Academic Edi o : Kenji Sai oh
Recei ed: 26 Sep embe 2024
Re ised: 20 Decembe 2024
Accep ed: 22 Decembe 2024
Published: 20 Janua y 2025
Ci a ion: Coya, R.; Rod íguez-Ruiz,
A.; Fueyo, Á.; O duna, C.; Mi alles, L.;
de Meo, I.; Pé ez, T.; Cid, J.R.;
Fe nández-Delgado, C.; Encina, L.;
e al. En i onmen al DNA and
Hyd oacous ic Su eys o Moni o ing
he Sp ead o he In asi e Eu opean
Ca ish (Silu us glanis Linnaeus, 1758)
in he Guadalqui i Ri e Basin, Spain.
Animals 2025,15, 285. h ps://doi.o g/
10.3390/ani15020285
Copy igh : © 2025 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
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A icle
En i onmen al DNA and Hyd oacous ic Su eys o Moni o ing
he Sp ead o he In asi e Eu opean Ca ish (Silu us glanis
Linnaeus, 1758) in he Guadalqui i Ri e Basin, Spain
Ru h Coya 1, Amado a Rod íguez-Ruiz 2, Ál a o Fueyo 1,3, Ca los O duna 4, Lau a Mi alles 1, Ila ia de Meo 4,
T inidad Pé ez 1, Juan Ramón Cid 4, Ca los Fe nández-Delgado 5, Lou des Encina 2,*, Yaisel J. Bo ell 1,*
and Ca los G anado-Lo encio 2
1Depa men o Func ional Biology, Gene ics, Uni e si y o O iedo, 33006 O iedo, Spain;
[email p o ec ed] (R.C.); [email p o ec ed] (Á.F.); [email p o ec ed] (L.M.);
[email p o ec ed] (T.P.)
2Depa men o Plan Biology and Ecology, Uni e si y o Se illa, 41012 Se illa, Spain; [email p o ec ed] (A.R.-R.);
[email p o ec ed] (C.G.-L.)
3En i onmen and Sus ainabili y A ea, Taxus Medio Ambien e, 33006 O iedo, Spain
4EcoFishUS Resea ch S.L.L., 41009 Se illa, Spain; [email p o ec ed] (I.d.M.);
[email p o ec ed] (J.R.C.)
5Depa men o Zoology, Uni e si y o Co doba, 14071 Co doba, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed] (L.E.); [email p o ec ed] (Y.J.B.)
Simple Summa y: In oduced in o he Eb o Basin in Spain in 1974, he in asi e Eu opean
ca ish is now p esen in almos all Spanish i e basins. This includes he Guadalqui i
Ri e , whe e i h ea ens na i e species and he ecological biodi e si y o he Doñana
Na ional Pa k. This s udy p o ides aluable in o ma ion on i s cu en dis ibu ion using
non-in asi e me hods such as hyd oacous ics and en i onmen al DNA and highligh s he
need o u gen con ol measu es.
Abs ac : The Eu opean ca ish (Silu us glanis Linnaeus, 1758) was in oduced in o he Eb o
Basin in Spain in 1974 o ec ea ional ishing. Since hen, he species has sp ead h oughou
he coun y’s i e basins, eaching he Iznája Rese oi (Guadalqui i Ri e Basin) in
2011. This a ea is o g ea ecological and economic ele ance, especially as i includes he
Doñana Na ional Pa k, one o he mos impo an na u e ese es in Eu ope. Recen ly,
he p esence o ca ish has been epo ed in he lowe eaches o he i e . In his wo k,
we used non-in asi e e ical and ho izon al hyd oacous ic su eys and en i onmen al
DNA i e sampling o un a el he ac ual dis ibu ion and dispe sal pa e n o he species
in he lowe Guadalqui i Ri e . The hyd oacous ic p o iles and he species-speci ic
de ec ions by eal- ime PCR (qPCR) and d ople digi al PCR (ddPCR) showed ha hese
non-in asi e me hods allow he de ec ion and quan i ica ion o ca ish and p o ide aluable
in o ma ion on he species’ p esence. We ha e con i med he p esence o ca ish in mos
o he s udy a ea, including downs eam a eas o he Guadalqui i Basin. The esul s
sugges he possibili y o o he isola ed in oduc ions and/o human-media ed mo emen s
o specimens, and imply ha a coo dina ed ca ish p e en ion and mi iga ion s a egy is
he e o e u gen ly needed.
Keywo ds: ea ly de ec ion; moni o ing; echosounde ; eDNA; Doñana Na ional Pa k
Animals 2025,15, 285 h ps://doi.o g/10.3390/ani15020285
Animals 2025,15, 285 2 o 20
1. In oduc ion
Aqua ic in asi e species (AIS) a e inc easingly h ea ening ecosys ems globally, caus-
ing biodi e si y imbalances and ha ing a subs an ial impac on economic en e p ises [
1
].
Among he ecological impac s, AIS can signi ican ly a ec local biodi e si y, po en ially
leading o he ex inc ion o na i e species [
2
]. These impac s can a ise om di ec bio ic
in e ac ions wi h he na i e auna, such as compe i ion and p eda ion, as well as indi ec
changes in habi a condi ions. In aqua ic en i onmen s, o ganisms a e cha ac e ised by
s ong ophic linkages ha can be al e ed by in oduced species. In asi e species in aqua ic
en i onmen s ha e s ong and ela i ely cons an ecological impac s [3].
Among in asi e species in con inen al eshwa e and low salini y (es ua ine) bodies
o wa e , he Eu opean ca ish (Silu us glanis, Linnaeus, 1758) s ands ou as he la ges
ish in Eu ope excluding he diad omous s u geon species [
4
]. Due o i s pisci o ous die ,
he ca ish poses a isk o na i e species and ecosys ems [
5
–
8
]. Ca ish we e in en ionally
in oduced in he Eb o Basin in Spain in 1974 and ha e since sp ead h oughou he
coun y’s i e basins, p ima ily in he con ex o ec ea ional ishing [
6
]. In July 2011, an
indi idual o his species was cap u ed in he Iznája Rese oi , ma king he i s eco d
o he Eu opean ca ish in oduced in o he Guadalqui i Ri e Basin (Figu e 1) [
9
]. In
2015, he species was ci ed in he Ri e a de Huel a Ri e , a ibu a y on he igh bank o
he Guadalqui i Ri e loca ed mo e han 200 km downs eam o he Iznája Rese oi
si e [10]. Since hen, he species has p og essi ely appea ed in di e en a eas o he lowe
Guadalqui i Ri e .
The lowe a ea o he Guadalqui i Ri e is home o a la ge numbe o aqua ic species
o g ea biological and economic alue, making i he mos impo an aqua ic biodi e si y
ho spo in Andalusia, Spain [
11
–
15
]. The si e co e s 50,720 hec a es o one o he la ges
we lands in Eu ope and is he las a ea o la gely undis u bed ma shland wi hin his
ecosys em [
16
,
17
]. This egion includes he lowe Guadalqui i es ua y, he sou he nmos
i e –es ua y–del a sys em in con inen al Eu ope. Inc easing human p essu e and limi ed
unde s anding o i s dynamics ha e exace ba ed socio-economic and en i onmen al con-
lic s in ecen yea s [
18
]. In addi ion, he biodi e si y o he Doñana Na ional Pa k and i s
associa ed ecosys ems is h ea ened by in ensi e ag icul u e, which causes an h opogenic
eu ophica ion o i s wa e s [
19
], as well as by high g oundwa e use, pollu ion, hyd ologi-
cal in as uc u e, clima e change, and in asi e species, which ha e signi ican ly al e ed he
ecological unc ioning o hese sys ems [
15
]. As an in asi e species, he ca ish h ea ens
biodi e si y, as well as local economies and cul u e [
20
–
22
]. Fo his eason, i is impo an
o s udy ca ish p esence, dis ibu ion, and dispe sal pa e ns in ecologically sensi i e
a eas such as he lowe Guadalqui i . This has been he objec i e o he ’STOPSILURO’
(www.s opsilu o.es, accessed on 20 Decembe 2024) p ojec , launched in 2023 and a ge ing
he de elopmen o con ol p og ams o his species ha minimise i s impac and p e en
i s sp ead in hese a eas o high ecological o socio-economic alue.
T adi ionally, aqua ic species moni o ing has depended on isual de ec ion and o en
he cap u e o o ganisms. Howe e , hese me hods a e cos ly and labou -in ensi e [
23
].
In ecen yea s, he e ha e been signi ican ad ances in echnologies o emo e and ea ly
de ec ion o in asi e species. Hyd oacous ics has p o en o be a highly sui able ool o he
quan i a i e cha ac e isa ion o ish popula ions and hei spa io- empo al dis ibu ion, as
i allows la ge olumes o wa e o be sampled wi h g ea e iciency and high speed o da a
acquisi ion, which is why i s use has inc eased in ecen decades [
8
,
24
,
25
]. Rega ding ea ly
de ec ion, en i onmen al DNA (eDNA) is conside ed one o he mos p omising analysis
and managemen ools due o i s high sensi i i y in species de ec ion [26–28].
Animals 2025,15, 285 3 o 20
Animals 2025, 15, x FOR PEER REVIEW 9 o 20
ca ish de ec ion in 13 si es (76.5%), while we ob ained 11 posi i e si es wi h ddPCR
(64.7%) om he 17 sample si es (Figu e S5). A compa ison o he esul s shows ha
76.5% o he si es we e conco dan in e ms o he p esence o ca ish eDNA be ween he
wo echniques es ed. The qPCR esul s showed an a e age o 5.5 × 105 ca ish eDNA
copies ( anging om 108.2 o 3.4 × 106). Using he ddPCR me hodology, he alues o
accep ed gene a ed d ople s we e on a e age 17,375 and anged om 12,284 o 19,592.
F om his, an a e age o 454.5 posi i e ca ish eDNA ddPCR copies we e es ima ed
( ange: 60 o 1260 copies). S a is ical analyses e ealed a signi ican posi i e co ela ion
be ween he loga i hmically ans o med copy numbe da a ob ained by qPCR and
ddPCR o ca ish en i onmen al DNA (p- alue < 0.01) (Figu e S6).
Figu e 1. De ec ion and quan i ica ion o Silu us glanis using eDNA and hyd oacous ic su eys in
he Guadalqui i Ri e Basin. (A1) Ibe ian Peninsula showing he Andalusian egion. (B1) The
Andalusian egion, showing he Guadalqui i Ri e Basin and including he Doñana Na ional Pa k
loca ion. (C1) De ailed map o he Guadalqui i Ri e Basin in he p o ince o Se illa showing he
loca ions sampled o hyd oacous ics and ca ish eDNA (qPCR and ddPCR). (C1.1) De ail o he El
Ge gal Rese oi in Se illa. (D1) De ailed map o he Iznája Rese oi in he Guadalqui i Ri e
Basin, p o ince o Có doba. In his igu e, he squa es ep esen ca ish eDNA qPCR quan i ica ion
esul s while he ci cles a ound hem ep esen ca ish eDNA ddPCR quan i ica ion esul s. Fo
bo h molecula me hods, a hea map om colo whi e o colo ed do s g adien indica es he
Figu e 1. De ec ion and quan i ica ion o Silu us glanis using eDNA and hyd oacous ic su eys in
he Guadalqui i Ri e Basin. (A1) Ibe ian Peninsula showing he Andalusian egion. (B1) The
Andalusian egion, showing he Guadalqui i Ri e Basin and including he Doñana Na ional Pa k
loca ion. (C1) De ailed map o he Guadalqui i Ri e Basin in he p o ince o Se illa showing he
loca ions sampled o hyd oacous ics and ca ish eDNA (qPCR and ddPCR). (C1.1) De ail o he El
Ge gal Rese oi in Se illa. (D1) De ailed map o he Iznája Rese oi in he Guadalqui i Ri e
Basin, p o ince o Có doba. In his igu e, he squa es ep esen ca ish eDNA qPCR quan i ica ion
esul s while he ci cles a ound hem ep esen ca ish eDNA ddPCR quan i ica ion esul s. Fo bo h
molecula me hods, a hea map om colo whi e o colo ed do s g adien indica es he numbe
o copies ob ained. Hyd oacous ic da a (agg ega e coun s) is also ep esen ed by a hea map om
whi e o g een indica ing he nume ical densi y o indi iduals g ea e han 1 m, while he numbe o
indi iduals pe s e ch is gi en nume ically in a yellow ci cle. In (C1.1) each indi idual is displayed
as a yellow do (some o hem o e lap in he middle) and whi e do s indica e he loca ion o he wo
nega i e wa e samples aken o eDNA analyses.
The po en ial o hyd oacous ic me hodology as a use ul ool o ish species manage-
men and con ol p og ammes, including exo ic species, has been shown [
8
], al hough
hyd oacous ic equipmen cu en ly lacks he abili y o iden i y species o he han by body
size o habi a dis ibu ion. Howe e , in ecosys ems whe e he e is a clea di e en ia ion o
species based on hei size, o whe e ish communi ies a e monospeci ic o he e is clea
Animals 2025,15, 285 4 o 20
habi a seg ega ion acco ding o species, mode n hyd oacous ic echniques ha e been used
as use ul e alua ion ools o ish species [
24
,
25
,
29
]. Rega ding he Eu opean ca ish, he
possibili y o using hyd oacous ic echniques o e alua e and moni o i s p esence and
abundance is o g ea ele ance gi en he di icul y o ca ching his species wi h adi ional
ishing me hods, which o en leads o i s unde es ima ion [
30
]. Ca ish a e cha ac e ised
by a high g ow h a e, especially du ing he i s yea o li e [
31
,
32
]. Thus, he la ge size
eached by his species a an ea ly age, combined wi h he smalle size o he o he species
p esen in he s udy a ea, enables adul ca ish specimens o be di e en ia ed om he es
o he species using hyd oacous ic me hods.
P e ious s udies ha e success ully applied he en i onmen al DNA (eDNA) me hod-
ology o he speci ic de ec ion o ca ish, demons a ing i s sensi i i y [
33
,
34
]. Pa ondo
e al. [
35
] p oposed he combina ion o comp ehensi e e iews o ci izen ale s wi h e i ica-
ions based on speci ic molecula echniques as a apid, cos -e ec i e, and uly accessible
s a egy o he ea ly de ec ion o ca ish in Spain. This could se e as a p elimina y
s ep be o e mo e comp ehensi e in o ma ion phases in managemen plans add essing
in asi e species [
35
]. Cu en ly, i is easible o design species-speci ic p ime s o apid
de ec ion, e en when DNA is pa ially deg aded. DNA deg ada ion can pose a challenge
in na u al en i onmen s o en i onmen al samples, bu ad ances in p ime design ech-
niques and DNA ampli ica ion o e come hese di icul ies [
36
]. By educing he ime and
esou ces needed o analyses, he implemen a ion o conse a ion and na u al esou ce
managemen s a egies is acili a ed, along wi h he s udy o biological popula ions and
communi ies [36,37].
En i onmen al DNA allows no only de ec ion bu also quan i ica ion o he species
s udied h ough wo molecula echniques: he quan i a i e polyme ase chain eac ion
(qPCR) and he d ople digi al PCR (ddPCR) [
38
]. The qPCR measu es he copy numbe
o he ampli ied a ge agmen using a calib a ion cu e de i ed om se ial dilu ions
o a sample wi h a known concen a ion. The h eshold luo escence le el is compu ed
om ini ial cycles, and he cycle numbe (CT alue) co ela es wi h he sample’s empla e
copy numbe [
39
]. The e o e, quan i ica ion ia qPCR elies on an indi ec measu e om
a calib a ion cu e, limi ing i s p ecision and ep oducibili y. In ecen yea s, a new
quan i a i e me hod, ddPCR, has eme ged, di ec ly quan i ying DNA wi hou e e ence
cu es. This in ol es pa i ioning he sample in o housands o d ople s and independen ly
conduc ing a PCR in each, de ec ing luo escence a he p ocess’s conclusion, as an end-
poin measu emen . P e ious s udies ha e demons a ed he e ec i eness o bo h qPCR
and ddPCR echniques in quan i ying DNA concen a ions o a ge species in eDNA om
mesocosm expe imen s wi h known abundances and biomasses [
40
–
42
]. Es ima es ia
ddPCR show less a ia ion han qPCR, sugges ing hey can calcula e eDNA concen a ion
mo e accu a ely, enhancing he abili y o es ima e species abundance/biomass in eDNA
s udies [
42
,
43
]. Howe e , no enough s udies ha e been conduc ed on en i onmen al
samples o de e mine species dis ibu ions in na u al habi a s and he po en ial p oblems
and bene i s o bo h echniques.
En i onmen al DNA-based echnologies, coupled wi h he use o echosounde s, can
p o ide accu a e and ea ly de ec ion o in asi e species such as ca ish, hus con ibu ing o
comp ehensi e moni o ing o biodi e si y and en i onmen al impac s in di e en aqua ic
en i onmen s [
44
]. In his s udy, a dual mapping s a egy was implemen ed along he
Guadalqui i Basin, using an en i onmen al DNA de ec ion me hod o moni o ing ca ish
based on he molecula echniques o eal- ime quan i a i e PCR (qPCR) and d ople digi al
PCR (ddPCR). The objec i e o his s udy was o de e mine he ange o expansion o
Eu opean ca ish in he lowe Guadalqui i and also o assess he complemen a i y and
Animals 2025,15, 285 5 o 20
po en ial syne gies be ween eDNA analysis and hyd oacous ic me hods o moni o ing
aqua ic in asi e species in i e ine en i onmen s.
2. Ma e ials and Me hods
2.1. Hyd oacous ic S udy
2.1.1. Hyd oacous ic Su ey
The acous ically su eyed a ea included h ee main zones, iden i ied based on i e
mo phology. The Alcalá del Río dam ep esen s he inal ba ie be o e he Guadalqui i
Ri e lows in o he sea. The e o e, we conside ed he sec ion o he i e ups eam o
he Alcalá del Río dam, ex ending o he Can illana dam, as a sepa a e a ea. This sec ion
o ms he Alcalá del Río Rese oi (Figu e S1). Nex , we su eyed he sec ion o he
Guadalqui i Ri e downs eam o he Alcalá del Río dam up o i s con luence wi h he
Guadaí a Ri e , co e ing a o al o 45 km. The no he n po ion o his sec ion, om
he Alcalá del Río dam o Sou h Se illa, is na owe , wi h a wid h o app oxima ely 150
m, while he sou he n po ion, om Sou h Se illa o he con luence wi h he Guadaí a
Ri e , is wide , a e aging a ound 300 m. Finally, we included he El Ge gal Rese oi ,
which connec s o he Guadalqui i Ri e h ough he Ri e a de Huel a ibu a y nea
No h Se illa.
Sampling was conduc ed using an ou boa d boa , wi h na iga ion always ca ied ou
ups eam and sailing speed main ained a a s eady 4 kno s. The sampling was conduc ed
h oughou he mon h o June o 2023, du ing dayligh hou s, be ween 8 a.m. and 9 p.m.
While he Eu opean ca ish is p ima ily noc u nal, he lack o adminis a i e pe missions
o nigh - ime na iga ion, due o he unsa e condi ions in he su eyed i e a ea, made
sampling du ing nigh imp ac ical. None heless, s udies ha e shown ha i can be e -
ec i ely de ec ed du ing he day because he highe day ime empe a u es keep hem
ac i e [
8
,
45
,
46
]. Fo he hyd oacous ic su eys, we used a Sim ad EK60 echosounde
(Sim ad Kongsbe g Ma i ime AS, Ho en, No way) wi h a spli -beam ci cula ansduce
ope a ing a 200 kHz (ES200-7 C). The ansduce was ins alled on a s ainless-s eel ame
(u ili y model numbe ES1279955, EcoFishUS and Uni e si y o Se illa, Se illa, Spain). The
s uc u e was moun ed on he side o he boa , p o iding he equipmen wi h good s abili y
du ing sailing.
The hyd oacous ic su ey was conduc ed bo h e ically and ho izon ally o co e
he en i e ecosys em, including he su ace and deepes zones. The su ace wa e laye
was su eyed using ho izon al hyd oacous ics, wi h he ansduce posi ioned 120 cm
below and pa allel o he wa e su ace. This ensu ed ha he acous ic beam’s opening
angle insoni ied a ep esen a i e olume o he i s 2 m o dep h, a oiding he po en ial
e ec s o engine noise and he nea - ield e ec [
47
–
49
]. Zones wi h a dep h g ea e han
2 m we e su eyed using e ical hyd oacous ics, wi h he ansduce posi ioned 40 cm
below he wa e su ace and o ien ed owa ds he bo om. This con igu a ion ensu ed
ha measu emen s aken below 2 m we e eliable and una ec ed by engine noise o he
nea - ield e ec [
47
–
49
]. The su ey was conduc ed while na iga ing ups eam in zigzag
ajec o ies, excep in a eas whe e he i e was oo na ow o ho izon al hyd oacous ic
su eys o insoni y a ep esen a i e olume. In such cases, pa allel ansec s along he
sho es we e pe o med, ensu ing he insoni ica ion o he en i e a ea.
Be o e sampling, he acous ic uni was calib a ed using a calib a ion coppe sphe e
ollowing he s anda d calib a ion me hod [
50
]. The pulse du a ion was 0.256 ms, and he
pulse mode was se o he maximum o e ed by he echosounde o he dep hs s udied.
The posi ion o he ansduce was geo- e e enced in eal ime using an Emlid Reach RS2
GPS (Emlid Tech K ., Budapes , Hunga y) placed on he ansduce s uc u e.
Animals 2025,15, 285 6 o 20
2.1.2. Hyd oacous ic Da a Pos -P ocessing
The da a s o ed du ing he echo su eys we e p ocessed using he hyd oacous ic
analysis so wa e Sona 5 P o 608.43 [
51
]. P io o p ocessing, he bo om line was se in all
sampling iles o be able o disc imina e be ween ish and bo om echoes. Subsequen ly,
a ious sou ces o noise ha could lead o e o s in ish de ec ion, such as bubbles, eg-
e a ion, pa ches o phy oplank on, zooplank on agg ega ions, and o he noise sou ces
we e emo ed.
2.1.3. Ca ish De ec ion
The acous ic signals we e analysed o de ec specimens ha could po en ially be ca ish
based on hei size. To di e en ia e ca ish om o he ish species in he s udy a ea, a h eshold
o 1 m in o al leng h was es ablished o de ec ed indi iduals. Among he species cohabi ing
he s udy a ea, some can each leng hs exceeding 50 cm, including he ca p (Cyp inus ca pio, Lin-
naeus, 1758), sou he n Ibe ian ba bel (Lucioba bus scla e i, Gün he , 1868), s iped mulle (Mugil
cephalus, Linnaeus, 1758), hinlip g ey mulle (Chelon amada, Risso 1826), wai e oli eshad (Alosa
allax, Lacépède, 1803), la gemou h bass (Mic op e us salmoides, Lacepède, 180), o Eu opean
eel (Anguilla anguilla, Linnaeus, 1758) (Table S1) [
6
,
52
–
54
]. While his size h eshold excludes
smalle ca ish, i minimises po en ial o e lap wi h o he species, ensu ing ha all de ec ed
specimens a e unequi ocally ca ish, which is he p ima y ocus o he su ey.
Fo he analysis o ansec s om e ical su eys, he echo coun ing me hod based on
single echo de ec ion (SED) was used. The Ta ge S eng h (TS)-leng h ela ionship equa ion
published by Lo e [
55
] and included in he s anda d UNE-EN 15910 [
56
] was employed
o he TS-size con e sion. In his case, he equa ion ela es he TS o he o al leng h (TL)
o he ish. Size anges wi h TS g ea e han
−
25.81 dB co espond o sizes g ea e han
1 m, so hey we e coun ed as ca ish. All de ec ions co esponding o indi iduals wi hin
hese size anges we e geo e e enced using he in o ma ion s o ed du ing simul aneous
GPS-acous ic su eying.
Fo he analysis o ansec s om ho izon al su eys, he ace coun ing me hod based
on ish acks was employed. The equa ion used o he TS-size ela ionship was de eloped
by Kubeˇcka and Duncan [
57
], as speci ied in he UNE-EN 15910 s anda d. This equa ion
conside s he di e en o ien a ions in which a specimen can be insoni ied du ing ho izon al
su eys. Unlike e ical su eys, whe e he ish is almos always insoni ied do sally, in a
ho izon al su ey, a ish can be insoni ied in di e en posi ions, anging om head- o- ail
o la e al. The head- o- ail posi ion gene a es a lowe TS by exposing less o he su ace
a ea o he ish o he acous ic beam han he la e al posi ion, whe e a la ge su ace a ea is
exposed. Howe e , bo h posi ions co espond o he same size, so i is c ucial o conside
he o ien a ion o he ish ela i e o he acous ic beam. In he ack analysis, he p og am
assigns he speci ic size co esponding o he specimen ha has gene a ed a pa icula
ajec o y based on he angle o he ajec o y ela i e o he acous ic beam. Each ack
wi h a size equal o o g ea e han 1 m was manually checked using he analysis so wa e
ools, which p o ide a isual ep esen a ion o he acks ac oss he di e en spa ial axes
o e i y hey o igina e om a ca ish. As in e ical sampling, all ca ish de ec ions we e
geo e e enced in he s udy a ea.
2.2. The eDNA P ocedu es and Analyses
2.2.1. Field Sample eDNA Collec ions
Wa e sampling was conduc ed in i e s and ese oi s o he Guadalqui i Basin
loca ed in he p o inces o Se illa and Có doba in sou he n Spain (Figu e S2) be ween
25 and 29 June 2023. The sampling si es we e mos ly loca ed in a eas ha a e exposed o
sunligh o many hou s and ha e no ege al canopy o p o ec hem om di ec UV adia-
Animals 2025,15, 285 7 o 20
ion, which can accele a e DNA deg ada ion. Sampling was conduc ed du ing dayligh
hou s bu was s uc u ed o a oid he cen al pa s o he day (12:00 o 15:00), as hese coin-
cide wi h he peak sola adia ion and highe empe a u es. A o al o 34 samples o 2 li es
each we e collec ed om 17 sampling si es (Figu e S2). Sampling poin s we e clus e ed
in o 3 s udy a eas: posi i e con ol a ea (Iznája , Pan alán, and De la Hoz), uppe i e a ea
(Via , El Ge gal, Lo a del Río, Can illana, B enes, Alcalá del Río, and La Rinconada) and
lowe i e a ea (No h Se illa, Pue o, Toma es, Sou h Se illa, Gel es, Co ia del Río and
La Señuela). Niskin- ype oceanog aphic bo les we e used o collec wa e samples o a
dep h o a leas one me e o a oid he shallowes laye (wi hou ouching he bo om),
which is he ho es and mos a ec ed by sola adia ion. A HannaR HI98494 (Hanna In-
s umen s, Woonsocke , RI, USA) wo ked o measu e hese pa ame e s in he wa e column
a 1–2 m a oiding con ac wi h he sedimen , including pH, oxida ion- educ ion po en ial
(mV), dissol ed oxygen pe cen age (%), dissol ed oxygen concen a ion (mg/L), elec ical
conduc i i y (
µ
S/cm), o al dissol ed solids (ppm), u bidi y (NTU), and empe a u e (
◦
C),
du ing sampling (Table S2).
Sampling was pe o med using s e ile equipmen and disposable ni ile glo es. All
he ma e ials we e s e ilised be ween sampling poin s using 10% / comme cial bleach
( inal [Cl
−
] 0.3% / ). Any esidual bleach was hen insed o wi h dis illed wa e and
he Niskin bo le was hen insed again wi h ese oi wa e a he nex sampling poin .
Benzalkonium chlo ide (BAC) was added o he samples o p ese e he DNA using a
pipe e wi h s e ile and il e ed ips o a inal concen a ion o 0.01% w/ BAC in he
sample [
58
,
59
]. Two samples we e collec ed a each sampling si e (R1 and R2) and sampling
was ca ied ou a emp ing o sepa a e he locali ies by a minimum o 2 km whene e
possible. A e sampling, he wa e samples we e s o ed in s e ile 2 L g adua ed bo les
(REF. 407008.O) (Del alab, Ba celona, Spain) a 4 ◦C un il u he p ocessing.
2.2.2. Fil a ion and DNA Ex ac ion
Wa e samples we e il e ed wi hin 24 h o collec ion in a decon amina ed labo a o y
using a il a ion amp wi h h ee il a ion uni s (Labbox Labwa e SL, Ba celona, Spain).
Two li es pe sample we e il e ed. Sample (2 L in o al): Sampling eplica e 1 (1 L)
−
Subsamples R1_1 (0.5 L) + R1_2 (0.5 L)/Sampling eplica e 2 (1 L)
−
Subsamples R2_1
(0.5 L) + R2_2 (0.5 L). We il e ed 2 L o each poin , wi h 1 L pe sample (0.5
×
4), using a
dual- il e s a egy o a oid clogging. The il e s used we e a cellulose ni a e memb ane
il e (CN) wi h a 1.2
µ
m po e size (SARTORIUS Cellulose Ni a e Fil e 11403—50----ACN)
and, below, a second CN il e wi h a 0.20
µ
m po e size (PRAT DUMAS F ance MCNE-
247-100). Fil a ion equipmen was s e ilised wi h 10% / comme cial bleach solu ion o
10 min and insed wice wi h s e ile dis illed wa e be ween samples. Fil e s we e olded
using s e ile weeze s and placed in bead ubes p o ided by he Powe Wa e
®
Ki (Qiagen,
Hilden, Ge many) and s o ed a
−
20
◦
C un il DNA ex ac ion. Fil a ion nega i e con ols
we e un once a day be ween he samples o check o con amina ion (n = 6). En i onmen al
DNA was ex ac ed using DNeasy Powe Wa e
®
Ki (Qiagen, Hilden, Ge many) ollowing
he manu ac u e ’s ins uc ions in a dedica ed clean oom o he p ocessing o eDNA
samples. Th ee ex ac ion nega i e con ols we e added.
2.2.3. The qPCR and ddPCR Analyses
P ime 3 so wa e implemen ed in Geneious P ime
®
2023.1.1 [
60
] was used o p ime s
and p obe design (see Supplemen a y Ma e ials o mo e de ails). Two speci ic p ime s
and a p obe (FAM-labelled) we e de eloped o a agmen o he 12S RNA mi ochon-
d ial gene o he genus Silu us: Sil1-F (5
′
-TTTTCCCCGCCTATATACCGCC-3
′
) Sil1-R
Animals 2025,15, 285 8 o 20
(5
′
-CTTCGGGCACTTACTTTCAAGG-3
′
) and Sil1-p obe (**FAM-AACGTCAGGTCGAGGT
GTAGCGTACG-MGB).
A e a s ic alida ion o he p ime s and p obe (SI.1), he qPCR eac ions we e
conduc ed in a dedica ed p e-PCR labo a o y whe e bench su aces we e s e ilised wi h UV
adia ion. Each PCR con ained 6
µ
L o empla e DNA, 1.2
µ
L o each p ime (10
µ
M), 0.8
µ
L
o p obe (5
µ
M), 10
µ
L o TaqMan
®
En i onmen al Mas e Mix 2.0 (The mo Fishe Scien i ic,
Wal ham, MA, USA), and DNase/RNase- ee wa e o a inal olume o 20
µ
L. Each PCR
pla e con ained a s anda d cu e consis ing o a 1:10 dilu ion se ies o gene a e a s anda d
cu e o known amoun s o DNA. Th ee echnical (PCR) eplica es we e pe o med o each
biological sample, esul ing in a o al o wel e eplica es pe sampling poin . In addi ion,
3 no empla e con ols (NTCs) pe pla e we e included, as well as il a ion and ex ac ion
con ols. The il a ion, ex ac ion and echnical con ols e u ned nega i e esul s. Samples
we e un on an Applied Biosys ems 7900HT Fas Real-Time PCR Sys em (The mo Fishe
Scien i ic) unde he ollowing he mal cycling condi ions: 2 min a 50
◦
C, 10 min a 95
◦
C,
50 cycles o 15 s a 95
◦
C, and 60 s a 60
◦
C. Sequence De ec ion So wa e SDS 2.4 (The mo
Fishe Scien i ic) was used o analyse he esul s. To include loca ions/si es o he es
o he analyses, wo o he h ee echnical eplica es, besides one o he wo subsamples
(e.g., R1_1 (0.5 L) + R1_2 (0.5 L)) in a leas one o he si es eplica es (R1 o R2), should
e eal ca ish eDNA (as ad ised ollowing a p ecau iona y amewo k ecommended by
Goldbe g e al. [61]).
Fu he op imisa ion and alida ion o he ddPCR assay we e pe o med (SI.1). In
his case, and due o logis ical es ic ions, subsamples (e.g., R1_1 (0.5 L) + R1_2 (0.5 L))
we e pooled, and o each loca ion/si e, wo eplica es (Replica e 1 and Replica e 2) we e
analysed. Each ddPCR eac ion mix (20
µ
L) comp ised 10
µ
L o sample, 900 nM o each
p ime , and 250 nM o TaqMan p obe, comple ed wi h Supe mix o P obes (No dUTP)
(1863023) (Bio-Rad, He cules, CA, USA) a a concen a ion o 1
×
. This mix u e was hen
combined wi h Bio-Rad’s d ople gene a ion oil and di ided in o 15,000–20,000 d ople s
using he QX-100 d ople gene a o (Bio-Rad). Indi idual sample d ople s we e placed
sepa a ely in o each well o a 96-well PCR eac ion pla e. PCR was pe o med in he sealed
96-well pla e using he Bio-Rad T-100 he mocycle (Bio-Rad). The inal PCR condi ions
we e 10 min a 95
◦
C, 40 cycles o dena u a ion o 30 s a 94
◦
C, and ex ension o 90 s a
57
◦
C wi h a empe a u e amp o 2
◦
C/second, ollowed by 10 min a 98
◦
C, and a hold a
4
◦
C un il pla e eading. A e PCR ampli ica ion, he pla e was ans e ed o he Bio-Rad
QX-200 d ople eade (Bio-Rad). Bio-Rad’s Quan aSo so wa e, e sion 1.7.4.0917, was
used o quan i y copies o he a ge DNA. To include loca ions/si es o he es o he
analyses, a leas one o he si e eplica es (R1 o R2) should e eal ca ish eDNA.
2.3. Mapping and S a is ical Analyses
Fo he map g aphics, we used QGIS . 3.32.3 Lima (h ps://qgis.o g/en/si e/,
accessed on 20 Decembe 2024) wi h hyd og aphy da a om he Spa ial Re e ence Da a o
Andalusia, Ins i u e o S a is ics and Ca og aphy o he Go e nmen o Andalusia. Wa e
physicochemical pa ame e s we e assessed o collinea i y using a co ela ion ma ix in
R so wa e . 4.3.3. [
62
]. As a esul , he ollowing a iables we e d opped om he inal
eg ession analysis: pH, dissol ed oxygen pe cen age (%), elec ical conduc i i y (
µ
S/cm),
and o al dissol ed solids (ppm). Pea son co ela ion analyses [
63
] we e conduc ed be ween
log- ans o med o al qPCR and ddPCR eDNA copy numbe s by si e.
P incipal componen analysis (PCA) [
64
,
65
] was pe o med on physicochemical a i-
ables and qPCR and ddPCR ca ish eDNA alues using PAST V.4.17 [
66
]. A PCA was also
pe o med using he o al ca ish eDNA alues ob ained by qPCR and ddPCR pe si e,
he hyd oacous ic ca ish de ec ions (agg ega e coun s) in i e sec ions be ween 1 and
Animals 2025,15, 285 9 o 20
10 km ups eam o he eDNA sampling si es ( he mid-poin be ween eplica es R1 and
R2 was aken as he e e ence poin ), and inally he dis ances om each eDNA sampling
si e o he i e mou h (km). S epwise linea eg ession analyses we e also pe o med
o iden i y associa ions be ween log- ans o med esponse a iables (eDNA ddPCR and
qPCR copy numbe s), and po en ially explana o y a iables in i e and ese oi habi a s
whe e e possible. Mo eo e , and despi e empo al and spa ial he e ogenei y be ween he
wo de ec ion me hodologies used in his wo k, Fishe ’s exac es (p esence/absence) [
67
]
and Spea man’s ank co ela ion we e used o assess possible ela ionships be ween he
abundance o ca ish de ec ed by hyd oacous ics (es ima ed as coun s ups eam o eDNA
sampling poin s as in he p e ious analyses), molecula echniques (qPCR and ddPCR
ca ish eDNA alues), and dis ances om he i e mou h (km).
3. Resul s
3.1. Hyd oacous ic Su ey Resul s and Spa ial Dis ibu ion o S.glanis
Bo h e ical and ho izon al su eys con i med he p esence o ca ish in he lowe
Guadalqui i as well as in he El Ge gal and Alcalá del Río Rese oi s (Figu e 1). A o al o
200 indi iduals we e posi i ely iden i ied as adul ca ish measu ing 1 m o mo e. In he El
Ge gal Rese oi , a o al o 11 indi iduals we e de ec ed, wi h he highes numbe in he
dam a ea (9 indi iduals); in he Alcalá del Río Rese oi , 54 ca ish de ec ions we e e enly
dis ibu ed ac oss he s udy a ea and, o e all, we e g ea e han in he El Ge gal Rese oi .
The highes numbe o ca ish was de ec ed in he i e , pa icula ly in he s e ch be ween
he Alcalá del Río dam and No h Se illa, wi h 78 de ec ions dec easing p og essi ely
downs eam, 54 de ec ions in he es o he Se illa a ea, and 3 in he Co ia del Río a ea, he
las loca ion whe e ca ish was de ec ed. Mos o he de ec ed indi iduals ell wi hin he
size ange o 1.0 o 1.2 m (Figu e S3).
3.2. En i onmen al DNA-Speci ic De ec ions, Quan i ica ions and Spa ial Dis ibu ion o S.glanis
The mean and s anda d de ia ion alues o each physicochemical pa ame e s om he
sampling loca ions we e es ima ed (Table S2): pH (
µ
= 8.2
±
0.45), oxida ion- educ ion po en ial
(ORP) (
µ
= 33.2
±
9.55 mV), dissol ed oxygen pe cen age (
µ
= 71.8
±
42.80%), dissol ed oxygen
concen a ion (
µ
= 5.5
±
3.23 mg/L), elec ical conduc i i y (
µ
= 1769.2
±
1118.84
µ
S/cm), o al
dissol ed solids (
µ
= 885.2
±
561.06 ppm), u bidi y (
µ
= 0.9
±
0.59 NTU), and empe a u e
(
µ
= 28.6
±
1.92
◦
C) showing high he e ogenei y among sampling si es o some o he a iables
and e iden co ela ion be ween some o hem (Figu e S4).
In he PCA analysis, PC1 and PC2 accoun ed o 74.8% o he o al a iabili y based on
he eigen alues ob ained (Figu e S4). S epwise linea eg ession analyses wi h loga i hmic
ans o ma ion we e pe o med among he a iables, showing ha in i e s, ddPCR copies
we e indeed posi i ely associa ed wi h dissol ed oxygen (mg/L) (p- alue < 0.01) and
nega i ely associa ed wi h u bidi y (NTU) (p- alue < 0.01). In ese oi s, copies de ec ed
by qPCR we e also weakly nega i ely associa ed wi h u bidi y (NTU) (p- alue < 0.05)
(Table S3).
O e all, bo h molecula assays (qPCR and ddPCR) demons a ed speci ic de ec ion
o ca ish. The e was no de ec ion in any o he nega i e echnical con ols using ei he
me hodology. All il e and ex ac ion con ols showed no ampli ica ion, indica ing he
absence o con amina ion a all s ages o he s udy. The qPCR da a epo ed posi i e ca ish
de ec ion in 13 si es (76.5%), while we ob ained 11 posi i e si es wi h ddPCR (64.7%) om
he 17 sample si es (Figu e S5). A compa ison o he esul s shows ha 76.5% o he si es
we e conco dan in e ms o he p esence o ca ish eDNA be ween he wo echniques
es ed. The qPCR esul s showed an a e age o 5.5
×
10
5
ca ish eDNA copies ( anging
om 108.2 o 3.4
×
10
6
). Using he ddPCR me hodology, he alues o accep ed gene a ed
Animals 2025,15, 285 16 o 20
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