Analysis of monoamines, adenosine and GABA in tissues of the land snail Helix lucorum and lizard Agama stellio stellio during hibernation
1
Laboratory of Animal Physiology, Department of Zoology, Faculty of
Biology, School of Science, Aristotle University of Thessaloniki, Thessaloniki
54006, Greece
2
Laboratory of Zoology, Department of Zoology, Faculty of Biology, School
of Science, Aristotle University of Thessaloniki, Thessaloniki 54006,
Greece
3
Department of Neurology, Athens National University, Aeginition Hospital,
74 Vas. Sophias Avenue, Athens 11528, Greece
* Author for correspondence (e-mail: michaeli{at}bio.auth.gr )
Accepted 22 January 2002
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Summary |
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Key words: land snail, Helix lucorum, lizard, Agama stellio stellio, neurotransmitter, hibernation, metabolic depression
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Introduction |
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In addition to GABA, some evidence seems to indicate a contribution of
adenosine to metabolic depression in vertebrates and invertebrates. Adenosine
is an inhibitory neuromodulator in the vertebrate brain, which decreases
neuronal excitability as well as neurotransmitter release. It has been
reported that adenosine is causally involved in the depression of neural
activity during entrance into hibernation
(Pakhotin et al., 1993;
Wang, 1993
;
Spangenberger et al., 1995
).
Moreover, adenosine reduces the rate of oxygen consumption in the marine
invertebrate Sipunculus nudus
(Reipschlager et al., 1997
)
and modulates monoamine release from the nervous system of the marine bivalve
Mytilus edulis (Barraco and
Stefano, 1990
). In anoxia-tolerant vertebrates, adenosine is
released in their brain during anoxia and might be involved in the metabolic
depression and anoxic survival of the brain
(Nilsson and Lutz, 1992
;
Lutz and Kabler, 1997
;
Pek and Lutz, 1997
).
Another group of neurotransmitters that seem to be implicated in metabolic
depression are the monoamines, serotonin (5-HT), dopamine (DA) and
norepinephrine (NE). The role of these monoamines in promoting metabolic
depression has been studied, especially in the anoxic brain of turtles and
crucian carp. The levels of 5-HT and NE are maintained in the brains of
turtles and crucian carp during extended periods of anoxia and they might
contribute to anoxic survival (Nilsson,
1989a,b
;
Nilsson et al., 1991
).
The role that neurotransmitters might play in promoting metabolic depression in several species during hibernation is not fully understood, nor is it well known whether these neurotransmitters could promote metabolic depression in vertebrates and invertebrates during hibernation. This prompted us to determine the levels of monoamines GABA and adenosine in the brain, heart and blood of the hibernating land snail Helix lucorum and the lizard Agama stellio stellio. These two species were chosen for this work as they both are found in northern Greek habitats facing the same environmental conditions. Also, their responses to harsh environmental conditions of winter are similar as they both hibernate buried into the ground for similar periods of time.
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Materials and methods |
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Animals and induction of hibernation
According to its biological cycle, Helix lucorum L. enters
hibernation at the end of October and is aroused at the end of March
(Staikou et al., 1988). Thus
adult specimens of Helix lucorum (largest shell diameter ranged
between 38 and 42 mm) were collected at the end of September 2000, in the
vicinity of Edessa, in northern Greece. The snails were kept in an active
state at a temperature of 25±0.5 °C and subjected to a 10.00 h:
14.00 h L:D photoperiod in large glass boxes, with a daily supply of lettuce
leaves and water. High humidity (85±1 %) was maintained by sprinkling
the interior of the boxes with water every day. To induce hibernation, the
animals were put into a cool room where the temperature was adjusted to 5
°C and exposed to the 10.00 h: 14.00 h L:D photoperiod. The animals kept
under these conditions are referred to as hibernating, because these
conditions are equivalent to the natural state. Hibernation started at the end
of October 2000 and lasted for 4 months. At the end of each month, samples of
hibernating animals were used for tissue sampling and analysis of
neurotransmitters.
The lizard Agama stellio stellio L. begins to enter hibernation
between late September and mid-October and emerges at the beginning of March
(Loumbourdis, 1984). Thus,
active animals were collected in mid-September 2000 and kept in an active
state at a temperature of 25±0.5 °C and subjected to a 10.00 h:
14.00 h L:D photoperiod in large glass boxes. Hibernation, which commenced in
late October 2000, was induced in a similar way to that described for the
snails and lasted for 4 months. At the end of each month, samples of
hibernating animals were used for tissue sampling and analysis of monoamines,
GABA and adenosine.
Tissue sampling and preparation of homogenates
After hibernation periods of 1, 2, 3 or 4 months, snails were decapitated,
haemolymph collected and brains (the circumesophageal ganglia) and hearts
dissected out, immediately frozen in liquid nitrogen and kept at -80 °C.
Similarly, after hibernation periods of 1, 2, 3 or 4 months, lizards were
decapitated, blood collected and brains and hearts rapidly removed, frozen in
liquid nitrogen and kept at -80 °C. 500 µl of haemolymph and blood were
mixed with 200 µl of ice-cold perchloric acid (PCA) containing 0.2 % EDTA
and 0.05 % sodium bisulfite and then centrifuged at 20 000 g
for 10 min. The supernatants were collected and used for analysis of
monoamines, adenosine and GABA. The brains and hearts from the snails and
lizards were homogenized in 4 % (w/v) ice-cold PCA containing 0.2 % EDTA and
0.05 % sodium bisulfite using a PotterElvehjem homogenizer
(Nilsson, 1990). The
homogenates (6 % w/v) were centrifuged (20 000 g for 10 min)
and the supernatants stored at -80 °C for 3 days for monoamine and
adenosine analysis and for several weeks for GABA analysis.
Brains, hearts and blood and haemolymph from active snails and lizards were treated as described above and used as controls.
Determination of monoamines
The monoamines tested were serotonin (5-HT) and its main metabolite
5-hydroxyindole-3-acetic acid (5-HIAA), dopamine (DA) and its metabolites
dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), norepinephrine
(NE) and epinephrine (E). The amounts of monoamines and monoamine metabolites
were quantified using reverse-phase ion-pair high-performance liquid
chromatography (HPLC), with electrochemical detection similar to the method
described by Reipschlager et al.
(1997). In brief, the system
for HPLC consisted of a Shimadzu solvent delivery unit with a double-plunger
reciprocating pump (LC-9A; Kyoto, Japan), a sample-injector fitted with a 20
µl loop (Model 7125; Reodyne, Cotati, CA, USA) a reverse-phase column
(C18 Nucleosil 120, 4.6x75 mm, 3 µm diameter,
Macherey-Nagel, Duren, Germany), thermostated to 40°C and an
electrochemical detector (model 141 Gilson, Middleton, WI, USA). The mobile
phase was prepared as described by Reipschlager et al.
(1997
) and it consisted of 100
mmol l-1 NaH2PO4, 0.5 mmol l-1
EDTA, 30 mg l-1 sodium octylsulfate and 6% methanol, pH 3.7, and
the flow rate was 1 ml min-1. The working electrode of the
electrochemical detector was a small disc of glassy carbon (3 mm diameter) and
the electrode potential was maintained at +750 mV set against an AgAgCl
reference electrode. The electrochemical detector was linked to a PC Pentium
IBM-compatible computer via a 14-bit AD-DA card. Standards were
dissolved in H2O and diluted 1:1000 with 4% (w/v) PCA containing
0.2% EDTA and 0.05% sodium bisulfite.
Determination of adenosine
Adenosine was determined in a similar way to that described by Reipschlager
et al. (1997) using HPLC
(isocratic) with spectrophotometric detection. In brief, a reverse-phase
column (C18 Nucleosil 120, 4.6 mm x 75 mm, 3 µm diameter,
Macherey-Nagel, Duren, Germany) was used and the mobile phase consisted of 10
mmol l-1 NaH2PO4, 0.25 mmol l-1
EDTA and 6 % methanol, pH 6.5, at a flow rate of 1 ml min-1. The
concentrations of adenosine in the homogenates were quantified by comparison
with standards prepared in the same neutralized PCA solution as the
extracts.
Determination of GABA
GABA levels were determined by reverse-phase HPLC (isocratic) with
fluorescence detection after derivation with o-phthaldialdehyde
(OPA), according to the method used by Kamisaki et al.
(1990), after modification. A
Symmetry C8 column, 4.6 mm x 250 mm and 5 µm diameter was
used. Excitation was set at 340 nm, and emission was measured at 456 nm. The
mobile phase consisted of sodium citrate, acetonitrile and methanole
(590:320:90 v/v) and the flow rate was set at 1.0 ml min-1. The
derivatization reagent was prepared by mixing 100 mg OPA, 2 ml methanol, 8 ml
borate buffer (pH 10.45) and 200 µl 2-mercaptoethanol. 60 µl of the
supernatant obtained after PCA extraction and centrifugation were mixed with
an equal quantity of derivatization reagent. After 2 min, 100 µl of the
mixture were injected into the sample loop and the run started at the same
time. Concentrations were calculated by comparison with standards prepared in
the same PCA solution as the extracts.
Statistical analysis
The results are presented as means ± S.E.M. Significance of
difference was tested with Bonferroni's test, which takes into consideration
multiple comparisons. The limit of significance was set at
P<0.05.
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Results |
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5-HT and DA were the only monoamines detected in the heart of active H. lucorum. The concentration of 5-HT was found to be at the same level (2.95±0.34 µg g-1 wet mass) as that measured in the brain, while the level of DA was much lower (0.27±0.032 µg g-1 wet mass) than that measured in the brain of active snails. During hibernation, changes in the levels of 5-HT were similar to those observed for the brain. Specifically, the concentration of 5-HT increased to 4.32±0.30 µg g-1 wet mass within the first 2 months, followed by a decrease thereafter (Fig. 2A). In contrast, a significant decrease in the level of DA was observed by the fourth month after the onset of hibernation (Fig. 2B).
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The concentration of adenosine and the changes in its levels in the brain and heart of hibernating H. lucorum are shown in Fig. 1E and Fig. 2C, respectively. As indicated, levels of adenosine decreased from 0.57±0.05 µg g-1 wet mass to 0.19±0.04 µg g-1 wet mass in the brain by the fourth month of hibernation (Fig. 1E), while the levels of adenosine did not change statistically in the heart of hibernating snails (Fig. 2C).
GABA was detectable only in the brain of active H. lucorum and its concentration was found to be 0.35±0.054 µg g-1 wet mass. Hibernation did not cause any significant change in its level. However, none of the neurotransmitters examined was detected in the haemolymph of active and hibernating snails.
Concentrations of monoamines, GABA and adenosine in the brain,
heart and blood of A. stellio stellio
As in H. lucorum, 5-HT and DA and their metabolites 5-HIAA and HVA
were detected in the brain of active lizard A. stellio stellio. In
contrast to H. lucorum, however, both NE and E and DOPAC, the
metabolite of DA, were found in the brain of A. stellio stellio.
Hibernation caused significant decreases in the levels of 5-HT and 5-HIAA. The
concentration of 5-HT decreased from 1.67±0.21 µg g-1 wet
mass to 0.25±0.032 µg g-1 wet mass and that of 5-HIAA
from 0.14±0.024 µg g-1 wet mass to 0.028±0.005
µg g-1 wet mass by the fourth month
(Fig. 3A, B, respectively). As
with 5-HT, the levels of DA decreased from 0.29±0.03 µg
g-1 wet mass to 0.042±0.006 µg g-1 wet mass by
the fourth month of hibernation (Fig.
3C), while the principal metabolites of DA, HVA and DOPAC, were
not detectable in the brain of hibernating lizards. The concentration of NE
measured in the brain of active lizards was higher (0.67±0.064 µg
g-1 wet mass) than that of DA (0.29±0.03 µg
g-1 wet mass). Within the first 2 months of hibernation, the levels
of NE decreased significantly (0.27±0.032 µg g-1 wet
mass), then increased within the next 2 months, reaching a value of
0.40±0.052 by the fourth month (Fig.
3D). In contrast, the concentration of E (0.11±0.015 µg
g-1 wet mass) remained at the same levels in the brain of lizards
during hibernation (Fig.
3E).
|
As in the brain, NE was found at higher levels (0.81±0.064 µg g-1 wet mass) than DA (0.039±0.004 µg g-1 wet mass) and 5-HT (0.13±0.02 µg g-1 wet mass) in the heart of active A. stellio stellio. During hibernation, all three monoamines showed approximately the same pattern of changes in the heart as those in the hibernating brain (Fig. 4). E was below the detectable limits in the heart of active A. stellio stellio, although it could be determined in the heart of hibernating lizards (Fig. 4D).
|
Adenosine was detected in the brain and heart of active A. stellio stellio, but the level of adenosine was about twofold higher in the brain (8.23±0.42 µg g-1 wet mass) (Fig. 3F) than in the heart (3.56±041 µg g-1 wet mass) of active lizards (Fig. 4E). Hibernation caused a marked decrease in the levels of adenosine in both brain and heart of lizards.
GABA was detected only in the brain of active A. stellio stellio at 420±22 µg g-1 wet mass, which is higher than those of the monoamines examined. In contrast to hibernating snails, however, GABA levels increased markedly in the brain of hibernating lizards. As shown in Fig. 3G, the levels of GABA increased fivefold in the brain within the first 2 months of hibernation (from 420±52 µg g-1 wet mass to 2.198±302 µg g-1 wet mass) and they remained high, reaching levels of 1.984±256 µg g-1 wet mass of brain in the fourth month.
In contrast to H. lucorum, NE, DA, E and 5-HT were detected in the blood of active A. stellio stellio and their levels were 0.15±0.023, 0.33±0.045, 0.018±0.0013 and 0.92±0.12 µg ml-1, respectively. During hibernation, the levels of NE, DA and 5-HT decreased significantly in the blood, while that of E increased (Fig. 5).
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Discussion |
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From our results presented here, DA does not seem to play any key role in
regulating hibernation in H. lucorum. The levels of DA remained
stable in the brain (Fig. 1B),
and no significant changes were observed in the levels of the DA metabolite
HVA (Fig. 1D). In contrast, the
levels of 5-HT increased by the second month, followed by a significant
reduction thereafter (Fig. 1A).
Moreover, the pattern of changes in 5-HIAA levels is similar to that of 5-HT
(Fig. 1C). It should be pointed
out, however, that these data do not necessarily imply an increase in
extracellular levels of 5-HT. The modulatory effects of 5-HT at peripheral and
central synapses during hibernation remain undetermined. Nevertheless, the
changes in the levels of 5-HT in the brain of hibernating H. lucorum
agree well with other data indicating an activation of serotonergic neurons in
the land snails during the first months of hibernation and a reduction in the
activity of these neurons thereafter. Specifically, biochemical measurements
have shown higher 5-HT content in the CNS of H. pomatia during the
first months of hibernations (Hiripi and
Salanki, 1973). Moreover, immunocytochemical studies have shown
that the number of serotonergic neurons changes in a seasonal manner in the
land snails, a smaller number being found during winter
(Hernadi et al., 1989
;
Bernocchi et al., 1998
). As
shown in previous studies, 5-HT is an excitatory agent
(Jones, 1983
) and enhances the
glycolytic rate in some tissues of land snails
(Michaelidis and Vasiliou,
1997
). Thus changes in 5-HT levels, depending on seasonal
conditions, might influence the rate of several physiological processes, in
this way contribute to metabolic depression in the hibernating land snails.
However, at peripheral sites in the snail, 5-HT is not always excitatory and
acts as a relaxing agent, e.g. in the penis retractor muscle of H.
pomatia (Wabnitz and Von Wachtendonk,
1976
).
In some hibernating vertebrates 5-HT seems to play a pivotal role in the
central neural control of hibernation. Brain 5-HT levels increased during
hibernation in a number of mammalian species, including the hedgehog
(Uuspaa, 1963), the ground
squirrel (Kudryavtseva and Popova,
1973
) and the Syrian hamster
(Novotna et al., 1975
). In
addition, the level of 5-HIAA was higher in some hibernating animals in
winter, suggesting an increase in the activity of their serotonergic system
(Duncan and Tricklebank, 1978
;
Novotna et al., 1975
).
GABA was found to be at low levels in the brain of H. lucorum, and
these results agree with those reported for H. pomatia
(Osborne et al., 1972).
Previous studies reported the existence of GABA-like immunoreactivity in the
CNS of several gastropods, including Helix species
(Hernadi, 1994
;
Richmond et al., 1991
;
Hatakeyama and Ito, 2000
).
However, the role of GABA as a transmitter in snails is poorly understood,
since the enzyme glutamic acid decarboxylase, involved in the synthesis of
GABA from glutamate, is detected in the nervous system
(Bradford et al., 1969
).
Moreover, pharmacological studies of GABA action on snail neurons have shown
that GABA may produce inhibitory or excitatory effects, depending on the
concentrations applied (Gerschenfeld and
Lasansky, 1964
; Walker,
1986
; Takeuchi,
1992
; Zhang et al.,
1997
). During hibernation, the concentration of GABA remained at
the same levels in the brain of H. lucorum, indicating that GABA may
have a minor role as an inhibitory neurotransmitter in hibernating land
snails.
To our knowledge, there are have been no previous reports of the levels of
adenosine in land snails. The levels of adenosine determined in the brain and
the heart of H. lucorum are comparable to those found in the
invertebrate Sipunculus nudus
(Reipschlager et al., 1997).
It has been reported that adenosine is causally involved in the depression of
neural activity during entry into hibernation
(Pakhotin et al., 1993
;
Wang, 1993
;
Spangenberger et al., 1995
).
Also, a recent investigation showed that the levels of adenosine increase in
S. nudus during anoxia, so it might be involved in metabolic
depression since it reduces the oxygen consumption of this worm
(Reipschlager et al., 1997
).
Several lines of evidence, including the existence of all requisite
adenylate-metabolizing enzymes (Lazou,
1989
) and adenosine receptors on the neurons of land snails
(Cox and Walker, 1987
), and
the inhibition of 5-HT and DA release by adenosine from the neurons of marine
bivalve Mytilus edulis (Barraco
and Stefano, 1990
), indicate a possible modulation of the nervous
system of invertebrates by adenosine. From the results presented, it is
difficult to speculate on the mobilization of monoamine stores by adenosine in
the brain of hibernating H. lucorum. It has been suggested that the
initial increase in the levels of adenosine in the brain of anoxia-tolerant
animals during anoxia is responsible for the decrease in energy utilization
(Nilsson, 1993
). Thereafter,
GABA seems to become responsible for the maintenance of depressed energy
consumption at a later stage of anoxia. Unfortunately, it was impossible to
determine the levels of adenosine in the brain of snails during the initial
stages of hibernation, because the animals responded quickly to handling
during the first stages of imposed hibernation, so it remains unclear whether
this type of initial increase in the level of adenosine takes place in the
brain of H. lucorum in response to hibernation.
In contrast to H. lucorum, in A. stellio stellio NE was
present in the brain, heart and blood, and the pattern of monoamine changes
was different from that of hibernating snails. In addition, DA and E were
found to be at lower levels than that of NE in the brain. These results seem
to agree with the distribution of catacholamine-containing neurons in the
brain of lizards. In particular, results from microspectrofluorometric and
pharmaco-histochemical analysis showed that the fluorescent substance in the
brain of lizards is mainly NE (Baumgarten
and Braak, 1968). The levels of NE in the brain of active lizards
A. stellio stellio are comparable to those found in other lizards
such as Anolis sagrei and Uromastix aegyptius
(Nilsson et al., 1991
;
Okasha et al., 1995
). During
hibernation, the levels of DA and 5-HT decreased significantly in the brain,
heart and blood of A. stellio stellio and remained at low levels
until the fourth month. In contrast, the levels of NE decreased within the
first 3 months, followed by an increase thereafter (Figs
3,
4 and
5). In agreement with our
results, decreases in the levels of monoamines NE, DA and 5-HT have been
reported for brain and blood of the hibernating lizard U. aegyptius
(Okasha et al., 1995
). In the
latter work, it was also reported that levels of NE increased significantly in
the brain and blood of the aroused U. aegyptius.
The above data clearly indicate a lower rate of catecholamine synthesis in the brain of lizards during hibernation. The decreases in catecholamine levels could be the result of reduced temperature and may contribute to the low neuronal activity and reduction of metabolic rate that characterize hibernating lizards. For example, the reduction in NE levels in the heart of hibernating lizards (Fig. 4C) indicates lower heart activity and a consequent decrease in the energy used by the contracting myocytes. Moreover, NE as a peripheral transmitter of sympathetic nervous system controls several mechanisms of cold defense. On the other hand, the slight increase in NE levels in the brain of A. stellio stellio after the second month of hibernation might be attributable to the peripheral and central functions of NE in thermoregulation, which are very important physiologically for the preparation of animals that are arousing from hibernation. This suggestion is also based on our results, showing an increase in NE levels on the heart (Fig. 4C).
In contrast to active lizards, E was detected in the blood
(Fig. 5D) and heart
(Fig. 4D) of hibernating A.
stellio stellio. The present results seem to coincide well with those of
earlier studies, which show that the adrenal gland is inactive in the early
stages of hibernation and becomes more active as hibernation proceeds
(Agid et al., 1961;
Duguy, 1963
). Adrenaline
(epinephrine) may be implicated in the reduction of glycogen content in the
liver and muscles of lizards during hibernation, through inducing
glycogenolysis via activation of phosphorylase. It is known that
oxidation of glycogen contributes to energy maintenance in hibernating lizards
and adrenaline exerts a hyperglycemic effect on reptiles
(Akbar et al., 1978
; Coulson
and Hernadez, 1983). According to recent findings, glucose seems to play a
cryoprotectant role in reptiles that are facing a cold winter
(Constanzo et al., 1995
;
Grenot et al., 2000
). It is
also interesting that adrenaline, in parallel with its hyperglycemic effect,
reduces oxygen consumption in some reptiles
(Coulson and Hernandez, 1986
).
However, it is not known whether adrenaline contributes in this way to
metabolic depression in hibernating A. stellio stellio or in other
lizards.
Based on the present results it is difficult to make any assumptions about
the physiological role that adenosine might play in metabolic depression in
the hibernating A. stellio stellio. Adenosine levels were maintained
in the brain during the first 5 days (data not shown), but decreased markedly
during prolonged hibernation. As in H. lucorum, A. stellio stellio
responded to handling during the first hours of imposed hibernation. Thus, it
remains unclear whether adenosine acts as an initial inhibitory
neurotransmitter in the hibernating lizards as it does in anoxia-tolerant
turtles (Nilsson and Lutz,
1992; Lutz and Kabler,
1997
).
The levels of GABA determined in the brain of active A. stellio
stellio are comparable to those measured in the brain of A.
sagrei and Varagunus griseus
(Abdel Raheem and El Mosallamy,
1979; Nilsson et al.,
1991
). GABA levels increased markedly in the brain of hibernating
A. stellio stellio and remained elevated during hibernation
(Fig. 3G). Similarly, levels of
GABA increased in the brain of hibernating V. griseus, while those of
the excitatory neurotransmitter glutamate decreased
(Abdel Raheem and El Mosallamy,
1979
; Abdel Raheem and Hanke,
1980
). As reported elsewhere, GABA is the major inhibitory
neurotransmitter in all vertebrates and it is suggested that increases in the
levels of GABA could mediate metabolic depression and, thus, anoxic survival
in ectothermic as well as endothermic vertebrates
(Nilsson, 1992
;
Nilsson and Lutz, 1993
). The
results presented indicate that GABA might be involved in reducing neuronal
activity and metabolic rate in hibernating lizards. We do not know, however,
whether an increase in the levels of brain GABA necessarily means an increase
in the percentage of bound GABA molecules on the membrane receptors at low
temperatures. Moreover, further experimentation is needed to illustrate the
mechanism by which a block or activation of GABA receptors modulates
hibernation in lizards.
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