73
Compa ing sampling p o ocols and esul s o be e de ec
he amphibian ungal pa asi e Ba achochy ium dend oba idis
in he wild
Anne e Taugbøl1
1 No wegian Ins i u e o Na u e Resea ch (NINA), Fakkelgå den, 2624 Lillehamme , No way
Co esponding au ho : Anne e Taugbøl ([email p o ec ed])
Copy igh : © Anne e Taugbøl.
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
The pa hogenic ungus Ba achochy ium dend oba idis (Bd) causes chy idiomycosis in amphibians
ac oss he wo ld, inc easing he isk o popula ion declines and species ex inc ions. Using a adi ional
sampling app oach wi h aps, g ea c es ed new s (T i u us c is a us) and smoo h new s (Lisso i on
ulga is) ha e been sampled in eigh ponds o popula ion es ima es since 2013. The p esence o Bd
has been in es iga ed by en i onmen al DNA (eDNA) in he ponds since 2017, bu esul s ha e o en
a ied be ween sampling me hods used du ing he same sampling pe iod. By compa ing esul s om
pond wa e using wo il e po e sizes (0.45 and 2.0 µm) and om amphibians ( il e ed ba hwa e , so
skin swabs, so sandpape ubbed on skin, and skin samples om be ween oes om oad ca casses),
he esul s showed ha il e ed ba hwa e samples o gen ly ubbing amphibian skin wi h so sand-
pape a e he mos eliable me hods o ob aining Bd DNA. Resul s om pond wa e il e ed h ough
he wo po e sizes did no di e signi ican ly ac oss posi i e si es. Resul s also indica ed ha a a ie y
o DNA concen a ions should be es ed in eplica ed qPCRs o ddPCRs o accoun o bo h po en-
ial inhibi ion and low le els o Bd DNA in he samples. Bo h c es ed new s and smoo h new s we e
iden i ied as in ec ed wi h a ying p e alence, bu no ends in popula ion declines we e obse ed o
any o he species du ing he sampling yea s 2013–2024. Howe e , he numbe o c es ed new s in
he moni o ing ponds was al oge he low and some imes spo adic, and ponds wi h highe numbe s
o c es ed new s should be included in u u e s udies on popula ion ends in in ec ed popula ions.
Key wo ds: Amphibians, Bd, eDNA, alse nega i es, me hod de elopmen , species de ec ion
In oduc ion
In asi e alien species a e ega ded as one o he mos impo an h ea s o na i e
biodi e si y because hey can lead o local ex inc ions and he collapse o habi a
ypes (Capinha e al. 2015; Ea ly e al. 2016; S and e al. 2019; Pysek e al. 2020).
In asi e alien species a e, howe e , no always easy o de ec : mos would ecognize
an elephan in he A c ic as an animal ha does no belong he e, bu ew, i any,
would spo in asi e mic oo ganisms li ing in hei ga den – e en hough i is highly
likely ha some ha e al eady mo ed in (Lo e e al. 2016; Sco -B own e al. 2018).
The smalle he alien species, he less likely i is o be pe cei ed by humans, bu accu-
a e ecogni ion o pa hogens is c i ical o wildli e disease esea ch and conse a ion
(Mö ne e al. 2002; Bu ke e al. 2015; Ba oso e al. 2021; Wes e al. 2024).
Academic edi o :
And ew (Sandy) Liebhold
Recei ed:
26 May 2025
Accep ed:
31 Oc obe 2025
Published:
19 No embe 2025
Ci a ion: Taugbøl A (2025) Compa ing
sampling p o ocols and esul s
o be e de ec he amphibian
ungal pa asi e Ba achochy ium
dend oba idis in he wild. NeoBio a
104: 73–93. h ps://doi.o g/10.3897/
neobio a.104.160230
NeoBio a 104: 73–93 (2025)
DOI: 10.3897/neobio a.104.160230
Ad ancing esea ch on alien species and biological in asions
A pee - e iewed open-access jou nal
NeoBio a
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
Amphibians ha e been declining h oughou he wo ld since a leas he 1950s
(Houlahan e al. 2000; Finn e al. 2023), whe e he easons o hei disappea ance
a e in e ac i e and complex (Blaus ein and Kiesecke 2002; Reid e al. 2019; Mi e
al. 2023). Diseases caused by in asi e alien pa hogens a e one o he ac o s d i -
ing hese e e -lowe numbe s (Fishe e al. 2012; Ma el e al. 2014; Fishe and
Ga ne 2020; Wes e al. 2024). In isible o he human eye, he gene alis ungal
amphibian skin pa hogen Ba achochy ium dend oba idis (he ea e e e ed o as
Bd) (Longco e e al. 1999) has sp ead om i s na u al ange in Asia (O’Hanlon e
al. 2018; Sun e al. 2025) o e e y con inen inhabi ed by amphibians (G eene
e al. 2020), mos likely h ough he pe and plan ade (Fu and Waldman 2022;
Lao den-Rome o e al. 2024). Bd was i s disco e ed in Eu ope ollowing a mass
mo ali y e en in 1997 (Bosch e al. 2001) and is now p esen in mos Eu opean
coun ies (Ga ne e al. 2005; Allain and Du us 2019). In No way, Bd was i s
de ec ed om en i onmen al DNA (eDNA) in 2017 om wa e collec ed om
new ponds (Taugbøl e al. 2021).
Species de ec ion om gene ic aces in he en i onmen is inc easingly used in
ou ine species su eys (Deine e al. 2017; Fediaje ai e e al. 2021; Sande cock
e al. 2023) and is also a commonly used non-in asi e me hod o de ec ing Bd
(Ches nu e al. 2014; B annelly e al. 2020a; Taugbøl e al. 2021; E e s e al.
2025). Howe e , he applica ion o eDNA species de ec ion is no s aigh o wa d,
and sampling om wild en i onmen s has a high chance o p oducing alse neg-
a i es (Fice ola e al. 2015; Cong am e al. 2022; Noguei a e al. 2025; Taugbøl
e al. 2025). Bd li es as a spo angium in he ke a inized skin o amphibians and
sp eads ia aqua ic zoospo es ha swim o abou 2 cm (Pio owski e al. 2004).
The e o e, when a ge ing only wa e samples, he likelihood o de ec ing Bd DNA
will depend on a ious ac o s, such as collec ing wa e nea in ec ed hos s ha
a e ac i ely shedding he pa hogen, he a e age in ec ion load o hos s (Longo e
al. 2023; Ha mann e al. 2024), and he quan i y o zoospo e elease a he ime
o sampling. Swabbing has been he ecommended (Hya e al. 2007) and mos
commonly used (Shin e al. 2014) sampling p o ocol o Bd since 2007. Howe e ,
s udies ha e since ound he me hod o yield inconsis en esul s, especially o an-
imals o si es wi h low in ec ion in ensi y (Shin e al. 2014). Fo species nega i ely
a ec ed by Bd in ec ion, alid in e ence o in ec ion s a us is key o enabling apid
esponses o p e en u he pa hogen pollu ion.
Li le is known abou how Bd sp eads om si e o si e once locally in oduced
(Johnson and Spea e 2005; Liew e al. 2017; P ado e al. 2023) o wha e ec he
pa asi e has once es ablished, as no all amphibians espond equally o in ec ion
(Schloegel e al. 2006; Ryan e al. 2008; C aw o d-Ash and Rowley 2021; Ha mos
e al. 2021). One me hod o measu e po en ial e ec s o Bd in he wild is o collec
da a on bo h Bd-in ec ed and Bd- ee popula ions wi hin he same geog aphic a ea
(Pilliod e al. 2010; Taugbøl e al. 2021; Wes e al. 2024). The p ima y compa i-
son be ween in ec ed and non-in ec ed ponds in No way in 2017 concluded ha
he e was li le e idence o popula ion decline o bo h new species in Bd-in ec ed
ponds, bu his inding may ha e been due o he inclusion o alse-nega i e ponds
in he “non-in ec ed” g oup, he eby basing he model on inco ec inpu da a.
Fu he mo e, he e we e no da a on how long Bd had been p esen in he ponds,
which species we e in ec ed, o he p e alence among he new s occupying he
Bd-in ec ed ponds (Taugbøl e al. 2021). Fu he , he in ec ed No wegian ponds
we e sca e ed and in e mingled wi h Bd- ee ponds, and al hough his is com-
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
monly obse ed in o he su ey a eas (Kä emo e al. 2018; Cong am e al. 2022),
subop imal Bd de ec ion me hods could esul in alse nega i es and an o e all un-
de es ima ion o he dis ibu ion ange. When he Bd-posi i e No wegian ponds
we e esampled in 2018, only one o he i e ponds p e iously ound o be in ec ed
in 2017 es ed posi i e h ough il e ed wa e samples (wi h mo e han wo- hi ds
o qPCRs es ing posi i e). Nega i e Bd esul s om h ee o he ponds con lic ed
wi h Bd-posi i e swabs collec ed om he same ponds, s eng hening he assump-
ion ha he 2017 da ase included alse nega i es ha may ha e con ounded he
popula ion modeling esul s.
This s udy compa es how Bd de ec ion a ies wi h a ange o sampling me hods
– including il e ed wa e samples om pond wa e and amphibian ba hwa e and
swabs om new s and oads (Bu o bu o) – om wild popula ions in sou heas e n
No way. The main objec i e was o iden i y a cos -e ec i e sampling s a egy ha
minimizes alse nega i es o he ex en possible. As he unding sou ces and goals
ha e a ied ac oss yea s, he di ec compa isons ac oss me hods a e kep wi hin
species and sampling yea s when mo e han one sample ype was collec ed.
Ma e ials and me hods
S udy a ea and limi a ions o sampling
Sampling was conduc ed in he sou heas e n pa o No way (Fig. 1a) in wo a eas
whe e Bd had p e iously been de ec ed (Taugbøl e al. 2021; S and e al. 2025).
New samples we e collec ed om eigh ponds sou h o Oslo. Pond wa e was il-
e ed in all sampling yea s o mos ponds, as one o he o iginal main aims was o
check whe he ponds wi h la ge popula ions o new s also had highe le els o new
eDNA and i eDNA could he eby subs i u e moni o ing wi h adi ional apping
(Taugbøl e al. 2025). The same DNA ex ac s we e also ou inely es ed o Bd, bu
as some ponds a ied in in ec ion s a us wi hin and ac oss yea s, addi ional al e na-
i e samples we e needed o be e de e mine he in ec ion s a us o he ponds. Due
o limi ed annual unding, compa able samples wi hin a yea we e collec ed only
du ing h ee o he eigh sampling yea s om new ponds: 2018, 2023, and 2024.
Toad samples we e collec ed om ca casses ound a a oad c ossing in Ni edal
(Fig. 1b). Ob aining sampling pe mi s o issue collec ion om li e anu ans (e.g.,
oes) is inc easingly di icul . The o iginal objec i e o he oad samples p esen ed
he e was o assess oad DNA quali y ac oss se e al ex e nal sampling me hod-
ologies and o es whe he he same samples could also yield eliable esul s o
indi idual Bd in ec ion s a us (Taugbøl 2024). As DNA om issue would ep e-
sen he ela i e DNA quali y agains which al e na i e samples we e compa ed,
all samples we e collec ed om ecen ly o e un oads om Ni edal, om a oad
popula ion ound o ha e a Bd in ec ion p e alence o 80% (S and e al. 2025).
Wa e il a ion p o ocol om new ponds
Pond wa e samples we e collec ed p io o ap placemen du ing all sampling
yea s. F om each pond, 15 subsamples (collec ed 1–4 m apa ) o 0.2 L su ace-sam-
pled wa e we e pooled in o a mixed sample (Fig. 2a). Subsampling inc eases he
likelihood o il e ing a sample ha mo e closely ep esen s he pond as a whole, as
eDNA is likely o be une enly dis ibu ed (T o h e al. 2021; Cong am e al. 2022;
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
Taugbøl e al. 2025). Due o high epea abili y in eDNA esul s om new s il e ed
om he same mixed wa e sou ce in 2018 (Taugbøl e al. 2025), only one sample
o 0.5 L was il e ed h ough a 0.45 µm cellulose ni a e il e (Fig. 2a; Nalgene CN
145-0045, The mo Scien i ic) using a acuum pump (Sa o ius Mic osa e.je )
connec ed o a h ee-place mani old (Pall il e mani old) in 2018. A e il a ion,
each il e was p ese ed in a 2 mL ube con aining 1440 µL ATL bu e (Qiagen).
Based on p e ious indings o seasonal inc eases in Bd eDNA in one pond iden i ied
as posi i e in 2017 (Taugbøl e al. 2021), pond wa e was sampled and il e ed ou
o i e imes om each pond in 2018: May 12 and 23, June 8, and July 3 and 13.
Addi ionally, o a subse o ponds and sampling imes in 2018 – as well as du ing
la e sampling yea s – wa e om he mixed sample was il e ed h ough 2.0 µm
glass ibe il e s (Me ck Millipo e; Fig. 2a) using a sel -p iming pe is al ic pump.
These la ge po e-size il e s allowed il a ion o a g ea e wa e olume (app oxi-
ma ely 1–1.5 L) and we e expec ed o inc ease he likelihood o de ec ing Bd DNA
(Fossøy e al. 2020). In 2024, mixed pond wa e was il e ed h ough duplica e se s
o 0.45 µm and 2.0 µm il e s du ing h ee ime poin s in May: May 13–14, 22–23,
and 27–28. Fo all sampling yea s, nega i e con ols consis ing o lake wa e and/o
ap wa e il e ed in he ield we e included o assess con amina ion isks (0.45 µm
il e s in 2018; 2.0 µm glass ibe il e s in all o he sampling yea s).
Sampling new s wi h aps, ba hwa e , and so swabs
G ea c es ed new s and smoo h new s we e caugh using unnel aps o iginally
designed o minnows. Fo each pond, 10 aps we e sys ema ically se along he
sho eline du ing he peak b eeding season each yea and le in place o app ox-
ima ely 24 hou s. Cap u ed new s we e placed in clean con aine s, unpacked a
he si e, and illed wi h pond wa e . In 2023 and 2024, 1–1.5 L o new ba hwa e
was il e ed h ough a 2.0 µm glass ibe il e (Fig. 2b; Me ck Millipo e) using a
sel -p iming pe is al ic pump. In 2018, 2020, and 2023, new s we e indi idually
Figu e 1. Sampling si es. a. The posi ions o he sampling si es in he sou heas e n pa o No way
a e shown as a g een squa e; b. The sampling loca ions close o he capi al o No way (Oslo), whe e
he squa e ma ked “ oad c ossing” indica es he si e whe e he oad samples we e collec ed, and he
enci cled numbe s in he sou h indica e new ponds: (1) Røe gå d, (2) Ga de enga, (3) Øs e Glenne,
(4) O a s ud, (5) Bellsjødammen, (6) Toke ud, (7) Solbe g, and (8) Øs e S økken. Bo h maps we e
c ea ed wi h A cGIS (En e p ise 11, 2022) in combina ion wi h Adobe Illus a o (Adobe Inc. 2019).
No way
1
2
4
5
78
6
3
Oslo
Toad-c ossing
New -
ponds
ab
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
selec ed and swabbed epea edly o e he ee and mou h wi h a so co on swab
(Fig. 2b). All swab samples collec ed in 2018 and 2020 we e s o ed indi idually in
ma ked 1.5 mL Eppendo ubes p e illed wi h 150 µL ATL bu e . In 2023, mul-
iple swabs pe pond (up o six) we e s o ed in 5 mL Eppendo ubes con aining
4 mL ATL bu e as collec i e samples o es o Bd p esence in i e o he ponds.
All cap u ed indi iduals we e eleased back in o he ponds. Fo each pond, a ca ch
pe uni e o (CPUE) was calcula ed o each species as he numbe o animals
di ided by he p oduc o apping ime and numbe o aps. Da a on CPUE o
he sampling yea s 2013–2016 we e collec ed om De o e al. (2017).
Sampling oad ca casses
All oad samples we e collec ed in Ap il 2022. The oad sampling si e (Fig. 1b)
was chosen o wo main easons: i s , oads om he si e es ed posi i e o Bd in
2021 (S and e al. 2025); second, nume ous oadkill casual ies allowed o issue
sampling om ca casses as a compa a i e me hod o oad DNA. All obse ed li e
oads we e ca ied ac oss he oad o he pond.
A o al o six esh ca casses wi h minimal ex e nal damage we e used o collec
he ollowing samples (Fig. 2b): so co on swabs – each o wo andomly selec ed
ee was swabbed 10 imes; so sandpape (P400) – he emaining wo ee we e
each swabbed 10 imes; swim skin – a piece o webbed skin be ween he wo lon-
ges oes om one o he hind legs; and oes om ca casses. The swab, sandpape ,
and skin samples we e s o ed in 150 µL lysis bu e . Replica es o so skin swabs
we e also s o ed d y in Eppendo ubes be o e DNA ex ac ion.
The ba hwa e sample was collec ed by il e ing 0.5 L o pond wa e in which
he ca cass o each animal had spen 10–15 minu es (Fig. 2b). Fil e ed ba hwa e
was always collec ed las o a oid in luencing he esul s o he o he sampling
me hods, in case exposu e o wa e al e ed Bd de ec ion. Toad ba hwa e was il-
e ed h ough a 2.0 µm glass ibe il e wi h binde (Millipo e) using a sel -p im-
ing pe is al ic pump (Maki a USA Inc.) and Nalgene™ Single Use Analy ical Fil e
Funnels (The mo Scien i ic™). Each il e was cu in hal , olded, and s o ed in wo
Eppendo ubes p e illed wi h 1500 µL lysis bu e . All samples we e kep a oom
empe a u e be o e being p ocessed in he labo a o y.
Figu e 2. O e iew o samples. a. Wa e il e ing: wa e was collec ed om se e al si es wi hin
each pond and mixed be o e being il e ed h ough ei he (1) a 0.45 µm il e o (2) a 2.0 µm il e ;
b. Amphibian samples: (3) il e ed ba hwa e il e s pond wa e con aining amphibians o 10–15
minu es (2.0 µL); (4) so skin swabs; (5) oads only, so sandpape ; (6) oads only, skin sample om
be ween he wo longes oes o one hind leg. All sampled oads we e oadkill casual ies. Illus a ions
we e c ea ed using Powe Poin (Mic oso ) and Adobe Illus a o (Adobe Inc. 2019).
1
2
B eeding pond
3
6
Holding ba h
4
5
b
a
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
Molecula me hods
DNA om he 0.45 µm il e s collec ed om he new ponds, as well as om all
swabs collec ed om new s, was ex ac ed using he DNeasy Blood & Tissue Ki
(Qiagen) ollowing he modi ied p o ocol o Spens e al. (2017). DNA a ached o
he 2.0 µm glass ibe il e s was ex ac ed using a modi ied NucleoSpin Plan II
Midi (Mache ey-Nagel) p o ocol. DNA om all oad samples was ex ac ed using
he sal -ex ac ion me hod de eloped by Aljanabi and Ma inez (1997).
A e DNA ex ac ions, he p esence o Bd DNA was es ed using duplica e
digi al d ople polyme ase chain eac ions (ddPCRs) in 2017 and 2018, wi h de-
ails as desc ibed in Taugbøl e al. (2021). Resul s we e classi ied based on d op-
le coun s: samples wi h h ee o mo e posi i e d ople s we e conside ed posi i e,
while hose wi h one o wo d ople s we e conside ed weakly posi i e (Dobnik e
al. 2015). Quan i a i e polyme ase chain eac ion (qPCR) was un in iplica e
wi h species-speci ic p ime s o Bd, ollowing he se up desc ibed by Boyle e al.
(2004). qPCRs we e conduc ed wi h 1 ng o inpu DNA ac oss all yea s. Samples
collec ed in 2024 (new s) and 2022 ( oads) we e also es ed wi h 5 ng o inpu
DNA. Fo all uns (ddPCR and qPCR), one o mo e nega i e con ols we e in-
cluded as empla es on each pla e, using dH2O and ish DNA as empla es, along
wi h a posi i e sample o Bd DNA. None o he nega i e ield samples o echnical
con ols ampli ied Bd DNA.
S a is ical analysis
All analyses we e pe o med in R 4.0.1 (R De elopmen Co e Team 2021). Di -
e ences in indi idual Bd concen a ions o each sampling me hod we e es ed
using wo- ailed - es s and Gene al Linea Models (GLMs) implemen ed in base
R. Visualiza ion o esul s was plo ed using a combina ion o base R g aphics and
ggplo 2 (Wickham 2016). Plo s we e u he combined using Adobe Illus a o
(Adobe Inc. 2019). All da a a e a ailable in Suppl.ma e ial 1.
Resul s
De ec ion o Bd om new ponds in ela ion o CPUE
T apping da a (2013–2024) and Bd esul s (2017–2024, excep 2021) a e summa-
ized o bo h new species in Fig. 3. Apa om pond 8, no ponds es ed consis-
en ly posi i e o Bd h oughou he sampling yea s, and Bd esul s om mixed
pond wa e il e s and samples om ba hwa e and so skin swabs we e con adic-
o y o ponds 1–4 and pond 7 o a leas one sampling yea (Fig. 3, hea map).
Due o he low numbe o ponds (n = 8) and he high likelihood o alse-nega i e
esul s, ca ch pe uni e o (CPUE) o he new s is p esen ed only as s acked ba
plo s (Fig. 3) and was no compa ed s a is ically based on in ec ion s a us.
Bd de ec ion om pond wa e and new swabs in 2022
In 2018, bo h mixed pond wa e and indi idual so skin swabs we e collec ed om
he i e ponds iden i ied as Bd-in ec ed in 2017. G ea c es ed new s we e iden i-
ied as posi i e in ou o he ponds (Fig. 4a). The p e alence o Bd DNA om he
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NeoBio a 104: 73–93 (2025), DOI: 10.3897/neobio a.104.160230
Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
indi idual swabs anged om 50% in ec ed c es ed new s a pond 8 (based on he
h ee-d ople cu o ) o 90% in he same pond when including samples wi h one
o wo posi i e d ople s (Fig. 4a). Using h ee o mo e d ople s as a cu o , only
pond 1 had posi i e samples o smoo h new s, whe eas all i e ponds had ddPCRs
wi h one o wo posi i e d ople s o his species (Fig. 4b). Fil e s om pond wa e
iden i ied wo ponds as posi i e wi h h ee o mo e posi i e d ople s – pond 7 and
pond 8 – whe eas pond 4 was posi i e wi h one o wo d ople s (Fig. 4c).
Bd de ec ion om pond wa e , new swabs, and new ba hwa e in 2023
A o al o h ee sample ypes we e collec ed and compa ed o Bd de ec ion in
2023: mixed pond wa e (2.0 µm), new ba hwa e (2.0 µm), and swab collec ions
(mo e han one swab included in he same ube as a collec i e “pond sample,” col-
lec ed only om ponds 6, 7, and 8). In o al, six o he ponds es ed posi i e o Bd
in one o mo e sample ypes (Fig. 3). Mixed pond wa e iden i ied h ee ponds as
posi i e (Fig. 5): pond 6 ( i s ime de ec ed as Bd posi i e, wi h i e o six posi i e
qPCRs), pond 7 (one o six qPCRs posi i e om 1 ng o DNA inpu ), and pond
8 ( h ee o six qPCRs posi i e, all a 1 ng o DNA inpu ).
Figu e 3. Bd in ec ion s a us ac oss yea s and es ima es o new popula ions pe pond. The uppe hea map o each pond illus a es
whe he he pond was iden i ied as in ec ed (ligh pink), ma ginally in ec ed (yellow; de ined as ewe han h ee posi i e d ople s in he
ddPCRs o less han 66% posi i e qPCRs), o no in ec ed by Bd (g een), based on wo sample ypes: (1) il e s om mixed wa e samples
and (2) samples collec ed om ba hwa e and/o so skin swabs. The lowe s acked ba plo illus a es ca ch pe uni e o (CPUE) o
smoo h new (ligh beige, uppe pa ) and c es ed new (ligh b own, lowe pa ) o he eigh ponds h oughou he yea s 2013–2024.
CPUE e e s o he numbe o indi iduals caugh in each pond, adjus ed o apping e o (numbe o aps × ime in wa e ). The ho i-
zon al blue line ep esen s a CPUE o 0.2 o illus a e di e ences in popula ion es ima es be ween ponds.
2013
2014
2016
2017
2018
2019
2020
2021
2022
2023
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
2013
2014
2015
2016
2017
2018
2019
2020
2021
2022
2023
2024
2024
1
2
1
2
Pond 1 Pond 2 Pond 3 Pond 4
Pond 5 Pond 6 Pond 7 Pond 8
Smoo h new
C es ed new
Posi i e
Low de ec abili y
No de ec ion
Bd eDNA
No sampling
CPUE
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Figu e 4. Bd de ec ion om swabs and pond wa e es ed wi h ddPCR o he i e sampled ponds
in 2018. a. Resul s (in pe cen age) om so skin swabs collec ed om g ea c es ed new s, wi h pos-
i i e (o ange) and nega i e (ligh blue) ou comes; b. Same as in (a), bu o smoo h new s; c. Resul s
om he mixed pond wa e il e (0.45 µm), whe e g een = nega i e Bd esul s, yellow = one o wo
posi i e d ople s o one o he eplica es, ligh o ange = one eplica e wi h h ee o mo e posi i e
d ople s, and da k o ange = bo h eplica es wi h h ee o mo e d ople s.
The DNA concen a ion in he sample was no ound o in luence Bd de ec ion
in he qPCR eac ions (Fig. 5). When compa ing he i e sample ypes collec ed
om ponds 6, 7, and 8, he a e age amoun o DNA ex ac ed o each sample
ype anged om 33.94 ng, collec ed wi h swabs om g ea c es ed new s in pond
7, o 2,456 ng o DNA collec ed om ba hwa e om smoo h new s in pond 6
(Fig. 5). Pond 8 had an o e all highe concen a ion o Bd compa ed wi h ponds 6
and 7, whe e he sample ype wi h he highes concen a ion had a mean Cq alue
o 29.9 and was ex ac ed om swabs collec ed om smoo h new s. Mixed pond
wa e had he highes concen a ion o Bd in he samples collec ed om pond 8.
Seasonal Bd de ec ion om pond wa e and new ba hwa e in 2024
In 2024, Bd was de ec ed om mixed pond wa e in ou o he ponds a all sam-
pling imes, while wo ponds had one posi i e sample a one o he sampling imes
(ponds 1 and 2; Suppl. ma e ial 2: ig. S1). The 2.0 µm il e had sligh ly ewe
Bd-posi i e qPCRs (77%) compa ed wi h he 0.45 µm il e (94%; Suppl. ma e ial
2: ig. S2), bu his di e ence was no signi ican ( = 0.49, p = 0.62). The amoun
o DNA inpu (1 o 5 ng o DNA) p oduced signi ican ly di e en esul s o he
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Anne e Taugbøl: Me hod de elopmen wi hin budge and ime limi a ions
Figu e 5. Cq alues (duplica es, y-axis) o Bd in ela ion o he o al DNA concen a ion o he sam-
ple (x-axis) om h ee ponds – pond 6 (da k g een bo de ), pond 7 (g ay bo de ), and pond 8 (black
bo de ) – whe e he esul s a e di ided in o he i e collec ed sample ypes (in blue, b own, co al, and
g ay) es ed wi h 1 ng (diamond) and 5 ng (ci cle) o DNA inpu in he qPCRs.
0 500 1000 1500 2000 2500
25 30 35 40 45
Cq- alue
DNA-concen a ion
Pond wa e
Ba hwa e smoo h
Swabs smoo h
Ba hwa e g ea c es ed
Swabs g ea c es ed
Pond wa e
Sample ype DNA inpu
5 ng
1 ng
Loca ion
pond 7
pond 8
Pond 6
Figu e 6. De ec ion o Bd DNA om pond wa e sampled a h ee di e en imes du ing sp ing 2024, whe e wo eplica ed wa e sam-
ples we e il e ed h ough ei he 0.45 µm o 2.0 µm il e s and he isola es we e es ed wi h bo h 1 and 5 ng o DNA inpu in iplica e.
Sampling ime 1 = May 13–14, sampling ime 2 = May 22–23, and sampling ime 3 = May 27–28. Da a a e shown only o ponds 6, 7,
and 8, as hese we e he only ponds wi h consis en posi i e indings ac oss all h ee sampling imes. Pond 2 was posi i e a sampling ime
2 (only o he 2.0 µm il e ), and pond 1 was posi i e a sampling ime 3 (only o he 0.45 µm il e ).
0.45
34
36
38
40
42
44
2.0
0.45
2.0
0.45
2.0
0.45
2.0
Pond 5 Pond 6 Pond 7
Pond 8
0.45
2.0
0.45
2.0
0.45
2.0
0.45
2.0
0.45
2.0
0.45
2.0
0.45
2.0
0.45
2.0
Pond 5 Pond 6 Pond 7 Pond 8
Pond 5 Pond 6 Pond 7
Pond 8
Sampling ime 1 Sampling ime 2 Sampling ime 3
5 ng DNA
1 ng DNA
0.45 µm il e , wi h 5 ng o inpu yielding a signal a 1.3 ewe cycles compa ed
wi h 1 ng o inpu ( = 2.52, p = 0.013). When pond 8 was analyzed sepa a ely, he
2.0 µm il e ga e posi i e signals a 2.04 ewe qPCR cycles compa ed wi h he
88
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Supplemen a y ma e ial 1
Da a o he sampling yea s 2018, 2023 and 2024
Au ho : Anne e Taugbøl
Da a ype: xlsx
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.104.160230.suppl1
Supplemen a y ma e ial 2
Supplemen a y igu es
Au ho : Anne e Taugbøl
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.104.160230.suppl2
