Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Abstract

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r2 = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.

Full text

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein
(Hsp70), in juvenile Concholepas concholepas (Mollusca)
Katherina Brokordt
a,b,
⁎, Nicolás Leiva
a,b
, Katherine Jeno
a,b
, Gloria Martínez
b
, Federico Winkler
b,a
a
Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Universidad Católica del Norte, Casilla 117, Coquimbo, Chile
b
Facultad de Ciencias del Mar, Universidad Católica del Norte, Casilla 117, Coquimbo, Chile
abstractarticle info
Article history:
Received 4 August 2008
Received in revised form 18 November 2008
Accepted 19 November 2008
Keywords:
Allozyme heterozygosity
Concholepas concholepas
Heat shock response
Hsp70
Thermal stress
Thermal tolerance
Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs).
Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate
elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to
elevated temperatures, increases energy demands. A positive association between multiple and single locus
heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In
molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas
concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these
young individuals are exposed both to thermal risks and the large metabolic costs required to cope with
thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced
(stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n =400) were acclimated at 16 °C
for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and
7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7
polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased
markedly (~90%) with temperature. We found a positive association between MLH and basal and induced
levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with
MLH (r
2
=0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with
intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles
with highest MLH (≥4 heterozygous loci). However, after 7 days of exposure to thermal stress, less
heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster
increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could
be less affected by thermal stress in the intertidal zone. We found an association between specific loci
genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous
juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in
juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme
loci could be more important in the period of initial response to high temperature and others can be more
important in the response to the chronic temperature stress.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Virtually all biological structures and biochemical and physiologi-
cal processes are affected by temperature. One of the main effects of
thermal stress is protein denaturation. One mechanism organisms use
to cope physiologically with extreme temperature is production of
heat shock proteins (HSPs). Specifically, expression of Hsp70 enhances
thermal tolerance and is a key strategy used by ectotherms to tolerate
elevated temperatures in nature (Hofmann and Somero, 1995;
Tomanek and Somero, 1999, 2000; Tomanek and Sanford, 2003).
Hsp70 is a molecular chaperone that decreases aggregation of
thermally unfolded proteins, helps in their refolding, and facilitates
channelling of irreversibly denatured proteins towards proteolytic
degradation (Parsell and Lindquist, 1993). Under “non stress”condi-
tions Hsp70 also play an important role in protein biogenesis by
preventing premature folding and aggregation of emerging polypep-
tides (Frydman et al., 1994; Hartl and Hayer-Hartl, 2002).
Intertidal marine invertebrates are constantly exposed to tem-
perature fluctuations and encounter thermal stress daily. For these
organisms, the heat-shock response is frequently induced. Studies
have shown a correlation between HSP`s concentrations and the level
of induced thermotolerance (Feder and Hofmann, 1999; Tomanek and
Somero, 2000; Osovitz and Hofmann, 2005; Brun et al., 2008). In the
Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26
⁎Corresponding author. Center for Advanced Studies in Arid Zones (CEAZA),
Universidad Católica del Norte, Casilla 117, Coquimbo, Chile. Tel.: +56 51 209929; fax:
+56 51 209782.
E-mail address: kbrokord@ucn.cl (K. Brokordt).
0022-0981/$ –see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2008.11.007
Contents lists available at ScienceDirect
Journal of Experimental Marine Biology and Ecology
journal homepage: www.elsevier.com/locate/jembe

intertidal mussel Mytilus trossulus, Hsp70 levels change seasonally,
being greater in summer- than in winter-collected specimens
(Hofmann and Somero, 1995). The magnitude of Hsp70 synthesis is
also correlated with the thermal niche occupied by congeners of the
intertidal-subtidal gastropod Tegula, species inhabiting upper inter-
tidal levels express quicker and reaches higher levels of Hsp70 than
species from lower intertidal levels (Tomanek and Somero, 1999,
2000). Most studies that have quantified Hsp70 in relation to increases
in environmental temperature have measured only induced levels of
Hsp70, since initial basal levels are used as control of the protein
expression. We think high basal levels of Hsp70 could be an adaptive
strategy. This could decrease the reaction time to thermal stress,
particularly in intertidal species that are constantly exposed to
environmental stress.
Intertidal species may spend considerable metabolic energy to
repair, replace, and restructure thermally sensitive biochemical
components of the cell (Somero, 2002). Energy is necessary for
many events in the heat-shock response, including activation of
transcription of heat-shock genes, synthesis of HSPs, and ATP that
HSPs require during chaperoning (Somero, 2002). Thus the metabolic
costs associated with maintaining the high concentration and activity
of HSPs may contribute considerably to cellular energy demands
(Hofmann and Somero, 1995; Krebs and Holbrook, 2001; Somero,
2002).
A positive association between allozyme heterozygosity at multi-
ple and single locus (MLH and SLH) and individual fitness has been
widely demonstrated (Koehn and Gaffney, 1984; Zouros and Foltz,
1987; Hansson and Westerberg, 2002; Brokordt, 2003). Molluscs are
one of the few groups in which positive correlations between multi-
locus heterozygosity and fitness has been frequently observed
although contradictory results exist (Gaffney, 1990). In various
bivalves, particularly sessile species such as mussels, efficiency of
protein synthesis rises and routine metabolic rate decreases as multi-
locus heterozygosity increases, thus the cost of maintenance is
reduced (Koehn and Shumway, 1982; Garton, 1984; Hawkins et al.,
1986; Tremblay et al., 1998). One possible mechanism underlying
heterozygosity-fitness correlations is that allozyme heterozygotes
may have improved biochemical efficiency compared to homozygotes
because they produce enzymes with different catalytic properties
whereas homozygotes can generate only one enzymatic form (Mitton,
1993). Biochemical properties of enzymes can affect the efficiency of
metabolic pathways and energy flow through an organism. The higher
metabolic efficiency in more heterozygous organisms leaves more
energy available for increasing performance of other fitness related
traits. It has been proposed that under the pressure of natural
selection species will evolve diverse ways of using this additional
energy, to obtain the greatest return in terms of fitness (Rodhouse
et al., 1986; Volckaert and Zouros, 1989). Sedentary bivalves show
tight heterozygosity-growth correlations during the juvenile stage,
which declines during the adult stage and is replaced by a he-
terozygosity-gonad size correlation (Rodhouse et al., 1986). However,
although mobile species like scallops do not consistently show a
relationship between multi-locus heterozygosity and growth rate
(Foltz and Zouros, 1984; Beaumont et al., 1985; Bricelj and Krause,
1992), more heterozygous animals have a higher scope for activity (i.e.
swimming and escape response) (Volckaert and Zouros, 1989; Alfonsi
et al., 1995). We propose that for intertidal molluscs that are con-
stantly exposed to thermal stress, allozyme heterozygosity should be
positively correlated with higher basal and induced levels of Hsp70.
Higher Hsp70 levels would increase thermal tolerance and thus sur-
vival chances of more heterozygous individuals.
The polygenic nature of most quantitative traits makes it difficult
to understand the genetic-biochemical basis of quantitative pheno-
typic variations (Krause and Bricelj, 1995). In the analysis of single
locus heterozygosity or genotype on the effects in the phenotype, the
use of allozymes whose metabolic function is well characterized may
contribute to such understanding. Specific allozyme variation may be
linked with thermal tolerance (Hsp70 expression) (Rank and Dahlhoff,
2002; Neargarder et al., 2003).
Concholepas concholepas is a commercially exploited gastropod
found along the Pacific coast of South America, from central Peru
(Callao, 12° 02′S; 77° 07′W) to southern most Chile (Cabo de Hornos
55° 55′S; 67° 16′W) (Stuardo, 1979). Juveniles of this species live in
rocky intertidal shores and migrate to the subtidal zone during their
adult phase (Castilla et al., 1979). The ontogenetic habitat change
implies that juvenile C. concholepas must cope with thermal stress
during the daily tidal emergence which in takes place during the
warmest hours along central Chile (Finke et al., 2007). Using juvenile
C. concholepas as a model organism, we evaluated the effect of
allozyme heterozygosity at multiple and single locus on basal and
induced levels of the Hsp70. It has been suggested that the potential
differences between organisms with different degrees of MLH an SLH
would be accentuated if the study is developed under physiologically
demanding conditions or environmental stress (Scott and Koehn, 1990;
Tremblay et al., 1998). Therefore, we exposed juvenile C. concholepas to
thermal stress using 2 and 7-day treatments. We reasoned that long
term exposures to thermal stress would increase potential differencesin
Hsp70 synthesis capacities between juveniles with different levels of
MLH an SLH.
2. Materials and methods
2.1. Sampling, laboratory acclimation and heat shock treatment
We determined body temperatures in the field using biomimetic
sensors's, deploying a Tidbit temperature logger (ONSET Corporation)
inside a Concholepas concholepas shell filled with gelatin and sealed
with silicon. These shells were attached to the rocks where juveniles
were found in the intertidal, during 15 days in the summer season.
For HSP induction experiments, we collected 400 live juvenile
C. concholepas (25-40 mm peristomal opening) from the rocky
intertidal at Coquimbo, Chile (30°5′S, 71°2′W). Previous to thermal
stress treatments juveniles were acclimated to laboratory conditions
for 2 weeks at 16 °C (local mean seawater temperature) and fed ad
libitum with the mussel Perumytilus purpuratus a common food item
in the diet of C. concholepas. One group (n = 100) was exposed to
thermal stress (24 °C) for 2 days, and another group (n = 100) for
7 days. In each experiment the temperature was gradually increased
(1 °C/h) and a control group (n = 100/experiment) was maintained for
the same periods (2 d and 7 d) at 16 °C. At the end of each experiment,
each individual was measured, and soft tissues (gills, digestive gland
and muscle) were dissected, weighted and deep frozen with liquid
nitrogen. Pieces of soft tissues were stored (-80 °C) for subsequent
allozyme electrophoresis analyses (digestive gland and muscle) and
for Hsp70 quantification (gills).
2.2. Quantification of allozyme variability
For each juvenile, the variability at 21 allozyme loci was analyzed
using starch gel electrophoresis. Small pieces of frozen (-80 °C)
adductor muscle and digestive gland were homogenized in an equal
volume of homogenization buffer (100 mM Tris-HCl,10 mM EDTA, pH
8.0), and centrifuged for 5 min at 5000 ×g. The supernatants were used
for electrophoresis. A total of 17 enzyme systems, which permitted the
identification of 20 allozyme loci, were assayed using three buffer
systems. Tris citrate pH 8.0 was used for Leucine aminopeptidase (LAP;
3.4.11.1), Octopine dehydrogenase (ODH; 1.5.1.11), Glycerol-3-phos-
phate dehydrogenase (G3PDH; 1.1.1.8), Hexokinase (HK; 2.7.1.1),
Esterase (EST; 3.1.1.-), Esterase-D (EST-D; 3.1.1.56) and Peptidase
(PEP). Tris citrate pH 6.3 was used for Mannose phosphate isomerase
(MPI; 5.3.1.8) and Catalase (CAT; 1.11.1.6). Tris citrate pH 5.1 was used
for Malate dehydrogenase (MDH; 1.1.1.37), Isocitrate dehydrogenase
19K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

Table 1
Electrophoretic data for juvenile Concholepas concholepas exposed for 2 and 7 days to
environmental (16 °C) and stress (24 °C) temperatures
Locus Alles 2 days 7 days Total
16 °C 24 °C 16 °C 24 °C
PGD⁎
100 0.949 0.948 0.984 0.972 0.965
147 0.051 0.052 0.016 0.028 0.035
Ho 0.074 0.104 0.033 0.056 0.063
He 0.098 0.010 0.032 0.055 0.067
F
IS
0.2563 -0.0480 -0.0112 -0.0234 0.0598
PGM-1⁎
80 0.006 0.002
100 0.828 0.950 0.905 0.865 0.887
162 0.172 0.050 0.089 0.135 0.112
Ho 0.180 0.033 0.167 0.135 0.133
He 0.287 0.129 0.173 0.235 0.202
F
IS
0.3787⁎⁎ 0.6631⁎⁎ 0.0222 0.4302⁎⁎ 0.1733⁎⁎
PGM-2⁎
70 0.044 0.022 0.005 0.028 0.024
100 0.919 0.94 8 0.957 0.966 0.949
111 0.037 0.030 0.038 0.006 0.017
Ho 0.153 0.104 0.043 0.056 0.085
He 0.162 0.101 0.084 0.063 0.098
F
IS
-0.0369 - 0.0202 0.2814⁎⁎ -0.0060⁎0.1541⁎
ODH⁎
100 0.985 0.985 0.989 0.94 4 0.975
106 0.007 0.002
110 0.007 0.015 0.011 0.056 0.024
Ho 0.029 0.030 0.022 0.090 0.046
He 0.029 0.030 0.022 0.107 0.052
F
IS
-0.0 001 -0.0 077 -0.0056 0.1589 0.0570
LAP⁎
100 0.610 0.679 0.647 0.579 0.629
159 0.382 0.313 0.342 0.414 0.363
202 0.007 0.008 0.011 0.007 0.008
Ho 0.426 0.507 0.446 0.329 0.422
He 0.485 0.444 0.467 0.496 0.474
F
IS
0.0640 -0.0552 0.0238 0.1730⁎⁎ 0.0533
G3PDH⁎
80 0.008 0.008 0.004
93 0.045 0.037 0.057 0.054 0.049
100 0.927 0.955 0.918 0.932 0.934
111 0.027 0.016 0.014 0.014
Ho 0.073 0.015 0.049 0.108 0.062
He 0.139 0.087 0.154 0.128 0.126
F
IS
0.3932⁎⁎ 0.4106⁎⁎ 0.5305⁎⁎ 0.4811⁎0.3434⁎⁎
HK⁎
70 0.023 0.012 0.010
100 0.828 0.864 0.921 0.905 0.885
118 0.175 0.121 0.045 0.083 0.098
135 0.015 0.011 0.0 07
Ho 0.254 0.231 0.079 0.143 0.167
He 0.297 0.231 0.149 0.175 0.207
F
IS
0.1467 - 0.0542 0.5801⁎⁎ 0.5412⁎⁎ 0.4182⁎⁎
PEP-F⁎
85 0.229 0.197 0.187 0.217 0.206
100 0.771 0.750 0.717 0.723 0.734
110 0.012 0.004
113 0.053 0.096 0.04 8 0.056
Ho 0.250 0.184 0.265 0.277 0.254
He 0.357 0.401 0.445 0.431 0.416
F
IS
0.3061⁎0.5425⁎⁎ 0.4285⁎⁎ 0.1235⁎⁎ 0.2423⁎⁎
PEP⁎
60 0.287 0.289 0.253 0.278 0.267
100 0.713 0.711 0.720 0.709 0.715
110 0.027 0.013 0.016
Ho 0.400 0.368 0.266 0.354 0.324
He 0.405 0.422 0.420 0.422 0.417
F
IS
0.0122 0.1351 0.4302⁎⁎ 0.0865 0.2745⁎⁎
Table 1 (continued)
Locus Alles 2 days 7 days Total
16 °C 24 °C 16 °C 24 °C
ME⁎
85 0.007 0.006 0.0 03
100 0.993 1 0.989 0.994 0.994
175 0.0 06 0.006 0.003
Ho 0.015 0.023 0.011 0.013
He 0.015 0.023 0.011 0.013
F
IS
0.0000 0.0000 0.0000 - 0.0016
LDH⁎
80 0.078 0.087 0.060
100 0.593 0.714 0.883 0.866 0.816
103 0.407 0.286 0.039 0.047 0.125
Ho 0.222 0.047 0.122 0.081 0.106
He 0.492 0.413 0.213 0.241 0.315
F
IS
0.5694⁎⁎ 0.9063⁎⁎ 0.5455⁎⁎ 0.5774⁎⁎ 0.6583⁎⁎
MPI⁎
90 0.074 0.026
100 0.926 1 1 0.974
Ho 0.149 0.051
He 0.139 0.050
F
IS
-0.0705 - 0.0226
EST-3⁎
85 0.063 0.202 0.190 0.182 0.164
100 0.937 0.798 0.810 0.818 0.833
Ho 0.127 0.224 0.380 0.365 0.288
He 0.149 0.324 0.310 0.330 0.278
F
IS
0.0603 0.3141⁎- 0.2307⁎-0.2184 -0.048
XDH⁎
86 0.012 0.138 0.096 0.079
100 0.512 0.607 0.730 0.697 0.664
103 0.463 0.393 0.132 0.208 0.257
Ho 0.123 0.641 0.275 0.090 0.283
He 0.529 0.477 0.546 0.465 0.505
F
IS
0.8273⁎⁎ -0.2386 0.5337⁎⁎ 0.8222⁎⁎ 0.568⁎⁎
IDH-1⁎
100 1 0.993 0.978 1 0.992
126 0.007 0.022 0.008
Ho 0.015 0.022 0.010
He 0.015 0.037 0.016
F
IS
0.0000 0.4970⁎0.250⁎
IDH-2⁎
74 0.007 0.002
100 0.940 0.985 0.970 0.903 0.950
139 0.052 0.015 0.030 0.083 0.044
164 0.014 0.004
Ho 0.090 0.030 0.037 0.139 0.078
He 0.114 0.029 0.059 0.179 0.103
F
IS
0.1275 -0.0077 0.3897 0.5558⁎⁎ 0.3999⁎
CAT
55 0.073 0.008 0.067 0.034 0.047
88 0.169 0.127 0.152 0.287 0.191
100 0.758 0.822 0.775 0.669 0.749
110 0.042 0.0 06 0.011 0.003
Ho 0.129 0.085 0.191 0.090 0.127
He 0.395 0.308 0.373 0.472 0.401
F
IS
0.6214⁎⁎ 0.5292⁎⁎ 0.2710⁎⁎ 0.8339⁎⁎ 0.487⁎⁎
P0.75 0.70 0.80 0.75 0.90
A2.20 2.10 2.45 2.45 2.90
Ho 0.135 0.131 0.121 0.117 0.112
He 0.205 0.171 0.175 0.191 0.162
F
IS
0.342 0.235 0.310 0.389 0.311
Allele frequency estimates, observed (Ho) and expected (He) proportion of
hetererozygote s and F
IS
statistic for fit to Hardy-Weinber g proportions, at 17
polymorphic loci.
P=polymorphism; A =mean number of alleles per locus; Hardy-Weinberg equilibrium
exact test: ⁎=Pb0.05; ⁎⁎ =Pb0.01.
20 K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

(IDH; 1.1.1.42), Phosphoglucomutase (PGM; 2.7.5.1) and 6-Phospho-
gluconate dehydrogenase (PGD; 1.1.1.44). Tris borate EDTA pH 8.0 was
used for Lactate dehydrogenase (LDH; 1.1.1.27), Malic enzyme (ME,
1.1.1.40) and Xanthyne dehydrogenase (XDH; 1.2.3.2). Tris borate EDTA
pH 9.0 was used for the Peptidase-F (PEP-F). Staining methods were
adjusted from Shaw and Prasad (1970),Bricelj and Krause (1992) and
Winkler (2000). Loci and allele nomenclature followed recommenda-
tions by Shaklee et al. (1990), in which the most common allele ateach
locus is assigned a value of 100 and alternative alleles are designed
based on their relative mobility to this in the gel.
Genetic variability was expressed as proportion of polymorphic
loci (P), mean number of alleles per locus (A), and observed (Ho) and
expected frequencies under Hardy-Weinberg equilibrium (He) het-
erozygosity (Nei,1973). A locus was considered polymorphic when the
most frequent allele has a frequency lower than 99%. To evaluate the
impact of MLH, juveniles with different number of heterozygous loci
were grouped into 4 categories (0, 1, 2, 3 and ≥4 heterozygous loci),
giving a representative number of juveniles per MLH level.
2.3. Quantification of Hsp70
We quantified expression of Hsp70 by Western blot analyses
modified from Tomanek and Sanford (2003). 30 mg of gill tissue were
homogenized in 300 µl of homogenization buffer (32 mM Tris-HCl pH
7.5, 2% (w/v) SDS, 1 mM EDTA, 1 mM Pefabloc, 10 µg ml
-1
pepstatin,
and 10 µg ml
-1
leupeptin). The homogenate was incubated for 5 min at
100 °C, homogenized and the procedure was repeated one more time.
The homogenate was centrifuged at 15,800 ×gfor 15 min. The super-
natant was removed and stored at - 80 °C. Protein concentrations were
determined using Micro-BCA.
Proteins (90 µg) were separated electrophoretically in a 10%
acrylamide gel, and subsequently transferred onto nitrocellulose
membranes in a transfer buffer (25 mM Tris-base, 20% (v/v) methanol,
0.1% (w/v) SDS,pH 8.3) during 55 min at100 mA. Then membranes were
treated with blocking buffer (TTBS; 25 mM Tris-HCl, pH 7.5,150mM NaCl
and 0.1% (v/v) Tween, plus 5% (w/v) nonfat dried milk) for 1 h,
subsequentlywashed (5 times/5 min) withTTBS (25 mM Tris-HCl, pH 7.5,
150 mM NaCl and 0.1% (v/v) Tween). Membranes were incubated with a
solution of a monoclonal rat antibody (IgG) against Hsp 70 (5A5 ab2787,
Abcam) (1:1000 in TTBS) overnight. After washing the membranes
(5 times/5 min) with TTBS, we incubated them for 1 h with a rabbit anti-
rat bridging antibody (IgG) (M7754, Sigma) solution (1:5000 in TTBS),
followed again by several washing steps. Membranes were washed and
overlaid with a solution of enhanced chemiluminescent (ECL) reagent
(Amersham GE Healthcare) for 1 min. We exposed membranes, under
dark room conditions, onto pre-flashed BioMax XAR Film (Kodak) for 1 h
after ECL treatment. All samples were run at least twice.
Film images were scanned, and digitized images were analyzed
with image analysis software (Image Pro Plus, ver. 4.5, Media
Cybernetics) to quantify Hsp70 band intensities. We estimated the
levels of Hsp70 (µg) using for each gel (internal control) a known
amount of bovine heat-shock cognate 70 (80 ng; H8285, Sigma).
2.4. Statistical analyses
Population genetic analyses were done using Genepop 4.0
(Raymond and Rousset, 1995; Rousset, 2007). Allele frequencies, ob-
served (Ho) and expected frequencies under Hardy-Weinberg equili-
brium (He) heterozygosity, and F
IS
statistics were estimated for each
locus using the Robertson and Hill (1984) procedures. The test for
Hardy-Weinberg equilibrium was conducted for each locus, tempera-
ture and exposure time using an exact Test (Haldane, 1954; Guo and
Thompson, 1992; Weir, 1996). Those loci that showed heterozygotes
deficiency were re-tested using a U-test (Guo & Thompson, 1992).
Only those loci where both tests showed significant differences were
consider having excess of homozygotes.
Two-way ANOVAs were used to assess the impact of MLH (0, 1, 2, 3
and ≥4 heterozygous loci) on the levels of Hsp70 expression in
juveniles under control (16 °C) and stress (24 °C) temperatures. These
analyses were run separately for the 2 days and 7 days experiments.
One-way ANOVAs were used to asses the effect of SLH on the levels of
Hsp70 expression in juveniles under control (16 °C) and stress (24 °C)
temperatures for both experiments (48 h and 7 d). When the as-
sumptions of normality and homoscedasticity were not met, the data
were ln-transformed. Normality was tested using a Shapiro-Wilk's
test (SAS, 1999) and homogeneity of variances using a Levene test
(Snedecor and Cochran, 1989). Multiple pairwise comparisons (LS
means) were used to test a posteriori for specific differences (P≤0.05)
(SAS, 1999).
3. Results
3.1. Allozyme variability
Of the 17 enzyme systems examined, 20 allozyme loci were
consistently detected (Table 1). 17 of them were polymorphic (99%
rule). Only EST-D⁎,MDH-1⁎and IDH-1⁎loci did not showed variability.
The analysis of the complete population (n = 400 individuals), showed
a large proportion of loci with a heterozygotes deficiency when com-
pared with Hardy-Weinberg (H-W) equilibrium expectations (with F
IS
ranging from 0.235 to 0.310, Pb0.05) (Table 1). Those loci were: PGM-
1⁎,PGM-2⁎,G3PDH⁎,HK⁎,PEP-F⁎,PEP⁎,LDH⁎,XDH⁎,IDH-1⁎,IDH-2⁎,
and CAT⁎). Among them, four loci (G3PDH⁎,PEP-F⁎,LDH⁎,CAT⁎)
showed consistent heterozygote deficiency across all treatments and
two other (PGM-1⁎,XDH⁎) showed the same tendency in three
combinations of temperature and exposure time. Only EST-3⁎showed
an excess of heterozygotes, but only in juveniles exposed to 24 °C for
7 days.
3.2. Effects of temperature and multi-locus heterozygosity (MLH) on
Hsp70 levels
The levels of Hsp70 increased markedly (~90%) when juveniles
were exposed to stress temperature (24 °C) (Fig. 1). Basal levels of
Hsp70 were similar between animals exposed during 2 and 7 days to
the mean environmental temperature (control: 16 °C) (2.6 μg× mg
protein
-1
and 2.9 μg× mg protein
-1
, respectively). Also the increase of
Hsp70 was very similar for both, juveniles exposed for 2 and 7 days to
the stress temperature (24 °C) (2.6 μg × mg protein
-1
and 2.2 μg×mg
protein
-1
, respectively), attaining the levels of 5.22 and 5.04 μg×mg
protein
-1
, respectively.
In juveniles exposed for 2 days to control and stress temperatures,
both temperature and MLH degree significantly affected the basal and
induced levels of Hsp70 (Fig. 2). However, the interaction between
both factors was not significant (two-way ANOVA, Table 2). Irrespec-
tively of temperature, concentration of basal and induced Hsp70
increased with MLH (r
2
=0.7 and 0.9, respectively in a polynomial
Table 2
Statistics for two way ANOVAs used to asses the effect of temperature (T°) and the
degree of multi-loci heterozygosity (MLH) on the levels of Hp70, in Concholepas
concholepas juveniles exposed during 2 and 7 days to environmental (16 °C) and stress
(24 °C) temperatures
Source df F P
2 days
T° 1 42.0 b.0001
Heterozygosity (HLM) 4 4.91 0.001
T° ⁎HLM 4 0.63 0.644
7 days
T° 1 34.3 b.0001
Heterozygosity (HLM) 4 0.71 0.587
T° ⁎HLM 4 0.12 0.976
21K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

fitting regression), attaining maximal levels in juveniles with inter-
mediate and high MLH (2 and 3 loci), and decreasing in juveniles with
highest MLH (≥4 heterozygous loci), but not significantly (Fig. 2). In
juveniles exposed for 7 days, only the increase in temperature had a
significant effect on Hsp70 levels (Table 2,Fig. 2). The degree of MLH
and the interaction of MLH and temperature did not affect the levels of
basal and induced Hsp70.
3.3. Effect of single locus heterozygosity (SLH) and allozyme genotype on
Hsp70 levels
For juveniles exposed for 2 days to the control (16 °C) and stress
(24 °C) temperatures SLH had a significant effect on 3 of 7 loci
analyzed (Table 3,Fig. 3). Heterozygous individuals for PGD⁎,PGM-1⁎
and PEP-F⁎locus showed higher levels of Hsp70 than homozygous
ones. Particularly, the highest basal or induced Hsp70 levels were
present in juveniles that had PGD
147
(basal and induced), PGM-1
162
(basal) and PEP-F
85
(induced) alleles. For juveniles that were exposed
during 7 days, we also observed a significant effect of allozyme SLH
and genotype on Hsp70 levels, but on different loci than for the 2 days
experiment (Fig. 4). Heterozygous individuals for IDH-2⁎and LAP⁎
showed the highest levels of induced Hsp70. The presence of the IDH-
2
139
and LAP
159
was associated with the highest Hsp70 levels. Half of
the loci that showed significant associations with Hsp70 levels had a
deficiency of heterozygotes, but the remainders were in H-W
equilibrium.
We did not consider all the possible genotypes in these analyses,
because the number of individuals representing the loci was
insufficient to test. This was mainly the case for homozygous for the
less common allele.
Fig. 1. Effect of temperature on the basal (control: 16 °C) and induced (stress: 24 °C)
levels of Hsp70, in juvenile Concholepas concholepas exposed for 2 and 7 days to thermal
stress. Values represent means ± S.E. (n=100 per temperature and time of exposure).
Means sharing the same letter are not significantly different (P≥0.05) as indicated by L S
means a posteriori multiple comparisons.
Fig. 2. Effect of multi-loci heterozygosity degree (number of heterozygous loci) on the
basal (control: 16 °C) and induced (stress: 24 °C) levels of Hsp70, in juvenile Concholepas
concholepas exposed for 2 and 7 days to thermal stress. Values represent means± S.E.
(n=100 per temperature and time of exposure). Means sharing the same letter are
not significantly different (P≥0.05) as indicated by LS means a posteriori multiple
comparisons.
Table 3
Statistics for one way ANOVAs used to compare the levels of Hsp70 in juvenile
Concholepas concholepas with different allozyme genotype
Source 2 days 7 days
df FP df FP
PGD 16 °C
Genotype 1 5.21 0.029 1 021 0.648
Error 32 47
PGD 24 °C
Genotype 1 4.71 0.036 1 0.73 0.397
Error 36 47
IDH-2 16 °C
Genotype 1 0.01 0.909 1 0.6 0.449
Error 32 36
IDH-2 24 °C
Genotype - - nd 1 4.58 0.038
Error - - 38
MPI 16 °C
Genotype 1 0.01 0.922 - - nd
Error 23 - -
MPI 24 °C
Genotype - - nd - - nd
Error - - - -
ODH 16 °C
Genotype - - nd 1 0.95 0.334
Error - - 44
ODH 24 °C
Genotype - - nd 1 0.74 0.394
Error - - 46
PGM-1 16 °C
Genotype 2 3.91 0.049 1 0.23 0.630
Error 30 45
PGM-1 24 °C
Genotype - - nd 2 0.19 0.831
Error - - 46
PGM-2 16 °C
Genotype 1 0.27 0.605 1 0.17 0.683
Error 32 38
PGM-2 24 °C
Genotype 1 1.3 0.261 1 0.0 0 0.986
Error 36 46
LAP 16 °C
Genotype 2 0.36 0.701 2 0.0 0 0.995
Error 32 42
LAP 24 °C
Genotype 2 1.26 0.296 2 3.89 0.029
Error 37 35
PEP-F 16 °C
Genotype 1 0.00 0.986 4 1.17 0.340
Error 20 38
PEP-F 24 °C
Genotype 1 6.59 0.018 3 1.69 0.183
Error 20 41
Juveniles were exposed to the mean environmental (16 °C) and stress (24 °C)
temperatures for 2 and 7 days.
22 K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

4. Discussion
Our results show that thermal stress markedly increases Hsp70
levels (~90%) in juvenile Concholepas concholepas.Moreover,
C. concholepas was able to maintain induced levels of Hsp70 for a
long period (7 days). Many studies of intertidal molluscs have
measured the induction of Hsp70 during short term exposure to
thermal stress (1-4 h) and found a level of induction similar to that we
found (Hofmann and Somero, 1995; Tomanek and Sanford, 2003; Sorte
and Hofmann, 2004; Brun et al., 2008). However it was surprising that
these animals were capable to maintain these high induced levels for
such a long period. Therefore, the induction of Hsp70 may be an
important strategy used by juvenile C. concholepas to tolerate thermal
stress. According to our field measurements the experimental increase
in temperature used (from 16 to 24 °C) was very similar (results not
shown) to that juvenile C. concholepas normally experience in their
natural habitat during low tide, mainly along spring and summer
seasons. Thus, the energy cost associated with maintaining and
Fig. 3. Effect of allozyme genotype on the basal (control: 16 °C) and induced (stress: 24 °C) levels of Hsp70, in juvenile Concholepas concholepas exposed for 2 days to thermal stress.
Values represent means ± S.E. Means sharing the same letter are not significantly different (P≥0.05) as indicated by L S means a posteriori multiple comparisons.
23K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

constantly increasing the Hsp70 may contribute substantially to the
individuals' energy demands in juvenile C. concholepas, as in other
intertidal molluscs (Hofmann and Somero, 1995; Somero, 2002).
Many studies have reported a positive correlation between multi-
locus heterozygosity (MLH) and fitness related traits, mainly in
molluscs like Mytilus edulis (Koehn and Gaffney, 1984; Gentili and
Beaumont, 1988) and Mulina lateralis, but also in fish like Salmo
gardnieri (Danzmann et al., 1985; Thelen and Allendorf, 2001). These
positive correlations have been attributed to a more efficient use of
energy in heterozygous individuals. For the gastropod C. concholepas,
2-days exposure experiments revealed a positive association between
MLH and basal and induced levels of Hsp70. These levels did not
increase lineally. Juveniles with intermediate-high MLH (2-3 hetero-
zygous loci) presented the highest levels of basal and induced Hsp70,
which augmented with the increment of MLH up to 3 heterozygous
loci and decreased slightly (but not significantly) in juveniles with the
highest MLH (≥4 heterozygous loci).
The numerous advantages of heat shock response suggest that
natural selection should maximize HSPs expression. However, HSPs
themselves are subject to strong auto regulation (Lindquist, 1993;
Krebs and Feder, 1997, 1998). An excess of Hsp70 decreases thermal
tolerance in Drosophila (Krebs and Feder, 1998), which may be
particularly unfavourable in faster growing developmental stages
(Krebs and Feder, 1997). Synthesis, action and degradation of HSPs
may consume large amount of the organism's energy stores (Somero,
2002) and occupy metabolic pathways which are necessary for other
Fig. 4. Effect of allozyme genotype on the basal (control: 16 °C) and induced (stress: 24 °C) levels of Hsp70, in juvenile Concholepas concholepas exposed for 7 days to thermal stress.
Values represent means ± S.E. Means sharing the same letter are not significantly different (P≥0.05) as indicated by L S means a posteriori multiple comparisons.
24 K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

synthetic and catabolic process (Koehn and Bayne, 1989; Hoffmann,
1995; Dhahbi et al., 1997). Thus the cost of excessive production of
HSPs may diminish the scope for growth and reproduction (Krebs and
Feder, 1998). The observed stabilization and slight reduction of Hsp70
levels in more heterozygous C. concholepas juveniles could be
associated with this required auto regulation of excess protein instead
of a reduced capacity for synthesis.
Hsp70 increases thermal stress tolerance (Tomanek and Somero,
1999; Tedengren et al., 1999; Tomanek and Sanford, 2003). According
to our results, juvenile C. concholepas with intermediate to high levels
of multi-loci heterozygosity would be less affected by the constant
thermal stress present in the intertidal zone potentially displaying
better possibilities for growing and survival. Interestingly, juveniles
with 2 and more heterozygous loci were the most abundant in our
population (~65%, data not shown).
Even though having high levels (but not an excess) of induced
Hsp70 enhances thermal tolerance and thus is a key strategy in
ectotherms, high basal amounts of Hsp70 could be also adaptive. For
an intertidal species that is cyclically exposed to thermal stress higher
basal levels of Hsp70 could make the heat shock response more
efficient through the decrease of reaction time to thermal stress.
Additionally, given that under “non stress”conditions, Hsp70 plays a
key role in protein biogenesis, preventing premature folding and
aggregation of emerging polypeptides (Frydman et al., 1994; Hartl and
Hayer-Hartl, 2002); higher basal Hsp70, especially in juveniles, could
make synthesis of new macromolecules, and consequently growth,
more efficient. Indeed, higher basal Hsp70, especially in juveniles, may
confer higher fitness under thermal stress and could positively affect
growth rate by reducing costs associated with protein biogenesis.
The association between MLH and Hsp70 levels was not observed
in juveniles exposed to thermal stress for 7 days. As has been proposed
(Rodhouse and Gaffney, 1984; Beaumont et al., 1985; Gentili and
Beaumont, 1988; Tremblay et al., 1998), we expected that the most
stressful conditions will accentuate potential energetic differences
between animals with low and high MLH levels. In contrast, our
results showed that Hsp70 levels stabilized at an elevated level when
temperature stress was maintained for 7 days; and individuals with
the lowest levels of MLH rose to similar Hsp70 levels to that observed
in individuals with intermediate and high MLH in the 2 days
experiment. This suggests that more homozygous juveniles
responded slower to thermal stress that more heterozygous indivi-
duals, possibly as a result of a less efficient synthesis of Hsp70.
In the 2-day thermal stress experiment, heterozygous juveniles for
the phosphogluconate dehydrogenase (PGD⁎) and phosphoglucomu-
tase (PGM-1⁎) loci showed higher basal Hsp70, and heterozygous for
PGD⁎and peptidase-F (PEP-F⁎) loci showed higher induced Hsp70
than homozygous juveniles. Thus PGD⁎genotype was strongly
associated with Hsp70 levels. This enzyme is important for biosyn-
thetic metabolic pathways, providing carbon compounds and reduced
NADP through the oxidative phase of the pentose phosphate cycle.
Thus its catalytic capacity could indirectly affect the energy available
for the synthesis and function of Hsp70. In turn, genotypic variation in
none of these allozymes was associated with differences in Hsp70
levels in the 7 days treatment, but IDH-2⁎and LAP⁎loci affected the
induced levels Hsp70. These data suggests that genotypic variation at
some allozyme loci could be more important in the period of initial
response to high temperature and others can be more important in
response to the chronic temperature stress.
Thermal tolerance is obviously a complex trait that may be affected
by genetic variation, the thermal history of the organism, and
genotype-environment interactions at multiple loci (Neargarder
et al., 2003). In the hopes of detecting links between allozyme
variability and stress response, we targeted several enzymes of
metabolic importance for biosynthetic pathways. In our study, the
loci that affected Hsp70 levels showed high allelic variability (PGD⁎,
PGM-1⁎,IDH-2⁎,LAP⁎,ODH⁎). Some of these allozymes catalyze
equilibrium reactions and have high specific activities, as typical for
bi-directional glycolytic enzymes (Hochachka et al., 1998). At a
simplistic level, a high specific activity suggests these enzymes are
in excess. However, the need to respond to changes in the mass action
ratio and maintain pathway flux requires highly efficient catalysts.
Metabolic control analysis has revealed that considerable control can
be vested in loci catalyzing equilibrium reactions (Kashiwaya et al.,
1994). As metabolic control is shared among the participants of a
metabolic pathway, functional variation at such equilibrium loci may
modify how the pathway responds to changing requirements (e.g.
thermal stress). The expression of more than one allele could permit a
greater range of performance than possible in homozygotes, poten-
tially explaining the cellular mechanism of allozyme heterozygosity
generating a faster heat shock response.
To our knowledge there are few studies relating enzyme genotype
with heat shock protein levels. Changes in allele frequencies of a
metabolically important enzyme (phosphoglucose isomerase, PGI)
have been related to environmental changes due to climate change or
latitudinal and altitudinal distribution in the montane beetle Chry-
somela aeneicollis (Dahlhoff and Rank, 2000; Rank and Dahlhoff, 20 02;
Neargarder et al., 2003). In both cases the distribution of the alleles
together with their functional characteristics suggest, but do not
specifically demonstrate, selection for these alleles.
Given the wide latitudinal distribution of C. concholepas (from
central Peru, 12° 02′S; 77° 07′W to southern most Chile, 55° 55′S; 67°
16′W), juveniles are exposed to a broad range of environmental
conditions. Future studies seeking to understand physiological
adaptation should associate distributional patterns (i.e. environmen-
tal conditions) with specific allozyme genotype (e.g. PGD and IDH) and
basal and induced levels of Hsp70.
Acknowledgements
This research was supported by FONDECYT (Fondo Nacional de
Desarrollo Científico y Tecnológico, Chile) #1050291 operating grant
to K. Brokordt, F. Winkler and G. Martínez. We are grateful to José
Pulgar and Giuliano Bernal for their help with the immunochemical
analysis. We specially acknowledge John Lawrence and Bernardo
Broitman for their insightful and detailed comments that helped us
improve this article. [SS]
References
Alfonsi, C.Y., Nusetti, O., Perez, J., 1995. Heterozygosity and metabolic efficiency in the
scallop Euvola ziczac (Linneaus, 1758). J. Shellfish Res. 14, 389–393.
Beaumont, A.R., Gosling, E.M., Beveridge, C.M., Budd, M.D., Burnell, G.M., 1985. Studies
on heterozygosity and size in the scallop, Pecten maximus. In: Gibbs, P.E. (Ed.), Proc
19th Eur. Mar. Biol. Symp. Cambridge University Press, Cambridge, pp. 443–454.
Bricelj, V.M., Krause, M.K., 1992. Resource allocation and population genetics of the bay
scallop, Argopecten irradians irradians: effects of age and allozyme heterozygosity
on reproductive output. Mar. Biol. 113, 253 –261.
Brokordt, K.B., 20 03. Integrando Genética y Ecofisiología: Efecto del Grado de
Heterocigosidad Sobre la Adecuación Biológica de Bivalvos Marinos. In: Bozinovic,
F. (Ed.), Fisiología Ecológica y Evolutiva de Animales. Ediciones Universidad Católica
de Chile, Santiago, Chile, pp. 59–76.
Brun, N.T., Bricelj, V.M., MacRae, T.H., Ross, N.W., 2008. Heat shock protein responses in
thermally stressed bay scallops, Argopecten irradians, and sea scallops, Placopecten
magellanicus. J. Exp. Mar. Biol. Ecol. 358, 151–162.
Castilla, J.C., Guisado, Ch., Cancino, J., 1979. Aspectos ecológicos y conductuales
relacionados con la alimentación de Concholepas concholepas (Mollusca, Gastro-
poda, Muricidae). Biol. Pesq. 12, 99–114.
Dahlhoff, E.P., Rank, N.E., 2000. Functional and physiological consequences of genetic
variation at phosphoglucose isomerase: Heat shock protein expression is related to
enzyme genotype in a montane beetle. Proc. Natl. Acad. Sci. U.S.A. 97,10056–10 061.
Danzmann, R.G., Ferguson, M.F., Allendorf, F.W., 1985. Allelic differnences in initial
expression of paternal alleles at an isocitrate dehydrogenase locus in rainbow trout
(Slamo gairdneri) Dev. Genet. 5, 117–127.
Dhahbi, J.M., Mote, P.L., Tillman, J.B., Walford, R.L., Spindler, S.R., 1997. Dietary energy
tissue-specifically regulates endoplasmic reticulum chaperone gene expression in
the liver of mice. J. Nutr. 127, 1758–1764.
Feder, M.E., Hofmann, G.E., 1999. Heat-shock proteins, molecular chaperones, and the
stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61,
243–282.
25K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26

Finke, G.R., Navarrete, S.A., Bozinovic, F., 2007. Tidal regimes of temperate coasts and
their influences on aerial exposure for intertidal organisms. Mar. Ecol. Prog. Ser.
343, 57–62.
Foltz, D.W., Zouros, E., 1984. Enzyme heterozygosity in the scallop Placopecten
magellanicus (Gmelin) in relation to age and size. Mar. Biol. Lett. 5, 255–263.
Frydman, J.E., Nimmesgern, E., Ohtsuka, K., Hartl, F.U., 1994. Folding of nascent
polypeptide chains in a high molecular mass assembly with molecular chaperones.
Nature 370, 111–117.
Gaffney, P.M., 1990. Enzyme heterozygosity, growth rate and viability in Mytilus edulis,
another look. Evolution 44, 204–209.
Garton, D.W., 1984. Relationship between multiple locus heterozygosity and physiolo-
gical energetics of growth in estuarine gastropod Thais haemostoma. Physiol. Zool.
57, 530–543.
Gentili, M.R., Beaumont, A.R., 1988. Environmental stress, heterozygosity, and growth
rate in Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 120, 145–153.
Guo, S., Thompson, E.A., 1992. Performing the exact test of Hardy-Weinberg proportion
for multiple alleles. Biometrics 48, 361–372.
Haldane, J.B., 1954. An exact test for randomness of mating. J. Genet. 52, 631–635.
Hansson, B., Westerberg, L., 2002. On the correlation between heterozygosity and
fitness in natural populations. Mol. Ecol. 11, 2467–2474.
Hartl, F.U., Hayer-Hartl, M., 2002. Molecular chaperones in the cytosol: from nascent
chain to folded protein. Science 295, 1852–1858.
Hawkins, A.J.S., Bayne, B.L., Day, A.J., 1986. Genotype dependent interrelationship
between energy metabolism, protein metabolism and fitness. Proc. R. Soc. Lond., B
229, 161–176.
Hochachka, P.W., McClelland, Burness, G.P., Staples, J.F., Suarez, R.K., 1998. Integrating
metabolic pathway fluxes with gene to enzyme expression rates. Comp. Biochem.
Physiol., B 120, 17–26.
Hoffmann, A.A., 1995. Acclimation: increasing survival at a cost. Trends Ecol. Evol. 10,
1–2.
Hofmann, G., Somero, G., 1995. Evidence for protein damage at environm ental
temperatures: seasonal changes in levels of ubiquitina conjugates and hsp70 in
the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198, 1509–1518.
Kashiwaya, Y., Sato, K., Tshuchiya, N., Thomas, S., Fell, D.A., Veech, R.L., Passonneau, J.V.,
1994. Control of glucose utilization in working perfused rat heart. J. Biol. Chem. 269,
25502–25514.
Koehn, R.K., Gaffney, P.M., 1984. Genetic heterozygosity and growth rate in Mytilus
edulis. Mar. Biol. 82, 1–7.
Koehn, R.K., Shumway, S.E., 1982. A genetic/physiological explanation for differential
growth rate among individuals of the American oyster, Crassostrea virginica
(Gmelin). Mar. Biol. Lett. 3, 35–42.
Koehn, R.K., Bayne, B.L., 1989. Towards a physiological and genetical understanding of
energetics of the stress response. Biol. J. Linn. Soc. 37, 157–171.
Krause, M.K., Bricelj, V.M., 1995. Gpi genotypic effect on quantitative traits in the
northern bay scallop, Argopecten irradians irradians. Mar. Biol. 123, 511–522.
Krebs, R.A., Feder, M.E., 1997. Deleterious consequences of Hsp70 overexpression in
Drosophila melanogaster larvae. Cell Stress Chaperones 2, 60–71.
Krebs, R.A., Feder, M.E., 1998. Experimental manipulation of the cost of thermal
acclimation in Drosophila melanogaster. Biol. J. Linn. Soc. 63, 593–601.
Krebs, R.A., Holbrook, S.H., 2001. Reduced enzyme activity following Hsp70 over-
expression in Drosophila melanogaster. Biochem. Genet. 39, 73–82.
Lindquist, S., 2003. Autoregulation of the heat-shock response. In: Ilan, J. (Ed.),
Translational Regulation of Gene Expression 2. Plenum, New York, pp. 279–320.
Mitton, J.B., 1993. Enzyme heterozygosity, metabolism, and developmental stability.
Genetica 89, 47–65.
Neargarder, G., Dahlhoff, E.P., Rank, N.E., 2003. Variation in thermal tolerance is linked
to phosphoglucose isomerase genotype in a montane leaf beetle. Funct. Ecol. 17,
213–221.
Nei, M., 1973. Analyses of gene diversity in subdivided populations. Proc. Natl. Acad. Sci.
U.S.A. 3321–3323.
Osovitz, C.J., Hofmann, G.E., 2005. Thermal history-dependent expression of the hsp70
gene in purple sea urchins: Biogeographic patterns and the effect of temperature
acclimation. J. Exp. Mar. Biol. Ecol. 327, 134–143.
Parsell, D.A., Lindquist, S., 1993. The function of heat-shock proteins in stress tolerance:
degradation and reactivation of damaged proteins. Annu. Rev. Genet. 27, 437–496.
Rank, N.E., Dahlhoff, E.P., 2002. Allele frequency shifts in response to climate change and
physiological consequences of allozyme variation in a montane insect. Evolution 56,
2278–2289.
Raymond, M., Rousset, F., 1995. GENEPOP (version 1.2): population genetics software for
exact tests and ecumenicism. J. Heredity 86, 248–249.
Rodhouse, P.G., Gaffney, P.M., 1984. Effect of heterozygosity on metabolism during
starvation in the American oyster Cassostrea virginica. Mar. Biol. 80, 179–187.
Rodhouse, P.G., McDonald, J.H., Newell, R.I.E., Koehn, R.K., 1986. Gamete production,
somatic growth and multiple-locus enzyme heterozygosity in Mytilus edulis. Mar.
Biol. 90, 209–214.
Rousset, F., 2007. GENEPOP'007: a complete reimplementation of the GENEPOP soft-
ware for Windows and Linux. Mol. Ecol. Res. 8, 103–106 .
Robertson, A., Hill, W.G., 1984. Deviations from Hardy-Weinberg proportions: sampling
variances and use in estimation of inbreeding coefficients. Genetics 107, 713–718.
SAS Institute,1999. SAS/STAT User's Guide, release, 8.02 ed. SAS Institute Press, Cary, N.C.
Shaklee, J.B., Allendorf, F.W., Morizot, D.C., Whitt, G.S., 1990. Gene nomenclature for
protein-coding loci in fish. Trans. Am. Fish. Soc. 119, 2–15.
Shaw, C.R., Prasad, R., 1970. Starch gel electrophoresis of enzymes –a compilation of
recipes. Biochem. Genet. 4, 297–320.
Scott, T.M., Koehn, R.K., 1990. The effect of environmental stress on the relationship of
heterozygosity to growth rate in the coot clam Mulinia lateralis (Say). J. Exp. Mar.
Biol. Ecol. 135, 109–116.
Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods. Iowa State University Press,
Ames, Iowa.
Somero, G.N., 2002. Thermal physiology and vertical zonation of intertidal animals:
optima, limits, and costs of living. Integr. Comput. Biol. 42, 780–789.
Sorte, C.J.B., Hofmann, G.E., 2004. Changes in latitudes, changes in aptitudes: Nucella
canaliculata (Mollusca: Gastropoda) is more stressed at its range edge. Mar. Ecol.
Prog. Ser. 274, 263–268.
Stuardo, J., 1979. Sobre la clasificación, distribución y variación de Concholepas
concholepas (Bruguiere, 1789): Un estudio de taxonomía Beta. Biol. Pesq. Chile 12,
5–38.
Tedengren, M., Olsson, B., Bradley, B., Zhou, L., 1999. Heavy metal uptake, physiological
response and survival of the blue mussel (Mytilus edulis) from marine and brackish
waters in relation to the induction of heat-shock protein 70. Hydrobiologia 393,
261–269.
Thelen, C.G., Allendorf, F.W., 2001. Heterozygosity-fitness correlations in rainbow trout:
effects of allozyme loci or associative overdominance? Evolution 1180–1187.
Tomanek, L., Somero, G.N., 1999. Evolutionary and acclimation-induced variations in the
heat-shock responses of congeneric marine snails (genus Tegula) from different
thermal habitats: implications for limits of thermotolerance and biogeography.
J. Exp. Biol. 202, 2925–2936.
Tomanek, L., Somero, G.N., 2000. Time course and magnitude of synthesis of heat-shock
proteins incongeneric marine snails (genus Tegula) from different tidal heights.
Physiol. Biochem. Zool. 73, 249–256.
Tomanek, L., Sanford, E., 2003. Heat-shock protein 70 (Hsp 70) as a biochemical stress
indicator: an experimental field test in two congeneric intertidal gastropods
(Genus: Tegula). Biol. Bull. 205, 276–284.
Tremblay, R., Myrand, B., Sevigny, J.-M., Blier, P.U., Guderley, H., 1998. Bioenergetic and
genetic parameters in relation to susceptibility of blue mussels, Mytilus edulis (L.) to
summer mortality. J. Exp. Mar. Biol. Ecol. 221, 27–58.
Volckaert, F., Zouros, E., 1989. Allozyme and physiological variation in the scallop Pla-
copecten magellanicus and a general model for the effects of heterozygosity on
fitness in marine molluscs. Mar. Biol. 103, 51–61.
Weir, B.S., 1996. Genetic Data Analysis II. Sinauer, Sunderland, Mass.
Winkler, F.M., 2000. Estimación de la variabilidad genética. In: Bustos, E.R. (Ed.),
Programa de selección para el mejoramiento genético del ostión del norte
(Argopecten purpuratus). Segundo informe de avance, FDI, Chile.
Zouros, E., Foltz, D.W., 1987. The use of allelic isozyme variation for the study of
heterosis. Isozymes: Current Topics in Biological and Medical Research, vol. 13,
pp. 1–59.
26 K. Brokordt et al. / Journal of Experimental Marine Biology and Ecology 370 (2009) 18–26