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First published online October 5, 2006
Journal of Experimental Biology 209, 4102-4114 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02484
Seasonal acquisition of chill tolerance and restructuring of membrane glycerophospholipids in an overwintering insect: triggering by low temperature, desiccation and diapause progression
1 Biology Centre AS CR, Institute of Entomology, Branisovská
31, 370 05 Ceské Budejovice, Czech
Republic
2 Faculty of Biological Sciences, University of South Bohemia, Ceské
Bud
jovice, Czech Republic
3 Department of Terrestrial Ecology, National Environmental Research
Institute, Silkeborg, Denmark
* Author for correspondence (e-mail: kostal{at}entu.cas.cz)
Accepted 10 August 2006
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Summary |
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Key words: membrane phospholipids, temperature, acclimatization, cold acclimation, desiccation, diapause, cold tolerance, ectotherm, Insecta, Heteroptera
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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; Steponkus,
1990; Cossins, 1994;
Hazel, 1995;
Hazel, 1997;
Wallis and Browse, 2002). Cold
acclimation characteristically leads to one or a combination of the following
adjustments as summarized by Hazel (Hazel,
1989; Hazel,
1995): (a) increased proportion of unsaturated fatty acids; (b)
restructuring of glycerophospholipid (GPL) molecular species; (c) elevated
relative proportions of glycerophosphoethanolamines (GPEtns) to
glycerophosphocholines (GPChols); (d) reduced proportions of plasmalogens
compared to diacyl GPLs; (e) adjustment of cholesterol content. Such changes
has been postulated to prevent perturbation of membrane structure and function
when the body temperature changes. Direct genetic proof for the role of
increased unsaturation in enhancing survival at decreasing ambient
temperatures has been obtained in cyanobacteria and plants
(Browse et al., 1994;
Murata, 1994;
Allakhverdiev et al., 1999;
Inaba et al., 2003). Also in
insects, acclimatory responses to cold has been shown to involve restructuring
of GPL composition in biological membranes (Bennet et al., 1997;
Ohtsu et al., 1998;
Kostál and Simek, 1998;
Hodková et al., 1999;
Hodková et al., 2002;
Bayley et al., 2001;
Holmstrup et al., 2002;
Slachta et al., 2002a;
Kostál et al., 2003;
Overgaard et al., 2005).
Decreasing ambient temperature is most often considered to be the
triggering factor for acclimatory changes in GPL composition. In insects,
however, the triggering factors and transduction cascades for GPL
compositional changes have not been analyzed in detail. Based on earlier
studies, at least three different triggering mechanisms might be involved:
ambient temperature, hydration state of the organism (mediated via
osmolarity/concentration of specific solutes) and endogenous factors such as
developmental hormones. In the field situation, all three factors may operate
in parallel, which makes it difficult to distinguish between their effects.
Changes in temperature intervene both daily and seasonally. Naturally
occurring diurnal variations in temperature were sufficient to induce the
nocturnal increase of cold tolerance in adults of Drosophila
melanogaster through the process of `rapid cold hardening'
(Lee et al., 1987) and this
was accompanied by small but significant changes in the GPL composition
(Overgaard et al., 2005). Many
insects that overwinter in temperate and polar habitats enhance their cold
hardiness during the acclimatization process, which starts upon entering to
diapause and continues during autumnal decrease of ambient temperatures
(Denlinger, 1991;
Slachta et al., 2002b).
Changes in GPL composition were observed both in the field and in the
laboratory studies during such winter cold hardening (Bennet et al., 1997;
Hodková et al., 1999;
Hodková et al., 2002;
Kostál et al., 2003).
Partial dehydration often represents an inseparable part of the complex
acclimatory change related to cold acclimation
(Ring and Danks, 1994;
Block, 1996;
Danks, 2000;
Williams et al., 2004). It
raises the question of whether the GPL changes observed during cold
acclimation may be triggered by changes in hydration state of the organism.
Indeed, typical membrane alterations resulting in a higher degree of
unsaturation were induced by mild desiccation stress applied to the
collembolan Folsomia candida
(Bayley et al., 2001;
Holmstrup et al., 2002). Many
insects change their hormonal milieu seasonally, in response to specific token
stimuli such as photoperiod, and enter diapause
(Denlinger, 1985;
Tauber et al., 1986;
Danks, 1987;
Kostál, 2006). Similar
remodelling of GPL composition was observed in response to cold exposure and
to transition from direct development to diapause in two insects, adult P.
apterus (Hodková et al.,
2002), and larvae of the fly Chymomyza costata
(Kostál et al.,
2003).
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;
Kostál and Simek,
2000). Fig. 1A
shows the air temperatures as recorded by the weather station of the Czech
Institute of Hydrometeorology in Ceské Budejovice. The insect samples
were transported to the laboratory (10 min) without changing their temperature
and were then processed immediately.
Laboratory-reared insects came from a culture that originated from adults
collected in Ceské Budejovice during spring 2005. F2 embryos
and nymphs were reared under conditions inducing reproductive diapause in the
adults, i.e. constant temperature of 25°C and a short-day photoperiod of
12 h:12 h, L:D (Hodek, 1968).
Dry seeds of the linden tree (Tilia parviflora Ehrh.) and water were
provided ad libitum. Adult F2 insects were kept at a
constant 20°C, with a short-day photoperiod and with access to food and
water, for 4 weeks. Such adults were considered to be in deep reproductive
diapause and their physiological state was considered as the initial
state. Three different treatments were then applied (depicted in
Fig. 1B). (1) Progression of
diapause: the insects were kept under constant conditions (access to food and
water) for another 8 weeks. (2) Desiccation: the insects were kept at
20°C, with a short-day photoperiod, and denied access to liquid water and
food for 14 days. Relative humidity of the air fluctuated between
40–60%. Such a treatment results in approximately 50% mortality
(Tollarová and V.K., unpublished data), and in the present study only
survivors were taken for analyses. (3) Cold acclimation: the insects were
exposed to gradually decreasing temperatures: 20°C (day)/10°C (night)
during the first week, followed by 15°C/5°C and 10°C/0°C
during the second and third weeks, respectively (all 3 weeks in short days),
followed by 5 weeks at constant 0°C and continuous darkness. Such an
acclimation protocol simulates the natural drop of temperatures during autumn
(see Fig. 1A) and the bugs
achieve higher chill tolerance than if constant temperatures (in contrast to
thermoperiodic regime) are used for acclimation
(Kostál et al.,
2001).
Physiological parameters
The fresh mass (FM) was measured in five males and five females
(individually) using a Sartorius balance with sensitivity of 0.1 mg. Dry mass
(DM) was measured after drying the specimens at 65°C for 3 days. Hydration
(in mg H2O mg–1 DM) was calculated from the
gravimetric data. Haemolymph samples for osmolality measurements were
collected from 10 insects (five males, five females) by cutting off one of the
antennae and allowing it to bleed into a calibrated capillary tube.
Osmolalities were measured in 10–15-nl droplets using the Clifton
Nanoliter Osmometer (Clifton Technical Physics, USA).
The concentrations of the two most abundant `winter' polyols (ribitol and
sorbitol) that typically accumulate in diapausing cold-acclimated P.
apterus adults (Kostál and
Simek, 2000; Kostál et
al., 2001), were measured in 70% ethanol extracts of whole body
samples (five males, five females, taken individually). The extraction,
derivatization and analytical procedures (gas chromatography coupled to mass
spectrometry) were the same as described by Kostál and Simek
(Kostál and Simek,
1995). All chemicals used for analysis were purchased from
Sigma-Aldrich Co. (St Louis, MO, USA).
The temperature of crystallization of body fluids (supercooling point, SCP)
was measured in individual insects (16 males, 16 females) at a cooling rate of
0.3° min–1 using the method described
(Nedved et al., 1995). Chill
tolerance was assessed by exposing groups of 20 insects (10 males, 10 females)
to a temperature of –15°C (maximum, –14.0°C; minimum,
–15.3°C) for various periods (1, 4, 7, 10, 13, 20 or 28 days). The
capacity for coordinated crawling at 25°C, 2 days after the exposure, was
used as a criterion for survival. Survival rate was described using the
parameter LT50, which gives the time of exposure that is subsequently survived
by 50% of the population sample. The parameter Lt50 was calculated from
nonlinear logistic regression with the sigmoid curve as described
(Kostál et al.,
2001).
Analysis of glycerophospholipids
Thoracic muscle and fat body tissues were rapidly (3 min) dissected from
the adults. Tissues from three individuals were pooled to obtain one sample,
and each sample was replicated six times (three times with males, three times
with females). Total lipids were extracted in ice-cold chloroform:methanol
(2:1) solution using the method of Folch et al.
(Folch et al., 1957) slightly
modified according to Kostál and Simek
(Kostál and Simek,
1998). After the extraction, the solvents were evaporated under a
stream of nitrogen, and lipids were stored at –80°C until
analysis.
High performance liquid chromatography (HPLC) combined with electrospray
ionisation mass spectrometry (ESI-MS) (Han
and Gross, 1995; Hsu and Turk,
2000; Brooks et al.,
2002; Wang et al.,
2005) was performed on a quadrupole ion LCQ mass spectrometer
(Thermo, San Jose, CA, USA) coupled to a Rheos 2000 ternary HPLC system (Flux,
Basel, Switzerland), equipped with a FAMOS autosampler and Thermos thermostat
(both LC Packings-Dionex, Amsterdam, The Netherlands). The stored dry samples
were dissolved in 1 ml of methanol and 5 µl aliquots were injected into a
150x2 mm i.d. 3.5 µm Synergi Polar HPLC column (Phenomenex, Torrance,
CA, USA). The mobile phase was composed of (A) 10 mmol l–1
ammonium acetate (NH4Ac) in methanol, (B): 10 mmol
l–1 NH4Ac in water and (C) isopropanol. A linear
gradient of A:B:C changing from 90:10:0 to 70:0:30 within 14 min was used with
a flow rate of 300 µl min–1. The column temperature was
maintained at 30°C. The mass spectrometer was operated either in positive
or negative ion detection modes at +4 kV or –3.6 kV, respectively.
Capillary temperature was 240°C; nitrogen was used as both the sheath and
the auxiliary gas. For MS2 and MS3 fragmentations, ion isolation windows were
5 and 2 Da, respectively. Maximum ion injection time was 100 ms, collision
energies were 30% (MS2) or 35% (MS3), mass range of 600–800 Da was
scanned each 0.5 s. All chemicals used for extraction and analysis were
purchased from Sigma-Aldrich Co.
Data processing
As there were no significant differences between males and females, the
data sets for both sexes were pooled. Owing to the complexity of instrument
response to particular GPL molecular species, which is dependent not only on
features of the chemical structure but also on experimental conditions,
results of GPL analysis were preferably expressed in relative values, i.e.
relative molar proportion of each lipid species from the total of 100%. Our
analysis was focused on major GPEtn and GPChol molecular species, which were
present at or above 1%. It is known that GPEtns plus GPChols represent more
than 80% of all glycerophospholipids in P. apterus tissues
(Hodková et al., 1999).
The ratio of total GPEtns to total GPChols (GPEtn/GPChol) was calculated for
each tissue/treatment and the relative proportions of individual fatty acyls
were calculated based on GPL data. Unsaturation ratio (UFA/SFA) was calculated
from the proportional composition of fatty acyls.
Unpaired two-tailed t-tests (parametric) were used to compare means of two variables with normal Gaussian distribution (F-tests were used to verify equality of variances). Mann–Whitney U-tests (non-parametric) were used to compare the medians of SCP variables (with skewed distribution of data). To investigate if there were any general trends in the changes of GPL composition caused by different treatments, all data were analysed together using a multivariate statistical analysis, Principal component analysis (PCA; Simca-P software, Umetrics, Umea, Sweden). Using this method, the data are represented in K-dimensional space (where K is equal to the number of variables) and reduced to a few principal components (two principal components, PC1 and PC2, in this study) that describe the maximum variation within the data. The principal components are then displayed as a set of scores, which highlight clustering. Differences between the mean scores for different treatments along the axes PC1 and PC2 were tested using one-way ANOVA followed by a post-hoc Student–Newman–Keuls test (SNK).
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There were no significant differences in the measured physiological parameters between the laboratory-reared insects during their initial state and those collected in the field in October (compare Tables 1 and 2; P>0.05 for all parameters, t-tests or U-test). When laboratory adults were subjected to various treatments, their dry mass did not change significantly (Table 2). Hydration remained stable during the progression of diapause at 20°C, but it significantly decreased during the desiccation and cold-acclimation treatments. Desiccating conditions caused the loss of 51% of initial water content and an approximate twofold increase in haemolymph osmolality. Cold acclimation was accompanied by the loss of 34% of initial water content and more than a threefold increase in osmolality. No winter polyols accumulated during the progression of diapause. Small amounts of polyols (slightly above the analytical thresholds: 2.5 or 1.0 µg of ribitol or sorbitol, respectively, in the sample) were found in desiccated insects and high amounts of polyols, i.e. 15.9 and 11.4 µg mg–1 DM of ribitol and sorbitol, respectively, were found in the cold-acclimated specimens. Supercooling capacity and chill tolerance at –15°C remained relatively low during diapause progression and desiccation while cold acclimation caused the SCP to decrease from –13.9 to –18.9°C and the Lt50 at –15°C to increase from <1 to 14.6 days.
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Glycerophospholipid composition
Gradient HPLC efficiently separated GPLs from other lipid classes,
particularly from triacylglycerols, which were dominant in the sample
extracts. In this way, highly reproducible full-scan positive ESI mass spectra
of the GPChol and GPEtn fractions were obtained with minimal outward peak
overlapping and ion suppression. As shown in
Fig. 2, [M+H]+ ions
dominated in the full-scan ESI spectra of GPEtn and GPChol fractions. Twelve
to 13 major GPEtn and GPChol molecular species were consistently detected in
the tissue samples of P. apterus (Figs
2,
3).
Fig. 3 shows a typical HPLC
chromatogram (upper trace), and extracted chromatograms of principal GPEtn and
GPChol species. The identity of each major ion was verified by fragmenting the
ion and studying the MS2 and MS3 spectra containing preferential losses of
headgroups and fatty acyls from the sn-1 or sn-2 positions
of the glycerol backbone. All GPL species were composed of only five different
fatty acyls (FAs): palmitoyl (16:0), heptadecanoyl (17:0), stearoyl (18:0),
oleyl (18:1) and linoleyl (18:2). The GPL compositions were qualitatively
similar in the wild (October) and laboratory-reared (initial state) insects in
both tissues (compare Fig. 4A
and Fig. 5A with
Fig. 6A and
Fig. 7A, respectively). There
were, however, quantitative differences at the level of individual GPL
species. The single largest difference was found in the proportion of
18:2/18:2-GPChol in the muscle tissue, which was 28.2% in the wild insects and
only 19.4% in the laboratory-reared insects (P<0.0001,
t-test). Wild specimens showed similar GPEtn/GPChol ratios in both
tissues (muscle, 1.0±0.03; fat body, 0.9±0.2). By contrast,
laboratory-reared insects had a ratio of 1.5±0.3 in the muscles and
0.5±0.1 in the fat body.
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Seasonal change in glycerophospholipid composition
The complex change in GPL composition during seasonal acclimatization in
the field is shown in Figs 4
and 5. Two most noticeable
alterations were: (1) the increase of 16:0/18:2-GPEtn by 6.6 or 5.3% (muscle
or fat, respectively), and (2) the decrease of 18:2/18:2-GPChol by 4.7% (both
tissues). The GPEtn/GPChol ratios significantly increased from October to
December: to 1.4±0.2 in the muscles (P=0.0011,
t-test), and to 1.2±0.2 in the fat body (P=0.0293,
t-test). FA composition changed relatively little during autumnal
acclimatization (Table 3).
Statistically significant increases in the proportion of palmitoyl (16:0) were
registered in both tissues, and the proportions of linoleyl (18:2) tended to
decrease. Such changes resulted in a slight, but statistically significant,
decrease in UFA/SFA ratio in both tissues.
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Changes in glycerophospholipid composition during laboratory acclimations
The diapause progression and desiccation treatments were accompanied by
relatively small changes in GPL composition of the muscle
(Fig. 6B,C) and fat body
(Fig. 7B,C). The relative
proportions of GPEtn/GPChol remained unchanged (P>0.05,
t-test): muscle after diapause progression, 1.4±0.3; muscle
after desiccation, 1.6±0.4; fat body after diapause progression,
0.6±0.1; and fat body after desiccation 0.6±0.1. Relatively
small changes were observed in FA composition, and the UFA/SFA ratios did not
change significantly (Table
4).
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The cold-acclimation treatment resulted in considerable restructuring of GPL composition. The general patterns of changes were similar in both tissues (compare Fig. 6D with Fig. 7D). The proportion of 16:0/18:2-GPEtn increased by 8.1% and 3.3% in the muscle and fat body tissue, respectively; 18:0/18:2-GPEtn was the second most increased species (by 5.9% and 3.5%). Also, 18:2/18:2-GPChol decreased by 8.8% and 6.5%, and 16:0/18:2-GPChol decreased by 3.5% and 2.5%. The GPEtn/GPChol ratios significantly increased in both tissues, to 3.6±1.1 in the muscle (P=0.0010, t-test), and to 0.7±0.1 in the fat body (P=0.0037, t-test). Changes were observed also at the level of FA composition in both tissues; the UFA/SFA ratios significantly decreased (Table 4).
PCA analysis
PCA identified two principal components (PC1 and PC2), which together
accounted for 82.8% of the total variation seen among different treatments
(field and laboratory data were analyzed in a single run).
Fig. 8A presents the scores of
the two principal components for individual treatments. In the two-dimensional
space described by PC1 and PC2 scores, vectors may by drawn, which connect an
initial state with a state after the application of a treatment. Similar
patterns in changes of GPL composition appear as similar orientations of the
vectors. The length of each vector is proportionally related to the
significance of the change. The vectors displaying the autumnal
acclimatization in the field and the cold acclimation in the laboratory were
relatively long and of similar orientation in both tissues. The differences
between the relevant scores of principal components were significant at
=0.05 (ANOVA followed by SNK test)
(Fig. 8B,C). In contrast, the
vectors related to diapause progression and desiccation (these were not drawn
in Fig. 8A) were relatively
short and the differences between the scores were mostly insignificant.
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;
Danks, 1996;
Renault et al., 2002;
Sinclair et al., 2003) of
which the membrane restructuring is just one. Hence, it is difficult to
estimate the effect of any single component in isolation. In P.
apterus, depression of SCP temperature down to between –15 and
–20°C is necessary to extend the range of survival temperatures.
This phenomenon was observed repeatedly in the field
(Kostál and Simek,
2000) (Table 1) and
in the laboratory cold-acclimation experiments (Hodková and Hodek,
1997; Kostál et al.,
2001) (Table 2).
The low SCP alone, however, does not guarantee high levels of chill tolerance
(Bale, 1987). About 25% of
diapausing P. apterus adults showed SCP lower than –15°C
already prior to cold acclimation (both in the field and in the laboratory,
see the values of 75 percentiles in Tables
1 and
2). The minimum SCP values in
such adults was –20°C. Despite that, none of those adults survived
longer than 1 day at –15°C (Tables
1,
2). This shows clearly that
additional adjustments are required to reach the maximum chill tolerance.
Among them, accumulation of ribitol and sorbitol play an important role
(Kostál and Simek,
2000; Kostál et al.,
2001; Kostál et al.,
2004a). Both compounds were also found in cold-acclimated P.
apterus in relatively high concentrations in this study (Tables
1,
2). Enhancing the capacity to
maintain ion gradients across cell membranes and epithelia at sub-zero
temperatures is another adjustment that probably contributes to increasing
chill tolerance of cold-acclimated P. apterus
(Kostál et al., 2004b).
The potential contribution of membrane restructuring to the increasing chill
tolerance of this insect was hypothesized first by Hodková et al.
(Hodková et al., 2002).
They described the differences in the GPL composition of membranes between
laboratory, warm-acclimated insects and those collected in the field during
February [fig. 4 in Hodková et al.
(Hodková et al.,
2002)]. We verified and further extended their results using more
advanced analytical techniques and comparing the responses in the field and
variously treated laboratory animals.
Autumnal acclimatization and changes in GPL composition
The increase in the relative proportion of
1-palmitoyl-2-linoleyl-sn-GPEtn at the expense of
1,2-dilinoleyl-sn-GPChol was the most prominent specific change
observed in the wild adults of P. apterus during their autumnal
acclimatization in the field. It was one component of a more general trend,
i.e. the increase in the total proportion of GPEtn species counteracted by the
decrease in the total proportion of GPChol species
(Fig. 3B,
Fig. 4B), which also can be
expressed as an increase in the GPEtn/GPChol ratio. Similar changes were
registered in two tissues, thoracic muscle and fat body. It should be noted
here that whereas the locomotory function of muscle does not change
seasonally, the physiology of fat body changes profoundly. The fat body is the
principal tissue for intermediary metabolism in insects
(Keeley, 1985). Its major
function in reproductively active females is the synthesis and release of
vitellogenic proteins for yolk formation during oocyte maturation
(Telfer, 2003). At early
phases of diapause, the fat body synthesizes large amounts of energy reserves
in the form of diapause proteins, triacylglycerols and glycogen. During the
later phases of diapause, the synthetic activity of the fat body declines and
the lipidic and glycogen reserves are slowly utilized
(Tauber et al., 1986). Upon
stimulation by low temperature, the fat body glycogen becomes the main source
for the rapid biosynthesis of polyol cryoprotectants
(Storey and Storey, 1991).
The extensive seasonal restructuring of GPL composition in P.
apterus occurred without major changes in the proportions of the FAs. A
moderate increase (by 2.4%) in the proportion of palmitoyl (16:0) was found in
both tissues (Table 3). The
most common response of ectotherm membranes to cold is to increase the
proportion of unsaturated FAs and/or increase the number of double bonds per
FA. This response was not seen in P. apterus. In fact, autumnal
acclimatization led to a slight decrease in the UFA/SFA ratio in both
tissues (Table 3). Our FA data,
however, were not obtained by direct analysis of FAs but were derived from
original analytical data of GPL species. Nevertheless, direct GC/MS analysis
of FAs in P. apterus [performed by Hodková et al.
(Hodková et al., 1999)]
confirmed a positive correlation between the unsaturation ratio and ambient
temperature, and a negative correlation between the proportion of palmitoyl
and temperature.
In the field, various factors triggering/stimulating GPL restructuring may not be easily distinguished. Our laboratory experiments allowed us to separate three potential factors: low temperatures, desiccation, and endogenous factors related to diapause progression. Whereas the low temperatures stimulated significant changes (Fig. 5D, Fig. 6D), the later two factors appeared to have relatively small effects on GPL composition in P. apterus membranes.
Triggering by low temperatures
The changes in GPL composition triggered by low temperatures in the
laboratory were similar to the acclimatization changes observed under the
field situation. The GPEtn/GPChol ratio increased, as a result of the
increases in 16:0/18:2-GPEtn and 18:0/18:2-GPEtn countered by the decrease in
18:2/18:2-GPChol. Results of multivariate statistical analysis (PCA) further
supported the overall similarity between the restructurings seen in the field
and laboratory and in both tissues (Fig.
8). This was so despite the fact that the initial states widely
differed between the two situations and between the two tissues as well. Such
uniformity in the response suggests a common adaptive value of the underlying
GPL changes in relation to cold.
The ratio of GPEtn/GPChol generally tends to be higher in cold-than in
warm-acclimated ectotherms (Hazel,
1989; Hazel, 1995;
Hazel, 1997). Molecules of the
two lipid classes differ in their geometries: GPChols assume a cylindrical
shape and pack efficiently in a lamellar gel phase; GPEtns adopt a more
conical form, which is readily accommodated in the reverse hexagonal phase.
Consequently, less efficient packing of GPEtns in the lamellar phase disrupts
the organization of the membrane and thus counters the effects of low
temperatures, which tend to organize the membrane in a gel phase
(Hazel, 1989). The increasing
GPEtn/GPChol ratio in a membrane may thus help to prevent the formation of a
gel phase in membranes at low ambient temperatures. In our earlier work
(Slachta et al., 2002a), we
detected a slight increase in the GPEtn/GPChol ratio in response to low
temperatures in the non-diapausing adults of P. apterus. It increased
from 1.0 to 1.1 (NS, t-test) in the muscles, and from 0.7 to 1.0
(P=0.0032, t-test) in the fat body. Non-diapausing adults
were not able, however, to accomplish the other cold-acclimation adjustments
seen in diapausing adults (SCP depression, polyol accumulation and ion
gradients regulation) and no, or only a weak, increase in their chill
tolerance was observed (Slachta et al.,
2002a).
It is more difficult to search for an adaptive explanation for the observed
changes in relative proportions of specific molecular species, such as the
16:0/18:2-GPEtn. The range of temperatures at which membranes composed of
GPEtns remain fluid, i.e. Tm–Th
(where Tm is a temperature of transition from lamellar gel
to liquid-crystalline phase and Th is a temperature of
transition from liquid-crystalline to reverse hexagonal phase) markedly
increases with a decrease in acyl chain length
(Lewis et al., 1989).
Hodková et al. (Hodková et
al., 2002) suggested that a relative increase of 16:0 FA and its
specific pairing with 18:2 FA in the GPEtn molecule may help to maintain the
ambient temperature within a suitable interval above Tm
yet below Th, and protect membranes from unregulated phase
transitions caused by either a drop of ambient temperature or partial
dehydration, or both occurring at the same time, as is normal in overwintering
insects. Generally, the phase behaviour, fluidity and other properties of
membranes are finely tuned to serve various purposes in different
environmental situations. Changing the lipidic composition is the main tool of
such tuning. Some changes may represent just a side effect without any
explicit adaptive value. However, increasing the relative proportion of
16:0/18:2-GPEtn, as a specific response to cold, might be of some adaptive
value as it was found in two tissues with different physiological functions,
and it was observed also in the fly, Chymomyza costata
(Kostál et al., 2003),
and water strider (M. Hodková, unpublished results), which are
ecologically and phylogenetically distant to P. apterus.
Triggering by desiccation
It has been proposed that some of the physiological adaptations to cold
might be derived from more ancestral adaptations to desiccation
(Ring and Danks, 1994;
Block, 1996;
Danks, 2000;
Williams et al., 2004).
Populations of P. apterus probably colonized central Europe from Asia
Minor and the Near East relatively recently, during the climate warming after
the end of the last glacial period. The ancestral habitats were prone to
drying, which could drive the evolution of physiological adaptations to
desiccation in this species. Moreover, P. apterus adults typically
lose up to one third of body water during cold acclimation and overwintering
(Kostál and Simek,
2000; Kostál et al.,
2004b) (Tables 1,
2). In this study, we attempted
to test whether desiccation triggers some changes in GPL composition. In the
collembolan, Folsomia candida, a mild desiccation stress was
sufficient to elicit the restructuring of membrane GPLs
(Bayley et al., 2001;
Holmstrup et al., 2002). In
addition to alteration of membrane lipids, Folsomia candida responded
to desiccating conditions by accumulating glucose and myo-inositol, which led
to the increase in osmolality, and subsequently influenced the rates of water
uptake/loss (Bayley and Holmstrup,
1999). Our experiments showed that the loss of one half of body
water, associated with doubling the haemolymph osmolality, induced relatively
small changes in GPL composition in P. apterus and that the overall
pattern of these changes differed from the changes induced by low temperatures
(Fig. 7). For example, a
decrease in the proportion of 16:0/18:2-GPEtn was noted in response to
desiccation whereas an increase was detected as a result of cold acclimation
(see above). No, or negligible, accumulation of polyhydric solutes was
observed in response to desiccation in P. apterus
(Table 2).
Triggering by endogenous factors related to diapause
The preparation for winter survival starts long before the autumnal decline
of environmental temperatures in P. apterus
(Kostál and Simek,
2000), and in many other insects as well
(Danks, 1987;
Kostál, 2006).
Hodková et al. (Hodková et
al., 2002) found that the relative proportion of 16:0/18:2-GPEtn
was higher in short-day reared (diapausing) than in long-day reared adults of
P. apterus (non-diapausing, reproducing) that were maintained in the
laboratory at 26°C. Moreover, when non-diapausing females of P.
apterus had the corpus allatum surgically removed (which prevents
juvenile hormone production and causes entry into a diapause-like state), the
GPL profiles in their muscle and fat body tissues changed in a similar manner
to those of females in which diapause was induced photoperiodically
(Hodková et al., 2002).
Similar results were obtained with larvae of the fly Chymomyza
costata, where relative proportions of 16:0/18:2-GPEtn in total GPLs
extracted from the muscle and fat body tissues significantly increased in
response to both low temperature stimulus and the transition from direct
development to diapause, i.e. without any change in temperature
(Kostál et al., 2003).
Such results suggest that the seasonal restructuring of GPL composition is
photoperiodically activated already during the entrance into
diapause, which typically happens (long) before the seasonal decline in
temperatures, and that the changes are hormonally mediated. By contrast, the
changes in GPL composition during the diapause progression, as
described in this paper, were relatively small. Moreover, the orientations of
the vectors of diapause-related changes
(Fig. 8A) were opposite in the
muscle and fat body tissues. Such a clear difference in the vector
orientations might reflect specific (changes of) physiological roles in two
different tissues during diapause, which may require synthesizing/maintaining
specific GPL species. In this light, relatively deep and uniform restructuring
of GPL composition of both tissues in response to cold implies an adaptive
mechanism assisting in survival at low temperatures.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Allakhverdiev, S. I., Nishiyama, Y., Suzuki, I., Tasaka, Y. and
Murata, N. (1999). Genetic engineering of the unsaturation of
fatty acids in membrane lipids alters the tolerance of Synechocystis
to salt stress. Proc. Natl. Acad. Sci. USA
96,5862
-5867.
Bale, J. S. (1987). Insect cold hardiness: freezing and supercooling – an ecophysiological perspective. J. Insect Physiol. 12,899 -908.
Bayley, M. and Holmstrup, M. (1999). Water
vapor ansorption in arthropods by accumulation of myoinositol and glucose.
Science 285,1909
-1911.
Bayley, M., Petersen, S. O., Knigge, T., Köhler, H.-R. and Holmstrup, M. (2001). Drought acclimation confers cold tolerance in the soil collembolan Folsomia candida. J. Insect Physiol. 47,1197 -1204.[CrossRef][Medline]
Bennett, V. A., Pruitt, N. L. and Lee, R. E., Jr (1997). Seasonal changes in fatty acid composition associated with cold-hardening in third instar larvae of Eurosta solidaginis.J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 167,249 -255.
Block, W. (1996). Cold or drought – the lesser of two evils for terrestrial arthropods? Eur. J. Entomol. 93,325 -339.
Brooks, S., Clark, G. T., Wright, S. M., Trueman, R. J., Postle,
A. D., Cossins, A. R. and Maclean, N. M. (2002).
Electrospray ionisation mass spectrometric analysis of lipid restructuring in
the carp (Cyprinus carpio L.) during cold acclimation. J.
Exp. Biol. 205,3989
-3997.
Browse, J., Miquel, M., McConn, M. and Wu, J. (1994). Arabidopsis mutants and genetic approaches to the control of lipid composition. In Temperature Adaptations of Biological Membranes (ed. A. R. Cossins), pp.141 -154. London, Chapel Hill: Portland Press.
Cossins, A. R. (ed.) (1994). Temperature Adaptations of Biological Membranes. London, Chapel Hill: Portland Press.
Danks, H. V. (1987). Insect Dormancy: An Ecological Perspective (Monograph Series No. 1),439 pp. Ottawa: Biological Survey of Canada (Terrestrial Arthropods).
Danks, H. V. (1996). The wider integration of studies on insect cold-hardiness. Eur. J. Entomol. 93,383 -403.
Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46,837 -852.[CrossRef][Medline]
Denlinger, D. L. (1985). Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 8 (ed. G. A. Kerkut and L. I. Gilbert), pp. 353-412. Oxford: Pergamon.
Denlinger, D. L. (1991). Relationship between cold hardiness and diapause. In Insects at Low Temperature (ed. R. E. Lee and D. L. Denlinger), pp.174 -198. New York, London: Chapman & Hall.
Folch, J., Lees, M. and Sloane Stanley, G. H.
(1957). A simple method for the isolation and purification of
total lipids from animal tissues. J. Biol. Chem.
226,497
-509.
Han, X. and Gross, R. W. (1995). Structural determination of picomole amounts of phospholipids via elctrospray ionization tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 6,1202 -1210.[CrossRef]
Hazel, J. R. (1989). Cold adaptation in ectotherms: regulation of membrane function and cellular metabolism. Adv. Comp. Environ. Physiol. 4, 1-50.
Hazel, J. R. (1995). Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu. Rev. Physiol. 57,19 -42.[Medline]
Hazel, J. R. (1997). Thermal adaptation in biological membranes: beyond homeoviscous adaptation. Adv. Mol. Cell Biol. 19,57 -101.
Hodek, I. (1968). Diapause in females of Pyrrhocoris apterus L. (Heteroptera). Acta Entomol. Bohemoslov. 65,422 -435.
Hodková, M., Simek, P., Zahradnícková, H. and Nováková, O. (1999). Seasonal changes in the phospholipid composition in thoracic muscles of a heteropteran, Pyrrhocoris apterus. Insect Biochem. Mol. Biol. 29,367 -376.[CrossRef]
Hodková, M., Berková, P. and Zahradnícková, H. (2002). Photoperiodic regulation of the phospholipid molecular species composition in thoracic muscles and fat body of Pyrrhocoris apterus (Heteroptera) via an endocrine gland, corpus allatum. J. Insect Physiol. 48,1009 -1019.[CrossRef][Medline]
Holmstrup, M., Hedlund, K. and Boriss, H. (2002). Drought acclimation and lipid composition in Folsomia candida: implications for cold shock, heat shock and acute desiccation stress. J. Insect Physiol. 48,961 -970.[CrossRef][Medline]
Hsu, F.-F. and Turk, J. (2000). Characterization of phosphatiylinositol, phosphatiylinositol-4-phosphate and phosphatiylinositol-4,5-biphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study. J. Am. Soc. Mass Spectrom. 11,9896 -9899.
Inaba, M., Suzuki, I., Szalontai, B., Kanesaki, Y., Los, D. A.,
Hayashi, H. and Murata, N. (2003). Gene-engineered
rigidification of membrane lipids enhances the cold inducibility of gene
expression in Synechocystis. J. Biol. Chem.
278,12191
-12198.
Keeley, L. L. (1985). Physiology and Biochemistry of the fat body. In Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 3 (ed. G. A. Kerkut and L. I. Gilbert), pp. 211-248. Oxford: Pergamon.
Kostál, V. (2006). Eco-physiological phases of insect diapause. J. Insect Physiol. 52,113 -127.[CrossRef][Medline]
Kostál, V. and Simek, P. (1995). Dynamics of cold hardiness, supercooling and cryoprotectants in diapausing and non-diapausing pupae of the cabbage root fly, Delia radicum L. J. Insect Physiol. 41,627 -634.[CrossRef]
Kostál, V. and Simek, P. (1998). Changes in fatty acid composition of phospholipids and triacylglycerols after cold-acclimation of an aestivating insect prepupa. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 168,453 -460.
Kostál V. and Simek, P. (2000). Overwintering strategy in Pyrrhocoris apterus (Heteroptera): the relations betwen life-cycle, chill tolerance and physiological adjustments. J. Insect Physiol. 46,1321 -1329.[CrossRef][Medline]
Kostál, V., Slachta, M. and Simek, P. (2001). Cryoprotective role of polyols independent of the increase in supercooling capacity in diapausing adults of Pyrrhocoris apterus (Heteroptera: Insecta). Comp. Biochem. Physiol. 130B,365 -374.[CrossRef][Medline]
Kostál, V., Berková, P. and Simek, P. (2003). Remodelling of membrane phospholipids during transition to diapause and cold-acclimation in the larvae of Chymomyza costata (Drosophilidae). Comp. Biochem. Physiol. 135B,407 -419.[CrossRef][Medline]
Kostál, V., Tollarová, M. and Sula, J. (2004a). Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus. J. Insect Physiol. 50,303 -313.[CrossRef][Medline]
Kostál, V., Vambera, J. and Bastl, J.
(2004b). On the nature of pre-freeze mortality in insects: water
balance, ion homeostasis and energy charge in the adults of Pyrrhocoris
apterus. J. Exp. Biol. 207,1509
-1521.
Lee, R. E. (1991). Principles of insect low temperature tolerance. In Insects at Low Temperature (ed. R. E. Lee and D. L. Denlinger), pp. 17-46. New York, London: Chapman & Hall.
Lee, R. E., Chen, C. P. and Denlinger, D. L.
(1987). A rapid cold-hardening process in insects.
Science 238,1415
-1417.
Lewis, R. N. A. H., Mannock, D. A., McElhaney, R. N., Turner, D. C. and Gruner, S. M. (1989). Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions. Biochemistry 28,541 -548.[CrossRef][Medline]
Murata, N. (1994). Genetic and temperature-induced modulation of cyanobacterial membrane lipids. In Temperature Adaptations of Biological Membranes (ed. A. R. Cossins), pp. 155-162. London, Chapel Hill: Portland Press.
Nedved, O., Hodková, M., Brunnhofer, V. and Hodek, I. (1995). Simultaneous measurement of low temperature survival and supercooling in a sample of insects. Cryo Letters 16,108 -113.
Ohtsu, T., Kimura, M. T. and Katagiri, C. (1998). How Drosophila species acquire cold tolerance. Qualitative changes of phospholipids. Eur. J. Biochem. 252,608 -611.[Medline]
Overgaard, J., Sorensen, J. G., Petersen, S. O., Loeschke, V. and Holmstrup, M. (2005). Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster.J. Insect Physiol. 51,1173 -1182.[CrossRef][Medline]
Renault, D., Salin, C., Vannier, G. and Vernon, P. (2002). Survival at low temperatures in insects: what is the ecological significance of the supercooling point? Cryo Letters 23,217 -228.[Medline]
Ring, R. A. and Danks, H. V. (1994). Desiccation and cryoprotection – overlapping adaptations. Cryo Letters 15,181 -190.
Sinclair, B. J., Vernon, P., Klok, C. J. and Chown, S. L. (2003). Insects at low temperatures: an ecological perspective. Trends Ecol. Evol. 18,257 -262.[CrossRef]
Sinensky, M. (1974). Homeoviscous adaptation
– a homeostatic process that regulates viscosity of membrane lipids in
Escherichia coli. Proc. Natl. Acad. Sci. USA
71,522
-525.
Slachta, M., Berková, P., Vambera, J. and Kostál, V. (2002a). Physiology of cold-acclimation in non-diapausing adults of Pyrrhocoris apterus (Heteroptera). Eur. J. Entomol. 99,181 -187.
Slachta, M., Vambera, J., Zahradnícková, H. and Kostál, V. (2002b). Entering diapause is a prerequisite for successful cold-acclimation in adult Graphosoma lineatum (Heteroptera: Pentatomidae). J. Insect Physiol. 48,1031 -1039.[CrossRef][Medline]
Steponkus, P. L., Lynch, D. V. and Uemura, M. (1990). The influence of cold acclimation on the lipid composition and cryobehaviour of the plasma membrane of isolated rye protoplasts. Philos. Trans. R. Soc. Lond. B Biol. Sci. 326,571 -583.
Storey, K. B. and Storey, J. M. (1991). Biochemistry of cryoprotectants. In Insects at Low Temperature (ed. R. E. Lee and D. L. Denlinger), pp.64 -93. New York, London: Chapman & Hall.
Tauber, M. J., Tauber, C. A. and Masaki, S. (1986). Seasonal Adaptations of Insects. Oxford: Oxford University Press.
Telfer, W. H. (2003). Vitellogenesis. In Encyclopedia of Insects (ed. V. H. Resh and R. T. Cardé), pp. 1171-1173. San Diego: Academic Press.
Wallis, J. G. and Browse, J. (2002). Mutants of Arabidopsis reveal many roles for membrane lipids. Prog. Lipid Res. 41,254 -278.[CrossRef][Medline]
Wang, C., Kong, H., Guan, Y., Yang, J., Gu, J., Yang, S. and Xu, G. (2005). Plasma phospholipid metabolic profiling and biomarkers of type 2 diabetes mellitus based on high-performance liquid chromatography/electrospray mass spectrometry and multivariate statistical analysis. Anal. Chem. 77,4108 -4116.[Medline]
Williams, J. B., Ruehl, N. C. and Lee, R. E.
(2004). Partial link betwen the seasonal acquisition of
cold-tolerance and desiccation resistance in the goldenrod gall fly
Eurosta solidaginis (Diptera: Tephritidae). J. Exp.
Biol. 207,4407
-4414.
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