Metabolic responses to cold in subterranean crustaceans
1 Ecologie des Hydrosystèmes Fluviaux, UMR CNRS 5023,
Université Lyon 1, 69622 Villeurbanne Cedex, France
2 Fonctionnement des Ecosystèmes et Biologie de la Conservation, UMR
CNRS 6553, Université Rennes 1, 35042 Rennes Cedex, France
3 Physiologie des Régulations Energétiques, Cellulaires et
Moléculaires, UMR CNRS 5123, Université Lyon 1, 69622
Villeurbanne Cedex, France
4 Osmoadaptation et Métabolismes de Stress, UMR CNRS 6026,
Université Rennes 1, 35042 Rennes Cedex, France
* Author for correspondence (e-mail: julien.issartel{at}univ-lyon1.fr)
Accepted 7 June 2005
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Summary |
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Key words: hypogean crustacean, cold hardiness, free amino acid, trehalose, life history
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Introduction |
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Although the literature on insect cold hardiness is overwhelming, very few
studies deal with cold adaptations in crustaceans. An accumulation of
trehalose and myo-inositol was found in the terrestrial isopod Porcellio
scaber (Tanaka and Udagawa,
1993). Moreover, the FAA composition is known to widely vary with
season in this taxon (Graney and Giesey, 1986). Alanine, arginine, leucine and
glycine are the major FAAs found in crustacean hemolymph and, together with
other amino acids, are involved in several metabolic processes including
protein synthesis/catabolism, gluconeogenesis and oxidative pathways
(Graney and Giesy, 1986
).
However, their roles in crustacean cold tolerance are still obscure.
In a previous study, Issartel et al.
(2005) undertook a comparative
study on the behavioural, ventilatory and respiratory responses in hypogean
(i.e. subterranean) and epigean (i.e. surface-dwelling) crustaceans when
exposed to different temperatures. Subterranean environments (mainly porous
and karstic aquifers) are basically energy- and oxygen-poor habitats, and
several complex behavioural and physiological adaptations to hypoxia (Hervant
et al., 1995
,
1996
,
1997b
,
1999a
;
Malard and Hervant, 1999
) and
food shortage (Hervant et al.,
1997a
;
1999b
;
Hervant and Renault, 2002
)
were previously found in hypogean crustaceans. Moreover, such biotopes are
also characterised by an extreme thermal stability
(Ginet and Mathieu, 1968
), and
hypogean organisms should theoretically be classified as stenothermal species
(Huey and Kingsolver, 1989
;
Angilleta et al., 2002). Unexpectedly, an opposite conclusion was found in the
aquatic subterranean crustacean Niphargus rhenorhodanensis. Indeed,
this species exhibited eurythermal characteristics (from 2 to
28°C), with particularly high survival times and a large capacity to
maintain its metabolism at cold temperatures
(Issartel et al., 2005
).
In the present study, we focused on the cold hardiness of N. rhenorhodanensis. We investigated the changes in polyol, sugar and free amino acid contents in the aquatic hypogean crustaceans N. rhenorhodanensis and N. virei and in a morphologically close aquatic epigean aquatic crustacean Gammarus fossarum acclimated at 12°C, 3°C and 2°C. We thus tried to determine whether biochemical mechanisms are involved in N. rhenorhodanensis cold-hardiness and if such mechanisms may also be found in another aquatic subterranean species.
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Materials and methods |
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Sample preparation
For each experimental condition, 1015 groups, each of three animals,
were weighed before being lyophilised for 6 h. Food was removed from
experimental tanks one week before sampling the animals to ensure that the
presence of food in the gut would not affect the results.
Metabolite extraction
Amino acids, sugars and polyols were extracted from dry material. Groups of
three animals were homogenised in 1.5 ml of 70% ethanol and Fontainebleau
sand, before adding 1.5 ml of 40% ethanol. The homogenate was centrifuged for
10 min at 4500 g and 4°C, and the supernatant collected.
The first pellet was re-suspended in 1.5 ml of 70% ethanol and centrifuged for
10 min at 4500 g and 4°C, and the supernatant collected.
The second pellet was re-suspended in 1.5 mlultrapure water and centrifuged
for 10 min at 4500 g and 4°C. The combined supernatant
(N=3) was pooled in a balloon flask and dried by evaporation using a
rota-vapor system. The insoluble residue was re-suspended in 1 ml of ultrapure
water. Samples were stored at 80°C until needed for metabolite
assays.
Analytical procedure
Free amino acids assay
Free amino acids were assayed as described by Bouchereau et al.
(1999). Amino acids were
characterized and quantified by HPLC after pre-column derivatization with
6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC) (using a Waters
Accq-Tag amino acid analysis system; Waters Corporation, Milford, USA) and
reversed-phase liquid chromatographic separation (see
Bouchereau et al., 1999
for a
full description of the method). 20 µl aliquots of the crude aqueous
extracts were assayed using the procedure optimised by Cohen and Michaud
(1993
).
Polyols and sugars assay
Derivatization was achieved according to Adams et al.
(1999). A known volume of
supernatant was transferred in a capped vial and lyophilised (12 Pa,
40°C). The dried residue was resuspended in pyridine containing
hydroxylamine (30 mg ml1). The solution was then heated at
75°C for 30 min, which allows conversion of sugars to their oximes. Sugar
oximes and polyols were converted to their TMS (trimethylsilane) derivatives
by addition of a mixture of HDMS (hexamethyldisilazane) containing
trifluoroacetic acid (10:1, v/v) and then sonicated at 50°C for 30 min
before being heated at 100°C for 60 min. 1 µl of this solution was then
injected into a gas chromatograph (model; Thermo Quest Trace GC 2000 series;
Milan, Italy) equipped with a 30 m HP-1 capillary column with 0.25 µm film
thickness. Injection was performed in the split mode (30:1) at 260°C.
Hydrogen was used as the carrier gas at a flow rate of 1 ml
min1. The HP-1 column was held in an oven at an initial
temperature of 60°C for 2 min and then heated to 150°C at a rate of 20
deg. min1 and to 300°C at 6 deg. min1
and finally held at this temperature for 20 min. The temperatures of the
injector and detector were kept at 260°C and 300°C, respectively.
Calibration plots were constructed using external standards, and compounds
were identified on the basis of retention time.
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Results |
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In the subterranean crustacean N. rhenorhodanensis (Fig. 2), arginine (18.36±1.11 µmol g1 fresh mass), alanine (8.32±0.56 µmol g1 fresh mass) and lysine (7.75±0.72 µmol g1 fresh mass) also constitute the three major FAAs in control animals. A decrease in the acclimation temperature induced a significant increase of the total FAA content by 23% at 3°C and 68% at 2°C (Table 1). At 3°C, six of the 17 FAAs detected in N. rhenorhonanensis increased: glycine and alanine rose significantly by 95% and 48% (P<0.01), respectively, while proline, valine and phenylalanine content also increased significantly, albeit less markedly (P<0.05). When N. rhenorhodanensis was acclimated at 2°C, all FAA body contents, with the exception of aspartate, glutamate, proline, threonine and lysine, were significantly higher (P<0.05) than in the controls and/or 3°C-acclimated individuals.
At the control temperature, the subterranean crustacean N. virei (Fig. 3) showed high body levels of arginine (26.63±3.50 µmol g1 fresh mass), glutamine (14.77±1.62 µmol g1 fresh mass) and alanine (5.68±0.76 µmol g1 fresh mass) compared with the other FAAs. No significant change in the total FAA content was observed after an acclimation at 3°C (Table 1). Glycine and alanine contents increased consistently (P<0.05), by 68% and 119%, respectively, with the decrease in acclimation temperature to 3°C.
Sugars and polyols
Among all sugars and polyols quantified by the method described, trehalose
was the only one that accumulated in crustaceans that had been cold-acclimated
(see Table 2). In G.
fossarum, no difference in the trehalose body content was observed
between groups acclimated at 12 and 3°C. However, the trehalose level was
found to be five times higher at 2°C than in control organisms
(P<0.01).
|
In N. rhenorhodanensis acclimated at 3°C and 2°C, trehalose values were 6-(P<0.01) and 12-fold higher, respectively, than in control individuals.
Finally, no variation in trehalose was observed in the hypogean N. virei between the control and 3°C acclimation temperature.
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Discussion |
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The three species investigated responded differently after being cold
acclimated: the subterranean crustacean N. rhenorhodanensis was the
only one showing a significant rise in the total FAA pool (see
Table 1). Such an accumulation
of FAAs during cold acclimation has previously been shown in insects
(Zachariassen, 1985; Fields et al.,
1998) and is believed to play a major role in cold hardening.
Increasing levels of proline, alanine and glycine seem to be the common
feature accompanying insect acclimation to cold
(Hanzal and Jegorov, 1991
;
Storey et al., 1993
).
Interestingly, alanine and glycine were largely accumulated in the hypogeans
N. rhenorhodanensis and N. virei during cold exposure,
whereas glycine level did not change in the surface-dwelling G. fossarum.
In vitro experiments have demonstrated that glycine and alanine act as
cryoprotectants by stabilizing enzymes and by preserving their activity at
cold temperatures (Carpenter and Crowe,
1988
; Carpenter et al.,
1990
). Thus, alanine and glycine may play a similar cryoprotective
function in both hypogean crustaceans during cold acclimation. Moreover,
glutamine and arginine were also significantly accumulated at 2°C
in N. rhenorhodanensis; as a result, we hypothesise that they play a
possible role in the cold hardening of this crustacean. Although the
implicated role of such FAAs has never been shown in cold-hardy ectotherms
before, Anchordoguy et al.
(1988
) suggested that amino
acids containing positively charged amine groups in their side chain, such as
arginine and glutamine, minimize membrane disruption by interacting directly
with negatively charged membrane phospholipids. Regarding these data, obtained
in vitro with artificial membranes, the authors suggest that these
two FAAs prevent the close apposition of two bilayers during low-temperature
exposures. However, the cryoprotective role of such complex side-chain amino
acids in in vivo conditions remains to be explored more deeply.
Furthermore, FAAs such as arginine play an important role in metabolism. As a
result, the accumulation of such molecules could also result from an
alteration (induced by low temperatures) of metabolic pathways
(Fields et al., 1998
).
Proline, which is generally found in large amounts in cold-exposed insects,
occurred at low levels in N. rhenorhodanensis, N. virei and G.
fossarum regardless of temperature. This suggests that this FAA is not as
essential as it is in insects for energy metabolism
(Auerswald and Gäde, 1999;
Yi and Adams, 2000
;
Gäde and Auerswald, 2002
)
or cold hardiness: a positive correlation has been found between proline level
and cold acclimation in insects (Hanzal
and Jegorov, 1991
; Fields et
al., 1998
). The constant low level of proline during temperature
decrease is surprising, as this amino acid increased markedly in several
stressed ectotherms (Danks,
2000
; Ramlov, 2000).
Among all sugars detected, trehalose was the only one that accumulated in
cold-acclimated crustaceans. Thus, with decreasing temperature, the hypogean
N. rhenorhodanensis gradually increased its trehalose level, reaching
the maximum concentration previously observed in cold-acclimated insects
(Fields et al., 1998). The
trehalose body content of the epigean G. fossarum was found to
increase only at 2°C, and no variation was observed in the hypogean
N. virei. An increasing trehalose content has already been detected
in cold-acclimated terrestrial crustaceans (but never in aquatic or hypogean
ones, as far as we know), as in the overwintering isopod Porcellio
scaber (Tanaka and Udagawa,
1993
). This organism inhabits a cold buffered environment below
the ice during winter, and its trehalose level increases consistently when the
temperature goes down to 0°C. Trehalose is widely recognized as a
compatible solute: it has been identified as a membrane and protein protectant
under desiccating conditions and thermal stress in a variety of organisms
(Crowe, 1998; Ring and Danks,
1998
; Fields et al.,
1998
). No variation in body water content was found in the studied
animals during these experiments, indicating that the trehalose rise measured
in cold-acclimated crustaceans is not due to desiccation stress.
In vitro experiments showed that this sugar appears to (1)
interact with polar head groups of membrane lipids to stabilize the bilayer
structure (Rudolph and Crowe,
1985) and (2) stabilize proteins by replacing the extensive shell
of water molecules around them and thus maintain their tertiary structure
(Carpenter and Crowe, 1988
;
Carpenter et al., 1990
).
In a previous study, Issartel et al.
(2005) found that N.
virei showed survival times (Lt50) of 2 days at 2°C
whereas G. fossarum and N. rhenorhodanensis had
Lt50 values of 21 and 55 days, respectively. The present study
clearly demonstrated that the distinct physiological responses exhibited by
the three crustaceans during cold exposure appeared to be correlated with
their survival at cold temperatures. N. rhenorhodanensis, which
combined high durations of survival at low temperatures and significant
accumulations of alanine, glycine and trehalose, may therefore be classified
as a cold-hardy crustacean, although it never experiences temperature
variations in its natural environment. We should hypothesise that these
elevations of FAAs and trehalose measured in cold-acclimated N.
rhenorhodanensis probably remain too low to involve a depression of the
supercooling point (Storey,
1997
). N. virei, which showed a very low survival time at
2°C, moderate levels of glycine and alanine, and no change in its
trehalose content, may be classified as a cold-susceptible crustacean. The
epigean G. fossarum showed an intermediate pattern, with significant
accumulations of FAA (mainly alanine) and trehalose (only at
2°C).
In subterranean biotopes, temperature is strongly buffered and generally
shows an annual variation of less than 1°C
(Ginet, 1960). Consequently,
the N. rhenorhodanesis cold hardiness does not make any ecological
sense in the present climatic conditions. However, the mechanisms pointed out
in our study may result from the biogeographic history of this species. It is
well established that glaciations represent one of the most important factors
explaining present hypogean species distribution
(Ginet, 1988
). In France,
N. rhenorhodanensis and N. virei are presently found inside
and outside the Pleistocene glaciation areas, respectively
(Ginet and Juberthie, 1987
).
Some biogeography studies provide proof of a sub-glacial survival and/or a
post-glacial recolonization of some subterranean amphipods in Europe and North
America (for a review, see Proudlove et
al., 2003
). Thus, we assume that N. rhenorhodanensis may
have survived the Pleistocene glaciations in refugium-habitats at the outskirt
of the glaciers, in a mixture of cold glacial water and cool groundwater, and
may have subsequently recolonized subterranean biotopes that were formerly
covered by the ice using river corridors. Such survival and recolonization
could have selected a eurythermal profile with high efficiency at cold
temperatures. After the last quaternary deglaciation, N. virei might
have shown a lower individual variability than N. rhenorhodanensis,
and thus no selection of any eurythermal profile occurred in this species. As
a result, N. virei's current distribution outside glaciation areas
may be explained by its inability to cope with cold temperatures.
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Acknowledgments |
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