On the nature of pre-freeze mortality in insects: water balance, ion homeostasis and energy charge in the adults of Pyrrhocoris apterus
tál1,*
1 Institute of Entomology, Academy of Sciences, eské
Bud
jovice, Czech Republic
2 Faculty of Biological Sciences, University of South Bohemia,
eské Bud
jovice, Czech Republic
3 Agricultural Faculty, University of South Bohemia, eské
Bud
jovice, Czech Republic
* Author for correspondence (e-mail: kostal{at}entu.cas.cz)
Accepted 4 February 2004
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Summary |
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Key words: chill tolerance, diapause, water loss, ion gradient, pre-freeze mortality, Heteroptera, Pyrrhocoris apterus
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Introduction |
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Despite the fact that chilling-injury is generally recognized as a
widespread and serious cause of death in insects that overwinter in a
supercooled state (Knight et al.,
1986; Bale, 1987
,
2002
;
Lee, 1991
;
Nedved, 2000
;
Ramløv, 2000
),
surprisingly few attempts have been made to characterize physiological
mechanisms involved in pre-freeze mortality. Several studies focused on
electrical activity of the nervous system during chilling. It has been
observed that, at a sufficiently low temperature, the insects enter a state of
chill-coma when excitability of muscles and nerves is severely altered or lost
(Anderson and Mutchmor, 1968
;
Bradfisch et al., 1982
;
Hosler et al., 2000
). The loss
of nervous membrane excitability was attributed to the preceding decrease of
resting potential due to the effect of low temperature on transport mechanisms
involved in ion balance (Heitler et al.,
1977
; Kivivouri et al.,
1990
; Cossins et al.,
1995
). Thus, the neuronal damage is one of the likely causes of
injury inflicted by chilling (Yocum et
al., 1994
). Pullin and Bale
(1988
) and Pullin et al.
(1990
) studied pre-freeze
mortality in the nettle aphid Microlophium carnosum. They found that
ATP content and energy charge declined relatively slowly at low temperature,
which they interpreted as insusceptibility of catabolic respiratory processes
to chill-injury. Nevertheless, chilled aphids displayed almost doubled levels
of ATP in comparison with the control (non-chilled) group during the first day
after the start of chilling (i.e. before any substantial mortality occurred).
This might mean that ATP could not be normally processed, which would indicate
mismatching among various metabolic pathways
(Hochachka, 1986
;
Knight et al., 1986
). The same
authors observed no evidence of rapid leakage of electrolytes during chilling
of aphids (Pullin and Bale,
1988
). However, the method they used (measuring conductivity of
water in which the aphids were submerged) was rather imprecise and might have
been influenced by the relative impermeability of the cuticle. Membrane
failure due to the phase transition in the membrane lipids
(Hazel, 1989
), oxidative
stress (Rojas and Leopold,
1996
) and protein denaturation or incorrect folding
(Yocum, 2001
) were suggested
as other potential mechanisms of chilling-injury.
The main purpose of the present study was to follow the changes in water
and ion balances and energy status during exposure to a sub-zero, but
non-freezing, temperature of 5°C in the adult bugs of
Pyrrhocoris apterus (Insecta: Heteroptera: Pyrrhocoridae). Such
changes were correlated with pre-freeze mortality and chilling-injury
(assessed as the time necessary for recovery from chill-coma). P.
apterus was a convenient model because relatively extensive knowledge on
its diapause and physiology of cold hardiness has been gathered
(Sláma, 1964; Hodek,
1968
,
1983
; Hodková and
Hodek, 1994
,
1997
;
ula et al., 1995
;
Socha et al., 1997
;
Ko
tál and
imek,
2000
; Ko
tál et
al., 2001
;
lachta et
al., 2002
; Hodková et al.,
1999
,
2002
). Here, bugs in three
acclimation groups (non-diapause, diapause and diapause, cold-acclimated),
which differed markedly in the level of chill-tolerance, were compared. We
found that during exposure to 5°C, the least chill-tolerant
(non-diapause) insects, in comparison with the other two groups, displayed
relatively rapid loss of water from the haemolymph compartment, an inability
to maintain ion gradients across the fat body membrane, an inability to
regulate ions into the hindgut fluid and no spending of total adenylate pools
in the fat body. These observations are discussed with respect to their
potential role in chilling-injury and pre-freeze mortality.
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Materials and methods |
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Insects from the three acclimation groups were then exposed to a constant
temperature of 5°C (fluctuations between 4.3°C and
5.7°C) for different periods of time (up to 60 days). The
temperature of 5°C was selected as it is well above the SCP level
of any acclimation group: LD, 9.4±0.4°C (N=154);
SD, 11.6±0.9°C (N=32); SDA,
15.9±0.8°C (N=32) (means ±
S.E.M.; taken from
Kotál et al.,
2001
;
lachta et al.,
2002
) and, thus, the occurrence of freezing events was minimized.
Exposure to a constant temperature of 15°C (fluctuations between
14.5°C and 15.5°C) was used for one additional
experiment with the SDA group. Samples (with equal proportions of males and
females) were taken at three time points: (1) just prior to the exposure (day
0); (2) after the exposure, which caused pre-freeze mortality in
20% of
the population sample (LD, day 4; SD, day 20); (3) after the exposure, which
caused mortality in
50% of the population sample (LD, day 8; SD, day 35).
In the SDA group, mortality rates did not exceed 10% during 60-day exposure;
therefore, the two samples were taken arbitrarily at days 40 and 60. The
following analyses were performed.
Size, hydration and osmolality of body compartments
Ten specimens of each acclimation group (sampled at day 0) were carefully
dissected on a cold stage at 0°C under a binocular microscope without
adding any buffer. The fresh mass (FM) of each single tissue/compartment was
measured using a Sartorius balance with a sensitivity of 0.1 mg. The following
tissues (compartments) were considered: gut (oesophagus and midgut), hindgut +
rectum (subsequently called hindgut), abdominal fat body (90% of the
tissue was collected), gonads (only in the LD group), `remains' after the
dissection (skeleton, epidermis, muscles, nervous system, etc.). Dry mass (DM)
was measured after drying the specimens at 65°C for 3 days. Hydration (in
mg water mg1 DM) and total water content (WC) were
calculated from the gravimetric data. Total volume of haemolymph was estimated
by subtraction of the sum of the water contents of all dissected
tissues/compartments from the value of the whole body-water content (see
below). Some additional haemolymph rested in the remains after the dissection;
thus, half (our estimation) of the amount of water from the remains was added
to the calculated total volume of haemolymph. Volume of the hindgut could be
measured only in some specimens of the SD and SDA groups (where the hindgut
was relatively big and spherical in shape) by measuring its radius
(r) and using it in a sphere volume (V) formula:
V=4/3
r3. In the insects with a small, tubular
hindgut (mostly LD group), its volume was estimated to represent 0.1
µl.
Whole body FMs and DMs were obtained from another 30 insects of each acclimation group. Loss of whole-body FM was measured in yet another 30 adults (per acclimation group) that were individually marked by a pencil on their elytra and weighed two (or three) times: prior to, (during) and at the end of the exposure to 5°C.
Haemolymph samples for osmolality measurements were collected from 10 individual insects by cutting off one of the antennae and allowing it to bleed into a calibrated capillary tube. Similarly, samples of the hindgut fluid were obtained by piercing the hindgut wall and collecting the fluid into a capillary tube. Osmolalities were measured in 1015 nl droplets using the Clifton Nanoliter Osmometer, according to the manufacturer's instructions (Clifton Technical Physics, Hartford, NY, USA).
Mortality and recovery time
Groups of 1030 insects were exposed to 5°C for different
periods of time. They were then transferred to 20°C, supplied with water
and linden seeds, and their survival was scored 5 days later. Only the
individuals capable of rapid coordinated crawling were considered to be
survivors (our preliminary experiments verified that such insects were later
capable of normal reproduction).
Recovery time was defined as the time necessary for resumption of locomotion after the chill-coma caused by the exposure to 5°C. Insects (10 per sample) that were previously exposed to 5°C for different periods of time were placed in glass Petri dishes on their backs and the time taken for them to turn over unaided and resume a normal position at 20°C was measured.
Na+ and K+ concentrations, equilibrium potentials
Concentrations of Na+ and K+ ions were determined in
the extracts of haemolymph samples taken as described above. The whole amount
of haemolymph that bled spontaneously (without pressing the animal) was
collected from each specimen. The haemolymph collected from 10 insects was
pooled, and the volume of each pooled sample was measured (depicted in
Fig. 3A) and used to estimate
the mean haemolymph volume in one specimen. Each pooled sample was taken in 6
(LD group) or 3 (SD and SDA groups) replications. Haemolymph was then
extracted in 100 µl of a 1 mol l1 solution of
trichloroacetic acid (Sigma Chemical Co., St Louis, MO, USA) in deionized
water (conductivity <0.1 µS cm1). Samples of hindgut
fluid (10 tissues pooled, three replications) were taken as described above.
Fat body tissues [10 tissues pooled, 6 (LD) or 3 (SD, SDA) replications] were
weighed (FM), dried and weighed again (DM) in order to calculate the amount of
water. Samples of hindgut fluid and fat body tissue were extracted in 100
µl of 65% nitric acid. After the extraction, the samples were centrifuged
at 20 000 g for 10 min and the concentrations of ions were
measured in supernatants by atomic emission spectrophotometry (Na+
at 589.0 nm; K+ at 766.5 nm) using a spectrophotometer SpectrAA 640
(Varian Techtron, Mulgrave, Australia).
|
The equilibrium potentials (E) across the fat body membrane were
calculated for each ion separately using the Nernst equation:
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Adenylates and energy charge
The abdominal fat body tissues from 35 specimens were pooled for
each sample (taken in four replications). They were collected in liquid
nitrogen and, upon nitrogen evaporation, weighed. Ice-cold HClO4
(6%) solution containing 1 mmol l1 EDTA was added and the
sample was quickly homogenized using a plastic pestle and a hand-held
battery-driven homogenizer. After centrifugation at 22 000 g
at 4°C for 5 min, the supernatant was neutralized to pH 7 by adding a
solution of 1.5 mol l1 KOH, 0.4 mol l1
imidazole and 0.3 mol l1 KCl. Precipitated HClO4
was removed by centrifugation at 22 000 g at 4°C for 5 min
and the supernatant was stored at 80°C until analysis.
The concentrations of ATP, ADP and AMP were measured using enzymatic
methods, which couple the interconversions between the adenylates with the
reduction/oxidation of NAD(P)/NAD(P)H
(Passonneau and Lowry, 1993).
Absorbance at 340 nm was measured using a Pye Unicam SP8-100
spectrophotometer.
The contents of ATP, ADP and AMP were used to calculate the adenylate
energy charge (AEC) according to the formula:
AEC=[ATP]+0.5[ADP]/[ATP]+[ADP]+[AMP]. AEC represents a linear measure of the
metabolic energy stored in the adenine nucleotide system
(Atkinson, 1968).
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Results |
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The differences found between the SD and SDA groups were considered to represent the physiological changes that accompany the process of cold acclimation. Partial dehydration was the most obvious change (Table 1) in SDA adults: (1) whole-body FM decreased, although not significantly; (2) the difference in WC indicated that an average of 8.6 µl (19.5%) of the whole-body water disappeared during acclimation (this amount practically equals the loss of whole body FM) and (3) total volume of haemolymph significantly decreased by 12.6 µl (52%). Insignificant decreases of water content were seen in the other compartments (gut, fat, remains). Haemolymph osmolality almost doubled during the cold acclimation. The volume of hindgut increased from 0.1 µl to 3.9 µl on average, its shape became spherical and the hindgut fluid was hypo-osmotic (195 mmol kg1) to the haemolymph (626 mmol kg1). Thus, the decrease in haemolymph volume (12.6 µl) could be fractioned almost perfectly between the evaporative/excretory loss of water (8.6 µl) and the transfer of water to the hindgut (3.8 µl).
Pre-freeze mortality and chilling injury
Fig. 2A documents the
considerable differences in the level of chill tolerance between three
acclimation groups of insects. While a sufficient time to kill 50% of LD
adults was only 7.6 days at 5°C, 35.6 days were required in the SD
group, and mortality did not exceed 10% after a 60-day exposure in the SDA
group. We believe that freezing events were very exceptional in our
experiments. First, the temperature of 5°C was well above the mean
SCP of any group; second, it was always possible to sample liquid haemolymph
from all specimens (sampling took place within 3 s after withdrawing the
specimen from 5°C); third, if stochastic freezing events occurred
with high frequency, the relationship between mortality and exposure time
would be expected to take a logarithmic rather than a sigmoid shape.
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Recovery time necessary for resumption of locomotion after chill-coma linearly increased with increasing time of exposure to 5°C. After exposure for a given duration, recovery time was always shortest in the most chill-tolerant variant (SDA) and longest in the least chill-tolerant variant (LD) of the three variants tested (Fig. 2B).
Changes in hydration and osmolality at 5°C
Insects of all acclimation groups tended to decrease their whole-body FM
during exposure to 5°C (Fig.
3A, inset). As we could observe no defecation during the exposure
and the loss of DM was probably negligible because of extremely low
metabolism, we suppose that the changes in whole-body FM primarily reflected
losses of water. The rate of water loss was clearly highest in the LD group.
The loss of 12.7% of water during the 8-day exposure would correspond to 4.4
µl when 34.3 µl (Table 1)
is taken as the initial total amount of water. It seems that the haemolymph
compartment was particularly prone to lose water in the LD group.
Fig. 3A shows that the mean
volume of haemolymph rapidly decreased in the LD group and, after 8 days,
dropped to the volume that was typically obtained from the SDA group (SDA
group was characterised by its low hydration and low haemolymph volume; see
Table 1). By contrast, no
significant change in hydration or water content was observed in the fat body
(in fact, an insignificant tendency to increase water content was registered
in the fat body; Fig. 2B).
Considering that all 4.4 µl of water were lost from the haemolymph
compartment, this would shrink from an initial 16.3 µl
(Table 1) to 11.9 µl, which
is 1.37-fold. Indeed, a 1.34-fold increase in haemolymph osmolality (from 370
mmol kg1 to 497 mmol kg1) was observed
concomitantly (Table 2). Thus,
we suppose that most of the water was really lost from the haemolymph
compartment in the LD group insects. The hindgut remained small and tubular in
all specimens of the LD group during the exposure to 5°C and its
volume was estimated to be 0.1 µl.
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Compared with the LD group, less rapid loss of whole-body FM was observed in the SD group: 6.66% (i.e. 2.9 µl) during 35 days of exposure (Fig. 3A, inset). The mean volume of haemolymph remained practically constant (Fig. 3A). No sign of dehydration was observed in the fat body, its total WC remained constant and hydration rate showed an insignificant increase (Fig. 2B). We presume that most of the water was again lost from the haemolymph compartment. But, because of its large initial volume of 24.2 µl (Table 1), the loss of 2.9 µl would correspond to a 1.14-fold decrease only. Haemolymph osmolality increased from 373 mmol kg1 to 737 mmol kg1, which is 1.98-fold (Table 2). The average volume of hindgut increased from 0.1 µl to 1.1 µl in the SD group (Table 2). In fact, the hindgut remained tubular in six out of 12 specimens, while it became spherical, with an average volume of 2.15±1.52 µl, in the other six specimens.
In the SDA group, the slowest rate of FM loss of the three groups was observed: 5.0% (1.8 µl) or 8.9% (3.2 µl) during 35 or 60 days, respectively (Fig. 3A, inset). The volume of average haemolymph sample remained constant (Fig. 3A). In contrast to the other two groups, a significant trend of fat body dehydration was observed in the SDA insects (Fig. 3B). The FM of fat body decreased from an initial 12.4 mg to 10.7 mg or 9.3 mg during 35 or 60 days, respectively. The hydration rate decreased from 1.12 mg water mg1 DM to 0.66 or 0.42 mg water mg1 DM. The total WC decreased from 5.6 µl to 4.3 or 2.8 µl. Thus, the amount of water lost from fat body tissue alone could almost completely explain the total loss of water from the whole body. This indicates that the haemolymph volume did not change significantly; nevertheless, its osmolality reached very high levels of 1065 mmol kg1 or 1247 mmol kg1 during 35 or 60 days, respectively (Table 2). No significant change in the hindgut volume was found in the SDA group. The osmolality of hindgut fluid increased significantly from 195 mmol kg1 to 344 mmol kg1 during the first 35 days of the exposure, and a further increase (statistically insignificant) to 523 mmol kg1 was observed during the additional 25 days (Table 2).
Na+ and K+ concentrations
The following changes in ion concentrations were observed in the haemolymph
and fat body tissue during the exposure to 5°C
(Fig. 4).
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Sodium in haemolymph (Fig. 4A). A significant decrease of the Na+ concentration was found in the LD group. In the other two groups, the concentrations were relatively constant. Considering the changes in haemolymph volume (see above) and the concentrations measured, we could estimate the changes in total pools of Na+: it decreased from 0.43 µmoles to 0.24 µmoles in the LD group; a relatively small decrease from 0.62 µmoles to 0.51 µmoles was observed in the SD group; and the pool was stable in the SDA group [from 0.43 µmoles to 0.37 µmoles (day 35) or 0.40 µmoles (day 60)].
Sodium in fat body (Fig. 4B). No significant changes in the concentration were detected with increasing time of exposure by linear regression analysis in any acclimation group. Also the pools were rather stable: no change in the LD group (0.03 µmoles); a small change in the SD group (from 0.07 µmoles to 0.08 µmoles); and a decrease in the SDA group [from 0.10 µmoles to 0.08 µmoles (day 35) or 0.05 µmoles (day 60)].
Potassium in haemolymph (Fig. 4C). The concentrations significantly increased with increasing time of exposure in all three acclimation groups. The total pools also increased in all groups: from 0.26 µmoles to 0.46 µmoles (LD group); from 0.37 µmoles to 0.46 µmoles (SD group); and from 0.13 µmoles to 0.21 µmoles (day 35) or 0.31 µmoles (day 60) (SDA group).
Potassium in fat body (Fig. 4D). A non-significant decrease in concentration was observed in the LD group. A significant decrease was observed in the SD group and a significant increase in the SDA group. The total pool was rather stable in the LD group (from 0.26 µmoles to 0.24 µmoles); it decreased in the SD group (from 0.84 µmoles to 0.58 µmoles) and also in the SDA group [from 0.67 µmoles to 0.55 µmoles (day 35) or 0.52 µmoles (day 60)].
In an additional experiment, the changes of haemolymph ion concentrations
were measured in the SDA insects when exposed to 15°C (instead of
5°C), where the duration of exposure that causes mortality in 50%
of the population sample (Lt50) shortens dramatically to 9.0 days
(Kotál et al.,
2001
). Under such conditions, the K+ concentration
increased significantly and rapidly while the Na+ concentration
significantly decreased. The rates of change in both concentrations were very
similar to the rates observed in the LD group exposed to 5°C
(Fig. 5).
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In the hindgut fluid (Table
2), no significant changes in the ion concentrations or total
pools were registered during the exposure to 5°C in the LD group,
and the pools were relatively small (0.002 µmoles of Na+
and
0.0050.006 µmoles of K+). In the SD group, the
initial pools of both ions were small (
0.002 µmoles of Na+
and
0.001 µmoles of K+). During the exposure, the
concentrations increased (significantly in the case of K+; see
Table 2) and the pools
increased too (to 0.04 µmoles of Na+ and 0.05 µmoles of
K+). In the SDA group, the highest increases (of the three groups)
of concentration and, especially, of the pools of both ions were observed.
After 35 days, 0.07 µmoles of Na+ and 0.24 µmoles of
K+ were accumulated in the hindgut fluid, and after 60 days it was
0.10 µmoles of Na+ and 0.17 µmoles of K+.
Na+ and K+ equilibrium potentials
Equilibrium potentials of sodium (ENa) across the fat
body membrane remained relatively constant at approximately +20 mV during the
exposure to 5°C in all the acclimation groups
(Fig. 6).
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Equilibrium potentials of potassium (EK) decreased from 49.5 mV on day 0 to 24.7 mV on day 8 in the LD group. A slower rate of decrease in EK was observed in the SD group (from 57.5 to 34.9 mV on day 35). The slowest rate of EK decrease, from 60.6 to 52.4 mV on day 60, was registered in the SDA group (Fig. 6). The values of EK taken at days 0 and 60 did not differ statistically (unpaired two-tailed t-test, P=0.2726).
Adenylates and energy charge
The concentrations of adenylates in the fat body cells showed no
statistically significant changes (in most cases) during the exposure to
5°C in all three acclimation groups
(Table 3). The trends of
decrease, nevertheless, were apparently indicated in the groups SD and SDA,
and these were significant in the case of AMP. Considering the decreasing FM
of fat body during the exposure of SDA insects to 5°C, there must
have been a significant decrease in the total content (pools) of all
adenylates in the fat body. By contrast, because the FM of the fat body was
stable in the LD and SD groups, the total pools probably did not decrease at
all (LD) or only slightly (SD).
|
The adenylate energy charge (AEC) was similar in all three groups (ranging between 0.83 and 0.85) prior to their exposure to 5°C and it was also fairly stable during the exposure (Table 3).
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Discussion |
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Initial physiological state prior to exposure to 5°C
The non-diapause insects (LD) were actively moving, feeding and reproducing
while the diapause insects (SD) had arrested reproduction, and their
locomotion and feeding activities were minimal. Such `overt' differences were
reflected in the sizes of gonads, gut and fat body and were undoubtedly based
on fundamental differences in gene expression and hormonal milieu that are
typical for each of the two alternative states in insects
(Tauber et al., 1986;
Danks, 1987
;
Flannagan et al., 1998
;
Denlinger, 1985
,
2002
). What concerns the
parameters assessed in this study are that no significant differences were
found between the LD and SD groups in their hydration, osmolality, ion
concentrations, adenylate concentrations and adenylate energy charge prior to
the exposure to 5°C. However, the ways in which individual
parameters changed during the exposure to 5°C differed
substantially between the two groups and will be discussed later.
Cold acclimation of the SD group of P. apterus is known to result
in various transformations, which lead to an increased level of
chill-tolerance (Hodková and Hodek,
1997; Hodková et al.,
1999
,
2002
;
Ko
tál et al.,
2001
;
lachta et al.,
2002
). Here, we showed that the process of cold-acclimation (SD
SDA) is also accompanied by the following changes: (1) partial
dehydration affecting especially the haemolymph compartment, which was reduced
to one half of its pre-acclimation level; preferential loss of water from the
haemolymph was reported previously in insects subjected to drying of their
habitats (Zachariassen and Einarson,
1993
; Hadley,
1994
; Zachariassen and
Pedersen, 2002
); (2) redistribution of water; a `reserve' (
4
µl) of hypo-osmotic fluid accumulated in the hindgut; the principal role of
the hindgut in insect water balance is widely recognised
(Hadley, 1994
;
Danks, 2000
;
Coast, 2001
); (3) regulation of
ion concentrations; despite the loss of 52% of water from the haemolymph, the
concentrations of Na+ and K+ were maintained almost
constant because the ion pools decreased by 0.19 µmoles or 0.24 µmoles
of Na+ or K+, respectively. Ion pools increased during
cold acclimation in the hindgut fluid (by 0.02 µmoles or 0.07 µmoles of
Na+ or K+, respectively) and they remained rather
constant in the fat body. Thus, we suppose that some fraction of ions from
dehydrating haemolymph could be excreted. No significant changes in the
concentrations of adenylates or in the AEC in the fat body were observed
during the cold acclimation of P. apterus.
Changes of physiological parameters during the exposure to 5°C
First, the changes observed in the LD group will be discussed. Although the
insects of all acclimation groups tended to lose body water during the
exposure to 5°C, the rate of water loss was clearly the highest in
the LD group. Such a difference between acclimation groups might be caused by
a different quality and/or quantity of the cuticular hydrocarbons, which form
the crucial barrier against water loss
(Lockey, 1985; Yoder et al.,
1992
,
1995
;
Hadley, 1994
). Respiratory
water loss was probably minor thanks to a deep depression of metabolism at
5°C. Nevertheless, we assume that the loss of body water per
se was not the likely cause of pre-freeze mortality in the LD group. Most
of the body water was lost from the haemolymph compartment while the
intracellular compartments remained normally hydrated. The final amount of
haemolymph after the 8-day exposure to 5°C in the LD group was at
least as high as in the SDA group prior to the exposure. And furthermore, the
SDA insects were substantially more dehydrated during their exposure to
5°C for 60 days but still did not die. During the exposure to
5°C, the LD insects displayed a dramatic decrease (to 56%) of the
Na+ pool in the haemolymph, which was counteracted by a similarly
massive influx of K+. As the volume of haemolymph decreased, the
efflux of Na+ did not result in a big change in its concentration
(but this was still significant) while the influx of K+ led to a
>2-fold increase in its concentration. At the same time, neither the
concentrations nor the pools of ions changed significantly in the fat body.
This was probably because the fat body represented a relatively small
compartment in the LD insects (
12.5% of total intracellular water). We
suggest that the other tissues (muscles, gut, gonads) served as more important
sinks for Na+ and as sources of K+. No significant
regulation of ions into the hindgut fluid (and no excretion by defecation) was
observed in the LD insects. These results might indicate that, at
5°C, the activity of Na+/K+-ATPases in the
membranes of tissues was not sufficient to counteract the inward movement of
Na+ down the electrochemical gradient, which caused a (partial)
depolarization of cell membranes followed immediately by an outward movement
of K+ (documented by a sharp decrease of EK
across the fat body membrane).
In comparison with the LD insects, the rates of water loss at
5°C were low in the SD and SDA insects. The initial water content
in haemolymph was considerably lower (by half) in the SDA than in the SD
insects. Perhaps, in order to protect a minimal haemolymph volume serving the
basic transporting function, the water gradually disappeared from the
intracellular compartments (fat body) of the SDA insects during their exposure
to 5°C. With respect to ion regulation, the SD insects showed ion
fluxes of a similar direction as detected in the LD insects but with much
lower rates. Thus, the Na+ pool decreased while the K+
pool increased, both 1.2-fold, in haemolymph. The opposite changes were
registered in the fat body (the fat body water represented 33% of the
total intracellular water pool in the SD group). In contrast to LD insects, at
least some (
50%) of the SD insects were able to build a reserve of
hindgut fluid and to regulate a certain fraction of ions into it. Such a
capacity was even better expressed in the SDA insects. They showed no decrease
of the Na+ pool and a slight increase of the K+ pool in
haemolymph during exposure to 5°C. Despite reduction of the water
content in the fat body to half, the Na+ concentration
there did not increase (its pool halved too) and the concentration of
K+ increased (despite its pool tending to decrease). Thus,
it seems that the capacity to prevent/counteract a leakage of
Na+ down the electrochemical gradient (from haemolymph to
the tissue cells) increased in the order LD<SD<SDA. As a result, the
rates of counteractive outward movement of K+, and of the
EK dissipation, decreased in the same order. Such a
capacity might also be supported by regulation of a certain fraction of ions
into the hindgut fluid (to counteract dehydration).
When the insects of the most chill-tolerant acclimation group (SDA) were exposed to a more severe low temperature of 15°C instead of 5°C, the rates of change of ion concentrations in their haemolymph became much faster and almost matched those observed in the LD group (the least chill-tolerant group) at 5°C. Interestingly, the Lt50 of the SDA group at 15°C shifted to 9.0 days, which also closely matched that of the LD group at 5°C (7.6 days).
The nature of chilling-injury
Most of the observations presented in this paper share one common
denominator and that is maintaining the ion concentrations and ion gradients
across membranes at sub-zero temperature. Entering into a state of chill-coma
in insects was previously attributed to the inability of
Na+/K+-ATPases to function at low temperatures and to
maintain/restore the nerve membrane electrochemical potential
(Heitler et al., 1977;
Kivivouri et al., 1990
;
Cossins et al., 1995
;
Hosler et al., 2000
). The
Na+/K+-ATPase is also responsible for maintaining
Na+ gradients across cell membranes in the other insect tissues
(Emery et al., 1998
). And,
finally, the overall ion balance in insects is maintained by the
gastrointestinal system including the hindgut and Malpighian tubules
(Maddrell and O'Donnell, 1992
;
Zeiske, 1992
). Here again,
ion-pumping ATPases coupled to various secondary ion transporters represent
the most important structural components
(Schweikl et al., 1989
;
Wieczorek et al., 1989
,
2000
;
Zeiske, 1992
). We found that
P. apterus adults of three different acclimation groups differed
significantly in all three aspects (recovery from chill-coma, ion gradients
across cell membranes, function of hindgut). The most chill-tolerant insects
(SDA group) showed the most rapid recovery from chill-coma, the best capacity
to maintain ion gradients across cell membranes and the highest level of ion
regulation into the hindgut. Thus, we suggest that the impaired function of
ion pumping systems together with the inability to prevent/restrict ion
leakage down the electrochemical gradient might be an important cause of
chilling-injury and pre-freeze mortality in non-diapause or non-acclimated
P. apterus adults.
To our knowledge, the ability of cold-hardy overwintering insects to
maintain ion gradients in a supercooled state has so far been reported only
briefly by Dissanayke and Zachariassen (1980) and Hanzal et al.
(1992) but without studying
the changes in non-acclimated specimens. It was also shown that freezing of
water in haemolymph of the wood fly Xylophagus cinctus at
10°C caused loss of function of the ionic pumps and rapid movements
(within a few hours) of Na+, K+ and Mg2+ to
electrochemical equilibrium across the cell membranes
(Kristiansen and Zachariassen,
2001
). There are examples of the effect of low temperature on ion
balance in other organisms. Prolonged hypothermic exposure in non-adapted
mammals may lead to dissipation of ion gradients across cell membranes,
partial membrane depolarization, the opening of voltage-dependent
Ca2+ channels and the influx of Ca2+, which activates
membrane phospholipid hydrolysis in a process that ultimately leads to cell
damage (for a review, see Hochachka,
1986
). Regulation of ion pumping and maintaining ion gradients is
considered to be one of the pre-requisites for successful cold acclimation in
overwintering ectothermic vertebrates
(Cossins and Kilbey, 1989
;
Boutilier et al., 1997
;
Guppy and Withers, 1999
;
Stinner and Hartzler, 2000
).
In plants, ion leakage is commonly recognised as a serious consequence and/or
cause of chilling-injury (Lyons,
1973
; Jennings and Tatar,
1979
). Maintenance of ion gradients requires active ion-pumping,
which requires energy in the form of ATP. Hence, the whole system is dependent
on the ability of an organism to maintain regulated metabolism, where ATP use
is balanced with ATP synthesis. We found that P. apterus adults of
all acclimation groups were able to keep stable adenylate energy charge (AEC)
in their fat body cells during exposure to 5°C. The trends to
decrease total adenylate pools, however, were registered in the SD and,
especially, SDA groups. This observation suggested that the energy could be
exploited for maintenance of ion gradients in SD and SDA groups. Thus, the
lack of energy does not seem to be the cause of the failure of
Na+/K+-ATPases to maintain the Na+ gradient
in the LD group. Rather, the ability of ionic pumps to exploit the energy
might be impaired. It is a question of whether the ionic pumps were able to
function at 5°C at all. Previously, we found that the oxygen
consumption rate at 0°C ranged between 8.4 and 17.1 µl O2
h1 g1 FM in cold-acclimated diapause
P. apterus adults (
lachta
et al., 2002
). Also, the biosynthesis of polyols rapidly proceeded
at 0°C (Ko
tál et al.,
2001
). These two examples showed that metabolic functions might
operate even at temperatures very close to that used in the present study. The
ability of SD and SDA insects to increase the concentrations and enlarge the
pools of ions in the hindgut fluid when kept at 5°C also indicated
that active transport might be still functioning at 5°C. It was
suggested (Hochachka, 1986
)
that maintaining low-permeability membranes (by means of downregulation of the
ion-specific channels) could represent an alternative or additional
compensating mechanism for the impaired function of ion pumps at a low
temperature. The function of ion-transporting systems is also significantly
influenced by the lipidic environment, in which the transporter molecules are
embedded (Cornelius, 2001
;
Cornelius et al., 2001
;
Haines, 2001
). Although the
cold-acclimation-related changes in lipidic composition of membranes have been
described in several insects, including P. apterus (Hodková et
al., 1999
,
2002
;
lachta et al., 2002
),
they have not been studied directly in relation to the function of ion pumps
or transporters.
Collectively, some pieces of correlative evidence have been presented in this paper, which suggest that further study on the (in)ability of insects to maintain ion gradients across cell membranes could bring better understanding of the causes of their pre-freeze mortality and, consequently, their success during overwintering or cold exposure.
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