Metabolism of the sub-Antarctic caterpillar Pringleophaga marioni during cooling, freezing and thawing
Spatial, Physiological and Conservation Ecology Group, Department of Zoology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
* Author for correspondence (e-mail: bjs{at}sun.ac.za)
Accepted 15 January 2004
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Summary |
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Key words: freeze tolerance, critical thermal minimum, chill-coma, metabolic rate, Pringleophaga marioni
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Introduction |
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Survival of internal ice formation is generally attributed to a process of
osmotic dehydration. Ice forming in the haemolymph creates concentrated fluid
pockets that dehydrate cells and restrict ice to the extracellular spaces
(Zachariassen, 1985;
Sinclair and Wharton, 1997
;
Ramløv, 2000
), although
intracellular ice formation probably occurs in some species
(Salt, 1962
;
Lee et al., 1993
;
Wharton and Ferns, 1995
;
Davis and Lee, 2001
). During
thawing, therefore, water must be redistributed and ion gradients
re-established. In the woodfly Xylophaga cincta, re-establishing the
ion balance takes several hours
(Kristiansen and Zachariassen,
2001
). Nonetheless, there are currently no explicit data about the
cause of lower lethal temperatures in freeze-tolerant insects. Although
mechanical stress (for example, ice crystals piercing cell membranes or lethal
recrystallisation of ice) has been proposed as a cause of mortality in frozen
insects (Salt, 1961
), the
relationship between water-to-ice conversion and temperature means that the
overall proportion of water converted to ice will increase continuously with
decreasing temperatures (Zachariassen,
1985
), and most hypotheses of causes of mortality tend to hinge on
an osmotic stress threshold being crossed. These hypotheses include the
inability of individuals to re-establish ion gradients in the nervous system
and transgression of a minimum cell volume resulting in lethal Ca2+
concentrations (Meryman, 1971
;
Kristiansen and Zachariassen,
2001
).
Although the hypothesised causes of lower lethal temperature act at the
sub-organismal level, it is reasonable to assume that the results are manifest
at higher levels of organisation. However, aside from the progression of ice
formation (see Ramløv,
2000 for a summary), there is little information about processes
at the organismal level during freezing and thawing of insects. Kozhantsikov
(1938
) suggested that
cold-hardy insects are those that are able to maintain a detectable level of
metabolism below 0°. This idea was challenged on methodological grounds by
Scholander et al. (1953
), who
pointed out that Kozhantsikov heated his animals to above 0°C to perform
measurements. Scholander et al.
(1953
) and Salt
(1958
) both showed that the
rates of change of metabolic rates with temperature (i.e. Q10) in
frozen and supercooled insects were extremely high at sub-zero temperatures
and that the rate of change in metabolic rate increases logarithmically as the
0° threshold is crossed. In later work, Zachariassen et al.
(1979
) directly addressed the
manifestation of freezing injuries by investigating the post-thaw metabolism
of freeze-tolerant Eleodes blanchardi beetles exposed to lethal and
sub-lethal temperatures. They concluded that metabolic rate was elevated after
freezing. Moreover, whole-organism metabolic rate did not differ between those
beetles that survived and those that were killed by freezing, suggesting that
(at least initially) mortality does not occur at the cellular level. Most
recently, Block et al. (1998
)
investigated the metabolic rates of freeze-tolerant larvae and adults of the
perimylopid beetles Hydromedion sparsutum and Perimylops
antarcticus on sub-Antarctic South Georgia. They found that, while
metabolic rates of both species did not differ pre- and post-chilling
(exposure to non-freezing sub-zero temperatures), metabolic rates of both
larvae and adults of P. antarcticus were significantly higher after a
brief freezing event but did not change after freezing in H.
sparsutum. They concluded that the elevated metabolic rate of the former
species is indicative of a rapid repair of slight freeze-induced injuries that
explains its greater cold tolerance but did not investigate the effects of
freezing injury or mortality within the frozen beetles.
All five of the preceding studies were conducted using closed-system
respirometry, which integrates instantaneous rate values over time periods
that are often prolonged. Therefore, this method generally does not reveal
activity on the part of the study animal nor does it provide the fine
resolution required to investigate short-term temporal variation in metabolic
rate (Lighton, 1991;
Addo-Bediako et al., 2002
). By
contrast, open-flow respirometry has the advantage that it enables the
investigation of rapid, real-time metabolic responses to thermal and other
stressors. To date, only Irwin and Lee
(2002
) have used open-flow
respirometry to compare the metabolic rate of frozen and supercooled insects.
They examined the larvae of the goldenrod gallfly Eurosta
solidaginis, concluding that the metabolic rate of frozen larvae was
lower than that of supercooled larvae, although they did not present metabolic
data from during the freezing, thawing or post-thawing processes. Here, we use
open-flow respirometry to explore the metabolic changes that occur during
cooling, freezing and thawing, using the sub-Antarctic caterpillar
Pringleophaga marioni as a model freeze-tolerant organism, and
compare metabolic patterns in individuals exposed to lethal and non-lethal
freezing stress.
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Materials and methods |
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Pringleophaga marioni larvae were collected from abandoned albatross nests in the vicinity of the Research Station on Marion Island during the April 2002 relief voyage. Caterpillars were placed in groups of five in 250 ml plastic jars filled with nest material and were returned under refrigeration to the laboratory in Stellenbosch within 10 days, where they were housed in the dark in a refrigerator (6.0±0.4°C) until they were used for experiments. Animals housed in this manner fed and moulted normally, and several individuals pupated.
Freezing and respirometry experiments
Caterpillars (0.202±0.016 g, mean mass ±
S.E.M., N=29) were frozen
individually in a 3 cm3 plastic respirometry cuvette. A 44-SWG
type-T thermocouple was affixed to the bottom of the cuvette's interior. The
cuvette was then placed inside a plastic bag in the bath of a Grant LTD-6
(Grant Instruments, Cambridge, UK) cooling bath controlled by a Grant PZ-1
temperature programmer. Synthetic air (21% O2, balance
N2) was scrubbed of CO2 with soda lime and of water with
silica gel and Drierite. The air flowed at 75 ml min1
(Sidetrack mass flow controller) through a narrow-diameter copper coil
(1.5 m total length) in the cooling bath before passing through the
cuvette to a LiCOR Li6262 CO2/H2O infra-red gas analyser
(IRGA). The respirometry system was controlled by DATACAN V software (Sable
Systems, Henderson, NV, USA), which also provided automatic baselining and
recorded the temperature inside the cuvette via a Sable Systems
TC1000 thermocouple thermometer. All initial analyses of CO2, water
and temperature traces were also performed with DATACAN V software. All
measurements were corrected to standard temperature and pressure and expressed
in ml CO2 h1.
At the start of each experiment, the caterpillars were removed from the
moist nest material, surface dried, weighed and quickly transferred to the
cuvette and placed in the water bath at 1.6°C to equilibrate for one hour.
Caterpillars were then cooled at 0.1 deg. min1 to the test
temperature, where they were held for 15 h (5 h for 18°C treatment,
which still resulted in 10 h frozen and always resulted in mortality)
before being rewarmed at 0.1 deg. min1 to 1.6°C, where
they were held for a further 25 h to enable the recovery from freezing
to be recorded by the respirometer. Preliminary experiments allowed us to
choose test temperatures that, under the experimental conditions, would be
survived by all the caterpillars (5.8°C, N=9), would
result in mortality of all caterpillars with recovery to a `moribund' state
(some uncoordinated movement; 6.0°C, N=8) and would kill
all caterpillars outright without any apparent recovery of movement
(18°C, N=5). After freezing, the caterpillars were weighed
again and removed to individual Petri dishes filled with nest material.
Recovery (coordinated movement and a righting response) was monitored for
seven days, after which all caterpillars were preserved in ethanol.
Data analyses
In the CO2 traces, a clear breakpoint in metabolism (b)
was visible (Fig. 1), and the
temperature at the start of this breakpoint was recorded. Freezing was evident
in the temperature traces (owing to the sensitivity of the fine thermocouple),
and the Tc the temperature immediately before the
exotherm was recorded. Freezing was also clearly discernible in the
CO2 (Fig. 1) and
water traces. Where CO2 production was averaged for metabolic rate
determination, temperature was averaged over the same period of recording.
Temperatures of crystallisation and breakpoint distributions were not
significantly different from normal, and means ±
S.E.M. are presented. All caterpillars whose
temperature dataset could yield breakpoint and Tc data
were included in these analyses, including those from preliminary experiments
to determine methods and test temperatures.
|
After baselining, mean CO2 production (ml CO2
h1) was calculated for a subset of data for each of four
stages during the freezethaw cycle: (1) the equilibration period before
freezing (24.6±1.2 min, N=16), (2) while the animal was frozen
(382.3±43.2 min, N=4 per treatment), (3) during the immediate
post-thaw period (the 34.5±2.9 min after cuvette temperature reached
1.6°, N=4 per treatment) and (4) >1 h post-thaw
(105.4±20.2 min, N=4 per treatment)
(Fig. 1). CO2
production was converted to µW, assuming a respiratory quotient of 0.72
(Withers, 1992). Minute
variations in the temperature of the IRGA chamber meant that some metabolic
rate measurements (while the caterpillars were frozen) were characterised by
regular (
30-min period) oscillations, in synchrony with small temperature
changes in the room. After we had taken steps to better regulate the IRGA
temperature, recordings where the range of metabolic rate (frozen) was greater
than the mean metabolic rate (frozen) were not used for any metabolic rate
comparisons, resulting in N=4 for each treatment for the majority of
metabolic rate comparisons (although the data files were still useable for
examination of b and Tc). Metabolic rate was
log-transformed and compared between treatments and stages of the
freezethaw cycle by a repeated-measures analysis of covariance (ANCOVA)
using the general linear model (GLM) procedure on Statistica 6.1 (Statsoft
Inc., Tulsa, OK, USA), using starting body mass of the caterpillar as the
covariate. For ease of display, metabolic rate data are presented graphically
as untransformed mass-specific data, although conclusions were drawn from the
procedures outlined above.
Q10 was calculated from the slope of a regression of
log10metabolic rate on temperature using the equation
Q10=10(10xslope)
(Cossins and Bowler, 1987).
Q10 was calculated at five points during the freezethaw
cycle: (1) during cooling before the breakpoint, (2) across the breakpoint,
(3) during cooling after the caterpillar had frozen (18°C treatment
only), (4) during rewarming between 15°C and 10°C
(18°C treatment only) and (5) during thawing (5°C to
2°C). Prior to cooling, metabolic rate was highly variable, and
relationships where the slope was non-significant (F-test,
P>0.05) were excluded from the analyses. Q10 was
compared between treatments using KruskalWallis Rank analysis of
variance (ANOVA) and between cooling and thawing using Wilcoxon's paired test
(Sokal and Rohlf, 1995
).
Water loss
Water loss rate measured by the IRGA was baselined and then integrated for
the 30-min period immediately prior to temperature decrease and for 240 min
(or as long as the trace allowed if less than this) to give total mg
H2O lost during these periods. For the post-thaw water loss, time
was standardised to 200 min to allow comparison between treatments. Water loss
was not normally distributed, despite transformation, and was compared between
treatments using a generalized linear model (GLZ) in Statistica 6.1 with
starting body mass as a covariate.
Mass loss during the course of the experiment was assumed to be entirely water (although several caterpillars did produce faecal pellets and/or vomit in the cuvette, the non-water mass of these was assumed to be negligible compared with the water lost). An ANCOVA with starting body mass as the covariate was used to compare total water lost between treatments.
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Results |
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In all treatments, metabolic rates before freezing were significantly higher than after thawing and, when frozen, caterpillars had significantly lower metabolic rates (F3,21=18.364, P<0.0001; Fig. 2). Metabolic rates measured in the first 30 min post-thaw did not differ significantly from those measured longer after thawing and did not differ between treatments (F2,7=3.624, P=0.08; Fig. 2).
|
There was no significant difference (KruskalWallis Rank ANOVA,
P>0.1) between treatments in pre-freezing Q10
(comparison only possible between 6.0°C and 18°C groups
due to noisy data; median test, 2=0.667, d.f.=2,
P=0.72), so these results were pooled. Q10 before the
breakpoint was highly variable (Table
1) but had a median of 2.20 (range 1.182.98, N=10)
whereas across the metabolic breakpoint it ranged from 4x102
to 1x105 (N=15). During cooling after the animal had
frozen, Q10 was low, as it was when the caterpillars were being
warmed between 15°C and 10°C. During thawing,
Q10 was significantly higher in the 6.0°C group
(KruskalWallis H=7.05, d.f.=2, N=11,
P=0.0294).
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Flow-through gas analysis indicated that water loss rate did not differ
between groups before freezing (Wald 2=1.42, d.f.=2,
P=0.49; Fig. 3A). The
18°C group lost considerably more body mass than the other two
groups (Wald
2=44.35, d.f.=2, P<0.0001;
Fig. 3B), and the IRGA results
suggest that this difference is attributable to the post-thaw period
(F2,18=11.313, P<0.001;
Fig. 3A).
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Discussion |
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Hosler et al. (2000) found
a steady decrease in the resting potential of flight muscle neurons of both
honey bees and Drosophila with decreasing temperature. They proposed
that the chill-coma temperature was the temperature at which the
Na+/K+-ATPase pump could no longer maintain nerve cell
polarisation to a level where action potential could be produced. Since the
contribution of transmembrane ion pumps to total metabolism is estimated to be
>55% of basal metabolic rate at normal temperatures
(Zachariassen, 1996
;
Hulbert and Else, 2000
), a
threshold temperature for pump activity is a plausible explanation for the
drop in metabolism we observed.
Because of its ecological significance, the CTmin (or
the closely comparable chill-coma temperature) has received attention in both
field and laboratory studies of several species of insects
(Chown, 2001;
Gibert and Huey, 2001
;
Kelty and Lee, 2001
;
Hoffmann et al., 2003
). For
example, Gibert and Huey
(2001
) found that the
chill-coma temperature of Drosophila differed between species and
populations from different latitudes (suggestive of natural selection) and
could be modified within those populations by developmental temperature
(indicative of developmental plasticity). Klok and Chown
(2003
) found that the
CTmin of sub-Antarctic weevils (Ectemnorhinus)
responded to acclimation, with higher acclimation temperatures resulting in
significantly higher CTmin values. Activity of the
Na+/K+-ATPase pump may be modulated to reduce energy
expenditure in response to heat shock and anoxia
(Hochachka et al., 1996
;
Wu et al., 2002
), and there is
a variety of isomeric sodium pumps produced by insects under different
environmental conditions, stages of development and tissues
(Emery et al., 1998
). This
variation could be adequate both as a substrate for natural selection and
possibly as a mechanistic explanation of acclimation responses in the
CTmin.
An alternative mechanism explaining variation in CTmin
has been proposed by Pörtner
(2001,
2002
), who suggested that the
CTmin is a consequence of a mismatch between oxygen demand
and delivery at low temperatures. However, because of the very large capacity
for oxygen delivery in tracheated insects
(Chapman, 1998
), it seems
unlikely that delivery failure sets limits to low temperature activity in
these animals (Chapman, 1998
;
C. J. Klok, B. J. Sinclair and S. L. Chown, manuscript submitted for
publication). Nevertheless, an oxygen-limited process (for example, ATP
production by mitochondria), rather than a failure in the kinetics of the
Na+/K+-ATPase pump at the membrane, would result in the
same depolarisation, so the working hypothesis presented here is not
inconsistent with Pörtner's
(2001
,
2002
) assertions. Further
investigation at the subcellular level is necessary to understand the
processes leading to the CTmin in insects.
Metabolic processes during freezing and thawing
The metabolic rate of frozen caterpillars (89.2±24.9 µW
g1) was low but not zero
(Fig. 2) and, during cooling
after freezing, showed a Q10 comparable with that measured
pre-freezing (pre-breakpoint) (Table
1). This suggests that the osmotically dehydrated cells are still
functional and that, although the tracheal volume is presumably greatly
reduced by the expansion of ice in the haemocoel, the tracheal system is still
open and continues to provide an atmospheric interface for the cells. Assuming
the animal's habitat allows access to atmospheric oxygen [see Scholander et
al. (1953) for examples of
chironomids frozen into lake ice; see Conradi-Larsen and Sømme
(1973
) for an example of
terrestrial beetles locked in ice; see Lighton
(1998
) for a discussion of the
hypoxic nature of below-ground habitats], this potentially allows the
caterpillars to respire aerobically when frozen. An alternative explanation
for this phenomenon is that the conversion of water into ice in
freeze-tolerant insects is a slow process, continuing towards an asymptote
over a period of hours (Lee and Lewis,
1985
; Ramløv and Westh,
1993
) and continuing to expand and expel CO2 from the
tracheal system. While this is probably a partial explanation, the rate of
CO2 does not attenuate to zero with increasing time frozen
(Fig. 1), suggesting that there
is a metabolic contribution to CO2 production.
The generally accepted model of the processes of freezing and thawing in
freeze-tolerant insects requires that ice formation begins in the haemocoel
and is confined to the extracellular spaces, resulting in osmotic dehydration
of cells (Zachariassen, 1985;
see Sinclair and Wharton
(1997
) for a demonstration of
this). This osmotic dehydration, and subsequent passive movement of ions,
means that water and ions must be redistributed and gradients re-established
upon thawing (Kristiansen and
Zachariassen, 2001
). It stands to reason from this model that the
freezing process is passive, but the apparent expulsion of CO2 from
the tracheal system during freezing did not allow us to test this part of the
model.
Redistribution of ions between intra- and extracellular compartments after
thawing is probably an active process taking a few hours
(Kristiansen and Zachariassen,
2001). There is a decrease in metabolic rate in P.
marioni in the four hours after thawing relative to before freezing
(Fig. 2), suggesting that the
initial redistribution of ions is not energetically expensive and that any
necessary tissue repair does not begin immediately. Joanisse and Storey
(1998
) suggest that it would
be advantageous for freeze-tolerant animals to depress post-freezing
metabolism to reduce oxidative damage caused by reactive species accumulated
while the animal is in the frozen state. The metabolic rate of Eleodes
blanchardi beetles is elevated 20 h after thawing
(Zachariassen et al., 1979
),
as is the metabolic rate of frozen Perimylops antarcticus beetles
from South Georgia (Block et al.,
1998
), and it seems that this elevation may be associated with the
repair of cellular or tissue damage sustained during the freezethaw
process. Assuming that some cellular or tissue damage is sustained by P.
marioni, metabolic rate might be expected to be elevated longer after
thawing. Measurements of free radicals and antioxidants (along with
longer-term monitoring of post-thaw metabolic rate) will be necessary to test
this hypothesis.
Causes of low temperature mortality in frozen P. marioni
Low temperature mortality in frozen cells that would otherwise survive
freezing has been hypothesised to be due to a threshold ice content resulting
in a lethal minimum cell volume (Meryman,
1971; Zachariassen et al.,
1979
; Zachariassen,
1985
). However, Zachariassen et al.
(1979
) did not find any
metabolic differences between beetles that were exposed to lethal or
sub-lethal freezing and concluded that mortality due to freezing might occur
at the organismal, rather than the cellular, level as a result of neuronal
membrane impermeability to sodium ions. Recent work by Yi and Lee
(2003
) on the gallfly
Eurosta solidaginis examined the levels of injury sustained by
different tissues at the lethal temperature of 80°C. Although nerve
tissue was not tested, there is differential mortality between tissues, with
gut tissue most tolerant to freezing and several tissues (intertegumentary
muscle, the distal segment of Malpighian tubules and haemocytes) least
tolerant, suggesting that damage to a specific tissue group such as nervous
tissue could account for mortality associated with freezing. We compared
metabolic rate and water loss in caterpillars that survived exposure to
5.8°C (which results in no mortality) to caterpillars exposed to
6.0°C (which results in moribund larvae that exhibit uncoordinated
movement but die after a few days) and 18°C (after which larvae
never show any response to stimuli). In common with Zachariassen et al.
(1979
), there was no
difference in post-freezing metabolism between the three groups
(Fig. 2), suggesting that, even
in the dead caterpillars, most cells remained intact and capable of
metabolism. The major difference between the three groups was that the
18°C group lost substantially more water than the other groups
(Fig. 3), and this water loss
is concentrated in the post-thaw period. This post-thaw water loss is likely
to be a direct consequence of a lack of muscle tone due to nervous system
disruption: thawed, dead caterpillars often vomit, and it is also likely that
the water from this, as well as from the open anus and spiracles, provides an
outlet for water loss from the caterpillar's body
(Wharton, 1985
). Immediate
water loss does not, however, provide an explanation for mortality in the
6.0°C group. The notable feature of this group is their elevated
Q10 during thawing (Table
1), which may suggest that the cellular processes associated with
recovery from freezing have been disrupted.
Conclusions
In this paper, we have used open-flow respirometry to explicitly examine
the metabolic processes associated with cooling, freezing and thawing in a
freeze-tolerant insect. We have demonstrated a pronounced decline in metabolic
rate that is not associated with freezing but is coincident with the
CTmin. This finding provides a plausible link between the
CTmin and mechanistic changes at the cellular and
whole-organism levels. It also indicates ways in which natural selection might
alter mechanisms at the cellular level, so giving rise to environmental
variation in the CTmin. We have also shown that frozen
caterpillars continue to respire when frozen, but we found no evidence that
either freezing or thawing were active metabolic events. Moreover, there were
no significant differences in metabolic rate between caterpillars that were
and were not killed by freezing, suggesting that mortality from freezing
occurs at an organismal, rather than cellular, level.
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Acknowledgments |
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References |
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