Pa asi ology
camb idge.o g/pa
Resea ch A icle
Ci e his a icle: Co eia S, Fe nández-Boo S,
Magalhães L, de Mon audouin X, Da e G,
Poulin R, Ve a M (2023). T ema ode gene ic
pa e ns a hos indi idual and popula ion
scales p o ide insigh s abou in ec ion
mechanisms. Pa asi ology 150, 1207–1220.
h ps://doi.o g/10.1017/S0031182023000987
Recei ed: 22 May 2023
Re ised: 2 Oc obe 2023
Accep ed: 7 Oc obe 2023
Fi s published online: 20 Oc obe 2023
Keywo ds:
Bucephalus minimus;Ce as ode ma edule;
clonal di e si y; COI; hos –pa asi e
in e ac ions; pa asi e; popula ion gene ics
Co esponding au ho s:
Simão Co eia;
Email: [email p o ec ed];
[email p o ec ed];
Manuel Ve a;
Email: manuel. e [email p o ec ed]
© The Au ho (s), 2023. Published by
Camb idge Uni e si y P ess. This is an Open
Access a icle, dis ibu ed unde he e ms o
he C ea i e Commons A ibu ion licence
(h p://c ea i ecommons.o g/licenses/by/4.0/),
which pe mi s un es ic ed e-use, dis ibu ion
and ep oduc ion, p o ided he o iginal a icle
is p ope ly ci ed.
T ema ode gene ic pa e ns a hos indi idual
and popula ion scales p o ide insigh s abou
in ec ion mechanisms
Simão Co eia1,2,3,4 , Se gio Fe nández-Boo2, Luísa Magalhães1,
Xa ie de Mon audouin5, Guillemine Da e6, Robe Poulin4
and Manuel Ve a3
1
Depa men o Biology, CESAM, Uni e si y o A ei o, 3810-193 A ei o, Po ugal;
2
Aqua ic and Animal Heal h
G oup, CIIMAR, Uni e si y o Po o, 4450-208 Ma osinhos, Po ugal;
3
Depa men o Zoology, Gene ics and Physical
An h opology, Campus Te a, Uni e si y o San iago de Compos ela, 27002 Lugo, Spain;
4
Depa men o Zoology,
Uni e si y o O ago, 9054 Dunedin, New Zealand;
5
CNRS, UMR EPOC, S a ion Ma ine, Uni e si y o Bo deaux,
F-33120 A cachon, F ance and
6
Uni e si é de Bo deaux, CNRS, Obse a oi e Aqui ain des Sciences de l’Uni e s,
F-33615 Pessac, F ance
Abs ac
Mul iple pa asi es can in ec a single hos , c ea ing a dynamic en i onmen whe e each
pa asi e mus compe e o e hos esou ces. Such in e ac ions can cause g ea e ha m o he
hos han single in ec ions and can also ha e nega i e consequences o he pa asi es
hemsel es. In hei i s in e media e hos s, ema odes mul iply asexually and can e en ually
each up o 20% o he hos ’s biomass. In mos species, i is unclea whe he his biomass
esul s om a single in ec ion o co-in ec ion by 2 o mo e in ec i e s ages (mi acidia), he
la e being mo e likely a p io i in a eas whe e p e alence o in ec ion is high. Using as
model sys em he ema ode Bucephalus minimus and i s i s in e media e hos cockles, we
examined he gene ic di e si y o he cy och ome c oxidase subuni I egion in B. minimus
om 3 dis inc geog aphical a eas and pe o med a phylogeog aphic s udy o B. minimus
popula ions along he No heas A lan ic coas . Wi hin locali ies, he high gene ic a iabili y
ound ac oss ema odes in ec ing di e en indi idual cockles, compa ed o he absence o
a iabili y wi hin he same hos , sugges s ha in ec ions could be gene ally o igina ing
om a single mi acidium. On a la ge spa ial scale, we unco e ed signi ican popula ion
s uc u e o B. minimus, speci ically be ween he no h and sou h o Bay o Biscay.
Al hough o he explana ions a e possible, we sugges his pa e n may be d i en by he
popula ion s uc u e o he inal hos .
In oduc ion
Wi h abou 45 000 species desc ibed in a wide ange o ecosys ems, ema odes a e one o he
mos common and widesp ead g oup o pa asi es (Ca lson e al., 2020). They can be ound a
almos ac oss all ophic le els o dynamic ood chains (Ba oli and Gibson, 2007). T ema odes
a e an impo an componen o ecosys em biodi e si y wi h signi ican impac s a he hos
indi iduals (Schul eOehlmann e al., 1997; Cu is e al., 2000; Thiel ges, 2004), hos popula-
ions (F edensbo g e al., 2005) and ecosys em communi ies (Poulin, 1999; Mou i sen and
Poulin, 2002; Goedkneg e al., 2016). Besides, by con ibu ing o he nu ien cycle, ac ing
as indica o s o en i onmen al changes o as p oxy o en i onmen al di e si y (due o mul i-
hos li e cycles), ema ode p esence may indica e a heal hy and esilien ecosys em (Johnson
e al., 2010; Ha che and Dunn, 2011).
T ema odes ha e a complex li e cycle ha al e na es be ween ee-li ing and pa asi ic
s ages. The mi acidium, he ema ode la a ha ched om an egg, in ec s he i s in e media e
hos (usually a mollusc) and ans o ms in o spo ocys s o ediae (pa asi ic s age). A his
s age, spo ocys s o ediae, h ough asexual mul iplica ion, p oduce ce ca iae ( ee-li ing
s age) ha eme ge om he i s hos o in ec he second in e media e hos (a e eb a e o
in e eb a e) whe e hey se le as me ace ca iae. In he ema ode’s inal hos (a e eb a e),
a e inges ion o he second hos , me ace ca iae de elop in o adul lukes, ep oduce sexually,
and comple e he li e cycle (C ibb e al., 2003; Ba oli and Gibson, 2007). In he i s in e -
media e hos , spo ocys s ages a e o e ly des uc i e, eplacing hos issue and eaching up
o 20% o he hos ’s biomass (Dubois e al., 2009; P es on e al., 2013), wi h di ec conse-
quences o hos ep oduc ion (Ca ballal e al., 2001), g ow h (Bowe s, 1969) and ene gy
demand (Jokela e al., 1993), leading o e en ual hos dea h (Thiel ges, 2006). In mos species,
i is cu en ly unknown whe he his spo ocys biomass esul s om a single mi acidium, ha
excludes o he mi acidia by p eda ion o in aspeci ic compe i ion, o om co-in ec ion. I
co-in ec ion is he ule, ema ode in asion may esul in a bu den ha he hos migh no
be able o bea (F edensbo g and Poulin, 2005; Mideo, 2009). On he o he hand, co-in ec ion
can, occasionally, bene i he hos by lessening he o e all bu den o in ec ion, by educing
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
pa asi e in ec ion success, o by s eng hening he hos ’s immune
esponse, leading o highe esis ance o in ec ion (Dumon e al.,
2007; Balme e al., 2009).
De e mining he gene ic di e si y o ema ode spo ocys s
wi hin and among indi idual i s in e media e hos s is he e o e
impo an o unde s and he biology, beha iou and e olu iona y
pa e ns o hese pa asi es. None heless, cu en knowledge
ega ding biology o hese pa asi es, and pa icula ly abou he
spo ocys li e s age, is s ill e y sca ce, despi e ema odes’wide
dis ibu ion and impo ance o he ecosys em. Few s udies ha e
ocused on he conspeci ic di e si y o ema ode spo ocys s
wi hin he i s in e media e hos (Rauch e al., 2005; Keeney
e al., 2007; Lag ue e al., 2007), showing ha he likelihood o
in ec ion by conspeci ics inc eased wi h he p e alence o he
pa asi e in he communi y (Keeney e al., 2008; Louhi e al.,
2013). Mo eo e , a he popula ion le el, he hos has a signi ican
impac on gene ic di e si y and popula ion s uc u e o pa asi es,
wi h subs an ial gene low occu ing in pa asi e species wi h e i-
cien dispe sion mechanisms (Agola e al., 2009; Feis e al., 2015).
Howe e , mos s udies o ema ode gene ic di e si y ha e ocused
on in e media e and inal hos s, wi h pa icula emphasis on e-
ma odes wi h ha m ul impac s on human (The on e al., 2004;
Bell e al., 2006; Balme e al., 2009) o socio-economically
impo an species, namely ish (Vilas e al., 2003; C iscione and
Blouin, 2006).
Bucephalus minimus is a ma ine ema ode pa asi e ha
occu s in se e al aqua ic sys ems along he No heas A lan ic
coas and Medi e anean Sea (Magalhães e al., 2015). In he
A lan ic a ea, his pa asi e in ec s he Eu opean edible cockle,
Ce as ode ma edule, which se es as he i s in e media e hos
when a mi acidium pene a es i s issue. In cockles, he p e alence
o his pa asi e a ies g ea ly among coas al sys ems and season;
depending on he ime since in ec ion, he pa asi e’s d y mass
in in ec ed cockles can ange om 1 o 20% o he o al li ing is-
sue wi hin he cockle shell (Magalhães e al., 2015;de
Mon audouin e al., 2021). Bucephalus minimus ini ially in ec s
he cockle’s gonad and diges i e gland bu p omp ly sp eads o
o he pa s o he hos , e en ually in ading he en i e body
(Desclaux e al., 2002; de Mon audouin e al., 2009). In ec ion
by B. minimus esul s in cas a ion (Ca ballal e al., 2001) and
ene gy consump ion (Dubois e al., 2009), leading o s a a ion
and au olysis o he hos ’s diges i e ac . This pa asi e is consid-
e ed as one o he mos ha m ul ema ode pa asi es in ec ing C.
edule (Magalhães e al., 2015; de Mon audouin e al., 2021). Inside
cockles, B. minimus p oduces spo ocys s and ce ca iae h ough
asexual mul iplica ion, which eme ge and in ec he goby
Poma oschis us spp. (second in e media e hos ), whe e hey encys
and de elop in o me ace ca iae. The inal hos , Dicen a chus lab-
ax, he Eu opean seabass, is in ec ed a e consump ion o pa a-
si ized gobies. Me ace ca iae de elop in o adul lukes and
p oduce eggs, h ough sexual ep oduc ion, o comple e he
cycle (Pina e al., 2009; Magalhães e al., 2015).
Due o he limi ed in o ma ion ega ding his ema ode’s
spo ocys s ages, he p ima y goal o he p esen s udy was o
assess he gene ic a iabili y o he cy och ome c oxidase subuni
I (COI) egion o B. minimus spo ocys DNA wi hin and among
he i s in e media e hos , C. edule. We es ed he hypo hesis ha
highe gene ic a iabili y a he hos indi idual scale ( esul ing in
2 o mo e haplo ypes among spo ocys s in a single cockle) a e
mo e common in locali ies wi h high p e alence o in ec ion,
whe e join in ec ions should be mo e equen by chance
alone. As ou second goal, a phylogeog aphic s udy o B. minimus
was also ca ied ou combining in o ma ion a ailable in he li e a-
u e and om samples aken on cockle beds ha we e examined
in his s udy o he i s ime (i.e. Ibe ian Peninsula and G ea
B i ain).
Ma e ials and me hods
Bucephalus minimus samples
The gene ic a iabili y o B. minimus spo ocys haplo ypes wi hin
he same hos was s udied by collec ing specimens p esen in he
i s in e media e hos , he edible cockle, a 3 di e en beds wi h
di e en p e alence along he Eu opean A lan ic coas : Ria de
A ei o, A ei o, Po ugal (lowes p e alence [Magalhães e al.,
2018]); de la Ramallosa Lagoon, Baiona, Spain (mode a e p e a-
lence [In ecma , 2021]); and Île aux Oiseaux, A cachon, F ance
(wi h high le els o p e alence [Magalhães e al., 2015])
(Fig. 1). Adul cockles (be ween 20 and 30 mm shell leng h)
we e haphaza dly collec ed a low ide and dissec ed in he labo a-
o y o mo phologically iden i y B. minimus in ec ion. The lesh
was hen ans e ed and obse ed unde a s e eomic oscope by
ca e ully comp essing i be ween 2 s e ilized glass slides. Fou
spo ocys eplica es we e ex ac ed pe cockle (in a o al o 5
cockles pe sampling si e) using o ceps, and p ese ed sepa a ely
in 100% e hanol a −20°C. All ma e ial was s e ilized be ween
samples. To enhance he possibili y o di e en clones, spo ocys s
we e aken om di e en in ec ed issues o he same cockle (i.e. 1
om he oo , 1 om he gills and 2 om he diges i e gland).
DNA isola ion, ampli ica ion and sequencing
Genomic DNA ex ac ion om B. minimus specimens was pe -
o med using E.Z.N.A Mollusc DNA ki (Omega Bio-Tek,
No c oss, GA, USA) in acco dance wi h he manu ac u e ’s
ins uc ions. Nanod op was used o assess DNA concen a ion,
and, i needed, aliquo s we e c ea ed o dilu e DNA o app oxi-
ma ely 30 ng μL
−1
.
The mi ochond ial COI agmen was ampli ied using he
Mpla COX1-dF (5′-TTW CIT TRG ATC ATA AG-3′) and
Mpla COX1-dR (5′-TGA AAY AAY AII GGA TCI CCA CC-3′)
p ime s (Moszczynska e al., 2009), esul ing in sequences o
587 bp. The polyme ase chain eac ion (PCR) was ca ied ou
in a inal olume o 20 μL composed o 1X eac ion bu e , 2.5
mMMgCl
2
, 100 μMdeoxynucleo ide iphospha es (dNTPs), 0.5
μMo o wa d and e e se p ime s, 0.65 uni s o The moFishe
AmpliTaq Gold DNA polyme ase (The moFishe , Wal ham,
MA, USA) and 60 ng o DNA. The PCR p og amme employed
had an ini ial dena u a ion s ep o 10 min a 95°C, ollowed by
35 cycles o 30 s a 94°C, 30 s a 50°C and 1 min a 72°C and a
inal ex ension o 10 min a 72°C. Following his ini ial PCR, a
second PCR was ca ied ou using he same condi ions p e iously
desc ibed bu using 2 μL o PCR p oduc ins ead o DNA. The
ampli ied PCR p oduc s we e analysed by elec opho esis h ough
a 1% aga ose gel dyed wi h SYBR sa e DNA Gel S ain (In i ogen,
Ca lsbad, CA, USA) and isualized unde UV ligh .
PCR p oduc s we e enzyma ically pu i ied wi h ExoSAP mix
(10 μL o PCR p oduc , 0.6 uni s o EXO I [DNA nuclease] and
0.3 uni s o sh imp alkaline phospha ase [SAP] o a inal olume
o 12 μL) unde he ollowing condi ions: 60 min a 37°C and 15
min a 85°C. Pu i ied PCR p oduc s we e sequenced using he
ABI P ism BigDye
TM
Te mina o 3.1 Cycle Sequencing Ki
p o ocol on an ABI P ism 3730 xl au oma ic sequence
(Applied Biosys ems, Fos e Ci y, CA, USA). All sequences
ob ained in his s udy we e deposi ed in GenBank (accession
numbe s: OQ625925–OQ625936; Table 1). Va iable si es we e
manually checked using he SEQSCAPE 2.5 p og am (Applied
Biosys ems) and aligned using Clus alW algo i hm implemen ed
on BioEdi .7.2.5 (Hall, 1999) wi h LaB haplo ype (GenBank
accession numbe : KF880429.1) as e e ence. Iden i ica ion o
he di e en haplo ypes in he B. minimus specimens analysed
was ca ied ou using he so wa e DNAsp 5.10 (Lib ado and
Rozas, 2009). Finally, o check o he p esence o p ema u e
1208 Simão Co eia e al.
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
s op codons, COI haplo ypes iden i ied we e ansla ed o amino
acid sequences using he la wo m m DNA code in he online
so wa e EMBOSS T anseq (Rice e al., 2000; Goujon e al., 2010).
Da a analysis
Bucephalus minimus gene ic a iabili y a hos le el
To de e mine he gene ic a iabili y o B. minimus specimens
wi hin he same cockle and among di e en cockles, haplo ypes
ound in he di e en issues o each analysed cockle we e com-
pa ed using BioEdi . A haplo ype ne wo k was buil using he
ob ained da a, iden i ying haplo ypes o each cockle wi h a di e -
en colou . The haplo ype ne wo k was cons uc ed by calcula ing
he dis ance (based on numbe o base pai di e ences) be ween
DNA sequences and de e mining he numbe o mu a ions
be ween haplo ypes using ‘pegas’and ‘ape’packages o R
S a is ical So wa e .4.2.2 (Pa adis, 2010; Pa adis and Schliep,
2019).
Phylogeog aphic analysis
Fo phylogeog aphic analyses, oge he wi h he specimens col-
lec ed in he p esen s udy, DNA ex ac ions o 13 specimens
om 7 cockle beds sampled as pa o he COCKLES In e eg
p ojec (h p://cockles-p ojec .eu/) we e sequenced as p e iously
desc ibed. Samples we e a ailable o A ei o, Po ugal (1 sample),
Noia, Spain (1 sample), A cachon, F ance (1 sample), Bay o
Somme, F ance (4 samples), The Dee, Wales (2 samples), Bu y
Inle , Wales (2 samples) and Wadden Sea, he Ne he lands (2
samples). Addi ionally, he analysis included ano he 54 COI
sequences a ailable in he GenBank da abase e ie ed in
Janua y 2023 (see Feis e al., 2015 and Table 1). In o al,
sequences ep esen ed specimens om 11 di e en cockle beds
loca ed in 7 coun ies, co e ing a la ge pa o he na u al dis i-
bu ional ange o cockles in he A lan ic a ea (Fig. 1).
Phylogene ic ela ionships we e s udied using di e en and
complemen a y app oaches. Fi s , a haplo ype ne wo k was com-
pu ed o he ull da ase as p e iously desc ibed, which included
in o ma ion ega ding he cockle bed in which he haplo ypes
we e ound and he equency o occu ence. Iden i ica ion o
unique haplo ypes p esen in he da ase was ca ied ou wi h
DnaSP .5.10 (Lib ado and Rozas, 2009). Using MEGA X so -
wa e (Kuma e al., 2018), he Hasegawa–Kishino–Yano nucleo-
ide subs i u ion a e (HKY) wi h a γ alue o 0.655 and
in a iable si es o 0.728 was iden i ied as he mos p obable
nucleo ide subs i u ion model o ou da a. Phylogene ic ees
we e cons uc ed using exclusi ely he di e en haplo ypes iden-
i ied and he nucleo ide subs i u ion model desc ibed abo e.
Maximum likelihood (ML) and neighbou -joining oo ed and
un oo ed ees we e cons uc ed using he R S a is ical So wa e
.4.2.2. The oo ed ee was c ea ed using he COI sequences o
Rhipidoco yle sp. (a ema ode om he same amily as B. mini-
mus, Bucephalidae, GenBank accession numbe : KM538111.1)
and Himas hla quisse ensis (a ema ode ha in ec s cockles as
second in e media e hos bu om a di e en amily,
Himas hlidae; GenBank accession numbe : MN272732.1) as ou -
g oups. The sequences we e immed o 540 bp o emo e missing
da a. The un oo ed ees we e cons uc ed using he ull 587 bp
sequences o B. minimus. The obus ness o he b anches o
Figu e 1. Geog aphical loca ion o he Ce as ode ma edule cockle beds sampled o he s udy o Bucephalus minimus gene ic a iabili y a hos le el (A ei o, Baiona
and A cachon [in i alics]) and o phylogeog aphic analysis. *Cockle beds sampled o he i s ime in his s udy.
Pa asi ology 1209
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
he phylogene ic ees was es ima ed wi h 1000 boo s ap epli-
ca es and a likelihood a io es was pe o med based on he min-
imum Akaike in o ma ion c i e ion alues o ML. All
phylogeog aphic analyses we e pe o med wi h he ‘ape’,‘pegas’,
‘gg ee’and ‘phango n’packages o R S a is ical So wa e .4.2.2
(Pa adis, 2010; Schliep, 2011;Yue al., 2017; Pa adis and
Schliep, 2019).
Gene ic di e si y pa ame e s, calcula ed as haplo ype di e si y
(h) and nucleo ide di e si y (π), we e es ima ed wi hin each cockle
bed s udied using A lequin .3.5.1.3 (Exco ie e al., 2005). The
HKY model is no a ailable in his so wa e. Fo his eason,
he Tamu a–Nei (TN) model wi h a γ alue o 0.639 (simila o
he HKY and iden i ied as he hi d bes op ion o MEGA X)
was used o πes ima ions. Gene ic s uc u e and popula ion di -
e en ia ion we e assessed wi h global and pai wise coe icien s o
popula ion di e en ia ion applying he TN wi h γ alue o 0.639
subs i u ion a e (ϕ
ST
alues) wi h A lequin. Analysis o molecula
a iance (AMOVA) applying di e en models o a p io i clus e -
ing (based on hos popula ion gene ics [Souche e al., 2015; Ve a
e al., 2022] and obse ed da a –see esul s) was ca ied ou o
s udy he dis ibu ion o gene ic a ia ion wi hin (ϕ
SC
) and
among (ϕ
CT
) bed g oups using A lequin. The signi icance o
all he ϕs a is ics was e alua ed wi h 10 000 pe mu a ions.
Resul s
Bucephalus minimus gene ic a iabili y a hos le el
Du ing his s udy, a o al o 210 cockles we e analysed, o which
17 we e ound o be in ec ed wi h B. minimus (5 each in A ei o
and Baiona and 7 in A cachon). The p e alence o B. minimus
in he cockle beds sampled a ied om 3.3% in A ei o
Table 1. Accession numbe o B. minimus COI gene DNA sequences
downloaded (wi h e e ence) and deposi ed (in bold) in GenBank
Haplo ype name GenBank accession numbe Re e ence
LaA KF880428.1 Feis e al.(2015)
LaB KF880429.1 Feis e al.(2015)
LaC KF880430.1 Feis e al.(2015)
LaD KF880431.1 Feis e al.(2015)
LaE KF880432.1 Feis e al.(2015)
LaF KF880433.1 Feis e al.(2015)
LaG KF880434.1 Feis e al.(2015)
LaH KF880435.1 Feis e al.(2015)
LaI KF880436.1 Feis e al.(2015)
LaJ KF880437.1 Feis e al.(2015)
LaK KF880438.1 Feis e al.(2015)
LaL KF880439.1 Feis e al.(2015)
LaM KF880440.1 Feis e al.(2015)
LaN KF880441.1 Feis e al.(2015)
LaO KF880442.1 Feis e al.(2015)
LaP KF880443.1 Feis e al.(2015)
LaQ KF880444.1 Feis e al.(2015)
LaR KF880445.1 Feis e al.(2015)
LaS KF880446.1 Feis e al.(2015)
LaT KF880447.1 Feis e al.(2015)
LaU KF880448.1 Feis e al.(2015)
LaV KF880449.1 Feis e al.(2015)
LaW KF880450.1 Feis e al.(2015)
LaX KF880451.1 Feis e al.(2015)
LaY KF880452.1 Feis e al.(2015)
LaZ KF880453.1 Feis e al.(2015)
LaAA KF880454.1 Feis e al.(2015)
LaAB KF880455.1 Feis e al.(2015)
LaAC KF880456.1 Feis e al.(2015)
LaAD KF880457.1 Feis e al.(2015)
LaAE KF880458.1 Feis e al.(2015)
LaAF KF880459.1 Feis e al.(2015)
LaAG KF880460.1 Feis e al.(2015)
LaAH KF880461.1 Feis e al.(2015)
LaAI KF880462.1 Feis e al.(2015)
LaAJ KF880463.1 Feis e al.(2015)
LaAK KF880464.1 Feis e al.(2015)
LaAL KF880465.1 Feis e al.(2015)
LaAM KF880466.1 Feis e al.(2015)
LaAN KF880467.1 Feis e al.(2015)
LaAO KF880468.1 Feis e al.(2015)
LaAP KF880469.1 Feis e al.(2015)
LaAQ KF880470.1 Feis e al.(2015)
LaAR KF880471.1 Feis e al.(2015)
LaAS KF880472.1 Feis e al.(2015)
(Con inued)
Table 1. (Con inued.)
Haplo ype name GenBank accession numbe Re e ence
LaAT KF880473.1 Feis e al.(2015)
LaAV KF880474.1 Feis e al.(2015)
LaAW KF880475.1 Feis e al.(2015)
LaAX KF880476.1 Feis e al.(2015)
LaAY KF880477.1 Feis e al.(2015)
LaAZ KF880478.1 Feis e al.(2015)
LaBA KF880479.1 Feis e al.(2015)
LaBB KF880480.1 Feis e al.(2015)
LaBC KF880481.1 Feis e al.(2015)
BmA OQ625925 This s udy
BmB OQ625926 This s udy
BmC OQ625927 This s udy
BmD OQ625928 This s udy
BmE OQ625929 This s udy
BmF OQ625930 This s udy
BmG OQ625931 This s udy
BmH OQ625932 This s udy
BmI OQ625933 This s udy
BmJ OQ625934 This s udy
BmK OQ625935 This s udy
BmL OQ625936 This s udy
1210 Simão Co eia e al.
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
(Po ugal) o 23.3% in A cachon (F ance). In Baiona (Spain), B.
minimus was p esen in 16.7% o he sampled cockles. Fi e
in ec ed cockles pe bed we e used o ex ac 4 spo ocys s pe
cockle, yielding a o al o 60 samples. Fi y-six samples we e suc-
cess ully sequenced, while 4 samples, om a single cockle om
A cachon (F ance), we e no success ully sequenced due o DNA
ex ac ion p oblems. F om he 56 sequenced spo ocys s, belonging
o 14 in ec ed cockles, 12 di e en haplo ypes we e iden i ied, wi h
5, 3 and 4 haplo ypes ound in A ei o, Baiona and A cachon,
espec i ely (Table 2). Six o he iden i ied haplo ypes we e cha ac-
e ized o he i s ime (named as BmA–BmF; see Table 1).
All spo ocys s o B. minimus om he same cockle had iden-
ical haplo ype, howe e B. minimus haplo ypes iden i ied in di -
e en cockles om he same bed we e di e en , excep in Baiona
whe e he haplo ype ound in 3 di e en cockles was iden ical
(haplo ype LaE; see Table 2 and Fig. 2). Mo eo e , haplo ypes
we e no sha ed among he 3 beds (Fig. 2).
Phylogeog aphic analysis
F om he 69 DNA sequenced samples (56 o he B. minimus
haplo ype gene ic a iabili y a hos le el s udy, and 13 om di -
e en Eu opean si es selec ed om he COCKLES p ojec ), 12
esul ed in no el haplo ypes (Table 1). No p ema u e STOP
codons we e iden i ied in hese sequences (da a no shown).
Thus, when GenBank esou ces we e included, a o al o 162
COI gene sequences o B. minimus specimens om 11 cockle
beds we e analysed. F om hese a ailable sequences, 66 ep e-
sen ed unique haplo ype sequences. Sha ed haplo ypes (i.e.
hose ound in mo e han 1 cockle bed) accoun ed o 17% o
he o al. The LaAQ haplo ype was he mos p e alen and abun-
dan haplo ype, occu ing 42 imes ac oss 6 di e en beds. On he
o he hand, 83% o he haplo ypes we e exclusi ely ound in a sin-
gle bed, wi h se e al epo ed only once (i.e. single ons). A cachon
p esen ed he highes numbe o di e en B. minimus haplo ypes
de ec ed (19, see Table 3).
Due o he high a iabili y ound in he COI egion, he ana-
lysis o B. minimus haplo ypes ac oss he a ious cockle beds
p oduced a complex ne wo k made up o se e al closely con-
nec ed haplo ypes and associa ed mu a ional s eps, wi h no
mo e han 5 mu a ions sepa a ing any 2 successi e haplo ypes
iden i ied. A common haplo ype (LaAQ), obse ed in se e al
beds si ua ed no h o A cachon (44°N), was loca ed in he cen-
e o he ne wo k, om which nume ous o he haplo ypes
di e ged in a s a -like pa e n. These haplo ypes we e exclu-
si ely ound in a single bed o sha ed be ween ela i ely close
beds (Fig. 3). None heless, haplo ype clus e s (i.e. haplog oups)
we e iden i ied in speci ic geog aphic a eas, and beds om he
Sou h (Me ja Ze ga, A ei o, Baiona and Noia) and No h (Bay
o Somme, English Channel, Cel ic Sea, Bu y Inle , The Dee
and Wadden Sea) did no sha e any haplo ype, wi h he
excep ion o he LaE haplo ype iden i ied in Baiona, A cachon
and he English Channel. Phylogene ic ela ionships obse ed
in he ne wo k we e also con i med wi h he phylogene ic
ees (Fig. 4).
The A cachon bed, loca ed in he cen e o cockle’s dis ibu-
ional ange be ween he no he n and sou he n geog aphic
a eas, exhibi ed he highes numbe o de ec ed haplo ypes (19),
sha ing haplo ypes wi h loca ions om bo h egions (Table 3).
Excluding Noia, whe e only 1 indi idual was analysed, haplo ype
di e si y anged om 0.3846 in Me ja Ze ga o 1.0000 in beds
whe e all indi iduals analysed had a dis inc haplo ype (A ei o,
Bu y Inle and The Dee). Nucleo ide di e si y anged om
0.0007 in Me ja Ze ga o 0.0076 in Baiona (Table 3). The high
di e si y and he haplo ype dis ibu ion among loca ions we e
also e lec ed in he ϕ
ST
alues. Global ϕ
ST
o he whole egion
was 0.2922 (P alue < 0.001). Many pai wise ϕ
ST
alues esul ed
in signi ican di e ences, al hough many compa isons be ween
close loca ions we e non-signi ican , mainly among hose in ol -
ing no he n beds (Supplemen a y Table S1). Mo eo e , all pai -
wise ϕ
ST
alues in ol ing Me ja Ze ga we e high and highly
signi ican (P alue < 0.001), sugges ing he singula i y o his
bed (global ϕ
ST
in he whole egion excluding Me ja Ze ga =
0.1706, P alue < 0.001). These esul s sugges he p esence o
one no he n g oup (composed by Bay o Somme, English
Channel, Cel ic Sea, Bu y Inle , The Dee and Wadden Sea)
mo e homogeneous gene ically (ϕ
ST
= 0.0295, P alue = 0.073)
han he sou he n one (composed by Me ja Ze ga, A ei o,
Baiona and Noia; ϕ
ST
= 0.6694, P alue < 0.001), wi h A cachon
ep esen ing a po en ial con ac egion be ween bo h geog aphic
a eas. The ϕ
ST
alue in he no he n g oup inc eased up o
0.0336 (P alue = 0.018) when A cachon was included, while
his alue dec eased in he sou he n g oup when his loca ion
was included al hough i emained qui e high (ϕ
ST
= 0.4184, P
alue < 0.001). Hence, hese esul s sugges a close ela ionship
o A cachon wi h he no he n g oup. AMOVA analysis assigned
34.58% o he gene ic di e en ia ion o di e ences be ween no -
he n and sou he n g oups (ϕ
CT
= 0.3458, P alue = 0.008), his
pe cen age being h ee imes highe han hose assigned o di e -
ences among beds wi hin g oups (ϕ
SC
= 0.2109, P alue < 0.001,
pe cen age o gene ic di e en ia ion = 13.80%). The AMOVA
model including A cachon in he no he n g oup yielded simila
alues (ϕ
CT
= 0.3100, P alue = 0.006, pe cen age o gene ic di e -
en ia ion = 31.00%; ϕ
SC
= 0.1567, P alue < 0.001, pe cen age o
gene ic di e en ia ion = 10.81%). This model assigned a highe
pe cen age o gene ic di e en ia ion among g oups and a lowe
pe cen age o di e ences among beds wi hin g oups han he
model including A cachon in he sou he n g oup (ϕ
CT
= 0.1327,
P alue = 0.048, pe cen age o gene ic di e en ia ion = 13.27%;
ϕ
SC
= 0.2299, P alue < 0.001, pe cen age o gene ic di e en ia ion
= 19.13%), sugges ing a mo e cohe en g ouping o he beds in he
o me model.
Table 2. Coo dina es o each cockle bed, he numbe o analysed cockles (N
cockles
), numbe o pa asi es sequenced (N
B. minimus
), p e alence o B. minimus, numbe o
haplo ypes (k), numbe o polymo phic si es (PS) and haplo ype composi ion (be ween pa en heses he numbe o indi iduals bea ing he same haplo ype when
di e en om one)
Cockle bed Coo dina es
N
Cockles B. minimus P e alence kPSHaplo ype composi ion
A ei o 40.710123, −8.704596 5 20 3.3% 5 8 BmA, BmB, BmC, BmD, BmE
Baiona 42.117020, −8.820283 5 20 16.7% 3 3 BmF, LaD, LaE (3)
A cachon 44.690111, −1.182944 4 16 23.3% 4 5 BmG, LaAE, LaAQ, LaAW
Gene al 14 56 8.1% 12 14
Pa asi ology 1211
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess

Discussion
Bucephalus minimus gene ic a iabili y a hos le el
Co-in ec ion by mul iple pa asi es, om he same o di e en spe-
cies, wi hin he same hos is a well- ecognized phenomenon in he
pa asi ological li e a u e (Poulin, 2001; Read and Taylo , 2001).
This pa e n has been ex ensi ely s udied o se e al pa asi e spe-
cies, namely wi h an impac on human heal h (The on e al.,
2004; Bell e al., 2006). Fo example, in he case o mala ia,
mo e han 5 s ains ha e been ound o be in ec ing he same
hos (Bell e al., 2006). The same end was obse ed o he
ema ode pa asi e Schis osoma mansoni wi hin hei second
and inal hos (The on e al., 2004). Simila o wha is obse ed
o me ace ca ial o adul s ages o ema ode pa asi es, i would
be an icipa ed ha di e en clones would in ec he same i s
in e media e hos when housands o eggs pe in ec ed de ini i e
hos a e shed in o he wa e column, i.e. housands o mi acidia
ha ching wi hin me es o each o he . This was obse ed o
some ema ode species (Rauch e al., 2005; Keeney e al., 2007;
Lag ue e al., 2007). In he p esen s udy, only 1 COI haplo ype
was ound inside each in ec ed cockle ( ega dless o he samples’
o igin), in con as wi h wha has been p e iously eco ded.
Ne e heless, i should be no ed ha in he p esen s udy only
he COI egion (ma e nally inhe i ed) was sequenced, while o
p e ious s udies, mic osa elli e ma ke s we e used o iden i y
indi idual a iabili y. In ac , mic osa elli e ma ke s a e mo e
accu a e o popula ion s uc u e analysis and indi idual iden i i-
ca ion since hey a e highly a iable polymo phic egions
Figu e 2. Haplo ype ne wo k o Bucephalus minimus samples om A ei o (Po ugal), Baiona (Spain) and A cachon (F ance) based on gene ic dis ance (numbe o
base pai di e ences) o cy och ome c oxidase subuni 1 (COI) gene sequences. Di e en haplo ypes wi h espec i e names a e ep esen ed by ci cles, wi h ci cle
size p opo ional o obse ed equency. In e ed mu a ion s eps a e shown by black do s. Colou s depic samples aken om he same cockle.
1212 Simão Co eia e al.
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
Table 3. Haplo ype equency o B. minimus COI gene pe cockle bed including haplo ype di e si y (h± s anda d de ia ion) and nucleo ide di e si y (π± s anda d de ia ion)
Haplo ype Me ja Ze ga A ei o Baiona Noia A cachon
Bay o
Somme
English
Channel Cel ic Sea Bu y Inle The Dee Wadden Sea Sum
LaA 000000000011
LaB20000200000022
LaC 000010000001
LaD 001010000002
LaE 003020100006
LaF 000000010001
LaG 000000100001
LaH000000000011
LaI 500000000005
LaJ 000000081009
LaK 000000010001
LaL 100000000001
LaM000000010001
LaN000000010001
LaO000010000001
LaP 000000000011
LaQ000000000011
LaR 000000110114
LaS 000000010001
LaT 000000010001
LaU000000000011
LaV 000000020002
LaW000010000001
LaX 000000100001
LaY 000000010001
LaZ 000000010001
LaAA 0 0 0 0 1 0 0 0 0 0 0 1
LaAB 0 0 0 0 1 0 0 0 0 0 0 1
LaAC000010000001
LaAD 0 0 0 0 0 0 0 0 0 0 1 1
LaAE 0 0 0 0 2 0 0 0 0 0 0 2
(Con inued)
Pa asi ology 1213
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
Table 3. (Con inued.)
Haplo ype Me ja Ze ga A ei o Baiona Noia A cachon
Bay o
Somme
English
Channel Cel ic Sea Bu y Inle The Dee Wadden Sea Sum
LaAF 0 0 0 0 0 0 4 1 0 0 0 5
LaAG 0 0 0 0 0 0 0 1 0 0 0 1
LaAH 0 0 0 0 0 0 0 1 0 0 0 1
LaAI 0 0 0 0 0 0 1 0 0 0 0 1
LaAJ 0 0 0 0 0 0 0 0 0 0 1 1
LaAK 0 0 0 0 0 0 0 0 0 0 1 1
LaAL 0 0 0 0 0 0 1 0 0 0 1 2
LaAM 0 0 0 0 0 0 0 1 0 0 0 1
LaAN 0 0 0 0 1 0 2 0 0 0 0 3
LaAO000000000011
LaAP 0 0 0 0 0 0 0 0 0 0 1 1
LaAQ 0 0 0 0 7 2 12 4 1 0 16 42
LaAR 0 0 0 0 0 0 1 0 0 0 0 1
LaAS 0 0 0 0 1 0 0 0 0 0 0 1
LaAT000000100012
LaAV000000000011
LaAW000020000002
LaAX 0 0 0 0 0 0 1 0 0 0 0 1
LaAY000010000001
LaAZ 0 0 0 0 1 0 0 0 0 0 0 1
LaBA 0 0 0 0 0 0 1 0 0 0 0 1
LaBB 0 0 0 0 0 0 2 0 0 0 0 2
LaBC 0 0 0 0 1 0 0 0 0 0 0 1
BmA010000000001
BmB010000000001
BmC010100000002
BmD010000000001
BmE010000000001
BmF001000000001
BmG000010000001
BmH000001000001
BmI000001000001
1214 Simão Co eia e al.
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess
(Abdul-Munee , 2014) and inhe i ed om bo h pa en s. Despi e
i s limi a ions, he high gene ic a iabili y o he COI egion
ound in his species (12 haplo ypes ou o 14 analysed cockles),
as well as in each o he s udied beds indi idually (no epea ed
haplo ypes in 2 ou o 3 s udied cockle beds –see Table 2), sug-
ges s ha possibly only 1 indi idual (i.e. 1 mi acidium) o B. mini-
mus in ec s he hos and/o p e ails inside i . Un o una ely, no
mic osa elli e ma ke s a e cu en ly de eloped o his ema ode
species o o he closely ela ed species (which could ha e been
used by c oss- alida ion). The e o e, u he s udies using nuclea
ma ke s ha a e ei he highly polymo phic (such as mic osa el-
li es) o in a high numbe (single nucleo ide polymo phisms,
SNPs) –which would educe he p obabili y o andom sha ing
o mul ilocus geno ypes among indi iduals –will be necessa y
o con i m ou esul s.
In he Ria de A ei o, gi en he low p e alence o B. minimus,
he p esence o only 1 pa asi e haplo ype pe hos was no su p is-
ing. The e is a well-known upwelling mechanism o sho e o his
coas al lagoon (Quei oz e al., 2012), esul ing in low wa e em-
pe a u e and consequen ly lowe p e alence and abundance o
ema ode pa asi es compa ed o o he coas al sys ems whe e
cockles a e dis ibu ed (Co eia e al., 2020). T ema odes a e
highly sensi i e o empe a u e, bo h in hei ee-li ing and pa a-
si ic s ages (Thiel ges and Rick, 2006; Selbach and Poulin, 2020).
Fo example, he p oduc ion and ha ching a e o ema ode eggs
a e posi i ely co ela ed wi h empe a u e, peaking unde ideal
he mal condi ions (Mo ley, 2012; Mo ley and Lewis, 2017).
The same happens wi h ce ca ial mul iplica ion wi hin and eme -
gence om he i s in e media e hos (Poulin, 2006;de
Mon audouin e al., 2016). Hence, Ria de A ei o may ha e
ewe ee-li ing s ages (mi acidia) in he wa e and migh ake
longe o he cycle o comple e. Adding o i he high densi y
o he hos ound in he a ea, a pa asi e in ensi y dilu ion e ec
(as occu s in o he egions, e.g. Magalhães e al., 2017) migh
also con ibu e o a single conspeci ic pa asi e in ec ion in each
indi idual hos , as we obse ed. Howe e , he same pa e n (i.e.
1 haplo ype pe hos ) was ound o cockles om de la
Ramallosa lagoon (Baiona) and Île aux Oiseaux (A cachon),
whe e he p e alence o B. minimus can exceed 20%. Double
in ec ion by ema ode spo ocys s in cockles is a e (Magalhães
e al., 2015,2020), mos likely due o a e exposu e o he hos
o a second mi acidium. Howe e , occu ence o co-clone in ec-
ion has been shown o a ise when he p e alence o hos s wi h
ema ode spo ocys in ec ion ises (Keeney e al., 2008; Louhi
e al., 2013). The e o e, he a ionale abo e ha ela es empe a-
u e and low p e alence as causes o single haplo ype in ec ion
lacks suppo in Baiona (Spain) and A cachon (F ance) and sug-
ges s again ha B. minimus in ec ion may o igina e om a single
mi acidium.
Al e na i ely, he gene ic di e si y a hos le el may be de e -
mined by he in ec ion mechanisms o hese pa asi es, such as
in aspeci ic compe i ion. The p esence o a single B. minimus
haplo ype pe hos could also be explained by he p oduc ion o
subs ances ha could change hos chemical a ac i eness
(Baiocchi e al., 2017) o be oxic agains compe i o s (Bu man,
1982; Sel a e al., 2009). This is ue o nema ode pa asi es,
bu he e is no in o ma ion on ema odes. Howe e , syn hesis
o ha m ul chemicals seems highly unlikely as i would impac
he ema ode’s own clones. None heless, o es hese p edic ions,
speci ic expe imen s would need o be conduc ed.
Ano he explana ion may be ha cockles wi h mul iple in ec-
ion (i.e. hose wi h mo e han 1 haplo ype) a e a e because hey
incu highe mo ali y a es han single in ec ions. This s age o
he ema ode li e cycle is highly dele e ious o he hos and
can lead o mass mo ali y e en s du ing ou b eaks (Thiel ges,
2006; de Mon audouin e al., 2021). Pe iods o high p e alence
BmJ000000000101
BmK010000000001
BmL000010000001
Sum 26 6 5 1 29 4 30 27 2 2 30 162
h0.3846 ±
0.1017
1.0000 ±
0.0962
0.7000 ±
0.2184
1.0000 ±
0.0000
0.9384 ±
0.0340
0.8333 ±
0.2224
0.8299 ±
0.0632
0.9003 ±
0.0461
1.0000 ±
0.5000
1.0000 ±
0.5000
0.7241 ±
0.0922
Π0.000688 ±
0.000732
0.007572 ±
0.005013
0.002082 ±
0.001834
0.0000 ±
0.0000
0.006253 ±
0.003626
0.003468 ±
0.002899
0.003077 ±
0.002031
0.005543 ±
0003282
0.001719 ±
0.002426
0.005170 ±
0.005961
0.003172 ±
0.002079
Pa asi ology 1215
h ps://doi.o g/10.1017/S0031182023000987 Published online by Camb idge Uni e si y P ess