Humai , P.F.O., Doue , V.R., Mo a´, F.M., Cadenas, M., Schouls, L.M., Van De Pol, I.,
Ge n, L., 2007. Molecula iden i ica ion o bloodmeal sou ce in ixodes icinus icks
using 12S DNA as a gene ic ma ke . J. Med. En omol. 44, 869–880.
Jaenson, T.G., T¨
alleklin , L., 1996. Lyme bo eliosis spi oche es in Ixodes icinus (Aca i:
ixodidae) and he a ying ha e on isola ed islands in he Bal ic Sea. J. Med. En omol.
33, 339–343.
Jaenson, T.G.T., Jaenson, D.G.E., Eisen, L., Pe e sson, E., Lindg en, E., 2012. Changes in
he geog aphical dis ibu ion and abundance o he ick Ixodes icinus du ing he pas
30 yea s in Sweden. Pa asi . Vec o s 5, 1–15.
Jah a i, S., Ruy s, S.C., F aze -Mendelewska, E., Jaa sma, R., Ve heyen, K., Sp ong, H.,
2017. Mel ing po o ick-bo ne zoonoses: he Eu opean hedgehog con ibu es o he
main enance o a ious ick-bo ne diseases in na u al cycles u ban and subu ban
a eas. Pa asi es Vec o s 10, 1–9.
Jones, K.E., Pa el, N.G., Le y, M.A., S o eyga d, A., Balk, D., Gi leman, J.L., Daszak, P.,
2008. Global ends in eme ging in ec ious diseases. Na u e 451, 990–993.
Jo e, S., Vanwambeke, S.O., Viljug ein, H., Isaksen, K., K is o e sen, A.B.,
Woldehiwe , Z., Johansen, B., B un, E., B un-Hansen, H., Wes e mann, S., La sen, I.
L., Y ehus, B., Ho shagen, M., 2014. Clima e and en i onmen al change d i es
Ixodes icinus geog aphical expansion a he no he n ange ma gin. Pa asi es
Vec o s 7, 1–14.
Jo e, S., Viljug ein, H., Ho shagen, M., B un-Hansen, H., K is o e sen, A.B., Nygå d, K.,
B un, E., O esen, P., Sæ ik, B.K., Y ehus, B., 2011. Mul i-sou ce analysis e eals
la i udinal and al i udinal shi s in ange o Ixodes icinus a i s no he n dis ibu ion
limi . Pa asi es Vec o s 4, 1–11.
Keesing, F., B unne , J., Due , S., Killilea, M., LoGiudice, K., Schmid , K., Vuong, H.,
Os eld, R.S., 2009. Hos s as ecological aps o he ec o o Lyme disease. P oc. R.
Soc. B 276, 3911–3919.
Kilpa ick, M.A., Dobson, A.D.M., Le i, T., Salkeld, D.J., Swei, A., Ginsbe g, H.S.,
Kjem up, A., Padge , K.A., Jensen, P.M., Du land, F., Ogden, N.H., Duik-Wasse , M.
A, 2017. Lyme disease ecology in a changing wo ld: concensus, unce ain y and
c i ical gaps o imp o ing con ol. Philos. T ans. R. Soc. B 372, 20160117.
Kim, H.J., Hame , G.L., Hame , S.A., Lopez, J.E., Teel, P.D., 2021. Iden i ica ion o hos
bloodmeal sou ce in o ni hodo os u ica a dug`
es (Ixodida: a gasidae) using DNA-
based and s able iso ope-based echniques. F on . Ve . Sci. 8, 620441.
Kjæ , L.J., Soleng, A., Edga , K.S., Linds ed , H.E.H., Paulsen, K.M., And eassen, Å.K.,
Ko slund, L., Kjelland, V., Sle an, A., S uen, S., Kjellande , P., Ch is ensson, M.,
Te ¨
a ¨
ainen, M., Baum, A., Kli gaa d, K., Bødke , R., 2019. P edic ing and mapping
human isk o exposu e o ixodes icinus nymphs using clima ic and en i onmen al
da a 2016. Eu osu eillance 24, 1800101.
S. Sipa i e al.
Ticks and Tick-bo ne Diseases 15 (2024) 102252
8
Klemola, T., So munen, J.J., Mojze , J., M¨
akel¨
a, S., Ves e inen, E.J., 2019. High ick
abundance and di e si y o ick-bo ne pa hogens in a Finnish ci y. U ban Ecosys .
22, 817–826.
Komonen, A., To niainen, J., Kiljunen, M., 2022. S able iso opes in moni o ing e es ail
a h opods. F on . Ecol. E ol. 10, 969595.
Laaksonen, M., Klemola, T., Feu h, E., So munen, J.J., Puis o, A., M¨
akel¨
a, S.,
Pen inen, R., Ruohom¨
aki, K., H¨
anninen, J., S¨
a¨
aksj¨
a i, I.E., Vuo inen, I., Sp ong, H.,
Hy ¨
onen, J., Ves e inen, E.J., 2018. Tick-bo ne pa hogens in Finland: compa ison o
Ixodes icinus and I. pe sulca us in sympa ic and pa apa ic a eas. Pa asi . Vec o s
11, 1–13.
Laaksonen, M., Sajan i, E., So munen, J.J., Pen inen, R., H¨
anninen, J., Ruohom¨
aki, K.,
S¨
a¨
aksj¨
a i, I., Ves e inen, E.J., Vuo inen, I., Hy ¨
onen, J., 2017. C owdsou cing-based
na ionwide ick collec ion e eals he dis ibu ion o Ixodes icinus and I. pe sulca us
and associa ed pa hogens in Finland. Eme g. Mic obes In ec . 6, 1–7.
Langel, R., Dyckmans, J., 2014. Combined 13C and 15N iso ope analysis on small
samples using a nea -con en ional elemen al analyze /iso ope a io mass
spec ome e se up. Rapid Commun. Mass Spec om. 28, 1019–1022.
LoGiudice, K., Ku chena, K., Ch is ophe , K., Sco , N., 2018. Explo a ion o s able
iso ope analysis o ick hos iden i ica ion. Ticks Tick Bo ne Dis. 9, 151–154.
LoGiudice, K., Os eld, R.S., Schmid , K.A., Keesing, F., 2003. The ecology o in ec ious
disease: e ec s o hos di e si y and communi y composi ion on lyme disease isk.
P oc. Na l. Acad. Sci. U. S. A. 100, 567–571.
Mannelli, A., Be olo i, L., Ge n, L., G ay, J., 2012. Ecology o Bo elia bu gdo e i Sensu
La o in Eu ope: ansmission dynamics in mul i-hos sys ems, in luence o molecula
p ocesses and e ec s o clima e change. FEMS Mic obiol. Re . 36, 837–861.
Medlock, J.M., Hans o d, K.M., Bo mane, A., De dako a, M., Es ada-Pe˜
na, A., Geo ge, J.
C., e al., 2013. D i ing o ces o changes in geog aphical dis ibu ion o Ixodes
icinus icks in Eu ope. Pa asi . Vec o s 6, 1–11.
Mo a´nmo a´, F., Cadenas, M., Rais, O., Humai , P.F.O., Doue , V.R., Mo e , J., Ge n, L.,
2007. Iden i ica ion o hos bloodmeal sou ce and bo elia bu gdo e i sensu la o in
ield-collec ed ixodes icinus icks in Chaumon (Swi ze land). J. Med. En omol. 44,
1109–1117.
Mys e ud, A., Hügli, C., Viljug ein, H., 2021. Tick in es a ion on medium–la ge-sized
mammalian hos s: a e all equally sui able o Ixodes icinus adul s? Pa asi . Vec o s
14, 1–6.
Nijho , A.M., Bodaan, C., Pos igo, M., Nieuwenhuijs, H., Ops eegh, M., F anssen, L.,
Jebbink, F., Jongejan, F., 2007. Ticks and associa ed pa hogens collec ed om
domes ic animals in he Ne he lands. Vec o Bo ne Zoono ic Dis. 7, 585–596.
Pe ulionien˙
e, A., Radziˇ
sauskien˙
e, D., Amb ozai is, A., ˇ
Caplinskas, S., Paulauskas, A.,
Venalis, A., 2020. Epidemiology o lyme disease in a highly endemic Eu opean Zone.
Medicina 56, 15.
Q ille , L., Risnes-Olsen, N., Bæ um, K.M., Meisingse , E.L., Loe, L.E., Y ehus, B.,
Viljug ein, H., Mys e ud, A., 2013. Landscape le el a ia ion in ick abundance
ela i e o seasonal mig a ion in ed dee . PLoS ONE 8, e71299.
R Co e Team. 2022. R: A language and en i onmen o s a is ical compu ing. R
Founda ion o S a is ical Compu ing, Vienna, Aus ia. URL h ps://www.R-p ojec .
o g/.
Randolph, S.E., 2004. Tick ecology: p ocesses and pa e ns behind he epidemiological
isk posed by ixodid icks as ec o s. Pa asi ology 129, S37–S65.
Rasgon, J.L., 2008. S able iso ope analysis can po en ially iden i y comple ely-diges ed
bloodmeals in mosqui oes. PLoS ONE 3, e2198.
Rizzoli, A., Hau e, H.C., Ca pi, G., Vou c’h, G.I., Ne ele , M., Ros`
a, R., 2011. Lyme
bo eliosis in Eu ope. Eu oSu eillance 16, 19906.
Schmid , O., Dau el, H., New on, J., G ay, J.S., 2011. Na u al iso ope signa u es o hos
blood a e eplica ed in moul ed icks. Ticks Tick Bo ne Dis. 2, 225–227.
Shaw, S.E., Day, M.J., Bi les, R.J., B ei schwe d , E.B., 2001. Tick-bo ne in ec ious
diseases o dogs. T ends Pa asi ol. 17, 74–80.
So munen, J.J., Kulha, N., Klemola, T., M¨
akel¨
a, S., Vesilah i, E.M., Ves e inen, E.J., 2020.
Enhanced h ea o ick-bo ne in ec ions wi hin ci ies? Assessing public heal h isks
due o icks in u ban g een spaces in Helsinki, Finland. Zoonoses Public Heal h 67,
823–839.
So munen, J.J., M¨
akel¨
a, S., Klemola, T., Alale, T.Y., Ves e inen, E., 2023. Voles, sh ews
and ed squi els as sou ces o ick blood meals and ick-bo ne pa hogens on an
island in sou hwes e n Finland. Ticks Tick-Bo ne Dis. 14, 102134.
S ense , N.E., Lu næs, P.N., Bja nad´
o i , V., Dahle, B.R., Fossum, K.H.I., Jigs ed, P.,
Swenson, J.E., 2016. Seasonal and annual a ia ion in he die o b own bea s u sus
a c os in he bo eal o es o Sou hcen al Sweden. Wildl. Biol. 22, 107–116.
S ock, B.C., Jackson, A.L., Wa d, E.J., Pa nell, A.C., Phillips, D.L., Semmens, B.X., 2018.
Analyzing mixing sys ems using a new gene a ion o Bayesian ace mixing models.
Pee J 2018, e5096.
Sykes, R.A., Makiello, P., 2017. An es ima e o Lyme bo eliosis incidence in Wes e n
Eu ope. J. Public Heal h 39, 74–81.
T¨
alleklin , Jaenson, 1997. In es a ion o mammals by Ixodes icinus icks (Aca i:
ixodidae) in sou h-cen al Sweden. Exp. Appl. Aca ol. 21, 755–771.
Vo , T., Ki ne , C., Hagedo n, P., Nied ig, M., Rühe, F., 2010. Tick bu den on Eu opean
oe dee (Cap eolus cap eolus). Exp. Appl. Aca ol. 51, 405–417.
Wodecka, B., Michalik, J., Lane, R.S., Nowak-Chmu a, M., Wie zbicka, A., 2016.
Di e en ial associa ions o Bo elia species wi h Eu opean badge s (Meles meles) and
accoon dogs (Nyc e eu es p ocyonoides) in wes e n Poland. Ticks Tick-Bo ne Dis. 7,
1010–1016.
S. Sipa i e al.