Intrapopulation Variation in the Standard ... - Roberto Nespolo

Intrapopulation Variation in the Standard Metabolism of a Terrestrial
Mollusc: Repeatability of the CO2 Production in the
Land Snail Helix aspersa
Paulina Artacho*
Roberto F. Nespolo
Instituto de Ecología y Evolución, Universidad Austral
de Chile, Casilla 567, Valdivia, Chile
Accepted 5/20/2008; Electronically Published 1/30/2009
ABSTRACT
During the past 2 decades, interest in interindividual variation
in performance traits has increased considerably among physiological
ecologists. A great deal of this interest has focused on
repeatability studies of physiological traits. One of the most
important physiological traits in animals is whole-animal metabolism
because it reflects several aspects of an organism’s
energy budget. However, in order to respond to natural selection
(ultimately), this variable should be consistent over most
of an individual’s life history. We studied energy metabolism
(CO2 production, ˙Vco2) in two of the southernmost populations
of Helix aspersa land snails, a cosmopolitan species that
colonized most of the human-inhabited world. Our results
show that H. aspersa exhibits a relatively lower than expected
˙Vco2 compared with that described in the few other published
studies on this species and that there is no significant difference
between populations (Valdivia ˙Vco2 p 0.21 � 0.01 mL CO2 h�1 ; Concepción mL CO2 h�1 ˙Vco2 p 0.20 � 0.01
; mean body
mass p 4.2 g). Repeatability of ˙Vco2 in land snails was significant
and was not statistically different in both populations
(Valdivia: t p 0.42; Concepción: t p 0.31).
These results sug-
gest that energy metabolism is repeatable and can eventually
respond to selection in land snails. We argue that land snails
are good, though underutilized, models for evolutionary physiology
studies.
Introduction
In the past 2 decades, interest in interindividual variation in
performance-related traits has increased greatly (e.g., Hayes et
al. 1992; Konarzewski and Diamond 1995; Chappell et al. 1996,
* Corresponding author; e-mail: paulinaartacho@gmail.com.
Physiological and Biochemical Zoology 82(2):181–189. 2009. � 2009 by The
University of Chicago. All rights reserved. 1522-2152/2009/8202-8006$15.00
DOI: 10.1086/590222
181
1999; Koteja 1996; Hayes and Jenkins 1997; Dohm et al. 2001;
Nespolo et al. 2003). This interest is due to the following two
factors: (1) interindividual variation represents the raw material
with which natural selection can act and (2) interindividual
variation represents the first—and certainly the most straightforward—step
toward addressing the potential for response to
selection in a trait. In evolutionary terms, it is of special importance
to distinguish between interindividual variation and
intraindividual variation. This distinction is summarized by
repeatability, that is, the consistent variation in a trait across
at least some part of the individual’s life (Hayes and Jenkins
1997). Although it is well known that repeatability is not the
sole condition for a trait to evolve by natural selection (because
it is not informative of any aspect of the relationship of the
trait with fitness), if a trait is not repeatable, it is unlikely that
it will change in response to selection (Hayes and Jenkins 1997).
One of the most common measures of repeatability is the
2 2 2 2
intraclass correlation coefficient, t p j A/(jA�j e) , where jA
is
2
the between-individual component of variance and je is the
residual variance component when multiple measurements are
performed in a sample of individuals (Falconer and Mackay
1996). This permits the separation of two important sources
of variation. The first is within-individual variance, which reflects
the effects of temporary differences between successive
tests and is sensitive to the precision of the measurement. The
second is between-individual variance, which is the result of
the external environment that permanently affect individuals
plus genetic differences (Falconer and Mackay 1996). Thus,
greater between- than within-individual variation indicates that
the trait is consistent within individuals, and in turn, it would
be able to respond to selection.
In general, morphological attributes such as body mass are
highly consistent over time (Falconer and MacKay 1996) because
they usually exhibit low residual variance. On the other
hand, physiological and behavioral traits show considerable variation
both between individuals (e.g., Bech et al. 1999; Rogowitz
and Chappell 2000; Angilletta 2001; Lindström and Klaassen
2003; Lardies et al. 2004; Terblanche et al. 2004) and within
individuals (Lindström and Rosen 2002; Lindström and Klaassen
2003; Marais and Chown 2003). Among physiological traits,
metabolic variables derived from respirometric records have
been copiously analyzed, indicating, for instance, significant
repeatability in the basal metabolic rate of endotherms (Bech
et al. 1999; Horak et al. 2002; Tieleman et al. 2003; Ksiazek et
al. 2004; Labocha et al. 2004; Rønning et al. 2005; Sadowska
et al. 2005) and in the standard metabolic rate (SMR) in ectothermic
vertebrates (Garland and Bennett 1990; McCarthy

182 P. Artacho and R. F. Nespolo
2000; Virani and Rees 2000). However, the repeatability of metabolism
in invertebrates is less well known, and most of this
work has focused on insects (Chappell and Rogowitz 2000;
Marais and Chown 2003; Nespolo et al. 2003; Terblanche et al.
2004; Nespolo and Franco 2007).
Some of the most environmentally constrained terrestrial
invertebrates are pulmonate snails. Terrestrial snails lose water
easily (e.g., Dallas et al. 1991; Arad et al. 1998), so their activity
periods, and therefore their capacity for reproduction and dispersal,
are restricted by environmental factors such as temperature,
humidity, and water availability (Guppy and Withers
1999; Arad et al. 2001; Cook 2001; Storey 2002). Moreover,
terrestrial snails have high costs of transport, and this further
restricts their dispersal ability (Denny 1980; Davies et al. 1990;
Kideys and Hartnoll 1991). Despite these characteristics, and
because of their association with humans, land snails have colonized
a wide range of environments, from arid to semiarid
regions and tropical areas (e.g., Arad et al. 1992; Zotin and
Ozernyuk 2002; Giokas et al. 2005), showing morphological,
behavioral, and physiological adaptations to a range of environmental
conditions (e.g., epiphragm formation, site selection,
and estivation; Withers et al. 1997; Giokas et al. 2005; Michaelidis
et al. 2007). Given that these mechanisms entail energetic
costs, it is reasonable to expect that they will influence
the overall energy budget (and probably also fitness); however,
very few studies have addressed the potential association in
these (e.g., Herreid 1977; Steinberger et al. 1982; Prior 1985;
Barnhart and McMahon 1987; Marshall and McQuaid 1991;
Zotin and Ozernyuk 2002).
Among terrestrial gastropods, Helix aspersa (Gastropoda,
Pulmonata) is one of the most successful species based on its
wide distribution range. Native to North Africa, since the Holocene,
it has colonized a large range of human-perturbed environments
across northwest Europe (agricultural and suburban
areas and domestic gardens) and has been dispersed mainly
by human movements (Guiller et al. 2001). Later, with the
development of human transportation, this land snail was introduced
in the majority of the temperate, Mediterranean, and
subtropical areas such as the Atlantic Islands, South Africa,
Haiti, New Zealand, Australia, Mexico, Chile, and Argentina.
In many places (e.g., Australia and California), this species has
achieved pest status. A handful of physiological studies have
Figure 1. Records of CO2 production in Helix aspersa. A, Five representative continuous records. The first and seventh records represent
the baselines. B, One like-discontinuous ˙Vco2 record (see “Results”). The first and third records correspond to the baselines. All records were
carried out at 20�C. During the measurements, the individuals remained without movement, according visual inspection.
˙Vco 2

Repeatability of Standard Metabolism in the Land Snail 183
Table 1: Means (�SE) and regression statistics between metabolic variables and M b
Variable (mL h�1 ˙Vco2 ) Mb (g) Slope Intercept r2 N
Concepción:
˙Vco2aver ˙Vco2min ˙Vco2max Valdivia:
˙Vco2aver ˙Vco2min ˙Vco2max .21 � .01
.08 � .01
.33 � .01
.20 � .01
.10 � .01
.31 � .02
4.10 � .12
4.10 � .12
4.10 � .12
4.24 � .15
4.24 � .15
4.24 � .15
.08 � .03*
.06 � .03*
.09 � .05 (NS)
.06 � .05 (NS)
.03 � .04 (NS)
.1 � .06 (NS)
.04 � .02 (NS)
�.004 � .02 (NS)
.07 � .03 (NS)
.06 � .03 (NS)
.02 � .02 (NS)
.05 � .04 (NS)
.14*
.10*
.08 (NS)
.05 (NS)
.02 (NS)
.09 (NS)
44
44
44
33
33
33
Note. Linear regression statistics were performed with the log 10-transformed variables. NS p not significant.
* .
P ! 0.05
been performed on H. aspersa, mainly in metabolic pathways
related to hibernation (Brook and Storey 1997) and estivation
(Giokas et al. 2005; Michaelidis et al. 2007). However, to our
knowledge, there are no studies dealing with the evolutionary
significance of these mechanisms or others related to the physiology
of this species.
In this study, we estimated the repeatability of SMR, the
obligatory energetic cost of maintenance in ectotherms, in two
populations of H. aspersa. SMR has generally attracted the attention
of physiological ecologists because it has an impact on
the amount of energy available for activity and production
(Steyermark 2002) and could contribute to the variation in
other life-history traits across a range of environments (Niewiarowski
and Waldschmidt 1992; Chown and Gaston 1999;
Angilletta 2001). Although there is evidence of an association
between energy metabolism and fitness in some vertebrates
(Hayes and O’Connor 1999; Jackson et al. 2001) and insects
(Crnokrak and Roff 2002), the demonstration of significant
repeatability is a preliminary step in the analysis of selective
significance. Despite the relative abundance of repeatability
studies in metabolic rate, we are not aware of any study in
taxonomic groups other than vertebrates and insects (Nespolo
and Franco 2007).
Material and Methods
Animals and Sampling
Forty-five juvenile Helix aspersa were collected under plants
and rocks in public parks in Concepción (36�47�S, 72�38�W)
and Valdivia (39�49�S, 73�15�W). These animals were of approximately
the same body mass ( ¯x � SD; Valdivia: 4.24 �
0.85 g; Concepción: 4.10 � 0.84 g) and were housed in plastic
cages filled with 10 cm humid soil. Snails were fed with corn
flour and maintained at 20�C with a 12L : 12D photoperiod for
1 mo before measurement. Relative humidity was maintained
at high levels by sprinkling the interior of the boxes with water
every day.
Respirometry
CO2 production was evaluated by open-flow respirometry using
a LiCor 6262 CO2 analyzer (LI-COR Bioscience, Lincoln, NE),
capable of resolving differences of 1 ppm CO 2 in air, connected
to a computerized data-acquisition system (Expe Data software,
Sable Systems) similar to that used by Lighton and Turner
(2004). The analyzer was calibrated periodically against two
kinds of gas (CO 2-free air and a commercial mix of 291 ppm
of CO 2). A Sable System eight-channel multiplexer was used
for performing the measurements, using five chambers with
individual snails and three chambers for baseline measurements
(before and after all records), which allowed us to correct for
possible drift (although it was almost nonexistent between baselines
and calibrations). The arrangement of the respirometry
system was as follows: air at 120 mL min �1 was pumped sequentially
through a Drierite–soda lime–Drierite column, a
flow meter that maintained flow rate within �1% of the desired
rate, and a transparent respirometry chamber with a volume
of 60 mL. CO 2-free air flowed through all chambers while one
of them was measured. Animal activity was visually monitored
for intervals of ca. 10 min, and measurements lasted 30 min.
Activity was uncommon, and data for active animals were discarded.
In order to achieve a postabsortive state, metabolic rate
was measured in individuals deprived of food for 18 h (Lighton
1989; Bradley et al. 2003). We believe this time was enough to
attain a postabsortive state based on preliminary measurements
of mean retention time in this species (P. Artacho et al., unpublished
manuscript). All metabolic trials were performed
during the day, when land snails are inactive, which corresponds
to the rest phase in this species (personal observation). Metabolic
records were automatically transformed by a macro program
recorded in the Expe Data software (Sable Systems) in
order to transform the measure from parts per million to milliliters
CO 2 per hour, taking into account the flow rate. The
respirometry equation used was
˙Vco pstp # (FeCO � FiCO )
2 2 2
FR
# ,
1 � FeCO 2 # [1 � (1/RQ)]
where FeCO 2 is the excurrent fractional concentration of CO 2,
FiCO 2 is the input fractional concentration of CO 2,FRisthe
flow rate (mL min �1 ), STP is the correction factor for standard
conditions of temperature and pressure (which for mass flow-

184 P. Artacho and R. F. Nespolo
Figure 2. Regressions between metabolic variables and body mass in individuals of Helix aspersa from Concepción.
meters is equal to 1), and RQ (respiratory quotient) is the
respiratory interchange ratio, which was assumed to be equal
to 0.85.
Also, we eliminated the first 10 min of the records (600
samples). From each individual record, we extracted three variables:
complete average of each transformed record ( ˙Vco2aver), the average of the 1-min steady state of minimum ˙Vco2 production
( ˙Vco2min), and the average of the 1-m steady state of
maximum Vco ˙ production ( ˙
2 Vco2max).
Precise measurements of repeatability can be accomplished
with two repetitions (Nespolo and Franco 2007), although three
or more are recommended for minimizing residual variance.
Because of logistical reasons, individuals from Concepciónwere
measured only twice. Individuals from Valdivia, however, were
measured three times. In both cases, the experiment was performed
during a 1-mo period.
Statistics
All statistics analyses were performed with Statistica 6.1
(StatSoft 2004). Common linear regressions were used for examining
the relationship between body mass (M b) and the three
metabolic variables. Repeatability was evaluated by the intraclass
correlation coefficient (t; Berteaux et al. 1996; Falconer
and Mackay 1996) by calculating the between- and withinindividual
variance component from one-way ANCOVA (M b
as covariable). Then, t p between-individual variance com-
ponent/(between-individual variance component � withinindividual
variance component). We checked normality and
homoscedasticity by Lilliefors and Levene tests, respectively, and
we log 10 transformed the variables whenever necessary.
Results
The pattern of ˙Vco2 production of Helix aspersa was clearly
continuous in 85% of the records (Fig. 1A). There were some
records exhibiting patterns of ˙Vco2 that seemed discontinuous
(Fig. 1B). However, after analyzing them, we found that these
records were of a nonperiodic nature and without the typical
pattern of zero gas exchange (Marais et al. 2005). According
to visual inspections, activity was unusual during respirometry
records. Linear regressions between metabolic variables and
body mass showed that only Vco ˙ and ˙
2 Vco2
were signif-
aver min
icant in the population from Concepción (Table 1; Figs. 2, 3).
A nested ANCOVA (using body mass as covariable) indicated
nonsignificant differences in ˙Vco2aver (between populations:
F1, 3 p 1.3, P p 0.3;
between measurements, within populations:
F , ), ˙
3, 186 p 0.2 P p 0.9 Vco2min
(between populations:
F1, 3 p 3.4, P p 0.07;
between measurements, within populations:
F , ), and ˙
3, 186 p 0.42 P p 0.74 Vco2max
(between populations:
F1, 3 p 1.3, P p 0.26;
between measurements, within populations:
F , ). Repeatabilities of ˙
3, 186 p 0.28 P p 0.84 Vco2aver,
Vco ˙ , and Vco ˙ (Table 2) were significant and remarkably
2min 2max

Repeatability of Standard Metabolism in the Land Snail 185
Figure 3. Regressions between metabolic variables and body mass in individuals of Helix aspersa from Valdivia.
consistent across metabolic variables and locality, averaging
0.33 � 0.06 (grand mean � SD).
Discussion
If we assume a respiratory quotient of 0.84 (Addo-Bediako et
al. 2002), the values of SMR of Helix aspersa from Chilean
populations obtained in this study are at least 50% lower than
measurements of the same species from Australia and Europe
(Vorhaben et al. 1984; Pedler et al. 1996). However, comparisons
with other studies in the same species and in other species
are complicated by the fact that most authors used different
methodological approaches (e.g., type of respirometry system,
measurement temperature, metabolism with respect to dry or
wet weight, with or without shell; Vladimirova 2001). Perhaps
this is one of the reasons why few generalizations have been
established in the bioenergetic and ecophysiological patterns of
terrestrial molluscs (with the probable exception of estivation
and hibernation; see Vladimirova 2001). Surprisingly, this fact
contrasts with the enormous quantity of information that exists
in other taxa (e.g., insects [Addo-Bediako et al. 2002; Chown
et al. 2007] and vertebrates [White and Seymour 2003;
McKechnie and Wolf 2004; Nagy 2005]). Most studies in the
energy metabolism of land snails have been performed with
stop-flow respirometry combined with the manometric method
(e.g., Vorhaben et al. 1984; Barnhart and McMahon 1987; Pedler
et al. 1996; Michaelidis 2002; Zotin and Ozernyuk 2002).
This procedure has several disadvantages because gas production/consumption
is averaged over relatively long periods without
taking into account movements, stress, and/or general discomfort
of the animal inside the chamber (Lighton 1991;
Lighton and Fielden 1995). Open-flow respirometry, on the
other hand, permits near real-time measurement of metabolic
rate and therefore facilitates identification and exclusion of periods
including activity. Indeed, stop-flow respirometry has
been shown to overestimate metabolic rate compared with
open-flow respirometry (Lighton 1991; Lighton and Fielden
1995; Addo-Bediako et al. 2002). Even with this difference in
technique, our results (∼259 mL O 2 h �1 ) appear lower than
predicted by body mass in snails (400 mL O 2 h �1 ; Guppy and
Withers 1999). Compared with results from individuals from
Australia and Europe, our results suggest that SMR is lower
than expected, which would be interpreted as a response of
these southern populations to the low temperatures they experience
(see also Clarke 1993; Partridge and French 1996).
However, a compilation of SMRs of three families of Gastropoda
indicated no relationship between the metabolic rate and
the latitude or climatic zone of the gastropod habitat (Vladimirova
2001).
To the best of our knowledge, this is the first report of
repeatability of metabolic rate in a land snail and the first study
of repeatability in any aspect of mollusc physiology (Nespolo
and Franco 2007). The fact that we selected individuals of

186 P. Artacho and R. F. Nespolo
Table 2: Repeatability as intraclass correlation coefficient (t) obtained from
variance components in a one-way ANCOVA using log 10 M b as covariable
Variable
Variance Component (Statistics in Parentheses)
Between Individuals
Within Individuals
(pResiduals)
Concepción:
Log ˙
10 Vco2aver
Log ˙
10 Vco2min
Log ˙
10 Vco2max
Valdivia:
Log ˙
10 Vco2aver
Log ˙
10 Vco2min
Log ˙
10 Vco2max
.0002 ( F43, 43 p 2.41, P p .002)
.0001 ( F43, 43 p 1.83, P p .03)
.0003 ( F43, 43 p 1.72, P p .04)
.0003 ( F32, 32 p 2.34, P p .002)
.0002 ( F32, 32 p 1.95, P p .01)
.0006 ( F32, 32 p 2.25, P p .003)
.0002
.0003
.0008
.0007
.0003
.0013
.42
.38
.30
.31
.25
.30
Note. Significant components in bold.
approximately the same body mass reduced probable residual
variation, which would be the explanation of the reduced scaling
that we found in metabolism. However, the results were
unchanged regardless of whether we included M b as a covariate.
This fact, together with the fact that repeatabilities were highly
consistent in the two studied populations, suggests that our
estimation was robust. Our repeatability estimations in SMR
were similar to what is known in other ectotherms (e.g., Chappell
and Rogowitz 2000; McCarthy 2000; Virani and Rees 2000;
Marais and Chown 2003; Nespolo et al. 2003), with some exceptions
when less than 2-wk periods were assayed, in which
higher estimations were obtained (Garland and Bennett 1990;
Chappell and Rogowitz 2000; Terblanche et al. 2004). However,
in general, there is no evidence of an effect of time between
measurements on the magnitude of repeatability (Nespolo and
Franco 2007).
Repeatability studies have gained considerable interest
among evolutionary physiologists because they might give insight
into the identification of traits that can ultimately respond
to natural selection (Arnold et al. 1995; Hayes and Jenkins 1997;
Hoffman 2000). Although the measurement of repeatability is
not necessarily a preliminary step before the analysis of selective
significance of a trait, it is generally regarded as the upper limit
of heritability (Falconer and Mackay 1996), although this assumption
remains to be tested (Hayes and Jenkins 1997; Dohm
2002; Konarzewski et al. 2005). Nevertheless, this interpretation
of repeatability remains useful because estimates of heritability
often cannot be obtained for natural populations (Dohm 2002).
In any event, we believe repeatability is a valuable tool for the
determination of environmental variance of a trait (Brown and
Shine 2007). The fact that SMR in a land snail is repeatable is
evidence that this trait could respond to natural selection; however,
it is clear that other conditions must be fulfilled (heritability
of the trait and consistent relationship between this trait
and fitness; Endler 1986). High repeatability, according to Falconer
and Mackay (1996), involves the potential existence of
genetic variation in this trait. Hence, the existence of genetic
variation on which natural selection can act would permit adaptive
responses in novel environments (Lee 2002). Clearly, a
single study of significant repeatability is not enough evidence
to support these assertions, and major insight into this hypothesis
is necessary for understanding the ecological success
of this widely distributed land snail. Several authors have indicated
that the definitive step to address the causal explanations
of the evolutionary potential of a species is phenotypic
selection studies combined with quantitative genetics tools
(Roff 1997; Kingsolver et al. 2001; Chown et al. 2006). However,
these kinds of studies are of very restricted application to wild
populations because few species can be both easily tracked in
the field and bred in the lab. Helix aspersa, on the other hand,
can be easily bred in the lab and has been successfully tracked
in the field (Dupont-Nivet et al. 1997, 1998, 2001). As such,
it represents an excellent and probably underutilized model
species in evolutionary physiology.
Acknowledgments
Financial support was provided by a CONICYT doctoral thesis
fellowship (AT-24060181) to P.A. R.F.N. thanks Proyecto Anillos
de Ciencia y Tecnología ACT-38.
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