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Does he eshwa e mussel Anodon a ana ina emo e he ish pa hogen
Fla obac e ium columna e om wa e ?
© The Au ho (s) 2021
Published e sion
Hajisa a ali, Mahsa; Aal onen, Sa i; Pulkkinen, Ka ja; Taskinen, Jouni
Hajisa a ali, M., Aal onen, S., Pulkkinen, K., & Taskinen, J. (2022). Does he eshwa e mussel
Anodon a ana ina emo e he ish pa hogen Fla obac e ium columna e om wa e ?.
Hyd obiologia, 849(4), 1067-1081. h ps://doi.o g/10.1007/s10750-021-04769-6
2022
PRIMARY RESEARCH PAPER
Does he eshwa e mussel Anodon a ana ina emo e
he ish pa hogen Fla obac e ium columna e om wa e ?
Mahsa Hajisa a ali .Sa i Aal onen .Ka ja Pulkkinen .Jouni Taskinen
Recei ed: 3 Feb ua y 2021 / Re ised: 19 No embe 2021 / Accep ed: 22 No embe 2021
ÓThe Au ho (s) 2021
Abs ac Global decline o eshwa e mussels
(Unionoida) is h ea ening biodi e si y and he essen-
ial ecosys em se ices ha mussels p o ide. As il e -
eeding o ganisms, eshwa e mussels emo e phy-
oplank on and suspended pa icles om he wa e . By
il e ing bac e ia, eshwa e mussels also dec ease
pa hogen loads in he wa e . The objec i e o his
s udy was o e alua e whe he he common eshwa e
bi al e Anodon a ana ina (duck mussel) could
emo e he bac e ial ish pa hogen Fla obac e ium
columna e om he wa e . Mussels educed bac e ia
in bo h o he wo expe imen s pe o med, so ha he
bac e ial concen a ion a he end o he 96-h moni-
o ing in mussel ea men s was only 0.3–0.5 imes
ha o he con ols. Su p isingly, mussels did no
educe algal cell concen a ion s a is ically signi i-
can ly. Mussel beha io (shell openness, oo posi ion,
and mo emen ) was no a ec ed by he p esence o
bac e ia o algae, excep o biodeposi ion o ma ion,
which was g ea es in algal- ed ea men s, ollowed
by bac e ial- ed ea men s and con ols, espec i ely.
The in es ines o bac e ia-incuba ed A. ana ina ha -
bo ed F. columna e, sugges ing ha mussels inges ed
he bac e ia. P esen esul s sugges ha eshwa e
mussels may also ha e a po en ial o mi iga e aqua-
cul u e pa hogen p oblems, as well as play a ole in
wa e quali y managemen .
Keywo ds Bio il a ion Columna is disease Duck
mussel Unionidae Bac e ia Ecosys em se ices
In oduc ion
F eshwa e mussels (Unionoida) a e one o he mos
h ea ened animal g oup in he wo ld (Lydea d e al.,
2004; Lopes-Lima e al., 2017). In he ecen decades,
a numbe o eshwa e mussel species ha e gone
ex inc (Haag & Williams, 2014). Mussel popula ions
a e declining due o pollu ion, habi a deg ada ion and
agmen a ion, in oduced species, and he loss o
obliga o y ish hos s (Bogan, 2008; Geis , 2010;
Fe ei a-Rod ı
´guez e al., 2019), educing he biolog-
ical di e si y in lakes and i e s. F eshwa e mussels
ca y ou impo an ecosys em se ices ia hei
il a ion and bu owing ac i i y, including nu ien
ecycling and he deposi ion o o ganic ma e om
he wa e column o he bo om in he o m o eces
and pseudo eces (e.g., Vaughn, 2018). The
Handling edi o : E ic R. La son
Supplemen a y In o ma ion The online e sion con ains
supplemen a y ma e ial a ailable a h ps://doi.o g/10.1007/
s10750-021-04769-6.
M. Hajisa a ali (&)S. Aal onen 
K. Pulkkinen J. Taskinen
Depa men o Biological and En i onmen al Science,
Uni e si y o Jy a
¨skyla
¨, P.O. Box 35, 40014 Jy a
¨skyla
¨n,
Finland
e-mail: [email p o ec ed]
123
Hyd obiologia
h ps://doi.o g/10.1007/s10750-021-04769-6(0123456789().,- olV)(0123456789().,- olV)
biodeposi ion o eces and pseudo eces ( he non-
inges ed pa icles loosely w apped in mucus and
ejec ed wi hou unde going he diges i e p ocess;
Be g e al., 1996) by eshwa e mussels p o ides a
nu ien - ich and easily assimila ed ood sou ce o
ben hic mic obes and in e eb a es, which suppo s
ish popula ions and links mul iple ophic le els in
aqua ic ecosys ems (Howa d & Cu ey, 2006; Vaughn
e al., 2008; Vaughn, 2018). The e o e, in addi ion o
biodi e si y educ ion pe se, he decline o eshwa e
mussels can ha e a ma ked e ec on he ecosys em.
Bi al es’ po en ial ole in wa e quali y manage-
men , hei ‘‘bi al e en i onmen al se ices’’, has
al eady been ecognized in ma ine sys ems (e.g.,
nu ien apping connec ed wi h mussel p oduc ion)
(Nielsen e al., 2016; Clemen s & Comeau, 2019;
Taylo e al., 2019; Ko a e al., 2020). Mussels’
e ec i eness in nu ien and pa icle emo al and in
was ewa e ea men has also been shown in esh-
wa e sys ems (S aye , 2014; Lumme e al., 2016;
Mezzano e e al., 2016; Hoellein e al., 2017; K eege
e al., 2018). Thus, eshwa e mussels can po en ially
be u ilized in wa e quali y managemen o educe
nu ien e luen s and eu ophica ion (K eege e al.,
2018). Depending on hei abili y o emo e bac e ia,
eshwa e mussels could also be used o educe
disease ou b eaks. Se e al esea che s ha e ound ha
he d eissenid mussel D eissena polymo pha (Pallas,
1771) can clea he pa hogenic bac e ium Esche ichia
coli (Migula, 1895) Cas ellani & Chalme s, 1919 om
wa e in labo a o y expe imen s (Sil e man e al.,
1995), in ield condi ions (Co ne e al., 1995), and
om ea ed e luen s o municipal was e wa e
(Mezzano e e al., 2016). Unionidae eshwa e
mussels a e also capable o il e ing bac e ia (Jø gen-
sen e al., 1984; K yge & Riisga
˚ d, 1988; Nichols &
Ga ling, 2002; Ch is ian e al., 2004Vande ploeg
e al., 2011b), including E. coli (Sil e man e al.,
1995; Ismail e al., 2015). Howe e , he abili y o
eshwa e mussels o elimina e bac e ial ish pa ho-
gens has no been in es iga ed.
The aim o he p esen s udy was o in es iga e he
capabili y o he duck mussel, Anodon a ana ina
(Linnaeus, 1758), o emo e he bac e ium Fla obac-
e ium columna e Be na de & G imon 1989 om he
wa e column in labo a o y condi ions. Anodon a
ana ina is a unionid mussel ha commonly occu s in
he lakes and i e s o Eu ope (Lopes-Lima e al.,
2016). This mussel has been shown o il e
Diplos omum pseudospa haceum Niewiadomska,
1984 la ae om wa e , he eby educing he ans-
mission o his ema ode pa asi e om aqua ic snails
o i s ish hos (Gopko e al., 2017). Mo e impo an ly,
A. ana ina has he po en ial o il e and inges colonial
and ilamen ous cyanobac e ia (Bon es e al., 2007).
Fla obac e ium columna e is a eshwa e bac e ium
and an oppo unis ic ish pa hogen ha causes colum-
na is disease (‘‘wa m wa e disease’’) in ish.
Fla obac e ium columna e can in ec a ange o ish
species h oughou he wo ld, such as salmon, ou ,
ainbow ou , channel ca ish, ca p, pe ch, pike, eel,
and ilapia (Decle cq e al., 2013). Columna is disease
is one o he mos ha m ul diseases in channel ca ish,
Ic alu us punc a us (Ra inesque, 1818) a ming in he
USA, leading o app oxima ely $30 million yea ly
losses (Wagne e al., 2006). I is also a e y di icul
p oblem in young salmonid cul u e, causing high
mo ali y and he use o an ibio ics (Pulkkinen e al.,
2010). This pa hogen can also be ound in na u al
lakes and i e s (Kun u e al., 2012) and has been
isola ed om issues o he unionid mussel Villosa i is
(I.Lea, 1829) (Clinch Ri e , USA; S a lipe e al.,
2008).
Ou hypo hesis was ha A. ana ina would be able o
emo e F. columna e om he wa e , mani es ed as a
lowe a e age bac e ial concen a ion in he wa e
wi h mussel ea men as compa ed o he con ol
wa e wi hou mussel. As a me hodological con ol o
he eeding o mussels, we es ablished a ea men
g oup ed wi h a comme cial mic oalgae p oduc
commonly used o mussel eeding. As he bac e ial
suspension may a ec he beha io o mussels,
especially he il a ion ac i i y, moni o ing o mussel
beha io was included o he s udy. In addi ion, as he
biodeposi o ma ion can indica e il a ion o ma e
om wa e , biodeposi ion o ma ion was also moni-
o ed du ing he expe imen . The p esen s udy is he
i s e o o e alua e he e iciency o eshwa e
mussels o emo e he ha m ul bac e ial ish pa hogen
om he wa e .
Ma e ials and me hods
Mussel collec ion and p epa a ion
In o al, 30 A. ana ina indi iduals (Table 1) we e
collec ed om Lake Koija
¨ i, Finland (60.97°N,
123
Hyd obiologia
23.73°E) du ing Sep embe 2016. The mussels we e
anspo ed o Konne esi Resea ch S a ion, Uni e si y
o Jy a
¨skyla
¨and we e kep in a 163 L low- h ough
ank wi h sand on he bo om un il he expe imen s
began. The mussels we e no ed, bu hey ecei ed
ood om incoming Lake Konne esi wa e du ing he
main enance pe iod. In Janua y 2017, he mussels
we e anspo ed o he labo a o y o he Depa men
o Biological and En i onmen al Science, Uni e si y
o Jy a
¨skyla
¨. The mussels we e placed in a la ge
bucke o well wa e , ae a ed wi h an aqua ium ai
pump, and kep on a 12 h ligh –da k cycle a *17 °
C. In he labo a o y du ing he p e-expe imen main-
enance, he mussels we e ed ad libi um wi h a
comme cial phy oplank on p oduc (Shell ish Die
1800Ò, SFD, Reed Ma icul u e, USA) con aining six
ma ine mic oalgae: Isoch ysis sp., Pa lo a sp., Te-
aselmis sp., Chae oce os calci ans (Paulsen)
H.Takano, Thalassiosi a weiss logii (G unow)
G.A.F yxell & Hasle, and Thalassiosi a pseudonana
Hasle & Heimdal.The mussels we e kep wi hou ood
o 24 h be o e s a ing he expe imen s un il hey no
longe eleased exc emen . The wa e empe a u e a
Konne esi Resea ch S a ion du ing he main enance
pe iod was ini ially 17 °C in Sep embe ; i g adually
declined o 3 °C in Decembe 2016, bu i was
a i icially g adually inc eased o 17 °C in he las
wo weeks be o e he expe imen s, which we e
conduc ed a 17 °C.
P epa a ion o bac e ial suspension, Shieh medium
and aga pla es
The F. columna e s ain (B613) used in he expe i-
men s was isola ed om a wa e sample aken om he
sho e o Lake Kynsi esi (62°2500 N, 26°1500 E) in 2014;
i was hen cul i a ed in a modi ied Shieh medium
(Song e al. 1988) and s o ed a -80 °C wi h 10%
glyce ol and 10% e al cal se um. In o de o p epa e
he bac e ial solu ion o he expe imen s, 20 ll om
he ozen s ock was added o 5 ml o modi ied Shieh
medium and incuba ed wi h con inuous s i ing a
120 pm o 24 h a oom empe a u e. One ml o he
o e nigh -g own bac e ial suspension was u he
added o 9 ml o modi ied Shieh medium, wi h
ano he incuba ion a oom empe a u e o e nigh .
The op ical densi y (OD) o he bac e ial suspension
was measu ed wi h a spec opho ome e a 595 nm
and con e ed o a bac e ial concen a ion using a p e-
es ablished ela ionship be ween OD and colony-
o ming uni s (CFUs); his was done o allow he
esea che s o adjus he ini ial bac e ia concen a ion
le els du ing he expe imen s.
Modi ied Shieh medium was p epa ed acco ding o
Song e al. (1988) o se e as a selec i e g ow h
medium o isola e F. columna e. Aga pla es we e
used o pla e-coun he iable bac e ial concen a ions;
he e, 10 g o aga powde we e added pe li e and
supplemen ed wi h ob amycin in o de o inhibi he
g ow h o bac e ia o he han F. columna e (Decos e e
e al., 1997).
Expe imen 1
The i s expe imen o e alua e A. ana ina’s abili y o
emo e F. columna e om wa e began in Feb ua y
2017 and included 5 ea men s wi h 5 eplica es each
( eplica e = 1 sedimen /sand- ee aqua ium illed wi h
5 L o well wa e wi h o wi hou a mussel indi idual;
see below). A schema ic p esen a ion o he expe i-
men al design is gi en in he Supplemen a y Fig. 1.
The aqua ia we e ae a ed and kep on a ligh –da k
cycle o 12 h:12 h a *17 °C. The expe imen al
ea men s we e as ollows:
Table 1 The numbe o mussels (Anodon a ana ina), a e age
leng h (mm, mean ±SD) and age (y, mean ±SD), sex
(F= emale, M= male), in ensi y o ema ode in ec ion
(Rhipidoco yle ennica) (ze o, ligh , mode a e, hea y; see ex
o ca ego iza ion), and occu ence o glochidia in mussels
used in he 1s and 2nd expe imen
Numbe o mussels Leng h Age Sex T ema ode Glochidia
Mean Mean F M Unknown Ze o Ligh Mode a e Hea y Numbe
1s expe imen 15 83 ±4 5.6 ±16 4 5 1 4 4 6 0
2nd expe imen 9 87 ±4 6.1 ±11 4 4 2 2 2 3 0
123
Hyd obiologia
(1) Bac e ia and mussel: A known concen a ion
(5 910
5
CFU ml
-1
)o F. columna e wi h an
indi idual mussel o e alua e he po en ial o A.
ana ina o il e ou F. columna e.
(2) Bac e ia con ol: A known concen a ion
(5 910
5
CFU ml
-1
)o F. columna e wi hou
a mussel was used o s udy changes in he
bac e ial concen a ion o he wa e in he
absence o he mussel.
The null hypo hesis was ha he e was no
di e ence in bac e ial concen a ion be ween
ea men s (1) and (2).
(3) Algae and mussel: A known concen a ion o
algae wi h a mussel; his se ed as a e e ence
ea men o s udy A. ana ina’s algal il a ion.
(4) Algae con ol: A known concen a ion o algae
wi hou a mussel was used o ack changes in
algae concen a ion in he wa e in he absence
o he mussel.
In ea men s (3) and (4), 35 ml o Shell ish Die
1800Òwas added o 1.1 L wa e ; hen, 100 ml
o his suspension was added o each aqua ium.
The null hypo hesis was ha he e was no
di e ence in algae concen a ion be ween ea -
men s (3) and (4).
(5) Mussel con ol: A mussel wi hou F. columna e
and wi hou algae was used o s udy mussel
beha io wi hou he p esence o bac e ia and
algae.
The null hypo hesis was ha he e was no
di e ence in beha io s be ween ea men s (1),
(3), and (5).
Expe imen 2
The second expe imen began in Ma ch 2017 o e i y
he esul s o Expe imen 1 in ega d o bac e ial
il a ion; i used a bac e ial inocula ion ha was an
o de o magni ude lowe han in Expe imen 1 (see
he Resul s sec ion and Fig. 1). A schema ic p esen-
a ion o he expe imen al design is gi en in he
Supplemen a y Fig. 2. The aqua ia we e ae a ed and
kep on a ligh –da k cycle o 12 h:12 h a *17 °C.
The expe imen al ea men s we e as ollows:
(1) Bac e ia and mussel: A known concen a ion o
F. columna e wi h an indi idual mussel o e alua e A.
ana ina’s po en ial o il e F. columna e; 9 eplica e
aqua ia wi h a single mussel in each we e used.
(2) Bac e ia con ol: A known concen a ion o F.
columna e wi hou a mussel was used o s udy changes
in bac e ial concen a ion in he absence o he mussel;
5 eplica e aqua ia we e used.
The null hypo hesis was ha he e was no di e -
ence in bac e ial concen a ion be ween ea men s (1)
and (2).
Bac e ial sampling om he wa e
Wa e samples we e aken om all he aqua ia p io o
s a ing each expe imen o exclude he possibili y o
p io con amina ion by F. columna e. The concen a-
ion o bac e ia was e alua ed om 100 ll wa e
samples om ea men s wi h added bac e ia (bac e ia
and mussel, bac e ia con ol) a ime poin s 0 h, 6 h,
and 24 h; 1 sample pe aqua ium was e alua ed. In
o de o dec ease he obse a ional e o associa ed
wi h educing bac e ial le els a ime poin s 48 h and
96 h, he numbe o epea ed wa e samples aken pe
aqua ium was inc eased o h ee. In Expe imen 2,
100 ll o wa e was aken a ime poin s 0 h, 48 h, and
96 h; 2 epea ed samples we e aken pe aqua ia. A
dilu ion se ies (10
-1
–10
-7
) o each wa e sample was
p epa ed and cul u ed on Shieh aga pla es; colony
o ma ion was checked a e 48 h. A he end o bo h
expe imen s, ea men s wi hou bac e ia we e
checked as desc ibed abo e (2 9100 ll wa e sam-
ples pe aqua ium) o exclude po en ial con amina ion
om added bac e ia du ing he expe imen .
Bac e ial sampling om in es ines and gonads
To examine i mussels can inges bac e ia and become
in ec ed wi h F. columna e, bac e ial samples we e
aken om in es ine and gonad o he mussels om all
ea men s a he end o bo h expe imen s. To a oid
bac e ial con amina ion om he aqua ium wa e ,
mussel shell su aces we e sp ayed wi h 70% e hanol
be o e dissec ion. A e opening he al es, he mussel
body was cu in 2 so ha a c oss-sec ion h ough he
gonads—and he in es ine winding h ough he
gonads—could be accessed. Then, he bac e ial sam-
ples we e collec ed wi h a s e ile loop and cul u ed on
Shieh aga pla es supplemen ed wi h ob amycin. In
Expe imen 1, bac e ial sampling was done i s om
he in es ine and hen om he gonads. In Expe imen
2, he sampling o de was e e sed o minimize
123
Hyd obiologia

possible con amina ion o he gonads om he
in es ine.
Algal cell sampling
To in es iga e A. ana ina’s il a ion capabili y, algal
cell concen a ion was moni o ed om 3 eplica e
wa e samples pe aqua ium a di e en ime poin s
(0 h, 6 h, 24 h, 48 h) using a mic oscope coun ing
chambe (Bu
¨ ke , Ma ien eld, Ge many, dep h = 0.1
mm, squa e wid h = 0.25 mm). A e 24 h, algal cell
concen a ion declined sha ply in bo h ea men s, and
moni o ing was discon inued.
Mussel beha io
In o de o check he possible e ec o he bac e ial
and algal die s on he mussels’ beha io ( o example,
a comple e inac i a ion o mussels when incuba ed
wi h bac e ia), in bo h expe imen s, A. ana ina
beha io was eco ded 3 imes pe day a 9:00,
12:00, and 15:00 h in a 2 min isual inspec ion o
99 h. The ai s ha we e moni o ed included shell
openness, oo posi ion (ex ension o con ac ion),
mo emen , and biodeposi ion ( eces and pseudo eces)
o ma ion since he p e ious moni o ing. Shell open-
ness was sco ed as ollows: ully closed (siphons
comple ely in isible) = 0, sligh ly opened (siphons
a e s ill in isible) = 1, hal open (siphons isible,
pa ly p o uding) = 2, ully open (siphons ully
isible, ully p o uding) = 3. Foo posi ion was
g aded as ollows: oo in = 0, oo pa ly ou = 1,
oo ou = 2. Mo emen (posi ion changes in he
mussel) was moni o ed by compa ing mussel posi ion
o a ma k on he lid o each aqua ium deno ing he
posi ion a he p e ious inspec ion. I was sco ed as
ollows: mo emen = 1, no mo emen = 0. Biodepo-
si ion p esence was classi ied in o 2 ca ego ies:
Fig. 1 Bac e ial
concen a ion (CFU ml
-1
)
(A) in aqua ia wi h mussels
and wi hou mussels a
di e en ime poin s (N = 5)
(mean ±SD) in he 1s
expe imen , in he 2nd
expe imen (B), and algal
cell concen a ion (cell
ml
-1
)(C) in aqua ia wi h
mussels and wi hou
mussels a di e en ime
poin s (N = 5) (mean ±SD)
123
Hyd obiologia
wi hou biodeposi ion = 0, and wi h
biodeposi ion = 1.
T ema ode pa asi ism, sex, and ep oduc ion
o mussels
Possible ema ode pa asi ism (pa asi e species, in en-
si y o in ec ion) in he mussels was examined a he
end o each expe imen when he bac e ial sampling
om he in es ine and gonads was pe o med; his was
done because ema odes can ha e a ma ked in luence
on mussel physiology (Taskinen, 1998; Jokela e al.,
2005). The gonads a e he main si e o ema ode
in ec ion in A. ana ina (Taskinen e al., 1997).
The e o e, a 2–3 mm slice ac oss he gonads was
dissec ed a e he bac e ial sampling was conduc ed,
p essed be ween la ge glass pla es, and examined
mic oscopically o ema ode spo ocys s using ans-
mi ed ligh . This was accompanied wi h he sex
de e mina ion o he mussel and an es ima ion o he
in ensi y o he in ec ion (Taskinen e al., 1994;
Taskinen & Val onen, 1995). The in ensi y o he
ema ode in ec ion was sco ed as ollows: unin-
ec ed = 0, ligh in ec ion (one spo ocys ubule o one
g oup o spo ocys ubules) = 1, mode a e in ec ion
om [1 g oup o spo ocys ubules o almos com-
ple e eplacemen o mussel gonad issue by spo o-
cys s = 2, and hea y in ec ion (gonad issue
comple ely eplaced by ema ode spo ocys s) = 3
(Taskinen e al., 1994).
S a is ical analyses
The s a is ical analyses o bac e ial and algal concen-
a ions we e pe o med using IBM SPSS so wa e
( e sion 24.0.0). Cell concen a ions ( he CFUs o F.
columna e and he coun s o algal cells) we e used as
esponse a iables; aqua ia we e used as he s a is ical
uni s. Gene al Linea Model (GLM) epea ed mea-
su es analyses we e pe o med o compa e di e ences
in bac e ial and algal concen a ion in ime ( he
wi hin-subjec ac o ) be ween ea men s wi h and
wi hou a mussel ( he be ween-subjec ac o ). The
di e ences be ween di e en ime poin s we e u he
examined using wi hin-subjec con as (‘‘simple’’).
Le ene’s es was used o assess he homogenei y o
a iance, and he G eenhouse–Geisse co ec ion was
applied o co ec he numbe o deg ees o eedom
when he sphe ici y assump ion was no alid.
Di e ences in mussel beha io (shell openness, oo
posi ion, mo emen , biodeposi ion o ma ion) a di -
e en ime poin s be ween he ea men s (mussel
only, mussel ?bac e ia, mussel ?algae) we e ana-
lyzed using mixed e ec s logis ic eg ession (GLMM)
in R ( e sion 4.0.2.) s a is ics so wa e and Lme4
package (R De elopmen Co e Team, 2015). The
in e dependence o epea ed obse a ions om he
same indi idual mussel du ing he expe imen was
accoun ed o by ea ing mussel iden i y as a andom
a iable. To be able o i he models using binomial
(logi ) dis ibu ion, all beha io al da a we e ecoded as
0 o 1 be o e he analysis. Thus, shell openness was
eca ego ized as 0 ( al es ully closed) and 1 ( al es
sligh ly, hal o ully open), and oo posi ion as 0 ( oo
in) and 1 ( oo pa ly o comple ely ou ).
The di e ences in he in ensi y o ema ode
in ec ion be ween ea men g oups in Expe imen 1
sco ed om ze o (unin ec ed) o 3 (hea ily in ec ed)
could no be es ed wi h a Chi-squa e es because
expec ed alues we e less han 5. Ins ead, we used
Fishe s exac es ex ended o 3 93 con ingency
able (McDonald , 2014). In addi ion, he di e ence in
he mean in ensi y o he ema ode in ec ion was
es ed be ween he ‘‘bac e ia and mussel’’ ea men s
in bo h expe imen s wi h a - es . Di e ences be ween
ea men s we e conside ed signi ican i p 0.05.
Resul s
E ec o mussel il a ion on bac e ial
concen a ion (Expe imen 1)
A he beginning o Expe imen 1, he mean concen-
a ion o bac e ia was simila in bo h ea men g oups
wi h F. columna e (Fig. 1A). Du ing he nex 48 h, he
bac e ial concen a ion inc eased in bo h ea men s,
bu he inc ease was mo e p onounced in he ea men
wi hou a mussel han in he ea men wi h a mussel.
Be ween 48 and 96 h, he bac e ial concen a ion
dec eased in bo h ea men s (Fig. 1A). The bac e ial
concen a ion was lowe in he ea men wi h a mussel
han in he ea men wi hou a mussel (Table 2,
be ween-subjec e ec s, F
1,8
= 16.31, p= 0.004).
Howe e , his di e ence became signi ican only a e
96 h o he expe imen , as indica ed by he signi ican
in e ac ion be ween ime and ea men and he
con as s be ween he le els o he wi hin-subjec
123
Hyd obiologia
ac o and ea men ( ime x ea men ) (Table 2).
Thus, i ook 48 h om he beginning o he expe -
imen be o e he emo al o he bac e ia by he
mussels was appa en (Fig. 1A). The ea e , he mus-
sels educed he bac e ia so ha he bac e ial concen-
a ion in mussel ea men was 0.7–0.5 imes ha o
he con ols a 48 and 96 h, espec i ely (Fig. 1A).
Thus, he mussels emo ed hal he bac e ia wi hin
96 h as compa ed o he con ols.
E ec o Mussel Fil a ion on Bac e ial
Concen a ion (Expe imen 2)
In Expe imen 2, a simila pa e n in bac e ial
concen a ions o hose in Expe imen 1 was obse ed.
The bac e ial concen a ions we e simila in he
beginning o he expe imen in he ea men s wi h
and wi hou a mussel, espec i ely (Fig. 1B). A 48 h,
he mean concen a ion o F. columna e had inc eased
in bo h ea men s, bu wi h a mussel he inc ease was
app oxima ely hal o ha in he ea men wi hou a
mussel. Be ween 48 and 96 h, he bac e ial
Table 2 Resul s o GLM epea ed measu es ANOVA o he mean concen a ion o Fla obac e ium columna e (CFU ml
-1
) in he
wo ea men s (wi h and wi hou mussel) a di e en ime poin s in he 1s and 2nd expe imen
Sou ce Fi s expe imen Second expe imen
d MS F pd MS F p
Be ween subjec s
In e cep 1 2.95E?12 654.47 0.001 1 4.75E?11 180.30 0.001
T ea men 1 7.36E?10 16.31 0.004 1 489E?10 18.55 0.001
E o 8 3.61E?10 12 2.63E?09
Wi hin subjec s
Time 2.335 1.78E?12 61.63 0.001 2 2.83E?11 61.17 0.001
Time 9 ea men 2.335 1.52E?11
a
5.26 0.012 2 2.74E?10 5.91 0.008
E o 18.676 2.88E?10
a
24 4.64E?09
Wi hin-subjec con as s
b
Time
Time 6 h s ime 0 h 1 8.26E?09 3.79 0.087
Time 24 h s. ime 0 h 1 1.55E?12 47.93 0.001
Time 48 h s. ime 0 h 1 5.95E?12 139.97 0.001 1 9.09E?11 107.46 0.001
Time 96 h s. ime 0 h 1 2.11E?12 61.64 0.001 1 4.22E?09 0.46 0.511
Time 9 ea men
Time 6 h s ime 0 h 1 6.76E?07 0.03 0.865
Time 24 h s. ime 0 h 1 1.37E?09 0.04 0.842
Time 48 h s. ime 0 h 1 1.72E?11 4.06 0.079 1 1.09E?11 12.92 0.004
Time 96 h s. ime 0 h 1 4.19E?11 12.21 0.008 1 3.36E?10 3.64 0.081
E o
Time 6 h s ime 0 h 8 2.18 E?09
Time 24 h s. ime 0 h 8 3.23 E?10
Time 48 h s. ime 0 h 8 4.25 E?10 12 8.46 E?09
Time 96 h s. ime 0 h 8 3.43 E?10 12 9.22 E?09
D = deg ees o eedom, MS = mean squa es
a
G eenhouse–Geisse co ec ed alues
b
Wi hin-subjec con as o he in e ac ion be ween ime (hou ) and ea men s. The con as is ‘‘simple’’, which compa es each ime
poin o he i s ime poin ( ime 0 h). No e ha o he 2
nd
expe imen measu emen s we e made a ime poin s 0, 48 and 96 h
123
Hyd obiologia
concen a ions dec eased in bo h ea men s (Fig. 1B).
The bac e ial concen a ion was lowe in he ea men
wi h a mussel han in he ea men wi hou a mussel
(Table 2, be ween-subjec e ec s, F
1,12
= 18.55,
p= 0.001). Con as s be ween he le els o he
wi hin-subjec ac o and ea men ( ime x ea men )
indica ed ha his di e ence was signi ican a e 48 h
bu no a e 96 h o beginning he expe imen
(Table 2). Mussels educed he bac e ial concen a ion
in mussel ea men o 0.5 and 0.3 imes ha o he
con ols a 48 and 96 h, espec i ely (Fig. 1B). Thus,
he mussels emo ed wo- hi ds o he bac e ia wi hin
96 h as compa ed o he con ols.
Gonad and In es ine Analysis (Expe imen s 1
and 2)
In Expe imen 1, F. columna e was isola ed bo h om
he mussels’ in es ines and gonads in he ea men
wi h added bac e ia, bu no in he o he ea men
g oups (i.e., he mussels wi h algae and he mussels
kep in clean wa e ). A he indi idual (mussel)
aqua ium le el, among he 5 eplica es o mus-
sel ?bac e ium ea men , 3 mussels had F. colum-
na e in bo h he in es ine and he gonads, 1mussel had
F. columna e in he in es ine bu no in he gonads, and
1 es ed nega i e (no bac e ium in ei he he in es ine
o in he gonads). In Expe imen 2, among he 9
eplica es o mussel and bac e ia, F. columna e was
only ound in he in es ines o 8 indi iduals; i was no
ound in he gonads.
E ec o Mussel Fil a ion on Algal Cell
Concen a ion (Expe imen 1)
The mean concen a ion o algal cells (Shell ish Die )
dec eased be ween he s a o he expe imen and 48 h
in bo h ea men s wi h and wi hou a mussel (Fig. 1C,
Table 3, wi hin-subjec e ec [ ime], F
3,24
= 83.20,
p= 0.001). Howe e , he p esence o A. ana ina did
no a ec algal cell concen a ion (Table 3,
F1,8 = 1.872, p= 0.208).
Mussel Beha io
Shell openness (Expe imen s 1 and 2)
In bo h expe imen s, he mussels in all ea men s had
open o pa ly open al es a he beginning o he
expe imen . The mussels g adually closed hei al es
du ing he i s 24 h bu s a ed opening hei shells
again owa d he end o he expe imen (Fig. 2A and
E). In Expe imen 1, he p esence o bac e ia did no
a ec he mussels’ shell openness as compa ed o he
mussels wi h algal cells o hose in clean wa e
(GLMM, p [0.05, Table 4).
Foo posi ion (Expe imen s 1 and 2)
Foo posi ion sco es e ealed ha he mussels kep
hei ee subs an ially con ac ed (Fig. 2B and F). In
Expe imen 1, he bac e ial p esence did no a ec he
oo posi ion as compa ed o he mussels exposed o
he algae o hose kep in clean wa e (GLMM
p[0.05, Table 4).
Mo emen (Expe imen s 1 and 2)
Anodon a ana ina in all ea men g oups in bo h
expe imen s exhibi ed a con inuous and cohe en
pa e n in mo emen beha io h oughou he expe -
imen (Fig. 2C and G). The p esence o bac e ia had a
sligh e ec in dec easing mussels’ mo emen ac i i y
(GLMM, he ela i e change in compa ison o mussel
only ea men 0.69 ±0.36, p = 0.053, Table 4),
while he mo emen ac i i y o mussels wi h algae
was lowe han mussels in clean wa e (GLMM, he
ela i e change in compa ison o mussel only ea -
men 0.75 ±0.36, p = 0.036, Table 4).
Table 3 Resul s o GLM epea ed measu es ANOVA o he
mean o algal cell concen a ion (cell ml
-1
) in he wo ea -
men s wi h and wi hou mussel a di e en ime poin s.
D = deg ees o eedom, MS = mean squa es
Sou ce d MS Fp
Be ween subjec s
In e cep 1 3.68E?13 1681.82 0.001
T ea men 1 4.10E?10 1.87 0.208
E o 8 2.19E?10
Wi hin subjec s
Time 3 2.46E?12 83.16 0.001
Time 9 ea men 3 8.33E?10 2.82 0.061
E o 24 2.96E?10
123
Hyd obiologia
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