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Mar Biol (2016) 163:131
DOI 10.1007/s00227-016-2905-z
ORIGINAL PAPER
Food regime modulates physiological processes underlying
size differentiation in juvenile intertidal mussels Mytilus
galloprovincialis
David Tamayo1
 · Katerin Azpeitia1
 · Pablo Markaide1
 · Enrique Navarro1
 ·
Irrintzi Ibarrola1
 
Received: 21 September 2015 / Accepted: 26 April 2016 / Published online: 9 May 2016
© Springer-Verlag Berlin Heidelberg 2016
Introduction
The energetic basis of interindividual differences in growth
rate has been analysed in several bivalve species, and the
somewhat conflicting results were systematized by Bayne
(1999) in the form of physiological models accounting for
growth differentiation. Discordant conclusions emerging
from these studies suggest that differential growth potential
is a consequence of multiple determinants. Accordingly,
different combinations of physiological growth components
have been reported to explain such endogenous differences.
Two different combinations of these models have
been used to describe the main trends reported in previous
studies:
1.	 Fast growth resulting from higher rates of ingestion
and absorption (acquisition model), usually coupled to
reduced metabolic costs of growth (sensu Parry 1983)
(metabolic efficiency model) (e.g. Holey and Foltz
1987; Toro and Vergara 1998; Bayne et al. 1999a, b;
Bayne 2000; Pace et al. 2006; Tamayo et al. 2011,
2013, 2014, 2015).
2.	 Endogenous differences in standard metabolic requirements
(allocation model) and metabolic costs of
growth (metabolic efficiency model) as key factors
explaining differences among specimens selected for
fast growth (e.g. Garton et al. 1984; Hawkins 1995;
Hawkins and Day 1996).
Overall, fast growth can depend on two different causes:
1) the ability to exploit available food sources by processing
larger amounts of food more efficiently (combination
1), even though this strategy may lead to higher net metabolic
outputs when growth rate differences are maximized
Abstract  Feeding and growth experiments were conducted
to test the hypothesis that physiological attributes responsible
for size differentiation among bivalves likely vary
with environmental conditions. Juvenile mussels (Mytilus
galloprovincialis) were collected from an intertidal population
in Biscay, Spain (43°24′42,462″N02°56′43,659″W),
in January 2007 and 2009. These mussels were maintained
in the laboratory under either restrictive or optimal feeding
conditions until fast- and slow-growing individuals on each
maintenance regime could be identified. After fast- and
slow-growing individuals were identified, the components
of energy balance responsible for such growth rate differences
were measured. The analysis of physiological traits
indicates that under optimal food supply conditions, the
capacity to ingest and absorb food and associated costs of
growth are the main factors underlying growth rate differences.
The set of physiological differences changed when
size differentiation took place under restrictive food conditions.
Higher rates of absorption coupled with reduced
rates of metabolism accounted for faster growth in this
case, especially under low food rations.
Responsible Editor: J. Grassle.
Reviewed by H. Guderley and an undisclosed expert.
*	 David Tamayo
	ectotermo@hotmail.com
1
	 Departamento GAFFA (Animal Physiology), Facultad de
Ciencia y Tecnología, Universidad del País Vasco/Euskal
Herriko Unibertsitatea, Apartado 644, 48080 Bilbao, Spain
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(Tamayo et al. 2011, 2013, 2014, 2015), or 2) the capacity
to save higher amounts of energy due to reduced metabolic
rates (combination 2).
A high degree of multi-locus heterozygosity is an
endogenous factor widely correlated with enhanced growth
rates among bivalves. Variable levels of heterozygosity
appear to underlie the physiological traits responsible for
fast growth in combination 1 (Holey and Foltz 1987; Toro
and Vergara 1998; Pace et al. 2006; Meyer and Manahan
2010) and in combination 2 (Garton et al. 1984; Hawkins
et al. 1986; Hawkins 1995; Bayne and Hawkins 1997).
Although heterozygosity–growth associations in bivalves
are widely accepted (see reviews: David 1998; Launey
and Hedgecock 2001; Meyer and Manahan 2010), correlations
reported in the literature seem intrinsically weak
(reviewed by David 1998) and frequently explain 20–30 %
of observed variance, at the most (Garton 1984; Koehn and
Gaffney 1984; Diehl and Koehn 1985; Gentili and Beaumont
1988; Koehn et al. 1988; Alvarez et al. 1989; David
et al. 1995). Increased multi-locus heterozygosity enhances
the energy budget of fast growers through improved efficiency
of protein deposition by reducing protein turnover
rates. This mechanism leads to more efficient standard and
routine metabolic rates (Hawkins et al. 1986, 1989; Bayne
and Hawkins 1997).
Interpreting those combinations would benefit by relating
the environment to the physiological characteristics
resulting in faster growth. In this respect, food conditioning
seems to affect the association between genetic and physiological
traits (Bayne and Hawkins 1997), lending weight
to the argument that benefits of a reduced protein turnover
are likely dependent on available food levels. As stated by
Bayne and Hawkins (1997), genetic factors promoting differential
protein turnover requirements become relevant
when food is abundant (when energy incorporation dominates
energy balance), whereas heterozygosity and growth
rate are uncorrelated at low food rations (when maintenance
requirements are the determinant).
The aim of the present study was to test the hypothesis
that the physiological components acting on size differentiation
of fast- versus slow-growing individuals differ
as a function of nutritional conditions during the selective
phase. For this purpose, juvenile Mytilus galloprovincialis
collected from a natural population were maintained in
the laboratory until size differentiation occurred under two
different experimental conditions: low and intermittent
phytoplankton supply involving restrictive feeding conditions
(Experiment 1, Expt. 1), and high and continuous
phytoplankton ration involving optimal feeding conditions
(Experiment 2, Expt. 2). In both experiments, individual
with lowest and highest growth rates were selected and the
physiological components of their energy balances determined
at different food rations. The goal was to identify
the specific physiological mechanisms involved in size
differentiation and to compare these mechanisms among
the different conditions applied to the selection of growth
groups.
Materials and methods
Collection, maintenance and selection of fast (F)‑
and slow (S)‑growing mussels
In both Expt. 1 and Expt. 2, juvenile (< 16 mm shell length,
SL) mussels (Mytilus galloprovincialis) were collected
from the rocky intertidal in the Plentzia estuary in Biscay,
Spain (43° 24′ 42,462″ N 02° 56′ 43,659″ W).
Expt. 1: restrictive feeding conditions
Two hundred freshly collected mussels (November 16,
2007), 7–16 mm SL umbo to margin, were measured to the
nearest 0.01 mm with a calliper and distributed in nine 1
mm SL groups (i.e. 7.00–7.99 mm, 8.00–8.99 mm and so
on). These groups were maintained separately for 5 months
with a low ration of phytoplankton (Isochrysis galbana,
T-ISO, given at a packed volume concentration of particles
of ≈0.5 mm3
L−1
; equivalent to 0.22 mg POM L−1
)
in a common feeding tank inside a recirculating sea water
system regulated at 14 °C (environmental temperature at
time of collection) and 34.0 salinity. In order to simulate
the discontinuous feeding imposed by the tidal regime at
the collection site (about 50 % emersion), mussels were fed
phytoplankton concentrations stated above for 12 h day−1
.
During the maintenance period, SL of individuals in each
size group was measured on nine occasions.
At the end of the maintenance period, F and S mussels
were selected by choosing the smallest and largest individuals
(~P25 and P75 percentiles) in each size group. Individual
growth rates were estimated as a daily increment (%)
using the following formula:
where final SL is the average SL after 5 months of maintenance,
and initial SL is the average SL of their size group at
the beginning. F and S groups used in physiological experiments
consisted of 30 fast- and 30 slow-growing mussels
selected following this methodology. These individuals
were between 12.25–19.29 mm SL in the F group and
10.70–16.17 mm SL in the S group.
Expt. 2: optimal feeding conditions
In this experiment, 200 mussels (March 19, 2009),
10–15 mm SL, were tagged for individual growth rate
100 × ([final SL−initial SL]/initial SL)/t (day)
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measurements. Mussels were fed a high ration of phytoplankton
cells (Isochrysis galbana, T-ISO, at a packed
volume particle concentration of ≈3 mm3
L−1
; equivalent
to 1.2 mg POM L−1
), supplied continuously for 42 days
at 14 °C. This food ration is similar to reported optimal
rations for mussels in terms of maximum growth efficiency
(Thompson and Bayne 1974). Compared to the conditioning
ration in Expt. 1, this ration was around 10.5 times
higher considering a 5.25-fold increase in food concentration
and twofold increase in dosing time (24 vs. 12 h
day−1
).
During the maintenance period, individual SL and
live weight were recorded on six occasions to determine
growth rate. Mussels were ranked according to growth
rates in terms of both live weight and SL, and each individual
was scored using a combination of points reflecting
the rankings by both measures. The 20 highest and lowest
scores were selected to form groups of F and S individuals.
This procedure ensured that the selected mussels were
those expressing the highest and lowest rates of increase in
both SL and wet weight. Mussels selected in this way were
20.81–25.77 mm SL for the F group and 13.07–21.55 mm
SL for the S group; their wet weights were 1.067–1.911 g
for group F and 0.340–1.143 g for group S.
Physiological experiments with selected F and S mussels
Selected F and S juveniles were subsequently used in shortterm
feeding experiments. The F and S groups obtained in
both Expt. 1 (restrictive feeding conditions, R) and Expt.
2 (optimal feeding conditions, O) were divided into two
groups that were fed, respectively, diets of low (L) and
high (H) particle concentrations, in order to determine the
physiological components of energy balance under different
food rations. Thus, the combination of conditioning and
measuring food regimes generated four different experimental
conditions (as shown in Table 1) henceforth termed
as RL and RH (for Expt. 1 conditioning) and OL and OH
(for Expt. 2 conditioning).
Diet characteristics
Diets used in measurements of physiological energetics
were prepared by mixing Isochrysis galbana (T-ISO) with
freshly collected and sieved (<67 µm) silt. The silt was
used as an inorganic tracer in measuring absorption efficiency,
following Conover (1986). Concentrated stocks of
the diets were dosed into the feeding tanks at stable particle
concentrations (given as packed volume: mm3
L−1
) that
were frequently checked with a Coulter Multisizer 3. For
further characterization of diets, samples of suspensions
were filtered onto ashed, pre-weighted GF/C filters and
processed for total (TPM: mg L−1
), inorganic (PIM: mg
L−1
) and organic (POM: mg L−1
) particulate matter concentration
according to standard procedures (e.g. Tamayo
et al. 2011). Organic content (OC) was estimated as POM
TPM−1
. These dietary characteristics are given in Table 1.
Physiological determinations
Energy acquisition
Gross energy acquisition corresponded to the organic ingestion
rate (OIR: mg POM h−1
) expressed in energy equivalents
using a conversion factor of 18.75 J mg POM−1
reported for
I. galbana (Whyte 1987). OIR was computed as the product
of clearance rate (CR: L h−1
) and POM concentration in the
diet (mg POM L−1
). Net energy acquisition corresponded to
the absorption rate (AR: J h−1
), which is the product of OIR
and the absorption efficiency (AE: decimal units).
For CR and AE determinations, mussels were placed in a
filtering device consisting of an inverted conical flask serving
as a filtration chamber and suited for the simultaneous
collection of faeces with minimal disturbance of mussels
(Tamayo et al. 2011, 2013, 2014; see Tamayo et al. 2015 for
further description). Water from a feeding tank containing
the diet was re-circulated through the chambers by means
of multichannel peristaltic pumps to produce flow rates
resulting in a 15–25 % reduction in particle concentration.
Table 1  Characteristics of experimental diets used in physiological
determinations in Expt. 1 and 2: TPM total particulate matter (mg
L−1
), PIM particulate inorganic matter (mg L−1
), POM particulate
organic matter (mg L−1
), OC organic content (decimal), Vol. packed
volume (mm3
 L−1
), n number of samples
Values are mean ± SD
Experiment Ration TPM PIM POM OC Vol. (mm3
L−1
) n
1
 Restrictive feeding condition Low (RL) 0.73 ± 0.07 0.06 ± 0.04 0.67 ± 0.04 0.92 ± 0.05 1.59 ± 0.11 5
High (RH) 1.32 ± 0.08 0.13 ± 0.05 1.19 ± 0.05 0.9 ± 0.03 3.2 ± 0.13 8
2
 Optimal feeding condition Low (OL) 0.59 ± 0.18 0.14 ± 0.04 0.45 ± 0.18 0.75 ± 0.07 0.79 ± 0.17 11
High (OH) 1.69 ± 0.25 0.52 ± 0.09 1.17 ± 0.18 0.7 ± 0.03 3.07 ± 0.32 9
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These flow rates were adjusted to ensure reliability of CR
measurements while precluding water re-circulation by the
mussels before leaving the system, which would cause CR
underestimation. The particulate suspension in the feeding
tank was maintained at the target concentration for each
ration by addition of the appropriate volume of particles
from concentrated stocks of the corresponding diet, while
concentration (mm3
L−1
) was frequently checked with a
Coulter Multisizer 3. A control chamber (without mussels)
served to correct particle sedimentation.
Mussels were given a 3-day acclimation period in these
chambers prior to measurements. During the fourth day,
CR measurements with each chamber were replicated 5–6
times and faeces (for AE determinations) were collected
on three occasions. Sample values represent the average of
these replicates.
In Expt. 1 (RL and RH conditions), 5 samples (chambers)
were used (n = 5) per condition and growth group,
each composed of 2 or 3 individuals of similar size with
similar growth rates. Physiological rates in this case were
referred to the mean individual weight of the group. In
Expt. 2 (OL and OH conditions), 10 individuals (n = 10)
were used per condition and growth group. In both experiments,
different F and S individuals were used to perform
measurements at high and low rations.
Clearance rates (CR: L h−1
) were calculated according
to Crisp (1971) with the following formula:
where F is the flow rate (L h−1
), n is the number of individuals
per sample, and Ci and Co are the particle concentrations
in the outflows of control and experimental chambers,
respectively.
Absorption efficiencies (AE: decimal units) were determined
following Conover (1966), by comparing the organic
content of food samples taken from the feeding tanks and
faeces collected from the bottom of the chambers.
Metabolic expenditure
Once CR and AE measurements were completed, mussels
were transferred to respirometers (150-ml capacity) filled
with filtered sea water and sealed with LDO oxygen probes
(cQ40d), and oxygen consumption was computed from
the decrease in oxygen concentration over time (1–2 h).
Controls (chambers without mussels) were used to check
the stability of oxygen concentration during the measurement
period. These measurements corresponded to the routine
metabolic rate (RMR: J h−1
). For standard metabolic
rate (SMR: J h−1
), mussels were starved for 48 h before
measuring their oxygen consumption again. The difference
between resting and active levels of metabolism is commonly
referred to as the scope for activity (see Bayne and
CR = (F/n) ∗ ((Ci −Co)/Ci)
Newell 1983, for discussion on using the concept in physiological
energetics of bivalves). Regarding our experimental
conditions for RMR and SMR, we have proposed (Tamayo
et al. 2013) the more specific term MSFG (metabolic scope
for feeding and growth) to denote the difference between
the two rates.
Rates of oxygen consumption were converted into
metabolic rates using an oxycaloric coefficient of 20.08 J
mLO2
−1
(Gnaiger 1983).
Energy balance
Resulting energy balance was computed as the scope
for growth (SFG: J h−1
), which represents the difference
between absorption rate (AR: J h−1
) and routine metabolic
rate (RMR: J h−1
).
Size standardization
After physiological determinations, mussels were dissected
out of the shells for flesh dry weight (DW) determination.
Clearance and metabolic rates were standardized to a common
size of 50 mg DW, which represents the average dry
weight of F and S mussels in Expt. 1 and 2, by using the
following expression:
where Yst and Ye represent the standard and experimentally
recorded rates, respectively, We the weight of the experimental
mussels, Wstd the standard weight and b the power
value that scales physiological rates to body weight in
this species, 0.583 for CR (Bayne and Hawkins 1997) and
0.724 for metabolism (Bayne et al. 1973).
Statistical procedures
Significant effects with respect to growth category (F vs.
S) and food ration on standardized physiological parameters
were tested using a two-factor analysis of variance
(ANOVA). Significant differences between values within
experiments were checked by means of t test comparisons.
A three-factor analysis of variance (ANOVA) was performed
to test significant differences among growth categories
(F vs. S), food rations and experiments.
Only in the case of RL, RMR and SMR measurements
were carried out on different individuals, and thus, MSFG
was computed without an associated variance. With the
aim of unifying the analysis of MSFG variation, significant
differences in metabolic rates promoted by activity
level (routine vs. standard) and growth category were
tested with a repeated measures two-factor ANOVA for
RH, OL and OH conditions, and a standard two-factor
ANOVA for RL.
Yst = (Wstd/We)b
∗ Ye
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Statistics were performed using SPSS 16, after homogeneity
of variances was evaluated with the Levene test.
Arc-sine transformation of ratios was performed when
necessary according to the results of a normality test
(Kolmogorov–Smirnov).
Results
Growth rates of mussels during size differentiation
phase
Mean growth rates (% SL increment day−1
) of F and S
juveniles selected under restrictive and optimal feeding
conditions (Expt. 1 and 2, respectively) are shown in
Fig.  1. Under restrictive feeding conditions, growth rate
of F mussels was 0.35 ± 0.17 % day−1
, in contrast to
0.048 ± 0.025 % SL increment day−1
in slow growers (S).
As for optimal feeding conditions, growth rate represented
1.24 ± 0.24 % and 0.442 ± 0.196 % SL increment day−1
,
which corresponded to 4.36 ± 1.42 % and 1.26 ± 0.53 %
weight increment day−1
in F and S groups, respectively.
Condition of mussels was analysed by means of power
equations relating DW (y) to SL (x), based on specimens
used in physiological experiments. This relationship was:
0.024 x2.62
(n = 55; r2
0.61) and 0.026 x2.64
(n = 42; r2
0.86) for Experiments 1 and 2, respectively. In the case of
Expt. 2, the relationship was 0.453 x2.55
(n = 21; r2
 = 0.75)
for F mussels and 0.251 x2.77
(n = 21; r2
 = 0.94) in the case
of S mussels. The similarity in exponents indicates an analogous
condition of mussels across experiments and growth
categories.
Physiological behaviour of F and S juveniles
Energy acquisition
Mean values for physiological components of energy balance
of F and S juveniles selected under restrictive feeding
conditions (Experiment 1) are given in Table 2, along with
a two-factor ANOVA to test significance of effects related
to juvenile type (F vs. S) and food ration (L vs. H). Concerning
clearance rate (CR), growth category, food ration
and an interaction term were significant in the ANOVA,
indicating that F juveniles had a significantly higher CR
than S juveniles at low ration and that a higher food ration
promoted a significant reduction in CR in both growth
groups, although it was greater in the F mussels (Fig. 2a).
CR declined with increasing POM, apparently preventing
the organic ingestion rate (OIR) from increasing above
optimal values, and in fact, no significant effects of food
ration on rates of ingestion were recorded (Table 2). Only
juvenile growth type had significant effects on OIR, with F
mussels having significantly higher values than S mussels
at low rations (28 % higher).
Absorption efficiency only changed significantly with
food ration, higher AEs being recorded with concentrated
diets in both growth groups (Table 2). Combined responses
of OIR and AE to the factors under study (growth category
and food ration) would account for significant effects of
both factors on rates of absorption (AR) (Table 2). F mussels
had, at low and high POM concentrations, respectively,
19 and 9 % higher absorption rates than S juveniles.
Mean values for physiological components of growth
measured in F and S mussels selected under optimal feeding
conditions (Expt. 2), together with results of a twofactor
ANOVA applied to these data for testing significant
effects of growth category and food ration, are given in
Table 3. Under optimal food conditioning, clearance rates
of F and S mussels were not significantly different and
were only affected by food ration (Fig. 2b). As in Expt.
1, clearance rate (CR) decreased with increasing POM
(Fig. 2), but this reduction was not great enough to preclude
the significant increase in organic ingestion rate (OIR) for
both growth groups (ANOVA Table 3). In contrast, rates
of ingestion were significantly higher in F mussels despite
the fact that the CR differences between the groups were
not significant (Fig. 2b). Differences in rates of ingestion
were significant at high food concentration, with OIR 31 %
higher in F than S mussels.
In Expt. 2, absorption efficiency was significantly
affected by food ration (AE increased with increasing
POM), whereas juvenile growth types were not significantly
different (Table 3). Given that absorption rate (AR)
0
0.25
0.5
0.75
1
1.25
1.5
1.75
Restrictive conditions
(Expt. 1)
Optimal conditions
(Expt. 2)
SLincrease(%d-1)
Fig. 1  Increase in SL (umbo to margin) (% day−1
) for F (filled
bar) and S (open bar) Mytilus galloprovincialis selected under
restrictive (Expt. 1) and optimal (Expt. 2) food regimes. Values are
means ± 95 % C.I. (n = 30 and n = 20 for experiments 1 and 2,
respectively)
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was calculated as the product of OIR and AE, it was significantly
affected by both growth category and food ration
(ANOVA, Table 3). Thus, overall, F mussels had a higher
AR than S juveniles even though their ration specific values
did not differ significantly (10 and 27 % in OL and OH,
respectively). Moreover, both groups increased their AR in
a similar fashion with increasing food concentration (2.2
and 1.9 times for F and S, respectively).
A summary of a 3-factor ANOVA to test for significant
effects promoted by growth category (F vs. S), food ration
(L vs. H) and experiment (Expt. 1 vs. Expt. 2), upon physiological
parameters of the energy balance, can be seen in
Table  4. This analysis offered, in general terms, an interpretation
of the results similar to those reported for the two
experiments separately. Namely, F mussels had higher food
processing capabilities that ultimately lead to higher absorption
rates (AR), and ration increments enhanced energy
acquisition. Interestingly, the factor termed experiment (i.e.
Expt. 1 vs. 2) interacted significantly with food ration, likely
due to the wider food range achieved in Expt. 2.
Metabolic expenditure
Mean values of metabolic rates under routine and standard
conditions (RMR and SMR) are given in Table 2 and 3
for mussels conditioned to restrictive and optimal rations,
respectively. Results of both experiments are compared
(Fig.  3), with metabolic scope for feeding and growth
Table 2  Expt. 1. Physiological variables in fast (F)- and slow (S)-growing mussel juveniles selected in a restrictive food regime, tested at low
and high rations
OIR organic ingestion rate, AE absorption efficiency, AR absorption rate, RMR routine metabolic rate, SFG scope for growth, SMR standard metabolic
rate, MSFG metabolic scope for feeding and growth. Physiological rates were standardized to an equivalent 50 mg dry soft-body weight.
Mean values are presented ± 95 % C.I. (n = 5 and 4 for F and S groups, respectively) together with a summary of two-factor ANOVA testing
significant effects of growth category and experimental food ration. Different letters beside each mean value indicate significant differences
among FL, SL, FH and SH (t test, p < 0.05)
Physiological parameter Growth category Low ration High ration Summary of two-factor ANOVA
Growth category Ration Interaction
OIR (mg h−1
)
F a 0.288±0.022 a b 0.265±0.034 DF = 1 DF = 1 DF = 1
S b 0.224 ± 0.023 a b 0.250 ± 0.014 F = 9.21 F = 0.013 F = 3.57
p = 0.009 p = 0.909 p = 0.080
AE (decimal)
F b 0.369 ± 0.052 a 0.547 ± 0.052 DF = 1 DF = 1 DF = 1
S b 0.398 ± 0.025 a 0.532 ± 0.025 F = 0.082 F = 47.51 F = 0.932
p = 0.779 p = 0.000 p = 0.351
AR (J h−1
)
F b c 1.979 ± 0.235 a 2.698 ± 0.361 DF = 1 DF = 1 DF = 1
S c 1.655 ± 0.087 a b 2.479 ± 0.025 F = 4.63 F = 37.44 F = 0.173
p = 0.049 p = 0.000 p = 0.684
RMR (J h−1
)
F a 1.076 ± 0.131 a 1.151 ± 0.205 DF = 1 DF = 1 DF = 1
S a 0.949 ± 0.118 a 1.147 ± 0.366 F = 0.34 F = 1.50 F = 0.30
p = 0.566 p = 0.241 p = 0.590
SFG (J h−1
)
F a b 0.903 ± 0.228 a 1.548 ± 0.402 DF = 1 DF = 1 DF = 1
S b 0.706 ± 0.180 a b 1.333 ± 0.389 F = 1.56 F = 14.88 F = 0.003
p = 0.232 p = 0.002 p = 0.957
SMR (J h−1
)
F b 0.635 ± 0.116 a b 0.773 ± 0.180 DF = 1 DF = 1 DF = 1
S a b 0.767 ± 0.085 a 0.973 ± 0.150 F = 5.14 F = 5.54 F = 0.22
p = 0.040 p = 0.034 p = 0.649
MSFG (J h−1
)
F 0.440 a 0.378 ± 0.339
S 0.182 a 0.174 ± 0.466
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(MSFG) values reported as a fraction of RMR. In Expt. 1,
there were no significant effects on routine rates of growth
category or ration. However, standard rates a) were higher
in S when compared to F mussels and b) increased with
the higher food ration in both types of mussels (Table 2).
In Expt. 2, no significant effects were recorded on standard
rates, while routine rates only differed significantly
between low and high rations (Table 3). A 3-factor
ANOVA (Table 4) confirmed that food ration was the only
factor exerting a significant effect on RMR. Regarding
the standard rate (SMR), both ration and the interaction
between growth category and experiment exerted significant
effects. These results account for the fact that, overall
(1) food ration tends to increase SMR, and (2) while
F individuals had similar SMRs in both experiments, S
mussels had higher rates than F mussels in Expt. 1 and
lower in Expt. 2.
With respect to metabolic scope for feeding and growth
(MSFG), no differences were found between F and S mussels
in Expt. 1 (RH rations) (Table 2). However, in Expt. 2
both growth category and ration were statistically significant.
While higher rations caused a significant increase in
MSFG for S mussels, similar values were recorded for F
individuals (Table 3). Further statistical analysis of MSFG
was done using four two-factor ANOVAs (see materials
and methods). ANOVAs were performed in order to compare
the four combinations of conditioning and experimental
rations (RL, RH, OL and OH; Table 5). Tested factors
were: 1) growth category (F vs. S) and 2) the level of metabolic
activity (SMR vs. RMR). The latter factor was used
to test whether metabolic differences between starved and
fed conditions of mussels (in fact, MSFG) were statistically
significant. Therefore, the interaction term in present analysis
accounts for significant differences in MSFG between F
and S mussels.
According to the ANOVA, feeding and growth activities
promoted significant metabolic increments in comparison
with starved conditions in all cases except for high ration
in Expt. 1 (restrictive food conditioning, RH), where it was
not statistically significant (p = 0.095; Table 5). However,
the interaction between metabolic level and growth category
was at the threshold of significance (p = 0.05) for
this RH experimental condition, suggesting that metabolic
behaviour during starvation differed between F and S mussels.
In fact, a t test to compare metabolic levels within
growth categories in this particular condition revealed significant
differences between routine (RMR) and standard
(SMR) metabolic rates for F mussels (t test, t8  = 2.781;
p = 0.024) and lack of significance for S mussels (t test,
t6 = -0.578; p = 0.584). Overall, analysis of factor interactions
(Table 5) reveals some features about the metabolic
behaviour of F and S mussels subjected to different food
conditioning regimes. As a general rule, although the magnitude
of MSFG differs between F and S mussels, the sign
of this difference is strongly dependent on food conditioning:
i.e. MSFG would be greater in F than in S mussels
if maintained under restrictive food conditions and vice
versa for F and S mussels conditioned to an optimal ration
(Fig. 3). Strict interpretation of the ANOVA in Table 5 indicates,
however, that this was significant only for metabolic
measurements performed in mussels at low rations.
Scope for growth
SFG of mussels conditioned to restrictive food conditions
was positively influenced by food concentration, effects
of ration being significant (Table 2). Although consistently
higher in F mussels, SFG differences between growth
groups were not significant (Table 2); the benefits to fast
growers of achieving significantly higher absorption rates
(especially at low POM) were counterbalanced by higher
metabolic expenditures.
Under optimal food conditioning, SFG was significantly
affected by both growth category and food ration (Table 3).
In addition to a positive influence of POM, large differences
between F and S mussels were recorded, reflecting
the combined effects of a greater capacity of F mussels to
0
0.1
0.2
0.3
0.4
0.5
0.4 0.7 1 1.3
CR(Lh-1
)
0
0.1
0.2
0.3
0.4
0.5
0.4 0.7 1 1.3
CR(Lh-1
)
POM (mg L-1
)
a
b
b
a
c
c
a
a
b
a b
Fig. 2  Size standardized clearance rate (CR: L h−1
) as a function of
particulate organic matter (POM: mg L−1
) for F (circles) and S (triangles)
Mytilus galloprovincialis selected under a restrictive food
regimes (Expt. 1, n = 5 and 4 for F and S groups, respectively); and
b optimal food regimes (Expt. 2 n = 8 and 10 for F and S groups,
respectively). Values are means ± 95 % C.I. CR was standardized to
an equivalent 50 mg dry soft-body weight. Different letters indicate
significant differences (t test)
	 Mar Biol (2016) 163:131
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absorb energy and reduce metabolic expenditure by having
significantly lower costs associated with growth represented
by MSFG (Table 5; Fig. 3).
Discussion
The present study used significantly size-differentiated
mussels maintained under food abundance or food deprivation
for sustained periods in the laboratory. Maintenance
of mussels under conditions of high phytoplankton supply
(Expt. 2) intensified the overall growth potential of organisms,
thus forcing the emergence of large interindividual
differences in growth and, consequently, allowing us to
form groups with very different growth rates (approximately
3 times higher for fast growers: 1.24 ± 0.24 vs.
0.44 ± 0.19 % increment in SL day−1
for F and S mussels,
respectively). In contrast, the low food conditions in
Expt. 1 promoted low growth rates reducing the scope for
size differentiation. Nevertheless, after 5 months, large size
differences were attained and the two groups selected for
the highest (F) and the lowest (S) growth rates achieved
0.35  ± 0.17 % and 0.05 ± 0.03 increment in SL day−1
,
respectively.
Comparison of physiological parameters involved in the
energy balance between the different growth groups in the
Table 3  Expt. 2. Physiological variables in fast (F)- and slow (S)-growing mussel juveniles selected in an optimal food regime, tested at low and
high rations
OIR organic ingestion rate, AE absorption efficiency, AR absorption rate, RMR routine metabolic rate, SFG scope for growth, SMR standard metabolic
rate, MSFG metabolic scope for feeding and growth. Physiological rates were standardized to an equivalent 50 mg dry soft-body weight.
Mean values are presented ± 95 % C.I. (n = 8 and 10 for F and S groups, respectively) together with a summary of two-factor ANOVA testing
significant effects of growth category and experimental food ration. Different letters beside each mean value indicate significant differences
among FL, SL, FH and SH (t test, p < 0.05)
Physiological parameter Growth category Low ration High ration Summary of two-factor ANOVA
Growth category Ration Interaction
OIR (mg h−1
)
F c 0.151 ± 0.016 a 0.307 ± 0.040 DF = 1 DF = 1 DF = 1
S c 0.144 ± 0.023 b 0.233 ± 0.048 F = 4.99 F = 44.76 F = 3.35
p = 0.032 p = 0.000 p = 0.077
AE (decimal)
F b c 0.542 ± 0.025 a b 0.594 ± 0.019 DF = 1 DF = 1 DF = 1
S c 0.520 ± 0.058 a 0.618 ± 0.015 F = 0.007 F = 16.23 F = 1.52
p = 0.935 p = 0.000 p = 0.226
AR (J h−1
)
F b 1.529 ± 0.156 a 3.407 ± 0.454 DF = 1 DF = 1 DF = 1
S b 1.409 ± 0.263 a 2.685 ± 0.542 F = 4.25 F = 59.62 F = 2.17
p = 0.047 p = 0.000 p = 0.150
RMR (J h−1
)
F b 0.837 ± 0.139 a 1.327 ± 0.243 DF = 1 DF = 1 DF = 1
S a b 1.044 ± 0.205 a 1.478 ± 0.259 F = 2.45 F = 16.36 F = 0.062
p = 0.127 p = 0.000 p = 0.805
SFG (J h−1
)
F b c 0.692 ± 0.161 a 2.080 ± 0.401 DF = 1 DF = 1 DF = 1
S c 0.364 ± 0.265 b 1.207 ± 0.614 F = 7.76 F = 26.79 F = 1.60
p = 0.009 p = 0.000 p = 0.215
SMR (J h−1
)
F a 0.687 ± 0.117 a 0.821 ± 0.159 DF = 1 DF = 1 DF = 1
S a 0.540 ± 0.126 a 0.692 ± 0.256 F = 2.177 F = 2.35 F = 0.10
p = 0.150 p = 1.135 p = 0.992
MSFG (J h−1
)
F b 0.150 ± 0.147 a b 0.507 ± 0.328 DF = 1 DF = 1 DF = 1
S a b 0.504 ± 0.181 a 0.785 ± 0.231 F = 7.29 F = 7.43 F = 0.103
p = 0.011 p = 0.010 p = 0.750
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contrasting scenarios represented by Experiments 1 and
2 will be discussed with respect to the hypothesis in the
Introduction, ‘that the physiological components of growth
acting in size differentiation of fast- versus slow-growing
individuals would differ as a function of nutritional conditions
occurring during the selective phase’.
In the presence of abundant food (Expt. 2), two physiological
processes in fast and slow growers differed significantly:
a) the capacity to ingest and absorb food and b)
the magnitude of metabolic expenditures in excess of the
standard rate, considered to represent the metabolic scope
for feeding and growth. In accordance with the assumed
use of this metabolic fraction, MSFG (= RMR-SMR) has
been reported to be proportional to absorption and growth
rates of mussels (M. edulis) measured in a wide range of
food concentrations, thus representing an increasing fraction
(26–45 %) of total metabolic expenses in a ~twofold
range of SFG (Bayne et al. 1989). There is a good agreement
between these and the increase in MSFG from low
to high food rations (32–45 % on average) observed in the
present study. However, this metabolic component also represents
a significantly variable fraction of total expenses
between growth groups (i.e. 27 and 50 % of RMR, on average,
for F and S mussels, respectively) (Table 3; Fig. 3b).
In summary, mussels that grew rapidly in an abundant
food supply combined an inherently higher capacity to
acquire food, which was especially evident at high food
rations (acquisition model) and energetically more efficient
food absorption, assimilation and incorporation into tissues
(metabolic efficiency model). These findings are similar to
Table 4  Comparison between Expt. 1 and 2: Summary of three-factor
ANOVA testing significant effects of growth category, measuring
ration and experiments on physiological parameters
Physiological
parameter
Source of vari-
ation
df F p Significance
CR (L h−1
) Growth category
(A)
1 7.96 0.007 **
Measuring
ration (B)
1 61.18 0.000 ***
Experiment (C) 1 1.40 0.242 n.s.
A × B 1 0.30 0.589 n.s.
A × C 1 0.18 0.670 n.s.
B × C 1 4.25 0.045 *
A × B × C 1 3.81 0.057 n.s.
OIR (mg h−1
) Growth category
(A)
1 8.38 0.006 **
Measuring
ration (B)
1 19.77 0.000 ***
Experiment (C) 1 11.82 0.001 **
A × B 1 0.10 0.756 n.s.
A × C 1 0.00 0.968 n.s.
B × C 1 18.81 0.000 ***
A × B × C 1 4.37 0.042 *
AE (decimal) Growth category
(A)
1 0.07 0.797 n.s.
Measuring
ration (B)
1 55.81 0.000 ***
Experiment (C) 1 47.88 0.000 ***
A × B 1 0.00 0.972 n.s.
A × C 1 0.03 0.874 n.s.
B × C 1 6.88 0.012 *
A × B × C 1 2.10 0.154 n.s.
AR (J h−1
) Growth category
(A)
1 5.09 0.029 *
Measuring
ration (B)
1 58.50 0.000 ***
Experiment (C) 1 0.13 0.724 n.s.
A × B 1 0.65 0.423 n.s.
A × C 1 0.24 0.628 n.s.
B × C 1 6.87 0.012 *
A × B × C 1 1.32 0.256 n.s.
RMR (J h−1
) Growth category
(A)
1 0.39 0.535 n.s.
Measuring
ration (B)
1 10.89 0.002 **
Experiment (C) 1 1.01 0.321 n.s.
A × B 1 0.03 0.856 n.s.
A × C 1 1.82 0.184 n.s.
B × C 1 3.22 0.079 n.s.
A × B × C 1 0.25 0.622 n.s.
SFG (J h−1
) Growth category
(A)
1 5.95 0.019 *
Measuring
ration (B)
1 28.05 0.000 ***
Table 4  continued
Physiological
parameter
Source of vari-
ation
df F p Significance
Experiment (C) 1 0.05 0.826 n.s.
A × B 1 0.72 0.399 n.s.
A × C 1 1.42 0.239 n.s.
B × C 1 2.11 0.153 n.s.
A × B × C 1 0.64 0.429 n.s.
SMR (J h−1
) Growth category
(A)
1 0.04 0.846 n.s.
Measuring ration
(B)
1 4.82 0.033 *
Experiment (C) 1 2.02 0.162 n.s.
A × B 1 0.09 0.765 n.s.
A × C 1 4.47 0.040 *
B × C 1 0.04 0.841 n.s.
A × B × C 1 0.03 0.863 n.s.
CR clearance rate (L h−1
), OIR organic ingestion rate (mg h−1
), AE
absorption efficiency (decimal units), AR absorption rate (J h−1
),
RMR routine metabolic rate (J h−1
), SFG scope for growth (J h−1
),
SMR standard metabolic rate (J h−1
). * p < 0.05; ** p < 0.01;
*** p < 0.001; n.s. non-significant differences
	 Mar Biol (2016) 163:131
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those in our previous studies on hatchery-selected juvenile
clams, Ruditapes philippinarum (Tamayo et al. 2011, 2013,
2015), and oysters, Crassostrea gigas (Tamayo et al. 2014).
Furthermore, and especially within the Ostreidae, similar
results have been reported for several different species.
It is noteworthy that these studies have been mainly performed
with individuals selected for their variable growth
and covering life stages from larvae to adults (Toro and
Vergara 1998; Bayne et al. 1999a, b; Bayne 2000; Pace
et al. 2006). This suggests that the physiological characteristics
described above could represent a general tendency in
bivalves when large growth rate differences are analysed.
In contrast, size-differentiated individuals did not exhibit
the same set of physiological differences after 5 months
under poor feeding conditions (Expt. 1). Primarily, selected
F and S mussels did not differ significantly in SFG at the
two food rations. Such a result is consistent with the weak
size differentiation achieved during the 5-month period of
maintenance at low food conditions and likely reflects the
fact that the RL ration was two to three times higher than
the maintenance ration in which mussels were allowed to
size differentiate. Although mean SFG was lower in S than
F mussels (especially at low ration), the difference was
not significant (Table 2). In spite of that, F and S mussels
differed significantly in CR and, hence, in ingestion and
absorption rates, indicating that fast growers were better
equipped for food acquisition than slow growers. However,
CR differences between F and S juveniles were maximized
at low POM concentration, and higher food rations cancelled
out significant differences in CR. In other words,
F juveniles would benefit from a higher capacity for food
acquisition only at low food concentrations, i.e. in conditions
where reduced energy incomes tend to magnify the
contribution of reduced metabolic expenditure to total
energy flux through the organism.
Another contrasting feature in comparing physiological
behaviour of mussels maintained under restrictive and
optimal feeding conditions concerns the metabolic aspects.
Unlike the mussels in Expt. 2, MSFG in F mussels in Expt.
1 was higher than in S mussels in the same experiment.
Since no significant changes in total metabolism (RMR)
were observed either between F and S groups or between
different food rations, it seems clear that differences in
metabolic scope in this case were a consequence of reduced
rates of resting metabolism (SMR) in fast growers (Table 2;
Fig. 3a). Considering the significance values of the interactions
in Table 5, the differences between growth groups
were especially relevant when measured at low ration—as
exemplified by the 46 % decline between RMR and SMR
in F mussels (from 1.07 ± 0.13 to 0.63 ± 0.11) compared
to the 20 % decline in S mussels (from 0.94 ± 0.11 to
0.76  ± 0.08)—even though RMR and SMR were barely
significant (p  = 0.05). Therefore, results point towards
the existence of differences between fast and slow growers
in their capacity to reduce metabolic expenditure under
starvation. Unlike the mussels conditioned to an optimal
ration, the fast growers under food limitation did not have
an increased efficiency of food processing and growth,
but their maintenance processes came at a reduced cost as
shown by lower SMRs. This feature has previously been
correlated with higher, although negative, SFG in mussels
0.0
0.5
1.0
1.5
2.0
F mussels S mussels F mussels S mussels
LOW RATION HIGH RATION
SMRandRMR(Jh-1)
0.0
0.5
1.0
1.5
2.0
F mussels S mussels F mussels S mussels
LOW RATION HIGH RATION
SMRandRMR(Jh-1)
0.40
0.19
0.32 0.15
0.17
0.48
0.38
0.53
a
b
Fig. 3  Size standardized standard metabolic rate (SMR: J h−1
; grey
bar) and routine metabolic rate (RMR: J h−1
; full bar) of F and S
Mytilus galloprovincialis recorded at high and low rations. Numbers
beside each bar correspond to the ratio of MSFG (open bar) to
RMR = (SMR–RMR)/RMR. Mussels were selected under a restrictive
food regimes (Expt. 1, n = 5 and 4 for F and S groups, respectively);
and b optimal food regimes (Expt. 2 n = 8 and 10 for F and S
groups, respectively). Values are means ± 95 % C.I. Metabolic rates
were standardized to an equivalent 50 mg dry soft-body weight
Mar Biol (2016) 163:131	
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under restrictive food levels (Hawkins 1995; Hawkins and
Day 1996).
Our study shows that food conditions during the size differentiation
phase might potentially alter the set of physiological
variables underlying differential growth potential.
This fact, that feeding conditions can affect the correlations
between endogenous (genetic) factors and physiological
rates, was previously suggested by Bayne and Hawkins
(1997). These authors analysed possible correlations
between growth rate and multilocus heterozygosity in
small mussels that were sequentially held at increasing
food levels. Increasing heterozygosity levels led to higher
efficiencies of protein deposition and, as a consequence,
lower costs of growth, thus allowing more heterozygous
individuals to achieve higher growth rates. These series of
correlations served to highlight the functional link between
protein turnover rate, metabolic efficiency and genetic factors.
However, Bayne and Hawkins (1997) also showed
that significant correlations between heterozygosity and
growth rate were only found at high food rations, while
at lower rations, corresponding to maintenance conditions
(low SFG), no significant correlations were found. It follows
that genetic factors promoting differential protein
turnover requirements become relevant under conditions of
food abundance (i.e. when energy incorporation dominates
the energy balance), whereas lack of correlation between
heterozygosity and growth rate at low rations suggests that
heterozygosity level does not modulate standard metabolic
expenditure. Therefore, it is very likely that interindividual
differences in SMR derive from causes other than differences
in protein deposition efficiencies. In this respect,
Pernet et al. (2008) reported that intra-specific differences
in SMR are related to the unsaturation index of membrane
fatty acids in Crassostrea virginica. These authors reported
that both SMR and membrane unsaturation were lower
in fast-growing hatchery oysters compared with slowgrowing
wild oysters. The higher unsaturation index in
the latter corresponded mainly with higher 22:6n-3 levels
whose importance in metabolic rate regulation of animals
had already been observed (reviewed by Hulbert and Else
2005). Wild oysters had higher SMRs even though they
were much more heterozygous than fast-growing hatchery
oysters with lower SMRs and higher CRs.
Very likely the possession of a reduced maintenance
metabolic output improves the probability of surviving
under stressful conditions. Diehl et al. (1986) grew juvenile
Mytilus edulis in a tidal salt marsh for 72 days and
then starved them in the laboratory for 2 months. Recorded
interindividual variability in rates of oxygen consumption
suggested that reduced rates of metabolism were associated
with increased resistance to weight loss during starvation
rather than with the capacity for weight gain during feeding.
An elegant experiment by LeBlanc et al. (2008) is even
more relevant. Juveniles of Mytilus edulis were submitted
to stressful conditions consisting in 11 h of air exposure
followed by 6 h of exposure to 32° C water, which caused
high mussel mortality. Survivors were used to determine
SFG and multilocus heterozygosity and compared with
control mussels. Higher heterozygosity in survivors than
controls indicated that heterozygotes were somehow more
resistant to stress. Measurements of SFG resulted in nonsignificant
differences between treated and control mussels,
but, meaningfully, there were significant differences in both
RMR and SMR which were, respectively, 40 and 30 %
lower for survivors than for control mussels.
A pertinent conclusion is that size differentiation of
juvenile mussels in the natural environment results from a
balance between opposing forces. These include a) periods
of good trophic conditions characterized by high POM concentrations
and long immersion times that would maximize
growth rates of individuals well equipped to acquire food
and process it at low costs, in spite of their relatively high
resting metabolic rates, and b) periods of restricted trophic
conditions of both low POM, low organic content of food
and/or prolonged emersion times that would have a more
adverse impact on those individuals unable to down-regulate
their SMRs. This dual impact of feeding conditions
upon the physiological profiles of individuals that are likely
to grow faster might help understanding contradictory
Table 5  Summary of two-factor ANOVA testing for significant
effects of a) level of activity (routine vs. standard) and, b) growth category
(F vs. S) on metabolic rates in mussels from the four combinations
of experimental conditions (RL, selected under restrictive food
conditions and fed low rations; RH, selected under restrictive food
conditions and fed high rations; OL, selected under optimal food conditions
and fed low rations; OH, selected under optimal food conditions
and fed high rations)
For RL conditions, independent groups of mussels were used for metabolic
determinations; thus, a standard ANOVA was performed. For
the rest of experimental conditions (RH, OL and OH), we performed
an ANOVA of repeated measures, as the same groups of mussels
were used in routine and standard measurements
Experimental condition Source of variation DF F p
R L Level of activity 1 26.550 0.000
Growth category 1 0.001 0.971
Interaction 1 4.580 0.050
R H Level of activity 1 3.710 0.095
Growth category 1 1.240 0.303
Interaction 1 0.506 0.500
O L Level of activity 1 28.030 0.000
Growth category 1 0.101 0.755
Interaction 1 8.220 0.011
O H Level of activity 1 42.130 0.000
Growth category 1 0.006 0.940
Interaction 1 1.960 0.181
	 Mar Biol (2016) 163:131
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131  Page 12 of 13
conclusions in previous bivalve studies regarding the prevalence
of energy input or output as the processes responsible
for size differentiation.
Acknowledgments This study was funded through the project
AGL2009-09981 of the Spanish Ministry of Science and Innovation.
D.T. was funded by a FPI grant from the Basque Government. Authors
are indebted to two referees for valuable comments and sugges-
tions. Finally, Tamayo D. wants to thank, in particular, the invaluable
support received from Muguruza C. over the years. Will you marry me?
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