ma s-07-577421 Sep embe 21, 2020 Time: 17:21 # 1
ORIGINAL RESEARCH
published: 24 Sep embe 2020
doi: 10.3389/ ma s.2020.577421
Edi ed by:
Samad Rahimnejad,
Uni e si y o Sou h Bohemia in ˇ
Ceské
Bud ˇ
ejo ice, Czechia
Re iewed by:
Vincenzo Pa ino,
Uni e si y o Messina, I aly
Weiqun Lu,
Shanghai Ocean Uni e si y, China
*Co espondence:
Daniel P ie o
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Aqua ic Physiology,
a sec ion o he jou nal
F on ie s in Ma ine Science
Recei ed: 29 June 2020
Accep ed: 03 Sep embe 2020
Published: 24 Sep embe 2020
Ci a ion:
P ie o D, A anz K, U u xu u I,
Na a o E, U u ia MB and Iba ola I
(2020) Va iable Capaci y o Acu e
and Ch onic The mal Compensa ion
o Physiological Ra es Con ibu es
o In e -Indi idual Di e ences
in G ow h Ra e in Mussels (My ilus
gallop o incialis).
F on . Ma . Sci. 7:577421.
doi: 10.3389/ ma s.2020.577421
Va iable Capaci y o Acu e and
Ch onic The mal Compensa ion o
Physiological Ra es Con ibu es o
In e -Indi idual Di e ences in G ow h
Ra e in Mussels (My ilus
gallop o incialis)
Daniel P ie o*, K is ina A anz, Iñaki U u xu u, En ique Na a o, Mi en Bego U u ia
and I in zi Iba ola
GIU 17/061, GI 609, Depa amen o de Gené ica, An opología Física y Fisiología Animal, Facul ad de Ciencia y Tecnología,
Uni e sidad del País Vasco/Euskal He iko Unibe si a ea (UPV/EHU), Bilbao, Spain
The aim o his s udy was o asce ain i he capaci y o acu e and ch onic compensa ion
o he mal e ec s on physiological a es ep esen s a ai con ibu ing o in e -indi idual
g ow h a e di e ences in he mussel My ilus gallop o incialis. Ju enile mussels (10–
11 mm shell leng h) we e collec ed, anspo ed o he labo a o y, and di ided in o wo
g oups: one g oup was main ained a 20◦C (wa m ea men ), and he o he a 10◦C
(cold ea men ). The mussels we e ea ed a hese wo empe a u es (con inuously ed),
un il clea size di e ences allowed us o selec as - (F) and slow (S)-g owing indi iduals
om bo h g oups (F20/S20 a 20◦C and F10/S10 a 10◦C). Selec ed F and S mussels
we e hen exposed o h ee expe imen al empe a u es (10, 15, and 20◦C), and he
ime-cou se o hei esponse, in e ms o clea ance a e (CR: L/h) and ou ine oxygen
consump ion (VO2: mLO2/h), was moni o ed. The o e all g ow h a e o mussels in
he wa m ea men g oup was signi ican ly highe han in he cold ea men g oup.
Fo bo h ea men s, signi ican di e ences we e ound in key physiological pa ame e s
be ween F and S mussels: F mussels had a highe CR and a la ge gill su ace a ea han
hei S coun e pa s. Al hough no signi ican di e ences in he he mal sensi i i y o he
clea ance o me abolic a es we e obse ed be ween F20 and S20 mussels ea ed a
20◦C, when exposed o acu e empe a u e changes, expe imen s wi h mussels ea ed
a 10◦C e ealed a di e en ou come: in esponse o acu e wa ming ( om 10◦C o 15
and 20◦C), F10 we e capable o compensa ing o he he mal e ec on CR and VO2;
howe e , no such compensa o y esponse was obse ed in S10. We conclude ha wo
signi ican ac o s con ibu e o endogenous di e ences in he g ow h a e o mussels:
(i) he capaci y o exhibi in ense il e ing ac i i y, which appea s o be unc ionally
co ela ed wi h he gill su ace a ea and (ii) he capaci y o compensa e o he e ec s o
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P ie o e al. Me abolic The mal Compensa ion in Mussels
empe a u e on il a ion and me abolic a e. The second ai does no appea o make
a signi ican con ibu ion o he in e -indi idual size-di e en ia ion obse ed in mussels
main ained in wa m en i onmen s, bu explains a signi ican p opo ion o in e -indi idual
g ow h a e di e ences in cold en i onmen s.
Keywo ds: g ow h, clea ance a e, me abolic a e, empe a u e, he mal compensa ion, My ilus gallop o incialis
INTRODUCTION
The physiological mechanisms unde lying di e ences in in e -
indi idual g ow h a e in bi al es ha e been ho oughly analyzed
in e ms o physiological ene ge ics in a wide a ie y o
s udies co e ing a b oad ange o species and en i onmen al
condi ions (To o and Ve ga a, 1998;Bayne, 1999, 2004;Bayne
e al., 1999a,b;To o e al., 2004;Pace e al., 2006;Pe ne
e al., 2008;Tamayo e al., 2011, 2015). Those wo ks epo ed
se e al di e ences be ween as - and slow-g owing indi iduals,
including di e en ial eeding a es, me abolic e iciency, and
ene gy alloca ion pa e ns. Such he e ogenei y indica es ha ,
a he han being associa ed wi h inna e di e ences in a single
physiological ai , in e -indi idual di e ences in g ow h a e
a e caused by di e ences in mul iple physiological p ocesses.
A se ies o s udies pe o med in ou labo a o y showed
ha he en i onmen al condi ions, and pa icula ly ophic
condi ions, p e ailing du ing he ea ing pe iod de e mine which
physiological pa ame e s con ibu e o di e ences in he in e -
indi idual g ow h a es o he mussel My ilus gallop o incialis
(Tamayo e al., 2016;P ie o e al., 2018, 2019). When ed
wi h a cons an supply o ei he high- o low-quali y ood, as
g owe s di e om slow g owe s in hei capaci y o exhibi
signi ican ly highe CR, a ea u e ha appea s o be unc ionally
linked o he possession, o de elopmen , o la ge gill su ace
a ea (P ie o e al., 2018, 2019, 2020). Howe e , unde se e e
eeding es ic ions, as g owe s di e om slow g owe s in hei
capaci y o de elop a lowe s anda d me abolic a e (SMR) du ing
pe iods o s a a ion. Such esul s led P ie o e al. (2018) o
de ine wo basic pheno ypes o as -g owing bi al es: as eede s
and ene gy sa e s.
Toge he wi h ood a ailabili y, wa e empe a u e s ongly
a ec s g ow h a e in ec o he mic o ganisms, including bi al es
(Newell e al., 1977;Bux on e al., 1981;Kang e al., 2016);
howe e , e y ew s udies ha e analyzed he possibili y ha in e -
indi idual di e ences in capaci y o he mal compensa ion o
physiological p ocesses could con ibu e o di e ences in in e -
indi idual g ow h a e. Ea ly s udies by Hawkins (1985) and
Hawkins e al. (1987) analyzed he p o ein u no e and ene gy
balance o My ilus edulis acclima ed o 10◦C, a e wa ming
hem o 20◦C. These au ho s ound ha he indi iduals ha
g ew as e had slowe p o ein u no e ; his esul ed in a lowe
me abolic a e and g ea e homeos a ic abili y unde condi ions
ha cause a ise in empe a u e. These au ho s concluded ha (i)
he capaci y o he mal compensa ion wi h ega ds o me abolic
a e has a signi ican e ec on he a e o p o ein u no e
in a gi en indi idual, and (ii) as e p o ein u no e a es
cons ain he scope o ac i i y, hus esul ing in a sho he mal
ange o posi i e ene ge ic scope o g ow h in he mussel.
In ano he s udy, Pe ne e al. (2008) analyzed he capaci y
o he mal adap a ion in a gene ically dis inc g oup o oys e ,
C assos ea i ginica, showing clea di e ences in g ow h a e.
These au ho s analyzed memb ane lipid composi ion, and ene gy
budge , ollowing acclima ion o di e en wa e empe a u es (4,
12, and 20◦C), and ound ha indi iduals om as -g owing
gene ic lines exhibi ed lowe SMRs; his was a ibu ed o he
educed unsa u a ion indexes o memb ane lipids. Tamayo e al.
(2013) measu ed he sho and medium- e m physiological
esponse o as (F)- and slow (S)-g owing clams (Rudi apes
philippina um) ollowing exposu e o cold (10◦C) and wa m
(24◦C) wa e empe a u es, and ound ha he g ow h a e o
he S g oup was cons ained by he signi ican ly highe he mal
dependency o he me abolic expendi u es which was ound o
p omo e a la ge inc ease in he ou ine me abolic a e (RMR) a
wa m empe a u es.
The aim o he p esen s udy was o asce ain i he
capaci y o acu e and ch onic compensa ion o he e ec s
o empe a u e change on physiological a es could po en ially
con ibu e o di e ences in in e -indi idual g ow h a es in he
mussel M. gallop o incialis. To his end, we ea ed ju enile
mussels in he labo a o y a 20 and 10◦C un il clea in e -
indi idual size-di e en ia ion allowed us o selec as - (F) and
slow-(S) g owing indi iduals om each ea ing empe a u e.
Selec ed F and S indi iduals we e hen used in a se ies o
expe imen s o (i) compa e he physiological p o iles o F and
S mussels unde di e en he mal egimes, and (ii) analyze he
acu e e ec s o empe a u e change on he il e ing ac i i y and
me abolic a e o F and S indi iduals.
MATERIALS AND METHODS
Collec ion and Selec ion o Mussel
Seeds
Mussel seeds (abou 500) o M. gallop o incilis we e collec ed
in Ap il 2015 om monolaye mussel beds g owing in a ocky
in e idal a ea loca ed in An zo as (Biscay, Spain, 43◦24029.100N;
2◦40051.000W), om a mussel popula ion ha has been used
in ou p e ious publica ions (P ie o e al., 2018, 2019, 2020).
Once a he labo a o y, mussels we e main ained imme sed in
anks whe e seawa e salini y (33PSU) and empe a u e (15◦C)
esembled hose measu ed a he mussel collec ion poin , and
we e ed Isoch ysis galbana du ing he ime (days) needed
o checking ha mussels we e appa en ly heal hy and ac i e,
and selec ing hose o be used in la e expe imen s. Du ing
his condi ioning pe iod, he shell leng h o each mussel was
measu ed wi h elec onic calipe s and 300 homogenously sized
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P ie o e al. Me abolic The mal Compensa ion in Mussels
indi iduals (shell leng h: 10.65 ±0.56 mm and li e weigh :
0.2 ±0.04 g) we e selec ed o pe o m he expe imen s.
Expe imen al Design
The selec ed 300 mussels we e di ided in o wo g oups o 150
indi iduals ha we e ea ed sepa a ely in wo anks, unde wo
di e en main enance empe a u es, 20 and 10◦C. We chose
hose wo empe a u es o being close o he maximum and
minimum seasonal alues eco ded in hei na u al en i onmen
(Iba ola e al., 2008;Albaina e al., 2009;A a ena e al.,
2009). All o he condi ions, wi h he excep ion o empe a u e,
we e iden ical du ing he ea ing pe iod: seawa e (33PSU) was
con inuously ae a ed and mussels we e ed a high o ganic con en
die (app oxima ely 70%) consis ing o cells o ou own cul u es
o I. galbana (T-Iso) mixed wi h Shell ish Die R
(a comme cial
mix o ou mic oalgae: Isoch ysis,Pa lo a,Te aselmis, and
Thalassiosi a weiss logii) and p e-sie ed pa icles o na u al sil .
The die was con inuously pumped o he seawa e anks om
concen a ed s ocks using pe is al ic pumps ha we e se o
p o ide o a s able ood concen a ion o 1.5 mm3/L in he
ank. F equen moni o ing o pa icle concen a ion in he
anks wi h a Mul isize 3 Coul e Coun e (Beckman Coul e )
allowed egula ing die pumping a es. To a oid accumula ion
o ammonia, he seawa e in he anks was o ally enewed
wice pe week. Du ing he wa e enewal he anks we e insed
wi h unning ap wa e and he mussels we e pulled apa om
one ano he by ca e ully cu ing he byssus. Such a p ocedu e
a oid mussel clus e ing, hus, ensu ing he o al absence o in e -
indi idual compe i ion o ood.
Addi ionally, once pe 2–3 weeks, he size o indi idual
mussels was de e mined by measu ing he shell-leng h using
elec onic calipe s (accu a e o 0.05 mm) and he li e weigh
by using a 10−5g p ecision balance. Mussels we e main ained
unde hese cons an condi ions un il clea in e -indi idual size
di e ences we e obse ed (a 2 and 5 mon hs o mussels
main ained a 20 and 10◦C, espec i ely) (Figu e 1). A e his
long-las ing ea ing pe iod, we selec ed in each acclima ion
empe a u e he 30 smalles and he 30 bigges mussels ha we e
conside ed o ep esen , espec i ely, he slow (S) and as (F)
g owing pheno ypes. Acco dingly, ou expe imen al g oups o
mussels we e c ea ed: as g owe s selec ed a 20◦C (F20), slow
g owe s selec ed a 20◦C (S20), as g owe s selec ed a 10◦C (F10),
and slow g owe s selec ed a 10◦C (S10). Mussels om hese
ou g oups we e hen used o he mal expe imen s ha we e
speci ically designed o analyze he acu e e ec s o empe a u e
change on physiological pe o mance (Figu e 1).
The mal Expe imen s Wi h Selec ed F and S Mussels
The selec ed F and S mussels om wa m (F20 and S20) and cold
(F10 and S10) ea men s we e exposed o h ee expe imen al-
empe a u es (Tex: 10, 15, and 20◦C). Mussels we e submi ed o
he expe imen al empe a u es wi hou any condi ioning pe iod,
his is, he wa m ea ed selec ed mussels we e di ided in o
h ee g oups and we e di ec ly imme sed a he co esponding
expe imen al empe a u e: one g oup a 10◦C, ano he one a
15◦C and he las one a 20◦C. The same p o ocol was used o
he cold ea ed selec ed mussels, ha we e also spli in o h ee
expe imen al g oups.
A any Texp, we moni o ed he ime-cou se o he clea ance
a e (CR) and RMR un il s able alues we e eco ded. As a ule,
CR was de e mined daily (we s a ed moni o ing he CR jus a e
he i s hou o exposi ion o any empe a u e change) and RMR
once e e y 2 days. We could no se in ad ance he leng h o
he moni o ing pe iod, being dependen on he esponse shown
by he expe imen al mussels, so we ex ended i un il obse ing
cons ancy; i ook be ween 8 and 22 days o he di e en
expe imen al condi ions. Once eeding and me abolic cons an
esponses we e obse ed a each expe imen al empe a u e, ood
deli e y was s opped and he educ ion in me abolic a e in
s a ed mussels was analyzed by moni o ing me abolic a e un il
i was obse ed o each o a s able minimum alue ha was
conside ed o ep esen he SMR.
Physiological Measu emen s
Clea ance Ra e
In o de o de e mine clea ance a es, we placed i e indi iduals
(n= 5) om each mussel g oup (F20, S20, F10, and S10) on o
il a ion chambe s. These chambe s we e 150 mL bo osilica e
glass bo les, wi h in low and ou low lines d illed in o he
plas ic lid. Wa e om a he mos a ic eeding ank con aining
he expe imen al die was pumped h ough he chambe s by
means o mul ichannel pe is al ic pumps ha we e egula ed o
p oduce low a es ha educed he pa icle concen a ion inside
he chambe s by 15–30%. A mix u e o I. galbana (T-Iso), and
sil pa icles (app oxima ely 2.5:1) was pumped in o he eeding
anks ( om concen a ed s ock solu ions) using pe is al ic pumps
a a es ha we e se o p o ide a cons an concen a ion o
20,000 pa icles pe mL (app oxima ely 1.5–2 mm3/L). Pa icle
concen a ion in he eeding anks was main ained a s able le els
by equen ly checking he concen a ion wi h a pa icle coun e
(Coul e Mul isize 3; Beckman Coul e Spain, Ba celona).
Clea ance a e (CR; L/h) was measu ed acco ding o he
o mula desc ibed by Hild e h and C isp (1976) [CR = F ×((Ci-
C0)/Ci)], in which “F” was he low a e (L/h), “Ci” was he
pa icle concen a ion in he con ol ou low, and “C0” was he
pa icle concen a ion in he expe imen al chambe ou low. The
concen a ion o pa icles was measu ed using a Coul e Coun e
Z1 (Beckman Coul e Spain, Ba celona). The daily clea ance a e
eco ded o each indi idual was de ined as he mean alue o
measu emen s aken e e y hou o a o al pe iod o 11–12 h.
Oxygen Consump ion
Bo h RMR and SMR we e de e mined as a es o oxygen
consump ion (VO2: mL O2/h). To de e mine hese a es, mussels
we e emo ed om he eeding chambe s and in oduced
in o 150 mL chambe s ha had been sealed wi h luminescen
dissol ed oxygen (LDO) oxygen p obes connec ed o oxime e s
(HATCH HQ40d; Hach Lange Spain, De io). The oxygen
consump ion alues we e ob ained om he calcula ion o he
a e o dec ease o he oxygen dissol ed in he wa e con ained
in he espi ome e s o e ime by using a linea eg ession.
Oxygen concen a ion was moni o ed e e y 5–10 min un il
alues showed a educ ion o 20–30% o he ini ial baseline
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P ie o e al. Me abolic The mal Compensa ion in Mussels
FIGURE 1 | Expe imen al design.
alues. A con ol chambe was used o de e mine he s abili y o
oxygen concen a ion.
The di e ence be ween RMR (a he onse o s a a ion) and
SMR ( he educ ion o me abolic a e du ing s a a ion), was
de ined as he me abolic scope o eeding and g ow h (MSFG).
Ene gy Balance
To iden i y po en ial di e ences in he physiological basis
unde lying di e ences in in e -indi idual g ow h a es be ween
mussels ea ed a di e en wa e empe a u es, we de e mined
he comple e se o pa ame e s needed o calcula e ene gy balance
in selec ed F and S indi iduals a hei co esponding ea ing
empe a u e (i.e., a an exposu e empe a u e o 20◦C o F20
and S20 mussels, and a 10◦C o F10 and S10 mussels). To
his end, we collec ed samples o wa e and eces om mussels
du ing he eeding pe iod. Wa e samples we e il e ed on o p e-
washed/p e-weighed GF/C glass- ibbe il e s, and subsequen ly
p ocessed o de e mine he concen a ions o o al pa icle ma e
(TPM) (mg/L), ino ganic pa icula e ma e (PIM) (mg/L), and
o ganic pa icula e ma e (POM) (mg/L). Re ained sal s we e
insed ou wi h a solu ion o ammonium o ma e (0.9%). TPM
and PIM we e hen es ima ed as he inc emen in d y and ash
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P ie o e al. Me abolic The mal Compensa ion in Mussels
weigh on he il e s, espec i ely. POM was hen calcula ed as he
di e ence be ween TPM and PIM. The o ganic con en o ood
( ) was hen es ima ed using a speci ic o mula ( = POM/TPM).
The inges ion a e o o ganic ma e by indi idual mussels
(OIR; mg POM/h) was also calcula ed using a speci ic o mula
(OIR = CR ×POM).
Samples o eces we e il e ed on o p e-washed/p e-weighed
GF/C glass- ibbe il e s and p ocessed in he same way as wa e
samples o de e mine o al ma e , ino ganic ma e , and o ganic
ma e in he eces. The o ganic con en o ecal ma e (e) was
es ima ed as he a io o o ganic/ o al ma e . We hen calcula ed
he abso p ion e iciency (AE) (decimal uni s) using he Cono e
(1966) exp ession (AE = ( −e)/(1 − ) ×e), in which “ ” and “e”
ep esen ed he o ganic con en o ood and eces, espec i ely.
Once AE had been de e mined, we calcula ed he indi idual
abso p ion a e (AR) (mg/h) using a speci ic o mula
(AR = OIR ×AE). The esul ing scope o g ow h (SFG)
(J/h) was hen de e mined as he di e ence be ween abso bed
ene gy (AR: J/h) and me abolic expendi u e (RMR: J/h). AR
(mg/h) was ans o med in o ene ge ic alues (J/h) using an
ene gy equi alence o 18.75 J/mg (Why e, 1987). Oxygen
consump ion (VO2) was ans o med in o ene gy alues using
an oxycalo ic coe icien o 20.08 J/mLO2(Gnaige , 1983).
Size S anda diza ion
Physiological a es a e exp essed in e ms o li e weigh . CR and
oxygen consump ion we e s anda dized o a common li e weigh
o 1 g, acco ding o a o mula desc ibed p e iously (Bayne and
Newell, 1983) (YSTD = (1/WEXP)b×YEXP) in which YSTD and
YEXP ep esen s anda d and expe imen al physiological a es,
espec i ely, and WEXP ep esen s he expe imen al weigh . The
powe alues used o scale physiological a es o body weigh (b)
o clea ance a e and oxygen consump ion we e 0.58 (Bayne and
Hawkins, 1997) and 0.724 (Bayne e al., 1973).
The mal Dependency o Physiological Pa ame e s
The mal dependency o physiological a es was de e mined
acco ding o he Van’ Ho equa ion [Q10 = (Y1/Y0)
(10/(T1−T0))], in which Y0and Y1 ep esen ed he physiological
a e de e mined a T0and T1 empe a u es, espec i ely. The
esul ing Q10 ep esen s he a e o a ia ion o he measu ed
physiological pa ame e as a consequence o a 10◦C inc ease in
he wa e empe a u e.
De e mina ion and S anda diza ion o
Gill Su ace A ea (GA, mm2)
A e he expe imen s we e comple ed, 15 indi iduals pe
expe imen al g oup we e dissec ed and placed on g aph pape o
allow sizing. A pho og aph o he in e nal issues o each mussel
was aken wi h a digi al came a, and he su ace a ea o he
gills om each indi idual was calcula ed using ImageJ so wa e
(Na ional Ins i u es o Heal h). All mussels we e isually heal hy
and no inju ies we e de ec ed. The da a gi en he ein co espond
o one side o a demib anch. Gill a eas we e s anda dized o
an equi alen 1 g o mussel li e weigh acco ding o a speci ic
o mula [GASTD = (1/WEXP)b×GAEXP], in which GASTD and
GAEXP ep esen he s anda dized and expe imen al gill a ea,
espec i ely, and WEXP ep esen s he expe imen al li e weigh
o he mussel. The powe unc ion used o scale gill a ea o
li e weigh was 0.66 (Vahl, 1973;Hawkins and Bayne, 1992;
Jones e al., 1992).
S a is ical Analysis
Signi ican di e ences in he g ow h a e o mussels du ing he
ea ing pe iod a wa m (20◦C) and cold (10◦C) empe a u es
we e iden i ied by es ing he slopes o linea eg essions (leas -
squa es me hod) o he mean shell leng h o mussels (Y)
s. ime (X). Slopes and ele a ions we e compa ed be ween
linea eg essions by analysis o co a iance (ANCOVA) as
desc ibed by Za (2010).
Signi ican di e ences in he ime-cou se o he mal
adap a ions (o CR and VO2) be ween F and S mussels we e
analyzed using a epea ed measu emen s wo-way analysis o
a iance (ANOVA). Time was deno ed as exposu e ime when
measu emen s we e pe o med unde eeding condi ions (CR
and VO2R measu emen s), and as s a a ion ime when VO2
was measu ed in s a ed mussels. P io o s a is ical analysis,
we es ed he no mali y o da a using he Shapi o–Wilk es .
Da a sphe ici y was es ed wi h he Mauchly es . Acco dingly,
subsequen analysis was ca ied ou wi h ei he a uni a ia e
app oach (assumed sphe ici y es ), o a mul i a ia e app oach
(Pillai’s ace es ). Mul iple compa isons we e ca ied ou
o all physiological pa ame e s wi h he leas signi ican
di e ence (LSD) es .
The e ec o g ow h condi ion (being F o S) and acclima ion
empe a u e (being ea ed a 10 o 20◦C) on he su ace a ea
o he gills, and he physiological pa ame e s ha de e mine
ene gy budge in mussels held a di e en ea ing empe a u es
(F20 and S20 a 20◦C; F10 and S10 a 10◦C) we e analyzed by
wo-way ANOVA. Homogenei y o a iance was e alua ed wi h
Le ene’s es ; he Games-Howell, o Tukey es , was applied o
mul iple compa isons, as app op ia e. All s a is ical analyses we e
pe o med using IBM SPSS S a is ics o Windows, Ve sion 19.0
(IBM Co p. Released 2010. A monk, NY: IBM Co p.).
RESULTS
G ow h Ra es
The g ow h a es o expe imen al mussels ea ed a acclima ion
empe a u es o 20 and 10◦C we e calcula ed by adjus ing
mean shell-leng h (mm) alues o linea eg ession models. The
esul ing equa ions we e as ollows:
20◦C:0.093 (±0.002)×day +10.653(±0.073),
F=2020.3,p<0.0001
10◦C:0.035 (±0.001)×day +10.237(±0.059),
F=1992.5,p<0.0001
Analysis o co a iance esul s e ealed signi ican di e ences
o bo h slope and ele a ion be ween mussels ea ed a 20◦C
and hose ea ed a 10◦C (Slope es : = 14.28, d = 1, 14,
p<0.05; ele a ion es : = 10.05, d = 1, 8, p<0.05). The
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P ie o e al. Me abolic The mal Compensa ion in Mussels
g ow h a e o mussels g own a 20◦C was almos h ee- old
highe han ha o mussels ea ed a 10◦C. Acco dingly, he size
di e en ia ion be ween indi iduals occu ed ea lie in he wa m
ea men (20◦C) g oup han in he cold ea men (10◦C) g oup
(60 s. 150 days, espec i ely). Table 1 shows he body size (shell
leng h and li e weigh ) and g ow h a es (g/day and mm/day) o
selec ed F and S indi iduals om he wo di e en acclima ion
empe a u es (F20 and S20 in he wa m ea men ; F10 and S10
in he cold ea men ). F indi iduals g ew signi ican ly as e ,
and we e app oxima ely 100% hea ie and 50% longe , han S
indi iduals; his was he case a bo h acclima ion empe a u es.
Moni o ing Changes in he Clea ance
Ra e and Oxygen Consump ion o F and
S Mussels a Di e en Exposu e
Tempe a u es
Mussels Rea ed a 20◦C
TEXP a 20◦C
Figu es 2A,B show he CR and oxygen consump ion o F20
and S20 a 20◦C, espec i ely. A summa y o he esul s de i ed
om epea ed measu emen s wo-way ANOVA wi h ega ds o
he e ec s o g ow h condi ion (F s. S), and exposu e ime,
on a ious physiological pa ame e s is p o ided in Table 2.
F20 mussels had a signi ican ly highe CR han S20 mussels,
indica ing ha g ow h condi ion exe ed signi ican e ec s on
CR (Table 2). Exposu e ime and in e ac ion did no ha e
any signi ican e ec s on CR. The mean CR o F20 and S20
mussels was 0.46 ±0.11 and 0.25 ±0.07, espec i ely. VO2R
was de e mined on h ee occasions (Figu e 2B); he e we e no
signi ican di e ences be ween F and S mussels (0.067 ±0.006
and 0.064 ±0.002 mLO2/h, espec i ely). S a a ion ime had
a signi ican e ec on oxygen consump ion, inducing a 35%
educ ion o VO2in bo h g oups o mussels.
Response o Cooling a 15◦C
When F20 and S20 mussels we e exposed o a empe a u e o
15◦C, he e was a subs an ial educ ion o CR (Figu e 2C)
compa ed wi h he CR a 20◦C. Du ing he i s 6 days o exposu e
o 15◦C, he e was no signi ican di e ence in CR alues be ween
he wo g oups (0.1–0.2 L/h). By day 7, he CR had s a ed o
inc ease in bo h g oups; howe e , he e was a la ge inc ease in
he CR o F20 mussels (0.558 ±0.220 L/h a day 11) han hei
slow-g owing coun e pa s (0.264 ±0.081 L/h a day 11); his
esul ed signi ican di e ences be ween he wo g oups o e he
TABLE 1 | Shell-leng h (L, mm), li e weigh (W, g), and g ow h a es (GR, g/day
and mm/day) in mussels selec ed as as - and slow-g owe s (n= 15) a e 60 and
150 days o main enance a 20 and 10◦C, espec i ely (mean alues ±SD).
W (g) L (mm) GR (g/day) GR (mm/day)
F20 0.8 ±0.11 18.6 ±0.8 0.011 ±0.002a0.135 ±0.013a
S20 0.4 ±0.07 13.2 ±1.1 0.003 ±0.001c0.045 ±0.014c
F10 0.9 ±0.12 19.4 ±1.0 0.005 ±0.001b0.060 ±0.007b
S10 0.3 ±0.06 11.9 ±0.8 0.001 ±0.001d0.011 ±0.005d
Le e s indica e signi ican di e ences be ween g oups o mussels.
las 3 days o he expe imen al pe iod (p<0.05). Acco dingly,
g ow h condi ion, exposu e ime, and he in e ac ion o hese
ac o s exe ed signi ican e ec on he CR o mussels (Table 2).
Al hough no ini ial alues we e measu ed on day 0, he CRs o
bo h F20 and S20 mussels on he las ew days o he expe imen al
pe iod we e simila o he CRs o mussels a 20◦C.
Rou ine me abolic a e was lowe a 15◦C han a 20◦C
(Figu e 2D); his was he case o bo h F and S indi iduals.
Th oughou he en i e exposu e pe iod, F20 mussels exhibi ed a
signi ican ly highe RMR han S20 indi iduals. In o he wo ds,
g ow h condi ion had a signi ican e ec (Table 2). Mo eo e ,
F20 and S20 mussels showed di e en ial pa e ns in e ms
o VO2R a ia ion: in F indi iduals, VO2R inc eased om
0.039 ±0.001 (day 1) o 0.049 ±0.006 mL O2/h (day 11),
whe eas S indi iduals main ained hei RMR a cons an alues
(0.028 ±0.008 on day 1; 0.029 ±0.007 mL O2/h on day 11).
Exposu e ime, and in e ac ion, hus exe ed a signi ican e ec
on he RMR o mussels when ans e ed om 20 o 15◦C
(Table 2). S a a ion ime did no induce a signi ican educ ion
in oxygen consump ion, and hus, only g ow h condi ion exe ed
a signi ican e ec upon s anda d VO2.
Response o Cooling a 10◦C
A change in empe a u e o 10◦C caused a se e e educ ion
o CR in bo h g oups o mussels (CR ell o app oxima ely
0.07 L/h). CR began o inc ease be ween he 7 h and 10 h days
o exposu e, eaching app oxima ely 0.3 L/h by he end o he
expe imen . No signi ican di e ences we e iden i ied be ween
F20 and S20 mussels. ANOVA iden i ied a signi ican e ec o
exposu e ime, bu no o g ow h condi ion (Table 2). Wi h
ega ds o VO2, (Figu e 2F), exposu e o 10◦C esul ed in
a educ ion o me abolic a e by mo e han 70%; his e ec
occu ed i espec i e o g ow h condi ion. Subsequen ly, he
RMR o bo h F20 and S20 mussels inc eased, and ollowing
a simila end o ha o CR. Acco dingly, ANOVA e ealed
ha exposu e ime exe ed a signi ican e ec on ou ine VO2.
The me abolic a e was sligh ly highe in S indi iduals ( om
0.013 ±0.004 o 0.039 ±0.006 mL O2/h) han in F indi iduals
( om 0.019 ±0.006 o 0.036 ±0.013 mL O2/h), and hus,
he in e ac ion (exposu e ime ×g ow h condi ion) also exe ed
a signi ican e ec (p= 0.035; Table 3). Du ing he pe iod o
s a a ion, he educ ion in SMR was simila in bo h mussel
g oups; he e o e, only s a a ion ime exe ed a signi ican e ec
on oxygen consump ion.
Mussels Rea ed a 10◦C
TEXP o 10◦C
Figu es 3A,B show he CR and VO2 o mussels g own a
10◦C and main ained a 10◦C, espec i ely. A summa y o
he esul s de i ed om wo-way ANOVA wi h ega ds o he
e ec s o g ow h condi ion (F s. S) and exposu e ime on
a ious physiological pa ame e s is p o ided in Table 3. CR
was signi ican ly highe in F10 (Mean CR = 0.384 ±0.100 L/h)
mussels han in S10 mussels (Mean CR = 0.141 ±0.070 L/h),
sugges ing ha g ow h condi ion exe ed a signi ican e ec
(Table 3). A sligh , bu signi ican , empo al change in CR
was also obse ed; in o he wo ds, exposu e ime exe ed
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P ie o e al. Me abolic The mal Compensa ion in Mussels
FIGURE 2 | Time-cou se o he clea ance a e (A,C,E) and oxygen consump ion (B,D,F) o F20 and S20 mussels a h ee exposu e empe a u es (20, 15, and 10◦C).
Fi e indi iduals om each g oup we e moni o ed in each expe imen al condi ion. As e isks indica e signi ican di e ences be ween as - and slow-g owing indi iduals
(ANOVA, p<0.05).
a signi ican e ec ). Wi h ega ds o RMR, he e we e no
signi ican empo al changes iden i ied, and F10 mussels had
a signi ican ly highe me abolic a e han S10 mussels (Mean
RMR = 0.053 ±0.002 and 0.020 ±0.001 mL O2/h, espec i ely).
Thus, only g ow h condi ions exe ed a signi ican e ec
(Table 3). Du ing s a a ion, F10 mussels exhibi ed signi ican ly
lowe oxygen consump ion; howe e , he VO2o S10 indi iduals
did no change signi ican ly. Consequen ly, ou analysis showed
ha g ow h condi ion and s a a ion ime bo h had signi ican
e ec s (Table 3).
Response o Wa ming a 15◦C
The change in CR a e inc easing he wa e empe a u e o 15◦C
di e ed be ween F10 and S10 indi iduals (Figu e 3C). The CR o
F10 mussels inc eased du ing he i s 3 days ( om 0.378 ±0.120
a day 0 o 0.828 ±0.085 L/h; a Q10 o 4.79), and hen a
compensa o y educ ion occu ed un il day 8 (0.198 ±0.136).
Subsequen ly, CR was main ained a simila alues un il he end
o he expe imen on he 16 h day. In con as , he CR o S10
mussels inc eased con inuously du ing he eeding pe iod ( om
0.157 ±0.026 o 0.433 ±0.131 L/h; a Q10 o 7.58). The e o e,
F10 mussels had a signi ican ly highe CR han S10 indi iduals
only un il he 5 h day o expe imen a ion. Then, because o he
compensa o y educ ion o CR in F10 mussels and he inc ease o
CR in S10 mussels, he CR u ned o be highe in S10 indi iduals
(signi ican di e ences we e ound a days 9, 10, 12, 13, and 14).
Acco dingly, exposu e ime and in e ac ion exe ed a signi ican
e ec on he CR o mussels; howe e , g ow h condi ion had no
e ec (Table 3).
Wa ming o 15◦C induced a sudden and in ense inc ease o
ou ine VO2in bo h F10 and S10 mussels (Figu e 3D). In F10
mussels, RMR inc eased om 0.048 ±0.007 o 0.089 ±0.009 mL
O2/h by day 4 ( ep esen ing a Q10 o 3.43). In con as ,
in S10 mussels, he VO2R inc eased om 0.026 ±0.010 o
0.053 ±0.013 mL O2/h by day 4 ( ep esen ing a Q10 o 4.15).
F10 mussels main ained a signi ican ly highe ou ine VO2 han
he S10 mussels un il day 6. The ea e , consis en wi h he ends
o CR, ou ine VO2dec eased in he as -g owing mussels (Q10
be ween he las day o eeding and he ini ial day = 1.77).
Because he RMR o S10 mussels was main ained a cons an
alues, signi ican ly highe oxygen consump ions we e eco ded
in S10 indi iduals a he end o he eeding pe iod. Acco dingly,
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P ie o e al. Me abolic The mal Compensa ion in Mussels
TABLE 2 | Two-way ac o ANOVA ( epea ed measu emen s) showing he e ec s
o g ow h condi ion (GC, F, o S) and exposu e ime (ET, days) on clea ance a e
(CR, L/h) and oxygen consump ion (VO2, mLO2/h) in ed and s a ed mussels
ea ed a 20◦C.
FED STARVED
CR VO2VO2
20◦C exposu e
GC 0.010 0.216 0.652
ET 0.231 0.266 <0.001
GC ×ET 0.448 0.463 0.931
15◦C exposu e
GC 0.041 0.001 0.014
ET <0.001 <0.001 0.262
GC ×ET <0.001 0.012 0.212
10◦C exposu e
GC 0.834 0.493 0.744
ET <0.001 <0.001 <0.001
GC ×ET 0.919 0.035 0.850
Bold alues deno e s a is ical signi icance (p <0.05).
TABLE 3 | Two-way ac o ANOVA ( epea ed measu emen s) showing he e ec s
o g ow h condi ion (GC, F, o S) and exposu e ime (ET, day) on clea ance a e
(CR: L/h) and oxygen consump ion (VO2: mLO2/h) in ed and s a ed mussels
ea ed a 10◦C.
FED STARVED
CR VO2VO2
10◦C exposu e
GC 0.014 0.004 0.031
ET 0.002 0.038 <0.001
GC ×ET 0.849 0.350 0.035
15◦C exposu e
GC 0.673 0.097 0.247
ET <0.001 0.010 <0.001
GC ×ET <0.001 0.001 0.121
20◦C exposu e
GC <0.001 <0.001 <0.001
ET 0.027 0.878 0.049
GC ×ET 0.059 0.771 0.069
Bold alues deno e s a is ical signi icance (p <0.05).
exposu e ime and in e ac ion be ween he es ed ac o s exe ed
signi ican e ec s on ou ine VO2. S a a ion p omo ed a simila
educ ion in he VO2o bo h g oups o mussels; consequen ly,
only s a a ion ime had a signi ican e ec on s anda d VO2.
Response o Wa ming a 20◦C
As an ini ial esponse o wa ming, mussels showed a educ ion
in CR (Figu e 3E). Subsequen ly, CR inc eased daily un il
day 11. Al hough he CR alues o F10 mussels we e highe
h oughou he expe imen , he di e ences be ween g oups we e
only signi ican o he i s 4 days, and on day 7. Al hough he
in a-g oup a iabili y in CR inc eased ma kedly om day 4, we
ound ha g ow h condi ion exe ed a signi ican e ec on CR.
Exposu e ime also had a signi ican e ec on CR; howe e , he
in e ac ion e m had no signi ican e ec .
In con as o he end exhibi ed by CR, he ini ial esponse
o wa ming led o a signi ican inc ease in RMR (Figu e 3F);
his was he case o bo h F10 and S10 mussels. By day 4, VO2R
alues we e double hose measu ed on day 0; in he F10 g oup,
VO2inc eased om 0.049 ±0.002 o 0.105 ±0.018 mL O2/h
(Q10 = 2.14), while o he S10, VO2inc eased om 0.026 ±0.010
o 0.057 ±0.026 mL O2/h (Q10 = 2.19). In he F10 mussels, oxygen
consump ion ell o 0.072 ±0.017 mL O2/h by he end o he
eeding pe iod (Q10 be ween he las day o eeding and day 1
was 1.77). Bo h g ow h condi ion and exposu e ime exe ed a
signi ican e ec on ou ine VO2. The onse o s a a ion induced
a educ ion o VO2in F10 mussels ( om 0.72 o 0.56 mL O2/h)
and in S10 mussels ( om 0.41 o 0.30 mL O2/h). F10 mussels
exhibi ed a signi ican ly highe SMR h oughou he s a a ion
pe iod. G ow h condi ion, s a a ion ime, and hei in e ac ion
all exe ed signi ican e ec s on SMR.
The mal E ec s on he Me abolic Scope
o Feeding and G ow h
Figu e 4 shows he mean RMR ( eco ded immedia ely be o e
s a a ion) and mean SMR ( eco ded a he end o he s a a ion
pe iod) o he ou expe imen al g oups o mussels (F20, S20,
F10, and S10) a each o he h ee expe imen al empe a u es (10,
15, and 20◦C). The Q10 alues o s anda d and ou ine oxygen
consump ion a e also indica ed in Figu e 4. Sho - e m changes
in he exposu e empe a u e p omo ed di e en ial e ec s upon
bo h RMR and SMR in as - and slow-g owing mussels, and
he esul ing MSFG. Figu e 4 shows ha i espec i e o ea ing
empe a u e, F mussels displayed highe MSFGs han S mussels
in he ange o empe a u es es ed. Fo bo h S10 and S20 mussels,
he MSFG was almos negligible a low empe a u es.
Ene gy Balance o Fas - and
Slow-G owing Mussels Rea ed a 20 and
10◦C
The physiological componen s o ene gy balance o F20, S20, F10,
and S10 mussels exposed o hei espec i e ea ing empe a u es
a e shown in Table 4, oge he wi h he su ace a eas o gills in
15 indi iduals om each mussel g oup. The e ec s o g ow h
condi ion and acclima ion empe a u e on he physiological
componen s o ene gy balance we e analyzed by wo-way
ANOVA (Table 4).
Wi hou excep ion, all pa ame e s we e signi ican ly highe
a 20◦C han a 10◦C; hus, acclima ion empe a u e exe ed
a posi i e and signi ican e ec on all pa ame e s. G ow h
condi ion exe ed a signi ican e ec on all physiological a iables
excep o abso p ion e iciency and SMR. F mussels had a
signi ican ly highe clea ance a e, abso p ion a e, and gill
su ace a ea, han hei S coun e pa s; his was he case a bo h
20 and 10◦C acclima ion empe a u es. Thus, ANOVA de ec ed
signi ican e ec s o bo h g ow h condi ion and acclima ion
empe a u e, bu no o hei in e ac ion. Howe e , in e ac ion
did ha e a signi ican e ec on me abolic pa ame e s and SFG,
hus indica ing he exis ence o dis inc pa e ns o in e -g oup
di e ences a di e en acclima ion empe a u es. Indeed, pos hoc
analysis indica ed ha signi ican di e ences in RMR be ween F
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P ie o e al. Me abolic The mal Compensa ion in Mussels
FIGURE 3 | Time-cou se o he clea ance a e (A,C,E) and oxygen consump ion (B,D,F) o F10 and S10 mussels a h ee exposu e empe a u es (20, 15, and 10◦C).
Fi e indi iduals om each g oup we e moni o ed in each expe imen al condi ion. As e isks indica e signi ican di e ences be ween as - and slow-g owing indi iduals
(ANOVA, p<0.05).
and S mussels only occu ed in mussels ea ed a 10◦C. In o he
wo ds, he di e ences in RMR (and hence, in SFG) be ween F
and S indi iduals we e compa a i ely highe in mussels ea ed
a 10◦C. A simila pa e n was also obse ed o SMR (ANOVA
iden i ied a signi ican e ec o he in e ac ion e m): in e -g oup
di e ences we e highe in mussels ea ed a 10◦C; his was due o
he educed SMR o S10 mussels.
DISCUSSION
The di e ences in g ow h a e be ween mussels ea ed in he
labo a o y a 20 and 10◦C (0.093 and 0.035 mm/d, espec i ely)
indica e ha he g ow h p ocess in mussels is highly dependen
on empe a u e (Q10 = 2.65). Posi i e co ela ions be ween
g ow h a e and empe a u e in bi al es ha e been epo ed
p e iously, pa icula ly wi h ega ds o ene gy balance (Widdows,
1978;Bayne e al., 1983;MacDonald and Thompson, 1985;Bei as
e al., 1995). In he p esen s udy, he physiological pa ame e s
o mussels ea ed a 20 and 10◦C (Table 4) clea ly illus a e he
ene ge ic basis o as e g ow h a highe empe a u es: ising
empe a u es exe ed a combined posi i e e ec on ene gy gain
p ocesses ( il e ing ac i i y and abso p ion e iciency) ha was
no o e come by he e ec o empe a u e on hei a es o ene gy
expendi u e (RMR). Mo e in e es ingly, he esul s showed ha
as e g owing indi iduals ea ed a 10◦C (F10) g ew sligh ly as e
han slow-g owing indi iduals ea ed a 20◦C (S20) (Table 1).
This indica es ha endogenously de e mined in e -indi idual
di e ences in g ow h po en ial exe an ou s anding con ibu ion
o he in a-popula ion a iabili y in he size dis ibu ion; hese
e ec s ha e also been epo ed p e iously (Bayne e al., 1999a,b;
Tamayo e al., 2011).
Fas -g owing indi iduals exhibi ed signi ican ly highe
clea ance a es han hei slow- g owing coun e pa s a bo h
acclima ion empe a u es in good ag eemen wi h p e ious
s udies showing ha il a ion a e is he main physiological
pa ame e unde lying as g owing in di e en bi al es, such as
mussels, (Pé ez-Camacho e al., 2000;Fe nández-Rei iz e al.,
2016;P ie o e al., 2018) clams (Holley and Fol z, 1987;Tamayo
e al., 2011, 2013) and oys e s (To o and Ve ga a, 1998;Bayne
e al., 1999b;Pace e al., 2006;Tamayo e al., 2014). In good
acco dance wi h ou p e ious s udies (Tamayo e al., 2011;
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