JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 312B:136–147 (2009)
Snail Phenotypic Variation and Stress Proteins:
do Different Heat Response Strategies Contribute
to Waddington’s Widget in Field Populations?
HEINZ-R. KÖHLER1!, RAIMONDO LAZZARA1, NILS DITTBRENNER1,
YVAN CAPOWIEZ2, CHRISTOPHE MAZZIA2, AND RITA TRIEBSKORN1,3
1
Animal Physiological Ecology, University of Tübingen, Tübingen, Germany
2
UMR 406, UAPV/INRA, Invertebrate Ecology– Environmental Toxicology Lab,
Site Agroparc, Domaine Saint-Paul, Avignon, France
3
Steinbeis-Transfer Centre for Ecotoxicology and Ecophysiology Rottenburg,
Rottenburg, Germany
ABSTRACT
On the basis of studies with laboratory strains of Drosophila and Arabidopsis, it
has been hypothesized that potential buffers to the expression of phenotypic morphological
variation, such as Hsp90 and possibly Hsp70, represent important components of Waddington’s
widget, which may confer capacitive evolution. As studies on field populations of living organisms to
test this hypothesis are lacking, we tested whether a heat response strategy involving high stress
protein levels is associated with low morphological variation and vice versa, using four natural
populations of Mediterranean pulmonate snails. In response to 8 hr of elevated temperatures, a
population of Xeropicta derbentina with uniform shell pigmentation pattern showed remarkably
high Hsp70 but low Hsp90 levels. In contrast, a highly variable population of Cernuella virgata kept
both Hsp90 and Hsp70 levels low when held at diverse though environmentally relevant
temperatures. Two other populations (Theba pisana and another X. derbentina population) with
intermediate variation in shell pigmentation pattern were also intermediate in inducing Hsp70,
though Hsp90 was maintained at a low level. The observed correlation of stress protein levels and
coloration pattern variation provide the first indirect evidence for an association of stress proteins
with Waddington’s widget under natural conditions. J. Exp. Zool. (Mol. Dev. Evol.) 312B:136– 147,
r 2008 Wiley-Liss, Inc.
2009.
How to cite this article: Köhler H-R, Lazzara R, Dittbrenner N, Capowiez Y, Mazzia C,
Triebskorn R. 2009. Snail phenotypic variation and stress proteins: do different heat
response strategies contribute to Waddington’s widget in field populations? J. Exp. Zool.
(Mol. Dev. Evol.) 312B:136–147.
A well-known dilemma in evolutionary biology
is associated with the principle that stabilizing
selection reduces variability even though variability is a prerequisite of evolution itself. Nowadays, it is accepted that cryptic genetic and
epigenetic variation (variation that is not expressed in the phenotype) exists in populations,
and this cryptic variation is shielded from being
purged by selection, allowing it to be maintained
in a population (Feder, 2007). Such a buffering
system is all the more important as it is reasonable
to assume that the evolutionary transition from
the absence to the presence of a complex adaptation to environmental challenges requires more
r 2008 WILEY-LISS, INC.
than a single nucleotide exchange but rather
requires ‘‘evolvability’’, which is an organism’s
capacity to generate heritable phenotypic variation: an accumulation of several random, neutral,
or deleterious mutations temporarily withdrawn
from selection, and a stabilization of their effects
Grant sponsor: German Research Council (Deutsche Forschungsgemeinschaft DFG); Grant number: KO 1978/5-1.
!Correspondence to: Heinz-R. Köhler, Animal Physiological Ecology, University of Tübingen, Konrad-Adenauer-Str. 20, D-72072
Tübingen, Germany. E-mail: [email protected]
Received 17 July 2008; Revised 9 October 2008; Accepted 30
October 2008
Published online 4 November 2008 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jez.b.21253
PHENOTYPIC VARIATION AND STRESS PROTEINS
(Lauter and Doebley, 2002; Feder, 2007). Under
novel environmental conditions these mutations
may be expressed to produce a superior phenotype
(Wagner and Altenberg, ’96; Rutherford et al.,
2007). The potential to facilitate rapid evolution
despite genetic and developmental constraints
necessitates a powerful system of capacitors of
cryptic variation, particularly in response to largescale environmental perturbations (Moczek,
2007).
In 1942, Conrad Waddington published a theoretical model of ‘‘capacitive’’ evolution in his
classic paper, ‘‘Canalization of development and
the inheritance of acquired characters’’ (Waddington, ’42). In it, Waddington considered that a
strong canalization of developmental pathways
could reduce the continuous spectrum of phenotypes, which potentially could be expressed by a
population of individuals. Under usual environmental circumstances, individuals then show the
same phenotype regardless of their genotype
unless canalization is disrupted by the action of
a strong stressor. Long before the eve of molecular
genetics, Waddington proposed that an unknown
mechanism exists that conceals phenotypic variation in organisms until they are stressed. Later,
this ‘‘unknown mechanism’’ was popularized in
the literature by the term ‘‘Waddington’s widget’’.
Indeed, an increase in phenotypic variation following stress has been frequently reported (e.g.
Hoffman and Parsons, ’97; Imasheva et al., ’97;
Kristensen et al., 2003), as has an increase in the
heritability of phenotypic traits (Waddington, ’53,
’56; Hoffman and Merilä, ’99; Bubliy et al., 2000).
This has led to the term ‘‘genetic assimilation’’.
However, to date the capacitor concept is still
controversial, the adaptive value of capacitors is
unclear, and it also has not been demonstrated
that genetic assimilation occurs in natural settings
(Flatt, 2005).
A number of studies have revealed the involvement of stress proteins in Waddington’s widget.
From a cell physiological point of view this seems
reasonable as central cellular processes are chaperoned by stress proteins. These processes need
to be protected from environmental or genetic
disturbances, such as disrupted developmental
signal transduction pathways or damaged protein
folding and trafficking. In studies of Drosophila
and Arabidopsis Rutherford and Lindquist (’98)
and Queitsch et al. (2002) showed that mutations
in the genes encoding for the stress protein Hsp90
or pharmacological inhibition of Hsp90 both reveal
previously hidden phenotypic variation. Quite a
137
number of the ‘‘novel’’ phenotypes could be
selected for several generations until they were
present (‘‘assimilated’’), even when Hsp90 activity
was restored to pre-stress levels. Rutherford and
Lindquist (’98) coined the term ‘‘genetic capacitor’’ (symbolizing a device storing cryptic genetic
variation) for Hsp90. Later experiments also
revealed an epigenetic capacitor function for
Hsp90 (Sollars et al., 2003; Ruden et al., 2005).
Despite the possibility that Hsp90-buffered variation can be unconditionally deleterious, the capacitor function of Hsp90 to facilitate adaptive
evolution in unpredictable environments nevertheless seems to be plausible (Gross, 2004;
Sangster and Queitsch, 2005; Rutherford et al.,
2007). However, other studies have rejected or
only partly supported the genetic capacitor role of
Hsp90 (Milton et al., 2003; Kellermann et al.,
2007).
Thus, it is still unclear whether Hsp90 represents an evolutionary capacitor and, if so, whether
it acts alone (Milton et al., 2003). Meiklejohn and
Hartl (2002) proposed that evolution should
produce a single mode of canalization that will
buffer the phenotype against all kind of perturbation, but recent findings rather support the
opinion that a large class of evolutionary capacitors might exist whose effects on phenotypic
variation complement the putative action of
Hsp90 (Bergman and Siegal, 2003). Members of
the 70 kDa stress protein family (Hsp70) chaperone a larger number of intracellular polypeptides
than Hsp90, and therefore Hsp70 may be a
potential candidate for a capacitor, as studies on
morphological variation in Drosophila suggest
(Roberts and Feder, ’99). Indeed, experimental
and theoretical studies suggest that not only is
stabilization of signalling performed by Hsp90 but
also stabilization of protein folding is undertaken
by Hsp70 and that numerous other components
are involved in the most substantial processes of
cell function and differentiation; they all may
suppress phenotypic variation (Bergman and
Siegal, 2003; Suzuki and Nijhout, 2006). However,
the role of capacitors in natural populations
exposed to environmental stress is still unclear
(Mitchell-Olds and Knight, 2002).
There are trade-offs between traits associated
with stress protein levels. A constitutively high
Hsp level should reduce the influence of environmental threats and thus allow the evolution of
complex characters beyond single nucleotide mutations. On the other hand, Hsp reduces phenotypic variation, which can be deleterious for
J. Exp. Zool. (Mol. Dev. Evol.)
138
H.-R. KÖHLER ET AL.
microevolution, and is anyhow remarkably costly
in energetic terms and therefore particularly
disadvantageous in the absence of an environmental stress (Roberts and Feder, 2000). Thus,
evolution should favor an ‘‘emergency service’’
comprising a highly effective molecular stress
response including the Hsp system, but not
expressed at too high a constitutive level. In fact,
evidence for selection of low Hsp70 levels was
found in multi-generation studies with Drosophila
(Bettencourt et al. ’99; Sørensen et al., ’99;
Lansing et al., 2000), long-term metal-polluted
isopod and diplopod populations (Köhler et al.,
2000; Arts et al., 2004), and naturally thermally
stressed Drosophila populations (Sørensen et al.,
2001; Zatsepina et al., 2001). Even though it may
be a general principle that long-term stress selects
for down-regulation of Hsp levels, stress-regulatory mechanisms in single species or populations
may differ according to their evolutionary history,
selective regimes and regulatory constraints.
For this study, we hypothesized that Waddington’s widget has an Hsp-based component and we
aimed to investigate the relevance of this mechanism for natural populations. On the basis of the
capacitor theory, i.e. that cessation or withdrawal
of Hsp90 (Hsp70) can lead to decanalization, i.e.
cause the release of phenotypic variation, we
tested the following hypothesis:
(a) A response strategy to environmental stress
that includes the potential to upregulate
Hsp90/Hsp70 levels should provide sufficient
capacitoring even under acute stress and,
thus, should be associated with low phenotypic
variation, whereas
(b) a heat response strategy that does not involve
considerable induction of Hsp90/Hsp70 may
not meet the organism’s needs for canalization
and, therefore, should be associated with high
phenotypic variation.
To test this hypothesis in field populations,
closely related species or populations living in an
identical habitat type, with sufficient physiological
capacity to heat stress, with easy-to-record phenotypic variation, and with a reasonable body size for
individual Hsp assays were sought. Mediterranean
pulmonate snails are hermaphroditic and fulfill all
the above requirements. Living as they do in dry
warm places without much vegetation, diverse
species are regularly exposed to full sunlight. For
our study, we selected populations of Cernuella
virgata, Xeropicta derbentina, and Theba pisana,
J. Exp. Zool. (Mol. Dev. Evol.)
which displayed inter- and intraspecific differences in variation of their shell morphology.
MATERIAL AND METHODS
Test animals
Four snail [Helicidae s.l.] populations from the
Vaucluse region, Southern France, were sampled
between May 26 and June 8, 2006: X. derbentina
(Krynicki, 1836) [Hygromiidae] from La Roquessur-Pernes (population 1; 431 590 N, 51 6.50 E), C.
virgata (Da Costa, 1778) [Hygromiidae] from a site
between Saint-Didier and Le Beaucet (population
2; 431 49.50 N, 51 70 E), T. pisana (O.F. Müller,
1774) [Helicidae s.s.] from a site between L’Islesur-la-Sorgue and Fontaine-de-Vaucluse (population 3; 431 55.50 N, 51 60 E), and X. derbentina
(Krynicki, 1836) from Malemort-du-Comtat (population 4, 441 1.50 N, 51 90 E). The maximum
distance of the locations from one another was
12 km. To ensure reliability of species identification, species were independently determined by
Wolfgang Rähle, University of Tübingen, Germany, according to morphological criteria, and by
Thomas Wilke, University of Giessen, Germany,
according to COI gene sequencing and the closest
match to GenBank sequences. Both methods of
determination yielded identical results: COI sequences of morphologically determined X. derbentina and C. virgata matched most closely the
GenBank COI sequences given for X. derbentina
and C. virgata (Steinke et al., 2004), respectively.
As the COI sequence for T. pisana is lacking, we
relied on morphology-based species determination.
Live snails were kept on moist filter paper,
supplied with food, transported into the laboratory, and subsequently kept there for a week
under constant conditions in transparent plastic
boxes on moist filter paper (size: 24.2 ! 20.7 !
6.4 cm, temperature: 251C, light regime: 12 hr/
12 hr, food: carrots from organic farming ad
libitum). The stress responses of snails to their
own habitat were compared: we selected those two
populations that resembled one another closest in
terms of animal size and, at the same time,
differed most in terms of shell coloration pattern
variation. Thus, additional specimens of X. derbentina from population 1 and of C. virgata from
population 2 (distance of the sites: 1 km) were
sampled randomly at the same date (June 8, 2006,
around noon at 261C temperature, but exposed to
the direct sun), morphometrically characterized
(see below), frozen in liquid nitrogen directly in
139
PHENOTYPIC VARIATION AND STRESS PROTEINS
the field, and stored frozen ("201C or lower) until
stress protein analysis (n 5 20 individuals each).
Heat exposure
High temperature exposure was carried out in
heating cabinets using the same plastic containers
as used for laboratory maintenance. Ten snails
each from populations 1, 2, and 4 were exposed to
temperatures of either 25, 33, 38, 40, 43, 45, 48,
50, or 521C for 8 hr. Because of the scarcity of
animals from population 3, snails from this
population were subjected to five different temperatures only: 25, 33, 38, 40, and 451C (also
n 5 10). Moribund individuals were detected by
their immobility, which was recorded for every
snail after prodding with a blunt needle after heat
exposure.
Morphometry and diversity
As measures of phenotypic variation, the following parameters were recorded for each individual:
(a) shell height, (b) shell width, (c) shell aperture
height, (d) shell aperture width (all measured by
vernier callipers, Fig. 1A), (e) coloration pattern of
the shells, and (f) average coloration intensity.
The coloration pattern was assessed by classification of each individual in one of four categories
according to the distribution of pigmentation (Fig.
1B): uniformly white or greyish shells (category 1),
shells with a single faintly pigmented band,
otherwise uniformly bright (category 2), darker
greyish or brownish shells with pigment concentrated in faint bands or spots (category 3), shells
with several dark and bright bands (category 4). In
order to quantify the diversity of these morphotypes within each population, Shannon–Wiener
indices (Hs) as
Hs ¼ "
s
X
i¼1
pi $ ln pi
(pi: quotient between the number of individuals in
category i and the total number of individuals
investigated for each population, s: number of
categories) were calculated. The average coloration
intensity of the shells was measured by densitometry: All shells were placed in an identical
orientation with the aperture to the ground on a
grey plastic plate. For densitometrical assessment
of the shell pigmentation, pictures of the shells
were taken with a digital camera (Canon Digital
Ixus 40, Tokyo, Japan) under identical artificial
illumination. To compensate for slight variation in
illumination brightness, the grey scale value of the
Fig. 1. Morphometry of shells. A: Indication of measured
distances (a) shell height, (b) shell width, (c) shell aperture
height, (d) shell aperture width. B: Symbolization of the
phenotypes typical for the four categories: uniformly white or
greyish shells (category 1), largely white shells with a single
faintly pigmented band only (category 2), darker greyish or
brownish shells with pigment concentrated in faint bands or
spots (category 3), shells with several dark and bright bands
(category 4).
background plate in direct vicinity of the shells
was used as a standard. Using Improvision Openlab
2.2.5. densitometric software, an average grey
value integrating both dark-banded and bright
shell portions was calculated for each individual
shell.
Stress protein analysis
For stress protein analysis, snails were individually frozen in liquid nitrogen. According to their
size, the samples were homogenized on ice in
600–800 mL of extraction buffer (80 mM potassium
acetate, 5 mM magnesium acetate, 20 mM Hepes
pH 7.5) and centrifuged (10 min, 20000g at 41C).
The total protein concentration in the supernatant was determined according to the method
of Bradford (’76). Constant protein weights (40 mg)
were analyzed by minigel SDS-PAGE (12% acrylamide, 0.12% bisacrylamide (w/v), 150 at 80 V, 900
at 120 V). Protein was transferred to nitrocellulose
by semi-dry blotting and the filter was blocked for
2 hr in 50% horse serum in TBS (50 mM Tris
pH 5.7, 150 mM NaCl). As the filters were not
stripped, Hsp90 and Hsp70 analyses were
conducted on separate filters. After blocking, the
J. Exp. Zool. (Mol. Dev. Evol.)
140
H.-R. KÖHLER ET AL.
filters were washed in TBS for 5 min and incubated in the first antibody solution (mouse antihuman Hsp90, Stressgen, Ann Arbor, MI, dilution
1:5000 in 10% horse serum in TBS or mouse antihuman Hsp70, Dianova, Hamburg, Germany,
dilution 1:5000 in 10% horse serum in TBS)
overnight, on a lab shaker. They were washed
again in TBS for 5 min and incubated in a second
antibody solution, goat anti-mouse IgG conjugated
to peroxidase (Jackson Immunoresearch, West
Grove, PA, dilution 1:1000 in 10% horse serum/
TBS) on the shaker at room temperature, for 2 hr.
After subsequent washing in TBS, the antibody
complex was detected by the staining solution
(1 mM 4-chloro(1)naphthol and 0.015% H2O2 in
30 mM Tris pH 8.5 containing 6% methanol).
Quantification of the optical volume (o.v.) of the
Western blot protein bands [o.v. 5 area (number of
pixels) ! average grey scale value] was with a
densitometric image analysis system (E.A.S.Y.
Win 32, Herolab, Wiesloch, Germany) after background subtraction. The optical volumes recorded
for the individual samples were related to a
standard sample (prepared from total homogenate
of the garden snail, Cepaea hortensis), which was
run in duplicate on every gel. Furthermore,
replicates of different experiments were run on
different gels to minimize the influence of methodological variation on mean grey value calculation. In an earlier study, the methodological
variability between identical samples on different
gels was72.7% (Köhler et al., 2005). Antibody
cross-reaction resulted in a single band per sample
lane, detecting a protein of about 90 or 70 kDa, as
determined with a molecular size marker. As
purified stress proteins were not available for the
investigated snail species, specificity of the antibodies was checked beforehand in a variety of
other species covering diverse taxa (Gammarus
fossarum, Oniscus asellus, Deroceras reticulatum,
Danio rerio). Linearity of the response was
assumed as the amount of total protein analyzed
was optimized to result in bands of moderate
intensity, and bands of high intensity were
avoided.
Regression analysis and statistics
Stress protein data were tested for normality
with the Shapiro–Wilk test. Subsequently, significance was tested either with Student’s t-test for
normally distributed data or with the Wilcoxon–
Mann–Whitney test (SAS JMP 6.0, SAS Institute
Inc., Cary, NC, USA). Analysis of the relationship
J. Exp. Zool. (Mol. Dev. Evol.)
between Hsp levels and morphometric data was
conducted using analysis of variance (ANOVA)coupled regression analysis (SAS JMP 6.0).
Response surfaces for Hsp levels vs. shell pigmentation intensity vs. temperature were calculated
using STATISTICA 7.0 (StatSoft (Europe) GmbH,
Hamburg, Germany). The level of significance was
set to Pr0.01.
RESULTS
Phenotypic variation
The investigated snail species/populations differed largely in the variation of their coloration
pattern. X. derbentina from population 1 hardly
revealed any variation as all individuals but one
were uniformly white or greyish. Most variation
was present in C. virgata from population 2, which
consisted of individuals of all four pattern categories, and 13% of all individuals displayed dark
and white bands. The pattern variation of the
other two populations was intermediate (Fig. 2).
Despite differences in coloration pattern between the populations, variation of the pigmentation intensity did not differ between populations.
Surprisingly, uniformly greyish individuals (category 1) and dark-and-bright-banded ones (category 4) could end up with equal densitometric
values for their pigmentation intensity. Within
each population, however, there was considerable
variation in pigmentation intensity among the
individuals and, therefore, pigmentation intensity
as a possible factor for Hsp induction had to be
investigated by regression analysis and response
surface calculation (see below).
There was almost no variation in the proportions of the shells and their apertures and,
% 100
90
80
70
60
50
40
30
20
10
0
category 4
category 3
category 2
category 1
Xeropicta derbentina
pop.1
pop.4
Cernuella
virgata
pop.2
Theba
pisana
pop.3
Fig. 2. Variation of the shell coloration pattern. Percentages of individuals classified in the four morphological
categories defined in the text and displayed in Figure 1.
141
PHENOTYPIC VARIATION AND STRESS PROTEINS
therefore, also no difference in variation between
the different populations and species (P40.05).
Height and width of the shells as well as of
apertures were highly related. For all populations
and regression analyses (shell height vs. shell
width, aperture height vs. aperture width),
Po0.000001. Individual shells were of different
size, with T. pisana containing the largest individuals and population 4 of X. derbentina the
smallest. Thus, the dependence of Hsp90 and
Hsp70 levels upon animal size was also investigated (see below).
Thermotolerance and Hsp induction
At the highest laboratory temperatures, snails
were less viable. Immobility, which was used as a
proxy for a moribund status, increased with
elevated temperature in all populations. The
populations differed in their thermotolerance
(immobility), which was largely reflected also by
their stress protein level patterns. At 501C and
higher, X. derbentina of population 1 showed 100%
immobility, and already 30% of the individuals
were immobile at 481C. Population 4 of the same
species, which consisted of smaller individuals,
only first exhibited 100% immobility at 521C; at
501C all individuals still remained mobile. C.
virgata also showed 100% immobility at 521C
and 30% at 501C, whereas T. pisana was completely immobile at 451C.
Concomitantly to this decrease in viability, the
stress protein levels decreased in all populations at
extremely high temperatures. Aside of this, however, the species/populations differed in their
strategy to respond biochemically to heat stress.
Both populations of X. derbentina showed a
gradual and significant increase in Hsp70 with
increasing temperature, at least as long as
temperatures remained environmentally relevant
(Fig. 3). The more tolerant population 4 even
managed to maintain its Hsp70 induction up to
the nonphysiological temperature of 481C. In
contrast, the Hsp90 levels remained rather low,
particularly in population 1. Population 4 exhibited a significant induction of Hsp90 from 45 to
501C, but at a much lower level than Hsp70.
Despite the limited dataset, the population of
T. pisana seemed to display a similar response.
T. pisana also raised its Hsp70 levels at environmentally relevant temperatures yet kept its
Hsp90 levels remarkably low.
A totally different response was observed in
C. virgata. Environmentally relevant tempera-
tures (at most 431C) failed to significantly induce
either Hsp90 or Hsp70. Only in response to
artificial, higher temperatures (451C–501C) did
the snails significantly up-regulate their Hsp90
level whereas Hsp70 remained low.
This fundamental difference between the closely
related species X. derbentina (population 1) and
C. virgata (population 2) was reflected by their
stress protein response in their own habitat when
20 individuals were randomly selected and frozen
in the field (Fig. 4). Hsp90 levels were low in both
species whereas the Hsp70 level was significantly
(Pr0.01) higher in X. derbentina. All stress
protein levels (which were related to the same
standard as those measured in the lab experiments) corresponded well with the experimental
data displayed in Figure 3.
Correlation of Hsp levels and morphology
Regression analysis did not reveal any significant relationship between stress protein levels and
either shell height, shell width, aperture height,
aperture width, or shell pigmentation intensity,
neither for Hsp90 nor for Hsp70, for any population, and at all tested temperatures. Among all
regression analyses, the P value never was lower
than 0.01, the significance criterion, and only in
three cases (about 1% of all analyses) lower than
0.05. Thus, the dependence of Hsp90 and Hsp70
levels on one of these morphological parameters
can be excluded. Also the calculation of response
surfaces for Hsp70 levels vs. temperature vs. shell
pigmentation intensity only revealed an influence
of temperature on the stress protein level, but did
not give any indication for the importance of shell
pigmentation intensity (Fig. 5).
However, a significant (Pr0.01) negative relationship between the shell pattern variation
(Shannon–Wiener diversity) and the maximal
inducible Hsp70 level (which characterizes the
respective stress response strategy) was found by
regression analysis (Fig. 6).
DISCUSSION
Our studies revealed a good correlation of heatinduced Hsp induction with shell pattern variation
both in the field and in lab experiments. At the
same time, we excluded a number of morphological criteria that might influence individual stress
protein levels. In this context, however, it is
relevant to ask the extent to which the applied
temperatures were physiologically and evolutionarily relevant, and the extent to which the
J. Exp. Zool. (Mol. Dev. Evol.)
142
H.-R. KÖHLER ET AL.
Relative
Hsp level
Relative
Hsp level
*
10
10
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
*
0
25
33
38
40
43
45
48
50
25
52
33
38
40
43
45
48
50
52
T [˚C]
*
Xeropicta derbentina, population 1
Relative
Hsp level
Xeropicta derbentina, population 4
Relative
Hsp level
10
*
10
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
0
0
25
33
38
40
43
45
48
50
52
25
33
38
40
43
45
48
50
*
Cernuella virgata, population 2
52
T [˚C]
Theba pisana, population 3
Fig. 3. Hsp90 (white columns) and Hsp70 (black columns) levels (relative to the standard) of the four snail populations in
response to different temperatures, means and standard deviations. Arrows indicate environmentally irrelevant temperatures
Z451C. Asterisks indicate significantly (Pr0.01) elevated stress protein levels vs. the respective controls (for Hsp90 under the
graphs: populations 4 and 2 at 45–501C; for Hsp70 above the graph: population 1: 38–431C, population 4: 43–481C, population 3:
38–451C). Hsp90 levels for population 4 at 381C and for population 3 at 431C were below the detection limit. Individuals of
T. pisana were not exposed to 40, 48, 50, and 521C (see Materials and methods).
relationship between the stress response and
phenotypic variation may have a mechanistic
basis.
For all our snail species and populations, solar
radiation and hence temperature is of crucial
importance. It is well known that all these snails
climb vertical objects during the day in order to
escape from the extreme heat on the soil surface
(Cowie, ’85; Arad and Avivi, ’98). Internal temperature measurements in 50 individuals of
J. Exp. Zool. (Mol. Dev. Evol.)
C. neglecta from Southern France showed the
distance from the ground, but not the shell
coloration, to be predominantly relevant for body
temperature (H. Köhler and R. Triebskorn, unpublished). Even though solar radiation in the
field may heat up darker or brighter shells to a
different degree, the particular aim of our study
was to experimentally achieve constant internal
temperatures throughout the individuals of every
exposure group, irrespective of their coloration.
PHENOTYPIC VARIATION AND STRESS PROTEINS
Relative
Hsp level
*
6
5
4
3
2
1
0
X. derbentina
pop 1
C. virgata
pop. 2
Fig. 4. Hsp90 (white columns) and Hsp70 (black columns)
levels (relative to the standard) of those two snail populations,
which differed most in terms of shell coloration variation, in
response to their own habitat. Means and standard deviations. The
asterisk indicates significant difference (Pr0.01) in the hsp70 level.
Relative
Hsp70
level
12
8
4
0
1.0
50
0.8
Shell
pigmentation
intensity
40
0.6
30
0.4
20
Temperature [˚C]
Fig. 5. Hsp70 level (band intensity relative to standard)
vs. shell pigmentation intensity (average densitometic value
relative to standard) vs. heat cabinet temperature. Individual
data and calculation of response surface (STATISTICA,
StatSoft). The stress protein level in the exposed snails
depends on the temperature but not on the pigmentation
intensity of the shell. At all shell pigmentation intensities, the
Hsp70 response to increasing temperature follows an optimum curve with a decline induced by extremely high stressor
impact as described by Eckwert et al. (’97).
To estimate the temperatures, which are evolutionarily relevant for these snail species, it is
necessary to consider this strategy of heat avoid-
143
ance and focus primarily on extremes. On a hot
summer day (August 28, 2007, 4:00 pm) in Southern France, we have measured the temperature
gradient above soil in the full sunlight at 73 kLx
(H. Köhler and R. Triebskorn, unpublished). Even
though the soil surface had a temperature of
48.970.41C, this has to be regarded irrelevant for
the snails as even in dense populations not a single
living snail remains on the ground. It has been
shown that in the natural habitat soil temperatures surpass the lethal temperature, and that
snails die when experimentally placed on the
ground (Kempster and Charwa, 2003). In our
unpublished measurements, the temperature at
2 cm above ground was 38.570.61C and decreased
to 37.370.41C at 1 m height. The body temperature of resident snails (C. neglecta) was a
maximum of 41.31C when they were located
30 cm or more above the ground, and only two
out of fifty individuals had a body temperature of
more than 431C. The absolute maximum air
temperature recorded in Avignon for the last 40
years was 40.51C in August 2003 (data provided by
AgroClim, INRA Avignon, France). All these data
fit well to the internal temperatures recorded for
snails that have been exposed in heating cabinets
in this study. According to a regression analysis by
Lazzara (2007), the internal temperature in specimens exposed to 451C for 8 hr was 40.21C,
indicating that this temperature is in the same
range as the internal temperatures of feral snails
on the hottest days in summer. Considering the
experimental exposure time of 8 hr to 451C, which
surpasses natural exposure by far, we have to
accept that this situation is rather extreme from
an ecological point of view and with high probability irrelevant for microevolution. Consequently, we have limited the field relevance of
the exposure experiments to a maximum of 431C
cabinet temperature for 8 hr. Nevertheless, we
include the data obtained for higher temperatures
in order to show that all investigated populations are
able to physiologically respond to short-term temperature peaks with an induction of stress proteins.
There is no indication that selection of phenotypes with a distinct coloration pattern is based on
different climatic conditions in these species. The
investigated populations were sampled within a
rather small spatial range and the microclimatic
conditions of the sites were identical. Nevertheless, the populations differed largely in terms of
shell coloration variation. Slight differences in the
shell surface temperature (DT 5 1.0–1.51C) between brighter and darker individuals exposed to
J. Exp. Zool. (Mol. Dev. Evol.)
144
H.-R. KÖHLER ET AL.
identical solar radiation seem to exist in C.
vindobonensis, C. nemoralis, or Littoria pallescens
(Heath, ’75; Cook and Freeman, ’86; Staikou, ’99),
but the effect on the internal temperature of living
animals remains unclear, and is irrelevant for our
experimental setup in heating cabinets. In freeliving populations, the selection of darker or
brighter morphotypes by different climatic conditions is not consistent across species and depends
rather on the specific situation of each population
(Richardson, ’74; Jones et al., ’77). Thus, the
frequency of bright individuals is not necessarily
higher in warmer regions than in colder ones.
Although C. nemoralis exhibits bright morphotypes specifically in Southern Europe, the frequency of bright individuals of the closely related
European species C. hortensis is highest in
populations from Northern Spain, North East
Germany, Northern England, and Iceland (summarized in Jones et al., ’77).
In our study, the relation between stress protein
levels under environmentally relevant temperatures with phenotypic variation, which has been
observed both in lab experiments and in the field, in
combination with the capacitor theory, indirectly
supports our hypothesis that low stress protein
levels may contribute to higher phenotypic variance
in the field. Maximum levels of Hsp70 induction in
this study and in a related experiment (Lazzara,
2007) are displayed in Figure 6. It has been reported
that the coloration pattern of the shells of terrestrial snails has a genetic basis involving
several alleles (Jones et al., ’77). On the other
hand, however, coloration can be also modified by
climatic conditions. Helix aspersa raised at low
temperatures (and thus presumably expressing only
low levels of Hsps) showed darker pigmentation
than usual (Lecompte et al., ’98). These observations may speak in favor of an epigenetic control of
genetically defined shell coloration characters,
which could well correspond to the current view of
phenotypic capacitating. With respect to our investigated populations and species, one may speculate that the genetic variation among these
populations may differ and, therefore, may influence phenotypic variation independent of Hsp
action. However, it is extremely unlikely that the
population that did not show variation in the shell
coloration pattern (population 1 of X. derbentina)
has become genetically impoverished for at least
three reasons:
(1) It is extremely abundant: the population
consists of hundreds of thousands, perhaps
J. Exp. Zool. (Mol. Dev. Evol.)
even millions, of individuals living very close
together, which should allow for considerable
gene flow. X. derbentina is the dominant
species in the entire area and forms consistently large, permanent populations. In contrast, the comparatively ‘‘small’’ population of
C. virgata (population 2) appears to be rather
isolated and thus, if at all, inbreeding should
rather be expected for this population. However, population 2 showed the highest morphological diversity in this study, indicating
the sufficiency of its size to prevent genetic
impoverishment.
(2) It has been shown that strong selection
pressures (temperature, heavy metal exposure) favor low Hsp70 responses to an individual’s own habitat (Bettencourt et al., ’99;
Köhler et al., ’99, 2000; Sørensen et al., ’99,
2001; Lansing et al., 2000; Zatsepina et al.,
2001; Arts et al., 2004), but population 1 of
X. derbentina showed high Hsp70 levels, both
in the lab and the field. This is indicative for
the absence of a strong selection pressure—
why then should this population be genetically
depauperate?
(3) At least one of Austin Bradford Hill’s criteria
of causation, the consistency criterion, can be
applied to the dataset (see Fig. 6): Repeated
observations of an association in different
populations increases the likelihood of an
effect. In our study, several populations of
Xeropicta, Cernuella, Theba, and Cepaea (the
latter investigated by Lazzara, 2007) consistently fit the Hsp70 level vs. shell coloration
pattern gradient, and there is consequently no
reason to believe that all these populations
may gradually have become genetically impoverished. Rather it seems reasonable that
distinct heat response strategies, which result
in higher or lower induction rates of Hsps,
have been evolved in different snail populations, and that these different strategies may
have gradually interacted with morphological
variation.
It has been shown for snails that not only
selection upon morphological characters (see
above) but also selection for stress protein induction patterns depend highly on the specific situation of a population (Tomanek and Somero, ’99,
2002; Tomanek and Sanford, 2003). It is known
that the expression of Hsp70 is effected not only
by the action of environmental stressors but
also by the genetic architecture of populations
145
PHENOTYPIC VARIATION AND STRESS PROTEINS
A
Hsp70level
Xeropicta derbentina, pop. 1:
601% (SD: 83%) at 43˚C
Xeropicta derbentina, pop. 4:
341% (SD: 104%) at 48˚C
Theba pisana, pop. 3:
335% (SD: 94%) at 38˚C
Cepaea hortensis (Lazzara
2007): 180% at 36˚C
Cernuella virgata, pop. 2:
152% (SD: 23%) at 38˚C
100%
Basal
Hsp70 level
at 25 ˚C
X. derbentina, pop. 1:
n for Hs = 700
Max.
Hsp70
level
Moderate
variation
High
variation
Maximum Hsp70
level
C. virgata, pop. 2:
n for Hs = 286
T. pisana, pop. 3:
n for Hs = 96
B
Almost
no variation
X. derbentina, pop. 4:
n for Hs = 736
800
600
ANOVA: p = 0.0066
400
95% confidence interval
(data points)
200
95% confidence interval
(regression line)
0
2
0
0.25
0.5
0.75
Linear regression, r = 0.9868
(Hsp70 = 636.18 – 749.47 Hs)
Hs
Fig. 6. Maximum Hsp70 levels induced by elevated temperatures in relation to the shell coloration pattern variation. A: The
graph includes the data obtained in this study and those of a study by Lazzara (2007). In the latter, individuals from a central
European population of Cepaea hortensis, which also displayed a highly variable shell coloration pattern, were experimentally
exposed to heat for 8 hr and analyzed in the same way, with the only difference being that foot muscle homogenate instead of
total body homogenate was used for stress protein analysis. B: Morphotype (categories 1–4) diversity, quantified by
Shannon–Wiener indices, vs. maximum Hsp70 levels in the four populations investigated. Linear regression analysis, 95%
confidence intervals for both the regression line and data points, and ANOVA. Significant (P 5 0.0066) association between these
two parameters. Whiskers represent standard deviation for the maximum Hsp70 levels.
J. Exp. Zool. (Mol. Dev. Evol.)
146
H.-R. KÖHLER ET AL.
(Pedersen et al., 2005). Apparently, also in the
populations investigated in our study, different
(and genetically fixed) strategies have evolved in
the trade-off between benefits (high protein
stability) and disadvantages (energy consumption)
of high stress protein levels. Certainly, other heatprotective traits like the capability to thermoregulation and avoidance strategies additionally
interact with constraints in Hsp activation, and
these ecophysiological aspects surely need to be
addressed in future studies. Independent of the
reasons for diverging biochemical stress responses
in these snail populations, however, it was striking
that gradually modified stress response strategies
to past and present climate corresponded very well
with a gradually modified morphological character. For sure, many more studies on this relationship in numerous other snail populations are
needed until a general principle can be formulated. Nevertheless, this is the first study that
suggests a possible contribution of Hsps to buffering morphological variation in free-living, natural
field populations.
ACKNOWLEDGMENTS
The authors are particularly grateful to Wolfgang Rähle, Tübingen, Germany, and Thomas
Wilke, Giessen, Germany, for the morphological
and molecular determination of the snail species.
Furthermore, we thank Magali Rault, Odile
Mascle, Tim Triebskorn, and Nik Triebskorn for
their help to collect snails in the field. Actual
climate data were gratefully placed at our disposal
by AgroClim of INRA Avignon, France. We are
grateful to Robert Paxton, Belfast, UK, and two
anonymous reviewers for improving style and
clarity of this paper. The study was financed
by the German Research Council (DFG, KO 1978/
5-1).
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