69
En i onmen al DNA-based de ec ion o Fascioloides magna
and Galba unca ula: a non-in asi e app oach o moni o ing
in asi e pa asi es
Ami eza Va zandi1, S e ania Zane 1, Elisa Rubele1, Anna T isciuoglio1, Ezio Fe oglio1
1 Depa men o Ve e ina y Sciences, Uni e si y o Tu in, La go B accini 2, G ugliasco, TO, I aly
Co esponding au ho : Ami eza Va zandi ([email p o ec ed])
Copy igh : © Ami eza Va zandi e al.
This is an open access a icle dis ibu ed unde
e ms o he C ea i e Commons A ibu ion
License (A ibu ion 4.0 In e na ional – CC BY 4.0).
Resea ch A icle
Abs ac
In asi e alien species, pa icula ly mic obial pa hogens and pa asi es, pose signi ican h ea s o biodi-
e si y, ecosys em s abili y, economies, ood secu i y, wildli e conse a ion, and public heal h. Thei in-
oduc ion can occu h ough human-media ed ansloca ions o en i onmen al ac o s such as wea h-
e pa e ns and ex eme e en s. To p edic , p e en , and manage eme ging in ec ious diseases caused
by in asi e pa asi es, a mul idisciplina y app oach is essen ial. In asion biology ocuses on p edic ing
po en ial in asi e pa asi es be o e in oduc ion, while wildli e e e ina y medicine emphasizes ea ly
de ec ion o e ec i e p e en ion and managemen . Bo h app oaches ely on con inuous moni o ing o
in asi e species and hei hos s. The gian li e luke (Fascioloides magna) has p o en o be a highly suc-
cess ul in asi e pa asi e, expanding i s ange h ough coe olu ion wi h na i e hos s, na u al mig a ion,
human- acili a ed anspo , and en i onmen al dispe sal mechanisms like looding and wa e bo ne
ansmission. E ec i e moni o ing s a egies a e needed o de ec i s p esence and ha o species in-
ol ed in i s li e cycle as ea ly as possible. In his s udy, a h ee-mon h en i onmen al DNA (eDNA)
sc eening was conduc ed in La Mand ia Regional Pa k (I aly) o de ec F. magna and i s lymnaeid snail
in e media e hos , Galba unca ula, using wa e and soil samples. By in eg a ing su ace wa e dynam-
ics o si e selec ion, collec ing mul iple en i onmen al ma ices, and u ilizing highly sensi i e molecula
me hods (ddPCR), he s udy success ully iden i ied bo h species wi hou elying on p io biological
dis ibu ion da a, highligh ing he e ec i eness o eDNA-based su eillance o in asi e pa asi es.
Key wo ds: eDNA, in asi e alien species, uno acking
In oduc ion
In asions by alien species inc easingly h ea en biodi e si y esou ces, ecosys em se -
ices, egional economies, ood secu i y, wildli e conse a ion and public heal h. The
isk o alien species in oduc ion is g owing apidly wo ldwide due o expanding ans-
po a ion co ido s, echnological b eak h oughs, geopoli ical dynamics, land use and
clima e change, (Galil e al. 2015; Mui head e al. 2015; Seebens e al. 2015; Ea ly e
al. 2016), (Fishe e al. 2012; Ma el e al. 2014). Alien species a e ca ied along wi h
hei symbiome (symbio ic mac o- and mic obiome) including i uses, bac e ia and
o he euka yo es among hem me azoan pa asi es (Fos e e al. 2021) ha may be i -
ulen o pa hogenic o na i e hos s (Lymbe y e al. 2014; Blackbu n and Ewen 2017).
Academic edi o : Ap il Blakeslee
Recei ed:
10 Ma ch 2025
Accep ed:
13 Augus 2025
Published:
10 Oc obe 2025
Ci a ion: Va zandi A , Zane S, Rubele
E, T isciuoglio A, Fe oglio E (2025)
En i onmen al DNA-based de ec ion
o Fascioloides magna and Galba
unca ula: a non-in asi e app oach
o moni o ing in asi e pa asi es.
NeoBio a 103: 69–84. h ps://doi.
o g/10.3897/neobio a.103.152667
NeoBio a 103: 69–84 (2025)
DOI: 10.3897/neobio a.103.152667
Ad ancing esea ch on alien species and biological in asions
A pee - e iewed open-access jou nal
NeoBio a
70
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
The eme gence o in asi e alien pa hogens and he challenge o de ec hei p esence
a e anked among he op issues in a ho izon scan o how u u e in asion p ocesses and
dynamics will be unde s ood and managed (Riccia di e al. 2017).
In asi e alien pa hogens, especially pa asi es, can cause eme ging in ec ious dis-
eases in wildli e h ough human in e en ion ( ia hos o pa asi e ansloca ions)
o na u al en i onmen al e en s. Pa asi es may be in oduced independen ly o
alongside in asi e hos s. These in asi e alien pa asi es can in ec na i e wildli e o
domes ic animals, igge ing new diseases (alien- o-na i e spillo e ) and po en ial-
ly endange ing na i e species. (C owl e al. 2008), (Daszak e al. 2000; Ta aschews-
ki 2006). In asi e pa asi es mus adap o na i e hos s by o e coming key in asion
ba ie s: in oduc ion (ini ial con ac wi h na i e hos s), es ablishmen (success ul
pe sis ence in na i e hos s), and sp ead (expansion ac oss hos s o geog aphic e-
gions), simila o ee-li ing in asi e species (Lymbe y e al. 2014). Pa asi e–hos
dynamics du ing in asion s ages depend on en i onmen al ac o s like pollu an s
(Bojko e al. 2020), empe a u e changes (La e y e al. 2017), biological in a-
sions (Dunn e al. 2012), and biodi e si y shi (F aine e al. 2018). Global en-
i onmen al change, ma ked by de ia ions om baseline condi ions and ex eme
wea he (La e y e al. 2017), may enhance he es ablishmen o in asi e pa asi es
by c ea ing condi ions simila o hei na i e habi a (Hulme 2009). D as ic en-
i onmen al e en s such as looding can also lead o biogeog aphical expansion
o in asi e pa asi es by ansloca ing pa asi e and species in ol ed in i s li e cycle
beyond hei es ablishmen ange (Ma inko ić e al. 2013).
Fascioloides magna is an in asi e li e luke in Eu ope, o iginally na i e o No h
Ame ica, which in ec s wild and domes ic ungula es. I s li ecycle in ol es wild
uminan s as de ini i e hos s and pond snails (speci ically eshwa e snails wi h
igh -handed (dex al) shell coiling, such as he lymnaeid snails Galba unca ula and
Radix pe eg a) as in e media e hos s. While in ec ion in ca le is usually subclinical, i
can be a al in sheep (Howell and Williams 2020). The pa asi e was i s desc ibed in
1865 nea Tu in, I aly, in impo ed wapi is (Bassi 1875), and i emains es ablished
he e, now in ec ing a b oade ange o hos s, including ca le, ho ses, and wild boa
(Balbo e al. 1989). O he s able Eu opean oci include a eas in he Czech Republic,
Poland, and he Danube loodplain o es s ac oss Aus ia, Slo akia, Hunga y, and
C oa ia (K álo á-H omado á e al. 2011; Filip-Hu sch e al. 2022).
T adi ionally, de ini i e hos s include ed dee , allow dee , and whi e- ailed
dee , which shed eggs in eces. Wild boa a e conside ed dead-end hos s, and oe
dee we e p e iously hough o be abe an hos s ha do no suppo pa asi e
ma u a ion (Pybus 2001). Howe e , ecen indings sugges ha oe dee can now
ac as de ini i e hos s, and sika dee ha e also been con i med as such, indica ing
hos ange expansion (Konje ić e al. 2021; Rehbein and Visse 2021).
F. magna causes li e damage, educing hos heal h. I s li ecycle includes egg
ha ching in o mi acidia in mois , wa m condi ions (24–28 °C), which hen in ec
snails. Inside he snail, he pa asi e de elops in o ce ca iae, which eme ge a nigh ,
encys on aqua ic plan s as me ace ca iae, and a e inges ed by g azing animals o
comple e he cycle (Csi incsik e al. 2023). F. magna has been demons a ed o be
a e y success ul in ade since i s expansion elies on a combina ion o in insic
ac o s such as coe olu ion wi h na i e hos s and ex insic ac o s such as hos -pa -
asi e encoun e a e (na u al mig a ion o by anspo a ion h ough humans), pas-
si e ansloca ions o in e media e hos o ee-s ages o pa asi e (eggs, mi acidia,
ce ca iae and me ace ca iae), by wa e , s ong wind ( a ely), looding, adhesion in
71
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
hai s o mammals and ea he s o bi ds as well as anspo a ion o soil and plan s
o comme cial easons (Sa mann e al. 2014; Rehbein and Visse 2021).
To e ec i ely p edic , p e en , and manage eme ging epidemiological h ea s - such
as he sp ead o in asi e alien pa asi es like F. magna - a mul idisciplina y app oach is
essen ial. In asion biology con ibu es by iden i ying pa asi e ai s (mo phological,
physiological, phenological, and beha io al) ha in luence success a a ious in asion
s ages: en ainmen ( he p ocess a ec ing he p obabili y ha a pa asi e is delibe a ely
o acciden ally selec ed o anspo om he po en ial sou ce pool o pa asi es s ill in
hei na i e ange (Pyšek e al. 2020)), anspo , in oduc ion, es ablishmen , sp ead,
and impac . Special a en ion is gi en o s ages be o e in oduc ion, as ea ly de ec ion
is key o p e en ion (Violle e al. 2007), (Ba well e al. 2023). Meanwhile, wildli e
e e ina y medicine ocuses on he ea ly de ec ion o pa hogens as he mos impo an
measu e o p e en ion and managemen om he ea ly in oduc ion s age. The co e
o bo h app oaches is he moni o ing o he p esence/absence o known o candida e
(p edic ed) species and o he species in ol ed in i s li ecycle.
To achie e he ea ly de ec ion aim o a moni o ing p og am wi hin wildli e
popula ions, an in eg a ed su eillance app oach whe e da a om pa hogen de ec-
ion me ges wi h hos communi y su eillance is equi ed (Ca doso e al. 2022).
En i onmen wi h i s bio ic and abio ic componen s could se e as a uni ying
amewo k o such in eg a ed moni o ing p og ams and eDNA me hods a e able
o po en ially cap u e da a om pa hogens (e.g. in asi e pa asi es) and hos com-
muni y (i.e. hos s in ol ed in he in asi e pa asi e’s li ecycle) a ea lies possible
(ENETWILD-conso ium e al. 2023). En i onmen al DNA/RNA (eNA)-based
me hods p o iding esea che s wi h in o ma ion on species p esence wi hou he
need o cap u e o di ec obse a ion a e inc easingly used o de ec ion, in es i-
ga ion, and su eillance o pa hogens (Bass e al. 2023). None heless, applica ion
o eNA me hods in he con ex o wildli e su eillance should imp o e owa ds a
g ea e s anda diza ion o sampling and p ocessing me hods. In his con ex , eNA
s udies should mo e om ad-hoc sampling si e selec ion based on species dis ibu-
ion da a (e.g. p esence/absence o pond snails in s udies aiming a ema ode de-
ec ion) o wa d owa ds me hods wi h lowe independence o species dis ibu ion
da a and aiming a collec ion o samples con aining eNA which ep esen wide
biodi e si y allowing inc eased downs eam de ec ion p obabili y (Ca a o e al.
2021). Due o dynamic hyd ological p ocesses, eDNA samples om unning wa-
e s and unde lying soil a e assumed o ep esen a b oade biodi e si y signal om
con ibu ing a eas ups eam. Such an app oach, pa icula ly in i e ine sys ems,
has p o en aluable bu also p esen s challenges, as p io hyd ological knowledge
is essen ial o meaning ul biological in e p e a ion (Ca a o e al. 2020, 2021;
URycki e al. 2024) Sampling poin selec ion based on su ace wa e dynamics
and ele a ion maps is a p omising app oach as i showed i sel capable o landscape
mammalian auna de ec ion, e en o mos species p esen a low densi y (Lye e
al. 2021). eNA me hods a e in insically non-in asi e and low cumbe some ela-
i e o di ec sampling me hods including a oidance o sampling ha is s ess ul
and des uc i e o hos s. Howe e , hey can be u he mo e simpli ied a sample
collec ion (e.g. sampling lowe olume o wa e ) when coupled wi h high sensi i -
i y molecula de ec ion app oaches such as d ople digi al PCR. ddPCR has been
demons a ed o ou pe o m qPCR in e ms o sensi i i y pa icula ly analyzing
eDNA samples in a ious biomoni o ing se ings (Mau isseau e al. 2019) enhanc-
ing de ec ion a es by educing alse nega i e.
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NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
Success ul in asion occu ing by F. magna equi es a moni o ing e o which will
be able o de ec he p esence o he in asi e pa asi e and/o species in ol ed in i s
li e cycle con inuously and a ea lies possible. eDNA me hods a e p omising in his
ega d and o his eason we pe o med a h ee mon h sc eening o en i onmen al
samples (wa e and soil) o p esence o F. magna and G. unca ula in i s I alian oci
o La Mand ia Regional Pa k (LMRP). Fo his s udy we 1. Hypo hesized sampling
si e selec ion based on su ace wa e dynamics would educe eliance on species (hos
o in e media e hos ) dis ibu ion da a and ad-hoc sampling designs, 2. Collec ing
wa e and soil samples con inuously would gua an y con inui y o sampling h ough
ime, 3. U ili y o highly sensi i e molecula de ec ion me hods (e.g. ddPCR) would
help dec easing sampling e o by collec ing lowe sample amoun s. 4. Selec ion o
sampling poin s based on su ace wa e dynamics coupled wi h simul aneous wa e
and soil sample collec ion would inc ease he eDNA cap u e ep esen ing mo e bio-
di e si y hus inc easing he possibili y o de ec ing he a ge s o in e es .
Ma e ials and me hods
S udy a ea
“La Mand ia” Pa k is a egional p o ec ed a ea since 1978, spanning app oxima ely
6,557 ha in he no heas o he me opoli an ci y o Tu in in Piedmon Region
(I aly). The pa k ea u es an in e nal co e a ea o abou 3,125 ha, su ounded by a
30 km-long wall om he ex e nal p e-pa k a ea. Pa k’s in e nal enced-o sec ion
is home o ou ungula e species: wild boa , ed dee , oe dee , and allow dee . This
a ea is egis e ed as an Alpine si e o communi y impo ance and a Special A ea o
Conse a ion (SAC) p ese ing he mos signi ican example o lowland o es in
Piedmon in which measu es o conse a ion o na u al and semi-na u al habi a s
and wildli e a e applied o achie e he aims o Na u a 2000 ne wo k in biodi e si y
sa egua ding in Eu ope (Ba is i e al. 2019). The mean annual ain all is 938 mm
and he mean annual empe a u e is 14.8 °C. I is ecognized as a p ima y ho spo
o F. magna in Eu ope. LMRP encompasses di e se eshwa e habi a s, including
a i icial lakes, s eams, and canals. The pa k’s en i e wa e ne wo k con ibu es o
he Ce onda Ri e in i s sou he n egion, which lows h ough he in e nal a ea
and me ges wi h he S u a di Lanzo Ri e downs eam be o e joining he Po Ri e .
Sampling si e selec ion
Wa e basins and s eam segmen s we e iden i ied using he wa e shed unc ion
o he GRASS plugin in QGIS (3.34.11) on an ele a ion map o he s udy a ea
(Fig. 1) (GRASS De elopmen Team 2024). Beginning, endings and con luence
junc ions among mul iple s eam-segmen s in ela ion o he iden i ied basin we e
ini ially iden i ied h oughou he s udy a ea and subsequen ly eigh sampling
poin s we e selec ed, h ee o which (poin s 1, 2, and 8) we e loca ed ou side he
enced-o a ea, while he emaining i e (poin s 3, 4, 5, 6, and 7) we e inside
(Fig. 1). Sampling poin s we e chosen based on hei loca ion a he beginning o
a s eam segmen (e.g., Poin 1) o a he con luence o mul iple s eam segmen s
(e.g., Poin 4). The iden i ied s eam segmen s included bo h pe manen and em-
po a y lo ic wa e bodies, uno lows, and a eas o land con aining len ic wa e
bodies such as empo a y wa e holes.
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NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
A each sampling si e, a isual su ey o he landscape was conduc ed o iden-
i y he p esence o su ace wa e uno o aces o pas uno (d ied pa hs).
The s a ing poin o a uno pa h was isually iden i ied, and he en i e pa h
was examined o de e mine whe he i led o a lo ic wa e body (e.g., a s eam
o i e ) o a len ic wa e body (e.g., a pond). This sampling app oach was
p ima ily based on he hypo hesis ha su ace wa e uno se es as he main
ehicle o en i onmen al eDNA (whe he cellula o ex acellula ) anspo -
ing gene ic ma e ial h ough he landscape and deposi ing i in o downs eam
wa e bodies. The selec ed s a ing poin o he uno was ma ked wi h a wood-
en s ick, and hal o he soil sample (25 mL) was collec ed om he su ace soil
by emo ing he op 0.5 mm laye wi hin a 25 cm diame e a ound he ma ke .
The sample was hen ans e ed o a clean, nuclease- ee 50 mL alcon ube.
The emaining hal o he soil sample was collec ed a wo addi ional loca ions
along he uno pa h, a a iable dis ances om each o he , ex ending owa d
ei he a wa e body (i p esen ) o he poin whe e he uno pa h disappea ed.
Collec ion o he second hal o soil samples was conduc ed a di e en poin s
on he iden i ied uno pa h be ween each sampling e en depending on he
isible uno pa h which a ied e e y mon h. I a wa e body was p esen a
he end o he uno pa h, 200 mL o wa e was collec ed in ou sepa a e nu-
clease- ee 50 mL alcon ubes (Fig. 2). All collec ed samples we e anspo ed
in cool bags (con aining ice bags) o he labo a o y. Upon a i al o he labo-
a o y, wa e samples we e il e ed using sy inge il e s wi h wo di e en po e
sizes (0.45 µm and 0.22 µm; Me ck KGaA, Da ms ad , Ge many), each wi h
Figu e 1. S udy a ea and selec ed sampling poin s.
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NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
100 mL o collec ed wa e . The sy inge il e s we e s o ed a -20 °C un il DNA
ex ac ion. Soil samples we e kep a 4 °C o e nigh , and he soil eDNA was
ex ac ed he ollowing mo ning.
DNA ex ac ion
Soil
Soil samples we e i s homogenized and subsampled o ob ain a educed bu
ep esen a i e po ion o he o iginal sample, since he downs eam DNA ex-
ac ion equi es a mos 250 mg o soil. This s ep was no in ended o en ich
any speci ic a ge o ganism o pa asi e s age, bu solely o ob ain he equi ed
sample amoun . Fo each soil sample (ini ially in a 50 mL alcon ube), he soil
was di ided in o wo equal pa s: hal o he soil om he o iginal ube was
ans e ed o a new 50 mL nuclease- ee alcon ube, and he same was done
o he emaining hal . Molecula -g ade nuclease- ee wa e was hen added o
each ube o b ing he o al olume o 50 mL. Each ube was o exed igo -
ously o 10 s, hen allowed o s and b ie ly (5 s) so ha la ge pa icles could
se le. A e his se ling pe iod, 25 mL o he supe na an om each ube was
ca e ully aspi a ed and ans e ed in o a esh 50 mL nuclease- ee Falcon
ube. All samples unde wen cen i uga ion o 80 minu es a 4000 × g. This
p e- ea men —comp ising he addi ion o wa e , supe na an collec ion, and
ex ended cen i uga ion—was adap ed wi h modi ica ions om he DNA ex-
ac ion p o ocol desc ibed by Douche e al. 2022. Following cen i uga ion,
he supe na an was disca ded, and DNA was ex ac ed om app oxima ely
250 mg o soil sedimen using he DNeasy Powe Soil P o Ki (Qiagen, Hilden,
Ge many) acco ding o he manu ac u e ’s p o ocol.
Figu e 2. Wa e and soil samples we e collec ed a ou dis inc poin s by acing uno pa hs a each
sampling si e. Yellow ma ke s indica e soil sampling loca ions along isually iden i ied uno pa hs.
The i s poin (No. 1) ep esen s a ixed soil sampling loca ion used consis en ly h oughou he sam-
pling pe iod. Poin s 2 and 3 deno e a iable soil sampling si es, selec ed based on he isible uno
pa h a each si e and sampling e en . The blue ma ke indica es a po en ial downs eam wa e sam-
pling loca ion a he end o he iden i ied uno pa h. A ows demons a e ou mo emen di ec ion.
75
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
Wa e
eDNA ex ac ion om wa e samples was adap ed wi h modi ica ions om a p e i-
ously alida ed p o ocol in ol ing sy inge il e s, o iginally used o collec wa e om
anks con aining F. magna’s mi acidia a di e en ime poin s (Va zandi e al. 2024).
In he o iginal p o ocol, lysis bu e was used o wash and backwash he sy inge il-
e s, and DNA was ex ac ed om he esul ing liquid. In ou modi ied app oach,
he lysis bu e p o ided wi h he ex ac ion ki was used o his s ep. Sy inge il e s
(0.22 µm o 0.45 µm) we e placed on he op o 1.7 mL mic ocen i uge ubes, and
200 µL o PW1 bu e om he DNeasy Powe Wa e Ki (Qiagen, Hilden, Ge ma-
ny) was added o he il e memb ane ia pipe ing. A e a 5-minu e incuba ion,
he liquid was aspi a ed om he opposi e side o he il e using a pipe e. This
memb ane-washing s ep was epea ed once mo e wi hou he incuba ion pe iod. Ad-
di ionally, wo ounds o backwashing we e pe o med by e e sing he liquid inpu
di ec ion, again wi hou he incuba ion pe iod. Th oughou hese s eps, he PW1
bu e was main ained a 55 °C. DNA was hen ex ac ed om he esul ing liquid
(con aining he PW1 bu e ) collec ed om he sy inge il e s using he DNeasy
Powe Wa e Ki , ollowing he manu ac u e ’s ins uc ions while omi ing he ini ial
o ex s ep in ended o pape memb ane dis up ion. Since he LMRP is known o
con ain bo h a ge o ganisms, e e y sample could po en ially yield a posi i e esul .
Conside ing ha we only collec ed samples in he ield and p ocessed hem in he
labo a o y, we included a nega i e con ol o moni o o po en ial alse posi i es.
This con ol consis ed o 100 mL o labo a o y ap wa e , il e ed h ough a 0.22 µm
sy inge il e and ex ac ed using he same p o ocol as he o he samples.
qPCR and ddPCR
qPCR eac ions we e pe o med in duplex using p ime s and p obes a ge ing ag-
men s o he ITS2 egion o ibosomal DNA, yielding amplicons o 69 base pai s o
F. magna and 82 base pai s o G. unca ula. The eac ion mix and p o ocol ollowed
hose p e iously desc ibed by Va zandi e al., using 30 ng o sample DNA as inpu . The
same p ime s and p obes we e used o ddPCR, wi h he modi ica ion o subs i u -
ing he luo opho e dye in he G. unca ula assay wi h HEX o ensu e compa ibili y
wi h ddPCR ins umen a ion. ddPCR eac ion mix we e p epa ed in a inal olume o
22 µL, including 11 µL o ddPCR Supe mix o P obes (No dUTP) (Bio ad Labo a o-
ies, CA, USA), 0.75 µM o each p ime , 0.25 µM o each p obe and 6.5 µL o sample
con aining 30 ng o inpu DNA (quan i ied using Nanod op). Fo he gene a ion o
d ople s 20 µL o eac ion was conside ed and used o he mocycling p og am in ol -
ing a 10-min hold a 95 °C, 40 cycles o 94 °C o 30 s and 60 °C o 1 min, ollowed
by 10-min hold a 98 °C o enzyme deac i a ion and a inal 30-min hold a 4 °C.
Pu i ied cloned ec o s con aining F. magna and G. unca ula amplicon inse s,
a concen a ions anging om 3 × 105 o 0.06 copies/µL, we e p epa ed o bo h
assays using he QIAGEN PCR Cloning Ki (Hilden, Ge many). The ollowing
o mula was used o calcula e he mass o he ec o -inse cons uc , which was
hen mul iplied by he desi ed copy numbe o gene a e he s anda d cu e.
m = n × 1.096 × 10–21 g/bp
whe e: n = plasmid size (bp) and m = mass .
76
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
In a p e ious s udy, we es ablished a limi o quan i ica ion (LOQ) o 0.6 copies/µL
o DNA o he qPCR assays, based on six 10- old se ial dilu ions o posi i e samples
and mul iple eplica es used o s anda d cu e de elopmen (Va zandi e al. 2024). In
he p esen s udy, we es ed posi i e samples con aining 6, 0.6, 0.3, 0.15, and 0.06 a -
ge copies/µL o plasmid DNA o compa e di ec quan i ica ion esul s ob ained ia
ddPCR wi h qPCR cycle h eshold (C ) alues. Addi ionally, we used hese samples
o de ine an ampli ude h eshold o he inclusion o posi i e d ople s (Suppl. ma e ial
1). This h eshold de e mina ion was pe o med using a single eplica e. The absolu e
quan i y o a ge genes pe µL o sample was calcula ed using he o mula below.
Whe e ddPCR copy numbe epo s he iden i ied copy numbe s in µL o e-
ac ion, eac ion inal olume was 20 µL and sample olume consis ed 6.5 µL o
eac ion’s inal olume.
Resul s
Se en samples we e excluded om he analysis: six wa e samples om sampling
poin h ee, whe e wa e bodies we e consis en ly absen h oughou he sampling
pe iod, and one soil sample om sampling poin se en, which was inaccessible in
Augus due o i s loca ion on p i a e p ope y. The la e was eplaced wi h i s ac u-
al loca ion in he subsequen mon hs. In o al, 65 samples we e ex ac ed and ana-
lyzed using ddPCR and qPCR, comp ising 23 soil samples and 42 wa e samples.
The qPCR analysis de ec ed wo posi i e samples ou o 65 (one posi i e o each
a ge assay). Howe e , quan i ica ion was no possible because he cycle h eshold
(C ) alues o F. magna (C 40 in a wa e sample il e ed wi h 0.22 sy inge il e )
and G. unca ula (C 36 in a soil sample) we e below he p e iously es ablished
limi o quan i ica ion (LOQ) o 0.6 copies/µL o plasmid DNA (Va zandi e al.
2024). In con as , posi i e con ol esul s demons a ed ha ddPCR could di ec -
ly quan i y samples wi h copy numbe s below he qPCR LOQ o app oxima ely
0.6 copies/µL. ddPCR success ully quan i ied posi i e samples es ima ed a 0.15
copies/µL, al hough he ac ual quan i ied copy numbe was highe han he es-
ima e; o example, a sample es ima ed a 0.15 copies/µL was measu ed as 0.5
copies/µL by ddPCR. None heless, he LOQ o qPCR emained a 0.6 copies/µL
o plasmid DNA (Suppl. ma e ial 1: able S1).
Subsequen ly, all samples we e es ed using he duplex ddPCR assay, which
iden i ied 12 and 17 posi i e samples o F. magna and G. unca ula, espec i ely,
using he same p ime s and p obes (Fig. 3A, B). Di ec quan i ica ion o samples
con aining 30 ng o inpu eDNA showed ha G. unca ula copy numbe s anged
om 1.7 o 2.14 in soil samples and om 1.8 o 19.8 in wa e samples (including
bo h il e ypes). Simila ly, he F. magna assay de ec ed copy numbe s anging
om 1.7 o 2 in soil samples and om 1.8 o 19.8 in wa e samples (including
bo h il e ypes).Al hough no s a is ically signi ican , an o e all dec easing end
in de ec ed copy numbe s was obse ed o e ime o bo h assays when all sample
ypes we e conside ed (Fig. 4A, B). Howe e , his end was no e iden when soil
samples we e analyzed sepa a ely (Fig. 4A, B). The highes copy numbe s we e
de ec ed exclusi ely in wa e samples, while he ange o de ec ed copy numbe s
emained low and ela i ely s able h oughou he sampling pe iod.
77
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
Figu e 3. De ec ed copy numbe s (in log scale o A. F. magna; B. G. unca ula) a e e y sampling poin du ing he h ee mon hs o all sam-
ple ypes ( he uppe numbe ) and o soil, wa e 0.22 and wa e 0.45 il e samples ( espec i ely in pa en heses). NAs indica e excluded samples.
Ou esul s demons a ed he p esence o F. magna and G. unca ula eDNA a
nea ly e e y sampling poin du ing a leas one sampling e en , excep o poin 5,
which emained consis en ly nega i e o F. magna. No ably, sampling poin s 1 and
2, loca ed ou side he enced-o a ea, we e consis en ly posi i e o F. magna and
G. unca ula, espec i ely. We de ec ed G. unca ula in bo h sample ypes (wa e and
soil) a h ee di e en sampling poin s (poin s 4, 6, and 8) ac oss wo di e en mon hs
84
NeoBio a 103: 69–84 (2025), DOI: 10.3897/neobio a.103.152667
Ami eza Va zandi e al.: eDNA-based de ec ion o in asi e pa asi e using su ace wa e dynamics and ddPCR
Pyšek P, Hulme PE, Simbe lo D, Bache S, Blackbu n TM, Ca l on JT, Dawson W, Essl F, Foxc o
LC, Geno esi P, Jeschke JM, Kühn I, Liebhold AM, Mand ak NE, Meye son LA, Paucha d A,
Pe gl J, Roy HE, Seebens H, an Kleunen M, Vilà M, Wing ield MJ, Richa dson DM (2020)
Scien is s’ wa ning on in asi e alien species. Biological Re iews o he Camb idge Philosophical
Socie y 95: 1511–1534. h ps://doi.o g/10.1111/b .12627
Rehbein S, Visse M (2021) Sika Dee (Ce us nippon) a e no “Dead-End Hos s” o he Gian Li e
Fluke, Fascioloides magna (Bassi, 1875) Wa d, 1917. Jou nal o Wildli e Diseases 58: 194–197.
h ps://doi.o g/10.7589/JWD-D-21-00004
Riccia di A, Blackbu n TM, Ca l on JT, Dick JTA, Hulme PE, Iaca ella JC, Jeschke JM, Liebhold AM,
Lockwood JL, MacIsaac HJ, Pyšek P, Richa dson DM, Ruiz GM, Simbe lo D, Su he land WJ,
Wa dle DA, Ald idge DC (2017) In asion Science: A Ho izon Scan o Eme ging Challenges and Op-
po uni ies. T ends in Ecology & E olu ion 32: 464–474. h ps://doi.o g/10.1016/j. ee.2017.03.007
Sa mann H, Hö weg C, Gaub L, Feix AS, Haide M, Walochnik J, Rabi sch W, P osl H (2014) Whe e-
om and whe eabou s o an alien: he Ame ican li e luke Fascioloides magna in Aus ia: an o e -
iew. Wiene Klinische Wochensch i 126: 23–31. h ps://doi.o g/10.1007/s00508-014-0499-3
Seebens H, Essl F, Dawson W, Fuen es N, Mose D, Pe gl J, Pyšek P, an Kleunen M, Webe E, Win-
e M, Blasius B (2015) Global ade will accele a e plan in asions in eme ging economies unde
clima e change. Global Change Biology 21: 4128–4140. h ps://doi.o g/10.1111/gcb.13021
Sindičić M, Da inack A, Bujanić M, Buga ski D, Mi če a J, Fe oglio E, Konje ić D (2023) A new
insigh in o gene ic s uc u e o Danube and I alian oci o ascioloidosis. Ve e ina y Pa asi ology
314: 109854. h ps://doi.o g/10.1016/j. e pa .2022.109854
Špakulo á M, Rajskỳ D, Sokol J, Vodňanskỳ M (2003) Gian li e luke (Fascioloides magna), an
impo an li e pa asi e o uminan s. PaRPRESS, B a isla a, Slo ak Republic.
Ta aschewski H (2006) Hos s and pa asi es as aliens. Jou nal o Helmin hology 80: 99–128. h ps://
doi.o g/10.1079/joh2006364
URycki DR, Ki ane AA, A ono R, A ila CC, Blackman RC, Ca a o L, E a d O, Good SP,
Hoyos J. DC, López-Rod íguez N, Mo a D, Schadewell Y, Schilling OS, Cepe ley NC (2024) A
new low pa h: eDNA connec ing hyd ology and biology. WIREs. Wa e 11: e1749. h ps://doi.
o g/10.1002/wa 2.1749
Va zandi A, Zane S, Rubele E, Occhibo e F, Vada R, Bena i F, Fe oglio E (2024) De elopmen o
a qPCR Duplex Assay o simul aneous de ec ion o Fascioloides magna and Galba unca ula in
eDNA samples: Moni o ing beyond bounda ies. Science o The To al En i onmen 916: 170338.
h ps://doi.o g/10.1016/j.sci o en .2024.170338
Violle C, Na as M-L, Vile D, Kazakou E, Fo unel C, Hummel I, Ga nie E (2007) Le he concep
o ai be unc ional! Oikos 116: 882–892. h ps://doi.o g/10.1111/j.0030-1299.2007.15559.x
Supplemen a y ma e ial 1
Supplemen a y in o ma ion
Au ho s: Ami eza Va zandi, S e ania Zane , Elisa Rubele, Anna T isciuoglio, Ezio Fe oglio
Da a ype: docx
Copy igh no ice: This da ase is made a ailable unde he Open Da abase License (h p://openda a-
commons.o g/licenses/odbl/1.0/). The Open Da abase License (ODbL) is a license ag eemen
in ended o allow use s o eely sha e, modi y, and use his Da ase while main aining his same
eedom o o he s, p o ided ha he o iginal sou ce and au ho (s) a e c edi ed.
Link: h ps://doi.o g/10.3897/neobio a.103.152667.suppl1
