Aquacul u e 555 (2022) 738147
A ailable online 24 Ma ch 2022
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Elemen al C and N balances e idence s oichiome ic adjus men s o die a y
p o ein con en in g ow h pheno ypes o he Manila clam
(Rudi apes philippina um)
K is ina A anz
*
, I˜
naki U u xu u, En ique Na a o
Depa amen o de Gen´
e 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, Apa ado 644, 48080 Bilbao, Spain
ARTICLE INFO
Keywo ds:
Nu ien balances
S oichiome y
G ow h pheno ypes
Rudi apes philippina um
C:N a io
Homeos asis
ABSTRACT
Ju eniles om wo sepa a ely b ed amilies o he Manila clam Rudi apes philippina um we e used o cons i u e
as (F)- and slow (S)-g owing g oups by size seg ega ion. The g ow h pheno ypes ha esul ed om hese 4
amily*g ow h ca ego y combina ions we e hen used o measu e elemen al C and N balances in he acu e and
ch onic esponses o wo di e en mic oalgal die s ha we e isocalo ic bu di e ed (x3) in hei p o ein:ene gy
a ios. Bo h die s we e based on he same phy oplank on species (Rhodomonas lens) ha we e ha es ed in he
exponen ial (low C:N a io) o s a iona y (high C:N a io) phases o an indoo cul u e. Aims we e o assess he
di e en ial acquisi ion and p ocessing o elemen al nu ien s acco ding o di e ences in hei die a y a ailabili y
and he a iable equi emen s se by he g ow h ends exhibi ed by di e en pheno ypes. Clams ed he high N
die achie ed he highes C and N balances h ough a combina ion o highe eeding a es and abso p ion e i-
ciencies o o e all o ganics. Howe e , he main di e en ial e ec s we e associa ed wi h inc eased abso p ion
e iciencies o N ela i e o C, which we e pa icula ly obse ed in clams subjec ed o ch onic N de ici in he
die . This occu ed in all g ow h pheno ypes and esul ed in he pa ial homeos a ic egula ion o nu ien
imbalance ope a ing a he p eabso p i e le el. Fu he adjus men s occu ed a he pos abso p i e le el wi h
inc eased N disposal in he o m o ammonia exc e ion and he esul ing dec ease o me abolic C:N indices
eco ded in clams ed he high N die s. The main pheno ypic di e ences we e obse ed in he in a amily (F s. S
g ow h g oups) a he han in he in e amily compa isons, wi h F clams exceeding he abso p ion a e o nu-
ien s (bo h C and N) o S clams by 50%. Physiological esponses o he acu e die a y change we e ound o be
as e in F clams, indica i e o a highe plas ici y o his pheno ype. Whe eas, s oichiome ic adjus men s by S
clams esul ed in highe N elease h ough exc e ion, sugges ing less e icien p o ein u no e .
1. In oduc ion
G ow h is an impo an a ibu e o animal indi iduals and pop-
ula ions o ma ine bi al es and a ac o o be accoun ed o in he
managemen and sus ainabili y o coas al ecosys ems, as well as an
economic a ge in he aquacul u e indus y. F om an ene ge ic poin o
iew, g ow h can be iewed as “ he ou come o acquisi ion and u ili-
za ion o nu ien s and ene gy” (Bayne, 2017), and his pe spec i e has
domina ed he ield since he ea ly g ow h models by on Be alan y
(1938) ha conside ed g ow h o esul om a balance be ween me a-
bolic p ocesses o assimila ion and ca abolism. Bioene ge ic models
cu en ly in use, ei he he classical scope o g ow h (SFG) app oach o
he dynamic ene gy budge (DEB) heo y (Kooijman, 2010), de i e om
he same p inciples, al hough elying on a mo e de ailed physiological
accoun o ene gy lows. These models ha e p o ided use ul ools o
p edic ing o e all g ow h ha a e o gene al in e es in aquacul u e
p oduc ion, while he expe imen al es ing o hese models has enla ged
ou knowledge o he mechanisms o g ow h wi h ega d o he speci ic
in luence o bo h endogenous and exogenous ( ood, empe a u e, e c.)
ac o s on he a ious componen s o he ene gy budge .
Howe e , he use o ene gy as he sole cu ency in hese budge s has
clea limi a ions s emming om he ac ha la ge di e ences a e o en
encoun e ed be ween consumed ood and consume body issues wi h
ega d o elemen al composi ion. This has special ele ance in
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (K. A anz).
Con en s lis s a ailable a ScienceDi ec
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h ps://doi.o g/10.1016/j.aquacul u e.2022.738147
Recei ed 17 Sep embe 2021; Recei ed in e ised o m 27 Janua y 2022; Accep ed 14 Ma ch 2022
Aquacul u e 555 (2022) 738147
2
he bi o ous o de i i o es elying on ege al esou ces, such as mos
bi al e molluscs, whe e o ins ance, he a he cons an C:N a ios e-
po ed o body composi ion (be ween 4.1 and 6.2) con as s wi h he
highly a iable alues o C:N a ios in hei die s, anging be ween 4.8
and 22.5 on a seasonal/expe imen al basis (Bayne, 2009; Bayne and
S ensson, 2006; Fielding and Da is, 1989; G an and C an o d, 1991;
Smaal and Vonck, 1997; summa ized in Bayne, 2017, Table 5.9 on p.
299). In he s oichiome ic app oach (S e ne and Else , 2002), hese
misma ches a e add essed in e ms o nu ien homeos asis ha in ol es
he esou ce in a sui o physiological mechanisms able o compensa e,
whene e necessa y, o he imbalance be ween he die and consume
issues. Nu ien limi a ion implies ha C o ene gy is in excess, and “ o
main ain o e all homeos asis, consume s should elease elemen s on
ood in excess o equi emen s while e aining mos o he limi ing
elemen …. On he o he hand, consume s may also be limi ed by ca bon
(ene gy), in which case i is he inges ed nu ien s ha a e subjec o
e icien ecycling” (Ande son e al., 2005). Acco ding o his desc ip-
ion, o s oichiome y o be ealized in g owing issues, any componen
o he die in excess o he app op ia e a io should be disposed o ,
whence impo an cons ain s on g ow h can be expec ed ha would
ha dly be iden i ied om only he accoun o ene gy low measu e-
men s. This app oach hus eplaces eliance on ene gy budge ing by
assessing nu ien (elemen al) balances based on physiologically de e -
mined componen s o g ow h. Se e al ea ly s udies emphasized he need
o an e alua ion o nu ien s, oge he wi h ene gy, in he analysis o
g ow h (C an o d, 1995; G an and C an o d, 1991; Bayne e al., 1993;
Hawkins and Bayne, 1985), and modelling e o s ha e ended o
inco po a e pa ame e s ha ake in o accoun he elemen al composi-
ion o die o i a be e app oach o ac ual g ow h (Schol en and Smaal,
1999; Schol en and Smaal, 1998; Smaal and Schol en, 1997; Bou l`
es
e al., 2009; B igolin e al., 2009; Emme y e al., 2011; G ange ´
e e al.,
2009).
Mechanisms o homeos a ic nu ien egula ion in bi al es can be
iden i ied a se e al di e en le els in he chain o physiological p o-
cesses connec ing eeding o g ow h. These include p einges i e as well
as p e- and pos abso p i e le els. The p einges i e selec ion o N- ich
pa icles has been b oadly epo ed in eeding expe imen s using na u al
ses on o mix u es o phy oplank on wi h suspended sedimen s con-
aining o ganic de i us (Hawkins e al., 1996; Iglesias e al., 1996;
Sole chnik e al., 1996; U u ia e al., 1996), a selec i e p ocess ha
would be mainly based on he abili y o gills and palps o disc imina e
be ween mic oalgae (low C:N a io) and de i al pa icles (high C:N
a io). In ac , highe selec ion e iciencies (SE) obse ed o chlo ophyll
ela i e o o e all o ganics we e ound o co ela e wi h highe SE o N
(SE
N
) compa ed wi h SE o C (SE
C
) in some o hese s udies (e.g.,
Iglesias e al., 1996; U u ia e al., 1996). Di e en ial selec ion o species
o phy oplank on acco ding o hei ood alue (Beninge e al., 2008;
Cognie e al., 2001; Pales-Espinosa e al., 2008; Pales-Espinosa e al.,
2007; Shumway e al., 1985) migh u he con ibu e o inges ed N
en ichmen . As e idence o he po en ial o hese selec i e mechanisms
o con ibu e o nu ien homeos a ic egula ion, Bayne (2009) epo ed
ha he a io o SE
N
o SE
C
is a posi i e unc ion o he nu ien imbal-
ance (N limi a ion) ep esen ed by he di e ence be ween he C:N a io
in he ses on and he C:N a io in oys e issues.
Simila selec i e mechanisms a he pos inges i e le el can also be
in oked o accoun o he N en ichmen o he abso bed a ion om
complex die s (i.e., ses on) since diges i e selec ion (Na a o e al.,
2016) has been shown o esul in he p e e en ial u iliza ion o mic o-
algae (low C:N a io) s. sedimen a y o ganic pa icles, including phy-
ode i us (high C:N a io). The expec ed inc emen in he abso p ion
e iciency o N compa ed o C in such scena ios has been ce ainly e-
po ed (Bayne, 2009; C an o d and G an , 1990; G an and C an o d,
1991; P ins and Smaal, 1989; Iglesias e al., 1996; U u ia e al., 1996).
E en in he absence o any scope o diges i e selec ion (e.g., mo e ho-
mogenous die o mula ions as hose used in he p esen s udy), he ne
N en ichmen may esul om he p e e en ial abso p ion o die a y
p o eins ela i e o ca bohyd a es and lipids epo ed in cockles (Ce a-
s ode ma edule) ed phy oplank on (Iba ola e al., 2000b). The almos
comple e abso p ion o labile p o ein om de i al pa icles (abo e he
AE o o e all POM) ound in oys e s (Adams e al., 2019) migh also
accoun o he p e e en ial abso p ion o N ela i e o C.
Pos abso p i e mechanisms conce n he me abolic a e o di e en
elemen s con ibu ing o inal adjus men s o he assimila ed a ion o
mee he speci ic equi emen s o issue biosyn hesis. The elease o
excess C (o o he nonlimi ing nu ien s) a his pos abso p i e le el may
ep esen a signi ican ac ion o he me abolic budge in animals
eeding on ela i ely unbalanced oods ha a e common in bo h
e es ial and aqua ic sys ems (Ande son e al., 2005). This has been
b oadly documen ed in bi al e molluscs (namely, mussels) wi h ega d
o se e al ea u es o p o ein u no e , whe e he e iciency o p o ein
deposi ion was epo ed o inc ease wi h a es o die a y p o ein ab-
so p ion (Hawkins, 1985). The inc eased a io o p o ein b eakdown o
p o ein syn hesis epo ed unde condi ions o educed N inpu (Haw-
kins, 1985) p obably e lec s he me abolic ole o die a y p o ein in
uelling he N pool h ough ansamina ion eac ions o p o ein syn-
hesis, wi h he consequen was e o mos o he p o ein C. This would
explain he disp opo iona ely highe e iciency o issue deposi ion o
he amino-N ac ion compa ed wi h he amino-C ac ion epo ed in
mussels (K eege e al., 1996; K eege e al., 1995) as he esul o a N
spa ing mechanism a he expense o ene gy (C) e en unde condi ions
o nega i e ene gy balance (Hawkins and Bayne, 1991). Con e sely, N
elease when N inpu is in excess o equi emen s occu s in he o m o
high a es o ammonia exc e ion, e lec ing he me abolic a e o p o-
eins in supplying ene gy h ough deamina ion eac ions. Reso o
expe imen al die s p o iding a ange o a ia ion in he ela i e p o-
po ion o di e en nu ien s, pa icula ly in he p o ein/ene gy a io,
ha e hus p o en use ul in s udying he con ibu ion o hese pos -
abso p i e p ocesses o homeos a ic nu ien egula ion in bi al es.
In a p e ious wo k (A anz e al., 2020), we analysed he e ec s o
wo di e en die s ha we e isocalo ic bu di e ed b oadly in he C:N
a ios on he physiological pe o mance o ju enile clams (Rudi apes
philippina um) measu ed in e ms o he ene gy budge s. These die s
consis ed o he same mic oalga species (Rhodomonas lens) cul u ed a
di e en g ow h s ages (i.e., exponen ial and s a iona y phases).
Di e en g oups o clams we e ood condi ioned (15 d.) wi h hese wo
die s and physiological componen s o g ow h and SFG measu emen s
we e hen pe o med in esponse o bo h he acu e and ch onic change in
die a y composi ion. Addi ionally, he s udy was pe o med on di e en
g ow h pheno ypes ha we e ob ained by combining selec i e b eeding
o wo amilies and size-g oup seg ega ion inside each amily in an
a emp o assess how physiological esponses o die composi ion we e
modula ed acco ding o he a iable g ow h demands o endogenous
o igin se by he occu ence o hese di e en pheno ypes.
In he p esen s udy, he same expe imen al design de eloped o
compu e ene gy balances (A anz e al., 2020) was ex ended o include
he elemen al balance o nu ien s (C and N), aiming o es he ollowing
hypo hesis conce ning he occu ence o compensa o y esponses o
di e en ial s oichiome ic imbalances be ween high and low N die s:
H1. Inc eased eeding a es ac ed o compensa e o dec eased N
a ailabili y in he die (o e eeding esponse).
H2. Ch onic N de ici o die a y o igin is compensa ed h ough he
p e e en ial abso p ion o N ela i e o C (ene gy): The p e-abso p i e
le el.
H3. Me abolic ac i i ies u he con ibu ed o he s oichiome ic ad-
jus men s, wi h me abolic C:N indexes e lec ing he di e en ial elease
o elemen s in excess: The pos -absop i e le el.
H4. Highe g ow h demands o endogenous o igin a e be e accom-
plished unde condi ions o high N a ailabili y which esul ed in he
maxima balances o bo h N and C (ene gy) being achie ed in as
g owing pheno ypes ed he N ich die .
K. A anz e al.
Aquacul u e 555 (2022) 738147
3
H5. Less e icien p o ein me abolism o slow g owe s is e ealed in
highe a es o ammonia exc e ion and educed alues o N ne g ow h
e iciency, ela i e o as g owe s.
H6. Time cou se o physiological adjus men s o die quali y di e
be ween ood condi ioned g oups and e eals di e en ial pheno ypic
plas ici y.
2. Ma e ial and me hods
2.1. Animal main enance, die cha ac e is ics and expe imen al design
Fea u es o die s, clam amilies, g ow h ca ego ies and he main e-
nance o hem, including expe imen al design, ha e been p e iously
desc ibed in A anz e al. (2020). B ie ly, specimens belonging o any o
wo ull-sib amilies (1 and 8) o he Manila clam Rudi apes philippina um
we e size-seg ega ed o cons i u e F ( as g ow h) and S (slow g ow h)
ca ego ies by choosing he la ges and smalles clams inside each amily.
The mean shell leng hs o he F and S ca ego ies we e 23.50 (1.67) mm
and 13.10 (1.65) mm, espec i ely. The di e en g ow h ca ego -
y* amily combina ions esul ed in ou g ow h pheno ypes (F1, F8, S1
and S8) ha we e used in subsequen expe imen s.
Main enance was ca ied ou unde cons an ambien condi ions in a
eci cula ing seawa e (34 PPT) sys em egula ed a 17 ◦C. Two g oups
o clams om each o he abo e pheno ypes we e each ed he same
a ion (~ 0.5 mg POM L
−1
) o he mic oalgal species R. lens ea ed in
ei he he exponen ial (C:N a io =4.94) o he s a iona y (C:N a io =
14.5) phases o he cul u e (A anz e al., 2020). The chosen no a ion
was N+ o die s based on mic oalgae in he exponen ial phase and N-
o hose in he s a iona y phase. These die s di e ed by ca. 3- old in N
con en and we e used o assess elemen al balances o clams in he
con ex o he acu e and ch onic physiological esponse o die a y
change.
Clam g oups we e condi ioned o 15 days o die s N+o N-.
Following ha “acclima ion” pe iod, physiological measu emen s we e
ca ied ou while subg oups o clams om each condi ioning g oup we e
exposed o ei he he condi ioning die (ch onic esponse) o he al e -
na i e die (acu e esponse). This design esul ed in he 16 expe imen al
g oups epo ed in Table 1.
2.2. Elemen al balances
Samples o die cha ac e iza ion we e pe o med wice a week in
quad uplica e du ing he acclima ion pe iod and 5–6 imes in quad u-
plica e du ing he exposu e expe imen s (A anz e al., 2020). Elemen al
analysis (CNH) o die s was conduc ed on samples collec ed o e p e-
weighed glass ib e il e s (GF/C) by il e ing a known olume o wa e
om he eeding anks and washing wi h 50 ml o il e ed seawa e .
Indi idual samples o aeces p oduced o e 8–10 h we e simila ly
collec ed o e p eweighed GF/C il e s and washed wi h 50 ml il e ed
seawa e . Bo h ypes o samples we e immedia ely ozen a −20 ◦C,
lyophilized, and main ained a −20 ◦C un il being analysed in a Eu o EA
Elemen al Analyse (CHNS) om Eu o Vec o , using ace anilide as a
s anda d. A subse o samples was calcined o 6 h a 450 ◦C and sub-
sequen ly measu ed in an elemen al analyse o be used as a con ol.
Physiological componen s o he scope o g ow h we e de e mined
as p e iously desc ibed (A anz e al., 2020), whe e in sho , i e ep-
lica es pe condi ion we e used o de e mine he clea ance a e unde he
low- h ough chambe me hod (C isp, 1971; Filguei a e al., 2006),
abso p ion e iciency (Cono e , 1966), ammonia exc e ion a e (Sol-
´
o zano, 1969) and me abolic a e (see mean alues in Table 1). Indi-
idual alues o his da ase we e combined wi h elemen al analysis o
die s and aeces o compu e N and C balances as ollows:
2.2.1. Inges ion a es
Pa icula e o ganic N and C (PON and POC, espec i ely) we e
calcula ed as he p oduc be ween POM and he p opo ion o o ganic N
and C p esen in he die in he ollowing way:
PON (mg L−1)=POM ×N%
100
POC (mg L−1)=POM ×C%
100
The inges ion a es o N and C (IR
N
and IR
C
: mg h
−1
) we e es ima ed
by mul iplying he clea ance a e by he pa icula e o ganic ma e o
each elemen , in hese e ms:
IRN=PON ×CR
IRC=POC ×CR
2.2.2. Abso p ion a es and abso p ion e iciencies
Abso p ion a e (AR
N
and AR
C
, mg h
−1
) cons i u es he di e ence
be ween inges ion (IR
N
and IR
C
: mg h
−1
) and eges ion a e (ER
N
and
ER
C
: mg h
−1
), which is calcula ed h ough he use o o ganic inges ion
a e (OIR), abso p ion e iciency (AE) and he p opo ion o o ganic N
and C p esen in he aeces, as ollows:
ERN=OIR × (1–AE) × N%( eces)
100
ERC=OIR × (1–AE) × C%( eces)
100
Abso p ion e iciency (AE
N
and AE
C
, decimal uni s) is hen gi en by
Table 1
Mean (SD) alues o clea ance a e (CR, L h
−1
), abso p ion e iciency (AE, decimal uni s), oxygen consump ion a e (VO
2
, ml h
−1
) and ammonia exc e ion a e (VNH
4
-
N,
μ
g h
−1
), om A anz e al. (2020), used o calcula e he p esen elemen al balances.
Food condi ioning Acu e exposu e G ow h ca ego y Family CR AE VO2 VNH
4
-N
N+
N+
F 1 1.22 (0.16) 0.84 (0.01) 0.06 (0.01) 4.26 (1.42)
8 1.1 (0.22) 0.85 (0.01) 0.07 (0.02) 6.31 (7.1)
S 1 1.1 (0.13) 0.85 (0.01) 0.06 (0.02) 12.58 (1.74)
8 0.72 (0.2) 0.85 (0.01) 0.08 (0.02) 15.22 (11.56)
N-
F 1 0.9 (0.13) 0.58 (0.04) 0.07 (0.04) 2.83 (1.35)
8 0.74 (0.13) 0.62 (0.02) 0.08 (0.03) 3.62 (1.48)
S 1 0.41 (0.11) 0.58 (0.02) 0.06 (0.03) 4.86 (1.21)
8 0.47 (0.11) 0.61 (0.02) 0.06 (0.03) 3.97 (1.06)
N−
N+
F 1 0.95 (0.09) 0.81 (0.03) 0.1 (0.04) 9.16 (2.64)
8 0.78 (0.41) 0.75 (0.06) 0.07 (0.02) 9.38 (3.25)
S 1 0.66 (0.16) 0.74 (0.03) 0.08 (0.04) 19.79 (7.36)
8 0.56 (0.22) 0.78 (0.03) 0.07 (0.02) 11.74 (2.18)
N-
F 1 1.1 (0.22) 0.44 (0.03) 0.05 (0.02) 1.29 (0.35)
8 0.83 (0.44) 0.48 (0.04) 0.07 (0.02) 0.95 (0.03)
S 1 0.9 (0.05) 0.53 (0.06) 0.04 (0.03) 0.82 (0.06)
8 0.63 (0.23) 0.65 (0.03) 0.04 (0.02) 0.63 (0.24)
K. A anz e al.
Aquacul u e 555 (2022) 738147
4
he quo ien be ween AR and IR o each elemen :
AEN=ARN
IRN
AEC=ARC
IRC
2.2.3. Ca bon and ni ogen losses
Ca bon loss due o espi a ion (R
C
, mg C h
−1
) was de i ed om
espi a ion a es (ml O
2
h
−1
), assuming a espi a o y quo ien RQ =0.9
mol CO
2
mol O
2−1
. Respi a ion a es we e ob ained wi h he aid o
oxime e s by moni o ing he decline in oxygen concen a ion in sealed
chambe s illed wi h seawa e whe e clams had been in oduced. Ni-
ogen loss due o exc e ion (E
N
, mg N h
−1
) was assessed h ough he
de e mina ion o inc emen s in ammonia concen a ion by he phenol-
hypochlo i e me hod (Sol´
o zano, 1969), employing lasks illed wi h
30 ml o il e ed seawa e (0.2
μ
m Millipo e memb anes) o 2–3 h. Fo
bo h de e mina ions, chambe s wi hou animals we e employed as
con ols.
2.2.4. Elemen al balances and g ow h e iciencies o ca bon and ni ogen
Elemen al balances o ca bon (SFG
C
, mg C h
−1
) we e calcula ed as
he di e ence be ween he amoun o C abso bed and espi ed:
SFGC=ARC–RC,
whe eas he elemen al balance o ni ogen (SFG
N
, mg N h
−1
) was
calcula ed as he di e ence be ween he amoun o N abso bed and
exc e ed:
SFGN=ARN–EN
The g ow h (deposi ion) e iciencies o bo h elemen s we e
compu ed as ollows:
NGEN=SFGN
ARN
NGEC=SFGC
ARC
A e concluding he expe imen s, clams we e dissec ed o compu e
he d y weigh o issues, and consequen ly, all a es we e s anda dized
o he common d y weigh o so issues (85.95 mg) using mass expo-
nen s o 0.6091 (CR), 0.6967 (R) and 1.00 (VNH
4
-N).
2.3. Da a analysis
Rela ionships be ween wo o mo e quan i a i e a iables we e
explo ed h ough eg ession analysis, while compa isons o quali a i e
ac o s, amily, g ow h ca ego y, acclima ion die and exposu e die
we e es ed h ough 4-way ANOVA using R (R Co e Team, 2018). These
analyses we e pe o med a e es ing o he no mali y (Shapi o–Wilk)
and homoscedas ici y (Le ene) o he da a.
3. Resul s
Resul s o he p esen expe imen s ha e been disposed in o de o i
he sequence o hypo hesis se in he in oduc o y sec ion.
3.1. Die e ec s
H1. Inc eased eeding a es ac ed o compensa e o dec eased N a ail-
abili y in he die (o e eeding esponse)
Conce ning elemen al (C and N) composi ion, acquisi ion pa ame e s
(i.e., a es o ood p ocessing and diges i e balances: IR, ER and AR)
we e mos ly in luenced by he composi ion o he die supplied o he
animals du ing physiological de e mina ions (exposu e die ) han o
p e ious condi ioning, al hough some e ec s conce ning he la e
a iable will be conside ed.
In his way, he inges ion a es o N we e signi ican ly highe when
clams we e exposed o he N ich die , doubling he alues eco ded in
hose ed he N poo die , while eges ion a es o N we e inc eased unde
N es ic ed condi ions (Tables 2 and 3 and Fig. 1). As a consequence,
abso p ion a es we e 3 imes highe when clams we e ed he N+die
compa ed o he N- die . P e ious condi ioning did no a ec physio-
logical pe o mance, al hough inges ion and abso p ion a es ended o
inc ease in hose indi iduals p e iously acclima ed o he N+die .
Inges ion a es o C eached he highes alues in hose g oups ed he
same die used in condi ioning (e.g., N +N+and N-N-; Fig. 1). Howe e ,
ends we e simila o C and N as ega ds he ne acquisi ion (highe
abso p ion a es o C in clams ed he N+die ).
Thus, maximiza ion o ene gy ob ained om high quali y oods
appea o be he ule in hese expe imen s, a he han compensa o y
o e eeding o N income egula ion on low quali y die s. An in e p e-
a ion o hese ea u es based on he occu ence o diges i e cons ain s
will be discussed in he co esponding sec ion.
H2. Ch onic N de ici o die a y o igin is compensa ed h ough he p e -
e en ial abso p ion o N ela i e o C (o ene gy): The p e-abso p i e le el.
Abso p ion e iciencies o bo h N and C we e clea ly supe io (p <
0.001) when exposed o he N+die ; acclima ion o his high quali y die
also had a posi i e e ec bu only on AE
C
. Di e en ial pe o mance o N
and C in he di e en acclima ion*exposi ion g oups o clams we e
analysed by plo ing elemen al AEs agains he o e all AE alues o
o ganics (Cono e ) epo ed in A anz e al. (A anz e al., 2020; Fig. 2).
As expec ed, he AE
C
closely i ed he Y =X line (AE
C
=1.04 AE – 0.003,
R
2
=0.997, p < 0.001), while AE
N
clea ly depa ed om his ela-
ionship, especially in he lowe ange o AE alues (AE
N
=0.88 AE +
0.13, R
2
=0.88, p < 0.001). As a gene al ule, AE
N
was abo e he AE o
o e all o ganics (equi alen o AE
C
), bu his disc epancy ended o be
highe in clams submi ed o ch onic N de ici (i.e., condi ioned o N-
die s). In ac , esul s o ANOVA es ing he di e ence be ween AE
N
and
AE (Cono e ),using acclima ion and exposu e die as ac o s, esul ed in
signi ican e ec s o he acu e esponse (F =9.6, p =0.005) and highly
signi ican e ec s o bo h he ch onic esponse (F =114.2, p < 0.001)
and he in e ac ion e m: (F =43.7, p < 0.001).
H3. Me abolic ac i i ies u he con ibu ed o he s oichiome ic adjus -
men s, wi h me abolic C:N indices e lec ing he di e en ial elease o ele-
men s in excess: The pos absop i e le el.
N exc e ion was highly a ec ed by die composi ion, such ha clams
exposed o N+exc e ed ca. 5 imes mo e ammonia-N han hose ed he
N- die (Fig. 3). This acu e esponse a ied depending on he condi-
ioning die , since clams acclima ed o N+and ans e ed o he N- die
educed hei a es o exc e ion bu o a lesse ex en han animals ully
acclima ed o N-. Con e sely, when clams condi ioned on he N- die
we e ans e ed o he N+die , exc e ion a es inc eased up o 50%
mo e han hose o N +-acclima ed clams. Ca bon loss due o me abolic
p ocesses e lec ed ewe luc ua ions be ween die s; clams exposed o
N+ eached app ox. 20% inc emen o espi ed C.
As a esul o he p e ious di e ences, elemen al balances o N and C
showed signi ican di e ences (Table 3) o condi ioning, exposu e and
hei in e ac ion. As be o e, acu e exposu e o die s wi h di e en N
con en s a ec ed he balance o bo h C and N o a g ea e ex en , and he
bes pe o mance was accomplished in clams ed he N+die (Fig. 3 and
Table 2). Condi ioning die was also impo an , bu i s e ec s on
elemen al balances we e only no iceable in animals ed he N+die ,
since eeding he N-poo die ended o educe he C and N balances o
he poin o cancelling ou he po en ial bene i s o high-quali y (N+)
condi ioning.
O e all, me abolic C:N indices (Table 2) e lec ed he di e en ial
s oichiome ic elease o bo h elemen s acco ding o he die supplied:
Bo h ch onic and acu e exposu e o die s o high N con en p omo ed a 2
o 4- old educ ion o his index (highly signi ican ; Table 3), ha was
mainly based on inc eased a es o ammonia exc e ion.
K. A anz e al.
Aquacul u e 555 (2022) 738147
5
The abo e was clea ly e lec ed in he ne g ow h e iciencies o C
and N. The e iciency o C (NGE
C
) was ai ly cons an ac oss die s, while
NGE
N
inc eased signi ican ly in clams ed he N- die (Tables 2 and 3)
poin ing o compensa ion o N de ici . Rega ding bo h elemen al
g ow h e iciencies, in e ac ion e ms o acclima ion and exposu e die s
(Table 3) we e signi ican , accoun ing o he ac ha he highes e i-
ciencies we e achie ed in he expe imen al g oups whe e condi ioning
and exposu e die s we e coinciden (N +N+and N-N-).
Physiological pa ame e s o each combina ion o acclima io-
n*exposi ion die s we e de e mined wi h a la ge s anda d de ia ion
because alues we e pooled means o di e en pheno ypes ep esen ed
by amilies and in a amily seg ega ed g ow h g oups. Compa isons
co esponding o hese endogenous sou ces o a ia ion will be consid-
e ed nex .
3.2. G ow h geno ype e ec s
H4. Highe g ow h demands o endogenous o igin a e be e accomplished
unde condi ions o high N a ailabili y, which esul ed in maxima balances o
bo h N and C (ene gy) being achie ed in as g owing pheno ypes ed he N
ich die .
Gene ally, signi ican e ec s on physiological pa ame e s in ol ed in
elemen al balances we e mainly due o g ow h ca ego y (Table 3),
al hough di e ences be ween amilies we e also de ec ed. Fas -g owing
ju eniles egis e ed highe alues o bo h inges ion and eges ion a es
o bo h N and C (Fig. 4). The esul ing diges i e balances ep esen ed by
abso p ion a es we e also signi ican ly highe . Indeed, inc emen s o
abso p ion in F compa ed o S clams anged om 35 o 40% o N and C,
espec i ely. Simila endencies be ween amilies we e no signi ican
(excep o N eges ion a e), al hough he esul s poin ed ou o a be e
pe o mance o Family 1 compa ed o Family 8, whose mean abso p ion
inc emen s amoun ed only o 4% in e ms o N and 8% o C. Conse-
quen ly, he combina ion o in e - and in a amilia e ec s led o a
g adual dec ease in p ocessing a es om he F1 o S8 g oups (Fig. 4).
Fig. 5 shows he dis ibu ion o AE alues (bo h C and N) ac oss di-
e a y condi ions and pheno ypes. The mains e ec on hese pa ame e s
consis s in AEs inc easing hei alues by ~70% in he change om N- o
N+die s. This combines wi h he anking dis ibu ion o ood p ocessing
a es among pheno ypes (Fig. 4) o esul in as g owe s ed he high
quali y die achie ing he highes alues o bo h C and N balances
(Tables 2; Fig. 7). In addi ion, bo h endogenous ac o s ( amily and
g ow h ca ego y) exe ed weak al hough signi ican e ec s on AE
N
and
AE
C
(Table 3), and wi h ew excep ions, hese e iciencies we e highe
o F8 specimens han o F1 specimens (<10% di e ence be ween
amilies).
H5. Less e icien p o ein me abolism in slow g owe s is e ealed in highe
a es o ammonia exc e ion and educed alues o ne g ow h e iciency o N.
Pheno ype e ec s on C espi a o y losses we e negligible, wi h only a
sligh end o a highe a e in F clams (Table 3). Ins ead, N exc e ion o
F ju eniles (mean: 5.56
μ
g h
−1
) was signi ican ly exceeded by S ju eniles
(mean: 8.02
μ
g h
−1
) (Table 3), which is mainly accoun ed (Fig. 6) o by
s ong F s. S di e ences eco ded wi h he N+die (low C:N index).
Pheno ype e ec s on elemen al balances o C and N we e es ic ed
o g ow h ca ego y, whe eas amily e ec s we e no signi ican (Fig. 7;
Table 3). F s. S di e ences we e highly signi ican (Tables 2 and 3) and
esul ed in elemen al balances o N ha we e ca. 3 imes highe o F
han o S clams, compa ed wi h wo old di e ences o C balances
(Table 2; Fig. 7). Ne g ow h e iciencies we e, in gene al; highe o F
clams han o S clams (Table 2; Fig. 8), bu only di e ences in NGE
N
a ained he signi icance le el (Table 3).
3.3. Acu e s. ch onic dynamics o N and C balances and ne g ow h
e iciencies
H6. Time cou se o physiological adjus men s o die quali y di e ed
be ween ood condi ioned g oups and e ealed di e en ial pheno ypic
plas ici y.
Conside ing he 4 ac o s (condi ioning, exposu e, g ow h ca ego y
and amily), N and C balances a e ep esen ed in Fig. 7 o accoun o he
in e - and in a amilia di e en ial pe o mances o acu e and ch onic
die a y changes. As bo h (N and C) balances we e subjec ed o he same
luc ua ions, ye each di e ed in he in ensi y o hese changes, he plo s
o Fig. 7 will be desc ibed oge he . When clams condi ioned o N+die
(N +N+g oups) we e ans e ed o N- (N +N- g oups), a sha p
dec ease o he ne balances o bo h N and C was obse ed in he ou
pheno ypes esul ing om g ow h g oup* amily combina ions (F1, F8,
S1 and S8), in ol ing he ac ha only abou one hi d o N and hal o C
was being e ained ollowing his change om high o low quali y die .
Full condi ioning o he N- die (N-N- g oups) ba ely a ec ed he as
g owe s pe o mance, while S clams nea ly doubled hei alues. The
acu e esponse o N- condi ioned clams o N en ichmen (N-N+g oups)
also in ol ed di e en ial esponses in as and slow g owe s: while F
clams pa ly eco e ed he ini ial e en ion o C and N, S clams main-
ained o e en lowe ed hei le els, as occu ed in Family 1 S specimens.
These di e en ial e ec s we e accoun ed o by he die exposu e*-
g ow h pheno ype in e ac ion e m (Table 3), which was only signi ican
o SFG
N
. Finally, compa ed o low N condi ioning, high N condi ioning
p omo ed a nea inc ease in he ne C and N balances o all g ow h
Table 2
Pooled mean alues (SE) o condi ioning, exposu e, g ow h ca ego y and amily o IR
N
and IR
C
(inges ion a es o N and C), ER
N
and ER
C
(eges ion a es o N and C),
AR
N
and AR
C
(abso p ion a es o N and C), AE
N
and AE
C
(abso p ion e iciencies o N and C), R
C
(C losses by espi a ion), E
N
(N exc e ed), M
C:N
(me abolic C:N
indices),SFG
N
and SFG
C
(elemen al balances o N and C) and NGE
N
and NGE
C
(ne g ow h e iciencies o N and C): Uni s a e
μ
g h
−1
o a es and SFG and decimal o
e iciencies.
Condi ioning Exposu e G ow h ca ego y Family
N+N- N+N- F S 1 8
IR
N
44.64 (5.3) 38.22 (3.82) 58.55 (4.14) 27.01 (1.79) 49.73 (4.29) 30.01 (3.47) 42.61 (4.5) 39.94 (4.7)
IR
C
329.48 (21.78) 313.78 (22.38) 316.88 (22.31) 324.86 (21.93) 377.29 (17.27) 246.53 (15.53) 337.13 (19.37) 305.38 (24.21)
ER
N
10.42 (0.87) 9.3 (0.8) 8.57 (0.58) 10.87 (0.91) 11.67 (0.77) 7.38 (0.49) 10.82 (0.82) 8.84 (0.8)
ER
C
99.96 (11.21) 124.71 (18.26) 61.07 (4.12) 155.77 (14.68) 132.67 (16.65) 86.6 (10.45) 123.88 (15.46) 101.97 (15.64)
AR
N
34.22 (5.33) 28.92 (3.64) 49.98 (3.76) 16.14 (0.98) 38.06 (4.52) 22.63 (3.38) 31.79 (4.55) 31.1 (4.5)
AR
C
229.51 (20.57) 189.07 (13.02) 255.81 (19.67) 169.1 (9.5) 244.62 (15.82) 159.94 (12.04) 213.25 (16.74) 203.41 (18.1)
AE
N
0.7 (0.03) 0.72 (0.03) 0.85 (0.01) 0.61 (0.01) 0.71 (0.03) 0.71 (0.03) 0.69 (0.03) 0.74 (0.03)
AE
C
0.68 (0.03) 0.63 (0.03) 0.8 (0.01) 0.53 (0.02) 0.65 (0.03) 0.65 (0.03) 0.64 (0.03) 0.67 (0.03)
R
C
35.81 (2.38) 37.17 (3.86) 41.7 (3.41) 32.24 (2.87) 39.93 (3.42) 31.98 (2.53) 35.93 (4.26) 37.12 (1.83)
E
N
5.97 (1.13) 7.2 (1.56) 11.57 (1.43) 2.53 (0.37) 5.56 (1.06) 8.02 (1.76) 6.4 (1.43) 6.83 (1.34)
M
C:N
9.94 (2.16) 17.01 (3.68) 5.04 (0.88) 20.75 (3.36) 14.79 (2.91) 12.12 (3.52) 12.56 (2.94) 14.72 (3.41)
SFG
N
28.25 (5.01) 21.72 (3.01) 38.41 (4.56) 13.61 (1.11) 32.5 (4.05) 14.6 (2.41) 25.39 (4.16) 24.27 (4.04)
SFG
C
193.7 (20.44) 151.9 (12.12) 214.11 (19.8) 136.86 (9.81) 204.69 (15.83) 127.96 (12.25) 177.32 (16.37) 166.29 (17.66)
NGE
N
0.77 (0.04) 0.78 (0.04) 0.71 (0.05) 0.82 (0.03) 0.85 (0.02) 0.67 (0.05) 0.78 (0.04) 0.76 (0.04)
NGE
C
0.82 (0.02) 0.79 (0.02) 0.81 (0.02) 0.80 (0.02) 0.82 (0.02) 0.78 (0.02) 0.82 (0.02) 0.79 (0.02)
K. A anz e al.
Aquacul u e 555 (2022) 738147
6
g oup* amily pheno ypes.
Simila acu e s. ch onic dynamics we e also obse ed o ne g ow h
e iciencies (Fig. 8), wi h di e en ial e ec s accoun ing o 3 d- and 4 h-
o de in e ac ion e ms (Table 3), which we e only signi ican o
ni ogen.
4. Discussion
One impo an issue om he s oichiome ic app oach conce ning
he e o ophic o ganisms is ha ophic ela ionships migh be
“misleading” i only ene gy balances a e conside ed. In he physiological
Table 3
Die condi ioning (C) and exposu e (E), g ow h ca ego y (G) and amily (F) e ec s on 4-way ANOVA able o he pa ame e s in ol ed in elemen al balances o nu ien s
(IR
N
and IR
C
: inges ion a es o N and C, ER
N
and ER
C
: eges ion a es o N and C, AR
N
and AE
C
: abso p ion a es o N and C, AE
N
and AE
C
: abso p ion e iciencies o N
and C, R
C
: C losses by espi a ion, E
N
: N exc e ed, M
C:N
(me abolic C:N indices), and SFG
N
and SFG
C
: elemen al balances o N and C).
IR
N
IR
C
ER
N
ER
C
AR
N
AR
C
AE
N
AE
C
C
F =3.986, p =
0.056
F =0.444, p =
0.511
F =1.975, p =
0.172
F ¼4.675, p ¼
0.04
F =4.018, p =
0.056
F ¼7.449, p ¼
0.011
F =2.819, p =
0.105
F ¼29.48, p <
0.001
E
F ¼100.671, p <
0.001
F =0.074, p =
0.788
F ¼7.585, p ¼
0.011
F ¼72.351, p <
0.001
F ¼169.552, p <
0.001
F ¼37.66, p <
0.001
F ¼422.92, p <
0.001
F ¼686.95, p <
0.001
G
F ¼23.435, p <
0.001
F ¼30.676, p <
0.001
F ¼31.768, p <
0.001
F ¼26.447, p <
0.001
F ¼17.565, p <
0.001
F ¼23.4, p <
0.001
F ¼5.44, p ¼
0.028
F ¼11.017, p ¼
0.003
F
F =1.663, p =
0.209
F =1.706, p =
0.203
F ¼5.492, p ¼
0.027
F =2.193, p =
0.151
F =0.746, p =
0.396
F =0.871, p =
0.359
F ¼10.34, p ¼
0.003
F ¼5.636, p ¼
0.025
C*E
F ¼4.341, p ¼
0.047
F ¼6.87, p ¼
0.014
F =1.959, p =
0.173
F =3.575, p =
0.07
F ¼8.716, p ¼
0.007
F ¼7.337, p ¼
0.012
F ¼14.84, p ¼
0.001
F =0.203, p =
0.656
C*G
F =0.306, p =
0.585
F =0.769, p =
0.388
F =0.984, p =
0.33
F =0.002, p =
0.968
F =0.14, p =
0.711
F =2.036, p =
0.166
F =2.425, p =
0.131
F ¼4.509, p ¼
0.043
E*G
F =0.974, p =
0.333
F =0.62, p =
0.438
F ¼5.879, p ¼
0.023
F ¼9.113, p ¼
0.006
F =3.715, p =
0.065
F =1.164, p =
0.29
F ¼9.569, p ¼
0.005
F ¼14.286, p ¼
0.001
C*F
F =0.141, p =
0.711
F =0.189, p =
0.667
F =0.077, p =
0.784 F =0, p =0.99
F =0.291, p =
0.594
F =0.492, p =
0.489
F =0.005, p =
0.946
F =0.832, p =
0.37
E*F
F =0.163, p =
0.689
F =0.004, p =
0.951
F =1.256, p =
0.273
F =1.022, p =
0.321
F =0.685, p =
0.415
F =0.464, p =
0.502
F ¼13.15, p ¼
0.001
F ¼8.746, p ¼
0.007
G*F
F =0.349, p =
0.56
F =0.347, p =
0.561
F =0.297, p =
0.591
F =0.657, p =
0.425
F =0.308, p =
0.584
F =0.097, p =
0.758
F =0.927, p =
0.345
F ¼7.022, p ¼
0.014
C*E*G
F =0.191, p =
0.666
F =0.224, p =
0.64
F =0.085, p =
0.773
F =0.219, p =
0.643
F =0.197, p =
0.661
F =1.243, p =
0.275
F ¼4.76, p ¼
0.038
F ¼7.834, p ¼
0.01
C*E*F
F =0.002, p =
0.967
F =0.023, p =
0.88
F =0.879, p =
0.357
F =0.308, p =
0.584
F =0.111, p =
0.742
F =0.034, p =
0.854
F ¼4.94, p ¼
0.035
F =3.53, p =
0.072
C*G*F F =0, p =0.998
F =0.393, p =
0.536
F =3.804, p =
0.062
F =3.434, p =
0.075
F =0.339, p =
0.566
F =0.188, p =
0.668
F =3.87, p =
0.06
F ¼12.416, p ¼
0.002
E*G*F
F =0.34, p =
0.565
F =0.031, p =
0.862
F =1.152, p =
0.293
F =0.041, p =
0.842
F =0.149, p =
0.703
F =0.189, p =
0.667
F =0.946, p =
0.34
F =0.017, p =
0.898
C*E*G*F
F =2.375, p =
0.135
F =2.813, p =
0.106
F =0.419, p =
0.523
F =1.689, p =
0.205
F =2.818, p =
0.105
F =2.768, p =
0.108
F =0.61, p =
0.441
F =0.066, p =
0.8
R
C
E
N
M
C:N
SFG
N
SFG
C
NGE
N
NGE
C
C
F =0.114, p =
0.738 F =1.313, p =0.262
F ¼5.596, p ¼
0.026
F ¼5.305, p ¼
0.03
F ¼7.027, p ¼
0.013 F =0.131, p =0.72
F =0.932, p =
0.343
E
F ¼5.385, p ¼
0.028 F ¼69.54, p < 0.001
F ¼30.271, p <
0.001
F ¼80.96, p <
0.001
F ¼26.77, p <
0.001 F ¼9.81, p ¼0.004
F =0.359, p =
0.554
G
F =2.963, p =
0.097
F ¼10.294, p ¼
0.004 F =2.79, p =0.107
F ¼26.25, p <
0.001
F ¼16.86, p <
0.001
F ¼32.399, p <
0.001
F =1.07, p =
0.311
F
F =0.038, p =
0.846 F =0, p =0.984 F =1.001, p =0.326
F =0.637, p =
0.432 F =0.86, p =0.362 F =0.108, p =0.746
F =0.89, p =
0.354
C*E
F ¼5.751, p ¼
0.024
F ¼8.387, p ¼
0.008
F ¼9.702, p ¼
0.004
F ¼14.82, p ¼
0.001
F ¼9.969, p ¼
0.004
F ¼29.558, p <
0.001
F ¼6.68, p ¼
0.016
C*G F =0.013, p =0.91 F =0.02, p =0.888 F =2.305, p =0.141 F =0.162, p =0.69
F =1.876, p =
0.182 F =3.07, p =0.092
F =0.63, p =
0.434
E*G F =0, p =0.989
F ¼7.509, p ¼
0.011 F =0.083, p =0.776
F ¼8.031, p ¼
0.009
F =1.035, p =
0.318 F =3.764, p =0.063
F =0.057, p =
0.814
C*F
F =0.253, p =
0.619 F =3.653, p =0.067 F =1.856, p =0.185
F =0.048, p =
0.828 F =0.282, p =0.6 F =0.327, p =0.573
F =0.598, p =
0.446
E*F
F =1.025, p =
0.321 F =0.005, p =0.942 F =1.346, p =0.257 F =0.64, p =0.431
F =0.146, p =
0.706 F =0.955, p =0.337
F =0.001, p =
0.981
G*F
F =0.557, p =
0.462 F =1.599, p =0.217 F =1.098, p =0.304
F =0.002, p =
0.968 F =0.01, p =0.92 F =1.526, p =0.228
F =0.532, p =
0.472
C*E*G
F =0.507, p =
0.483 F =0.207, p =0.653 F =1.09, p =0.306
F =0.343, p =
0.563 F =0.75, p =0.394
F ¼5.983, p ¼
0.022
F =0.246, p =
0.624
C*E*F
F ¼5.023, p ¼
0.034 F =3.674, p =0.066 F =2.68, p =0.114
F =0.171, p =
0.682
F =0.158, p =
0.694 F =0.836, p =0.369
F =0.829, p =
0.371
C*G*F
F =0.794, p =
0.381 F =0.661, p =0.424 F =1.751, p =0.197
F =0.723, p =
0.403
F =0.403, p =
0.531 F =0.444, p =0.511
F =1.175, p =
0.288
E*G*F F =1.988, p =0.17 F =0.632, p =0.434 F =1.113, p =0.301
F =0.004, p =
0.953
F =0.591, p =
0.449 F =0.125, p =0.727
F =1.14, p =
0.295
C*E*G*F
F =2.998, p =
0.095 F =1.307, p =0.263 F =2.644, p =0.116
F =3.993, p =
0.056
F =1.258, p =
0.272
F ¼4.963, p ¼
0.035
F =0.378, p =
0.544
Signi ican di e ences (p <0.05) a e shown in bold.
K. A anz e al.
Aquacul u e 555 (2022) 738147
7
desc ip ion o g ow h, conside a ion o speci ic nu ien balances be-
comes essen ial o accoun o ea u es om eeding, diges ion and
me abolic beha iou ha appea o be associa ed wi h he necessa y
coupling be ween he a ailable ood and he g owing issues (Bayne,
2017). The compu a ion o C and N balances p o ides a i s app oach in
he oad o a mo e de ailed nu i ional cha ac e iza ion o hese in-
e ac ions o he o ganism wi h he ood en i onmen .
In he p esen s udy, hese balances we e add essed in a double
expe imen al con ex : 1) he analysis o he e ec s o acu e and ch onic
changes be ween wo di e en die s (N+and N-) ha we e isocalo ic
bu main ained a ~ 3- old ange o a ia ion in he C:N a io and 2) he
use o di e en pheno ypes o p o ide o a b oad ange o g ow h
a iabili y o endogenous o igin.
4.1. Die e ec s
In his sec ion, he esul s o nu ien (N and C) balances epo ed
wi h clams main ained wi h N+and N- die s will be discussed wi h
e e ence o po en ial mechanisms o nu ien homeos a ic egula ion,
p o iding a means o ma ching he composi ion o he assimila ed
a ion and hose o he clam issues. Since he C:N a ios o ood in he N-
die (14.54) la gely depa om he co esponding alues o body
composi ion ha closely esemble ins ead he composi ion o ood in he
N+die (C:N a io =4.94), a use ul app oach will be o analyse physi-
ological esponses in he shi om N+ o N- die s as po en ial mecha-
nisms o o se die a y N limi a ions. As desc ibed in he In oduc ion
sec ion, he p e e en ial inges ion o N based on so ing be ween
di e en ypes o pa icles du ing pseudo aecal p oduc ion has p o en o
be an ex emely e ec i e mechanism o he compensa ion o nu i-
ional de ici s (Bayne and S ensson, 2006), bu no choice o such se-
lec ion was a ailable in he p esen expe imen s whe ein ood pa icles
we e homogeneous and eeding condi ions p e en ed pseudo aeces.
Howe e , he possibili y o p einges i e selec ion playing an impo an
ole in homeos a ic nu ien egula ion in clams ed mo e he e ogeneous
die s (e.g., ses on) canno be disca ded, as i has been epo ed in
di e en species o bi al es, including oys e s, mussels and cockles
(Hawkins e al., 1996; Iglesias e al., 1996; Sole chnik e al., 1996;
U u ia e al., 1996).
As p oposed by S e ne and Else (2002), ano he le el in he s oi-
chiome ic coupling be ween he animal and he ood en i onmen
migh consis o adjus men s o he assimila ion pa e ns whe ein he
inges ion/diges ion/assimila ion a es o a gi en elemen in de aul
migh be up egula ed. Bayne (2009) conside ed he possibili y o an
inc ease in inges ion a es o compensa e o low nu ien a ailabili y as
a s oichiome ic mechanism o main ain elemen al homeos asis wi hou
he need o selec i e eeding. In ac , he esul s o expe imen s, e y
simila o he p esen s udy, showed ha mussels (M. edulis) ed
phy oplank on cul u es wi h a low N con en inc eased eeding a es
compa ed wi h mussels ed phy oplank on wi h a high N con en (Bayne,
2017; see his Fig. 5.38). Howe e , no such eso o an inc emen o he
eeding a es was obse ed in he p esen wo k, which esul ed in
inges ion a es o N being educed by hal on a e age when clams we e
exposed o he N-poo die (see Table 2). In a p e ious analysis, we e-
po ed diges i e cons ain s in he p ocessing o N- die s, esul ing in
Fig. 1. Acu e and acclima ed esponse o die a y N changes o inges ion (IR), eges ion (ER) and abso p ion (AR) a es o N (le ) and C ( igh ).
Fig. 2. Rela ionship be ween AE
N
and AE
C
and Cono e AE, whe ein ci cles
ep esen he AE
C
alues, iangles ep esen AE
N
om clams condi ioned on
he N+die , and squa es ep esen AE
N
om clams condi ioned on he N- die .
Fo AE
N
da a poin s, black symbols ep esen animals exposed o N+, while
g ey symbols a e om hose exposed o N-.
K. A anz e al.
Aquacul u e 555 (2022) 738147
8
poo AE ( o o e all o ganics), u he hinde ed by a s ong nega i e
dependence be ween AE and he inges ion a e ha does no occu wi h
he N+die s (A anz e al., 2020). Al hough we p esen ly lack an
explana ion o he compa a i ely poo diges i e pe o mance o clams
ed R. lens in he s a iona y phase ( he o ganic componen o N- die s), i
becomes clea ha hese cons ain s would end o p eclude he e icacy
o any s a egy based on “o e ea ing” o compensa e o educed die a y
N. Con e sely, he highe diges ibili y o N+die s can be in e ed om
signi ican ly inc eased alues o he abso p ion e iciency o bo h C and
N exhibi ed by clams exposed o hese die s o high N con en (Tables 2
and 3).
Howe e , he di e en ial inc ease in AE
N
o e AE and AE
C
when ed
a N-poo die (Fig. 2) was he ele an mechanism employed o
compensa e o he nu ien imbalance, which ag ees wi h he p e ious
li e a u e desc ibed in he In oduc ion sec ion (C an o d, 1995; C an-
o d and G an , 1990; G an and C an o d, 1991; Hawkins, 1985;
Hawkins and Bayne, 1985; K eege e al., 1996; Smaal and Vonck,
1997). Simila ly, Iba ola e al. (2000a) epo ed ha cockles (C. edule)
ed cul u es o Te aselmis suecica in he s a iona y phase (low N con en )
imp o ed he abso p ion e iciency o p o eins ela i e o ca bohyd a es
and lipids compa ed wi h he diges i e beha iou in cockles ed hese
cul u es in he exponen ial phase (high N con en ). While he pe ec i
be ween AE
C
and o e all AE o o ganics (Cono e ) sugges s a lack o
limi a ions conce ning his nu ien ac oss he di e en combina ions o
exposu e*acclima ion die s, nea ly consis en depa u es o AE
N
om
he gene al ela ionship s ongly poin o s oichiome ic compensa ion.
Fig. 3. Acu e and acclima ed esponse o die a y N changes o abso p ion a es (AR), N exc e ion (E; le ) o C espi a ion (R; igh ) and elemen al balance (SFG)
a es o N (le ) and C ( igh ).
Fig. 4. G ow h pheno ype compa ison o elemen al acquisi ion pa ame e s (inges ion (IR), eges ion (ER) and abso p ion (AR) a es) o N (le ) and C ( igh ).
K. A anz e al.
Aquacul u e 555 (2022) 738147
9
On he o he hand, signi ican di e en ial e ec s on AE
N
a e associa ed
wi h condi ioning a he han exposu e o low N die s. Fo ins ance,
clams condi ioned o N- showed a clea disc epancy o AE
N
wi h espec
o o e all AE ha is no seen in hose condi ioned o N+, e en when
exposed o he N- die . The e o e, ega dless o he diges i e mechanism
in ol ed in he p e e en ial abso p ion o N, i becomes clea ha ime is
equi ed o elici a unc ional esponse, e y likely o a no iceable N
de ici o be de eloped. Addi ionally, acclima ion ime migh be
necessa y p o ided ha diges i e adap a ion p oceeds h ough diges i e
enzyme induc ion (Iba ola e al., 1999; Iba ola e al., 1998). Bayne
(2009) epo ed AE
N
:AE
C
a ios in oys e s ha ange be ween app ox.
0.8 in July o 1.0 in No embe and 1.3 in Ma ch, sugges ing ha
seasonally a iable equi emen s o speci ic nu ien s migh be me by
enzyme induc ion. When conside ing diges i e balances (ne abso p-
ion), one al e na i e/complemen a y explana ion o he p e e en ial
abso p ion o N may be he C en ichmen o aecal ma e ials con ibu ed
by endogenous ma e ials in he o m o me abolic aecal losses (MFL;
Hawkins and Bayne, 1985) ha we e ound o consis mainly o lipids in
cockles (e.g., up o 66% compa ed wi h 25%o p o eins and 6% ca bo-
hyd a es; Iba ola e al., 2000a).
Finally, he egula ion o nu ien balances can be achie ed in he
pos abso p i e phase by adjus ing he p opo ion o C and N eleased in
me abolic ac i i ies acco ding o di e en ial income in he die . This is
seemingly achie ed h ough a shi in he composi ion o he pool o
subs a es uelling me abolic ene gy p oduc ion. High le els o N con-
sump ion would enhance p o ein deg ada ion, and consequen ly, high
amoun s o N a e exc e ed as a consequence o deamina ion eac ions
esul ing om p o ein u iliza ion o ene gy pu poses, whe eas low N
a ailabili y may igge biochemical mechanisms p ese ing his
nu ien o p o ein syn hesis ha migh , howe e , be ene ge ically
expensi e (e.g., high a es o p o ein ecycling ha e been ela ed o a
dec ease in p o ein abso p ion in mussels; Hawkins, 1985). Acco ding o
he p esen esul s (see Table 2), he N+die p omo ed a gene al in-
c ease in me abolic ac i i ies, bo h ammonia exc e ion and espi ed C,
bu he impo an poin is ha he C:N a io o me abolism (Table 2)
changes om 5 ( ully compa ible wi h he me abolic b eakdown o
p o eins) in clams eeding on he N+die s o 20 in hose eeding he N-
die s, which sugges s he majo i y use o o he ene gy subs a es. These
di e ences could become e en la ge i a mo e p ecise RQ ( espi a o y
quo ien ) alue e lec ing p o ein-based ca abolism (i.e., RQ =0.8) was
applied o N+die s ins ead o he common RQ =0.9. A u he sou ce o
ene gy expendi u e associa ed wi h N-p ese ing mechanisms, mo e
p ominen wi h he N- die s, could ha e been dis ega ded in he p esen
pos abso p i e measu emen s, since C disposal co esponding o p o ein
me abolism in he o m o he amino-C ac ion was ound o occu in
mussels mainly by he exc e ion o dissol ed o ganic ma e (DOM; no
de e mined he e) a he han in he o m o espi ed CO
2
(i.e., 60 and
10%, espec i ely, o he amino-C assimila ed) (K eege e al., 1996).
Taking in o accoun he obse ed impac s o die quali y on he pa-
ame e s o he ene gy balance, a easonable conclusion is ha he
acquisi ion p ocesses con ibu ed o a la ge ex en o di e ences in he
elemen al balances be ween N+and N- die s. Elemen al balances o
bo h C and N we e inc eased unde he low C:N condi ions, despi e he
sligh dec ease in C a ailabili y unde he N+die . This beha iou co-
incides wi h he elemen al balances calcula ed om he da a shown in
Bayne (2009), whe e bo h SFG
C
and SFG
N
dec eased p opo ionally as
Fig. 5. AE
N
(le ) and AE
C
( igh ) o e e y combina ion o ac o s: condi ioning (N+: da k egion, N-: ligh egion), exposu e (see x axis), g ow h ca ego y (see x axis)
and amily (Fam 1: da k boxes, Fam 8: whi e boxes).
Fig. 6. Ammonia-N exc e ion o as (solid ci cles) and slow (open ci cles)
g owing ju eniles in ela ion o he die dosed.
K. A anz e al.
