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First published online May 29, 2009
Journal of Experimental Biology 212, 1859-1868 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027987
Do current environmental conditions explain physiological and metabolic responses of subterranean crustaceans to cold?
1 Equipe `Hydrobiologie et Ecologie Souterraine', CNRS, UMR5023, `Ecologie des
Hydrosystèmes Fluviaux', Université de Lyon, Université
Lyon 1, Villeurbanne, F-69622, France
2 Equipe `Paysages-Changements climatiques-Biodiversité', CNRS, UMR6553,
`Ecosystèmes-Biodiversité-Evolution', Université de
Rennes 1, Rennes, F-35042, France
3 INRA, UMR118, `Amélioration des Plantes et Biotechnologies
Végétales', Le Rheu, F-35653, France
4 Institut Universitaire de France, Paris, F-75005, France
* Author for correspondence (e-mail: celine.colson{at}ens-lyon.org)
Accepted 26 March 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: cold hardiness, free amino acid, hypogean crustacean, thermal variation, trehalose
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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),
in which 97% of the worlds liquid freshwater can be found
(Castany, 1998). Depending on
the geological substratum, water can percolate through the limestone,
producing karsts with caves; it can also run out from faults in volcanic or
metamorphic systems (fissured habitats) or circulate in alluvial sediments
(porous habitat) (Gibert,
2001). Despite their diversity, all these subterranean biotopes
share several properties, highly reduced indigenous primary production being
one of the most important from a biological standpoint. Indeed, as sunlight
does not enter these environments, primary production through the process of
photosynthesis is absolutely impossible and thus all supplies of oxygen and
most supplies of organic matter and nutrients come from the surface. These
supplies are unpredictable (Malard and
Hervant, 1999). The lack of oxygen production and the scarcity of
nutrient supplies (Malard and Hervant,
1999) restrict the development of life and lead to the
characterisation of the subterranean biotopes as extreme environments
(Culver et al., 2004). Nearly
constant temperature is another characteristic of these environments with, in
most systems, groundwater temperature varying by only one degree around the
annual average temperature of the region
(Freeze and Cherry, 1979;
Ginet and Decou, 1977). In
these unusual environments, 7000 species are described as obligatory
groundwater inhabitants (also called stygobionts)
(Botosaneanu, 1986;
Juberthie and Decu, 1994a;
Juberthie and Decu, 1998;
Juberthie and Decu, 2001). All
of them are ectotherms, and among them crustaceans account for the most
diverse taxa, representing more than 43% of these stygobionts
(Gibert and Deharveng, 2002).
Subterranean organisms are characterized by limited dispersal abilities and
significant geographic isolation (Gibert
et al., 1994; Humphreys,
2000). As many hypogean organisms have inhabited underground
ecosystems for several million years, we expect them to be well adapted to
their biotope, their physiology reflecting selective pressure exercised by
environmental conditions.
The thermal characteristics of the habitats are of prime importance when
working on ectotherm species because they are one of the major abiotic
parameters that influence their physiology
(Addo-Bediako et al., 2000;
Chown, 2001). Several studies
have shown that the level of thermal tolerance in many organisms is
proportional to the magnitude of the temperature variation they experience in
their natural habitat (Addo-Bediako et al.,
2000; Farrell et al.,
2008; Ghalambor et al.,
2006; Pörtner,
2006; Stevens,
1989). For example, Gaston and Chown studied the thermal tolerance
range of 26 species of dung beetle along an elevation transect (approximately
2500 m) in Southern Africa (Gaston and
Chown, 1999). They showed evidence for an increase in the
temperature tolerance range of these scarabs with elevation: individuals
living at 500 m of altitude had a thermal tolerance range of 28°C whereas
organisms living at 2865 m exhibited a 38°C thermal tolerance range, the
minimum critical temperature decreasing steeply with altitude. These
functional differences can even be found between populations of the same
species, as shown for two salmon populations from the Fraser River in British
Columbia, Canada (Farell et al., 2008). Both populations displayed a large
thermal tolerance range but their optimal and upper critical temperatures
differed by 2°C and 3°C, respectively. Moreover, Peck et al. showed
that Antarctic marine species cannot survive a temperature increase of
2°C, corroborating once again the model outlined above
(Peck et al., 2004). In
groundwater habitats, where temperature is highly buffered, hypogean
crustaceans experience minimal to null thermal fluctuations. Thus, we
hypothesised that they would not exhibit any particular thermal adaptations to
cold or heat, or any particular thermal plasticity.
Yet surprisingly, Issartel et al.
(Issartel et al., 2005a;
Issartel et al., 2005b) showed
that a population of the subterranean aquatic amphipod Niphargus
rhenorhodanensis survived low-temperature exposures by developing
adaptations to cold (such as accumulation of cryoprotectants) and even
displayed an eurythermal profile as defined by Huey and Kingsolver
(Huey and Kingsolver, 1989).
These observations indicate that subterranean environments do not always
follow the above-mentioned theory, i.e. the thermal plasticity of some
subterranean species does not match the thermal characteristics of their
current natural environments. To address this hypothesis and corroborate this
counter-example, we focused on the cold-hardiness ability of seven populations
of N. rhenorhodanensis inhabiting karst environments (of all
groundwater bodies, karstic ones are the most thermally buffered) within
aquifers of similar altitude and latitude. We then investigated whether these
organisms, which usually do not endure thermal variations, can acclimate to
cold exposures and present adaptive features to low temperatures. As a side
issue, previous studies on N. rhenorhodanensis
(Issartel et al., 2005a;
Issartel et al., 2005b) were
conducted on a population inhabiting a porous environment for which the exact
yearly thermal variations are unknown
(Ginet and Mathieu, 1968;
Issartel et al., 2007).
Therefore, we here investigated whether this population suitably represents
the physiological responses of all populations of N.
rhenorhodanensis.
The three levels used to characterize a biological response to stressful
conditions, i.e. behaviour, metabolism and biochemistry
(Hochachka and Somero, 2002),
were studied at two temperatures: the mean temperature of the subterranean
aquifers considered here (10°C) and a cold temperature (3°C). The
first expected response of an organism under stress is for it to flee. As
subterranean biotopes are very fragmented
(Gibert et al., 1994), N.
rhenorhodanensis is unlikely to be able to cope efficiently with stress
using this strategy, but monitoring locomotory activity may be a good
indicator in determining whether this is an intrinsic response or not. Second,
organisms have to develop metabolic responses, by modifying their catabolism
and anabolism, to cope with new thermal conditions. This can be measured by
examining both ventilatory activity and oxygen consumption. Finally,
biochemical responses such as production and accumulation of cryoprotectants
(specific carbohydrates, polyols, amino acids) help organisms to survive cold
stress. Recently, Issartel et al. showed that trehalose was the only
carbohydrate that was accumulated in cold-acclimated crustaceans, with glucose
and glycerol being key molecules for its metabolism
(Issartel et al., 2005a;
Issartel et al., 2005b). Here,
we will thus focus our study on these three compounds, as well as on free
amino acids that Issartel et al. found to be involved in cold-acclimation in
N. rhenorhodanensis (Issartel et
al., 2005b). Moreover, since cold exposure can induce a switch
from aerobic metabolism to anaerobic metabolism
(Hochachka and Somero, 2002),
we measured lactate body content.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), we
were able to choose springs or caves between 300 and 600 m of altitude, with a
mean yearly temperature of approximately 10±1°C. To ensure the
validity of the model, we measured temperature several times in the chosen
sites. Fifty organisms were collected in each location in order to conduct all
the experiments. All known locations for the Niphargus
rhenorhodanensis species were considered and we only retained the seven
locations that allowed us to collect enough living material in a maximum of
four sampling days (Table
1).
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Animal rearing and acclimation conditions
Niphargus rhenorhodanensis Schellenberg (hypogean amphipods) were
collected from seven different subterranean areas, as described in
Table 1. In all locations,
N. rhenorhodanensis were collected by filtering water of the
resurgence spring; except for Cormoran, where individuals were caught directly
using baited traps in small pools within the cave. Individuals were kept in
aquaria placed in the dark, in thermostatted chambers as described previously
(Hervant et al., 1997). Tanks
contained mesh and leaves in order to provide the organisms with a support to
hang onto and under which to hide. Animals were fed with food for aquarium
fishes once a week (TetraRubin, Tetra, Melle, Germany). All the animals were
acclimated to laboratory conditions at 10°C (±0.3°C) for one
month before the experiments. They were then separated into two groups: the
first group was acclimated at a cold temperature (3°C) and the second
group was kept at 10°C. These two groups were acclimated at their
respective temperatures for another month. Water was changed once a week and
all physico-chemical parameters (pH, oxygen concentration, temperature) were
kept the same during acclimation and experiments. We chose to concentrate our
multi-population study on cold responses rather than on both cold and heat
responses. The rational for this was first to be able to increase the number
of replicates (subterranean crustaceans are very difficult to catch in large
quantities, and testing both responses would have meant less replicates) and
secondly to be able to compare our results to those of previously published
works. The cold temperature was determined following results obtained by
Issartel et al. (Issartel et al.,
2005b), where the authors demonstrated the freeze-tolerance of a
population of N. rhenorhodanensis (experiment at –2°C) but
also recorded clear evidence of cold response (experiment at 3°C). For the
present study, we chose the latter temperature since it enabled us to avoid
the difficulties involved in working at sub-zero temperatures.
Measurement of oxygen consumption, ventilatory and locomotory activities
Food was removed from the experimental tanks one week before sampling the
animals, to ensure that the digestive tract was empty and that an overshoot in
O2 consumption due to digestive metabolism would not affect the
results. It is noteworthy that these organisms have been shown to be highly
tolerant to fasting (Hervant et al.,
1997), which suggests that a fasting period of one week should not
affect the biological significance of our results. In order to evaluate the
whole-body metabolic response, oxygen consumption was measured on four
replicates for each population with an oxygen sensor (Oximeter Oxi 330i; WTW,
Weilheim, Germany) in special plastic respirometers of 65 ml that can be
hermetically closed. Measurements of oxygen concentration were taken at the
beginning of the experiment and 24 h later, without agitation because it
stresses N. rhenorhodanensis individuals
(Hervant et al., 1995;
Hervant et al., 1996). Four to
nine organisms were pooled together to give the same total fresh mass in each
respirometer. The temperature sensitivity of oxygen consumption was determined
using the Arrhenius relationship for the decrease in physiological rate with
temperature (Q10). This rate typically decreases two- or
threefold every 10°C for ectotherms (Q10=2–3)
(Schmidt-Nielsen, 1997).
Q10 values were calculated between 3°C and 10°C
using the Van't Hoff equation: Q10=(R2
R1)10/(T2-/T1),
where R1 and T1 represent the average
low-temperature oxygen consumption and the low temperature (3°C),
respectively; R2 and T2 are the
average high-temperature oxygen consumption and the high temperature
(10°C), respectively (Hochachka and
Somero, 2002; Schmidt-Nielsen,
1997).
Ventilatory activity was determined by recording the frequency of pleopod
(ventilatory appendages of malacostracean crustaceans) beats during 1 min as
described previously (Hervant et al.,
1997).
Locomotory activity was assessed visually by counting the number of moving animals in 1 litre tanks containing 10 individuals. During 45 min, the experimenter entered the dark thermostatted chamber every 5 min and used a very low energy red light to count moving individuals. The mean value of these 10 measurements was calculated in order to determine the percentage of individuals in movement. Great care was taken to not disturb animals (no vibration and no light stress).
Sample preparation for metabolic analysis
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. For each experimental condition, 10 pools of three organisms were
weighed before and after being lyophilised, and then stored at
–80°C. We pooled organisms in order to suppress individual
variability and maintain interpopulation variability.
Metabolite extraction
Free amino acids, sugars, lactate and polyol were extracted from
lyophilized organisms according to Renault et al.
(Renault et al., 2006). Pools
of three animals were homogenized 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 4500g and 4°C, and the supernatant was
collected. The remaining pellet was re-suspended in 1.5 ml of 70% ethanol and
centrifuged for 10 min at 4500g at 4°C, and the
supernatant was collected. The second pellet was re-suspended in 1.5 ml of
ultrapure water and centrifuged for 10 min at 4500g at
4°C. The combined supernatants (N=3) were pooled in a balloon
flask and dried by evaporation using a rota-vapour system (Speed Vac
Concentrator, SavantTM, Ramsey, MN, USA) whereas the last pellet was
discarded. The solid residue obtained after evaporation of the three
supernatants was re-suspended in 1 ml of ultrapure water and used in the
following analytical procedure.
Analytical procedure
Free amino acids assay
Samples (5µl) of the crude aqueous extracts were assayed [see Bouchereau
et al. (Bouchereau et al.,
1999) for a full description of the method]. Free amino acids were
characterized and quantified by ultra-performance liquid chromatography (UPLC;
Waters, Waters Corporation, Milford, MA, USA) after pre-column derivatization
with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (using a Waters
Accq-Tag amino acid analysis system) and reversed-phase liquid chromatographic
separation.
Lactate, glycerol and sugars assays
Lactate, glycerol, trehalose and glucose were quantified using specific
kits (K-GCROL for glycerol and lactate, K-TREH for trehalose and glucose;
Megazyme International, Bray, Co. Wicklow, Ireland). Analyses were performed
with a spectrophotometer (Versamax, Molecular Devices, Sunnyvale, CA, USA)
using the software SoftMax Pro 4.8 (Molecular Devices) at 340nm.
Statistical analyses
All values are presented on graphs as means ± 95% confidence
intervals. A canonical discriminant analysis was performed to examine the
deviation of each mean of the replicate samples by temperature and population
from the mean of each metabolite. In this analysis, our primary matrix was the
metabolites, as columns, and replicate samples by temperature and populations,
as rows. This analysis enabled us to determine the main metabolites that
discriminate populations at each temperature. The multivariate analysis was
run with Tanagra 1.4.17 (Rakotomalala,
2005), and graphs were displayed with ADE-4 2001
(Thioulouse et al., 1997). The
statistical differences in metabolite concentrations observed between the
control temperature and 3°C were investigated using a Student's
t-test for two-sample comparisons. Significance levels were adjusted
using the Bonferroni correction. The degree of significance of the results was
represented with one, two or three asterisks, which correspond to significance
levels of 0.05, 0.01 and 0.001, respectively, for data that do not require
Bonferroni adjustment. Data were log- or square-root-transformed to homogenize
variances when homoscedasticity was not observed. Statistical analyses were
performed with Statistica software (version 7) (Statsoft, Tulsa, OK, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Temperature decrease had a strong effect on ventilatory activity (Fig. 1A); values were significantly lower at 3°C in all the populations (P-values ranged between 0.001026 and 0.000023, which corresponds to significance levels of ** and ***, respectively, after Bonferroni correction; see Materials and methods for definition of asterisks). During cold exposure, ventilatory activity showed a minimum decrease of 20% (Volognat population) and a maximum decrease of 48% for the Alex population (–34.0±8.7%, mean of decrease ± s.d.).
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Lactate body levels
No lactate was detected in the seven populations acclimated at either
temperature (3°C and 10°C), suggesting that this compound was either
absent or present at an undetectable level for the enzymatic method used
(under 0.003 g l–1 according to manufacturer
information).
Influence of the temperature treatment on metabolite body contents
The F1 and F2 axes of the canonical discriminant analysis
(Fig. 2A) explained 71.5% of
the variability contained in the data matrix, with 40.5% on the first axis.
This analysis allowed us to better characterize apparent differences observed
among populations during cold acclimation and to define the main metabolites
that were affected by cold exposure. Populations at a temperature of 10°C
were regrouped and had positive scores on both F1 and F2 axes
(Fig. 2A) whereas
cold-acclimated populations had negative scores on F1 and/or F2. All
cold-acclimated populations were characterized by reduced concentrations of
glycerol and glucose and increased concentrations of trehalose, proline and
alanine (Fig. 2A,B). At
10°C, overlap between populations occurred and reflected a low variability
in metabolite compositions within the seven populations. By contrast, at
3°C, ellipses were highly dispersed
(Fig. 2A) and, for all
populations, the ellipses' surfaces were larger than the corresponding ones at
10°C (Fig. 2A). This
pattern is the graphical consequence of both inter-population and
inter-individual heterogeneous responses. Cold exposure seems to have a
stronger effect on eight metabolites: glycerol, glucose, arginine, alanine,
proline, trehalose, glutamine and lysine.
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Glycerol and glucose levels were significantly reduced in cold-acclimated organisms, except for the Volognat population (Fig. 3A,B). These decreases ranged from 34% in Cormoran to 85% in Charabotte 2 populations (–62±20%, mean of decrease ± s.d.) for glycerol (Fig. 3A) and from 30% in Charabotte 1 to 56% in Alex populations (–46±9%, mean of decrease ± s.d.) for glucose (Fig. 3B). By contrast, trehalose concentrations significantly increased (Fig. 3C). This increase was the least pronounced for the Charabotte 1 population (71%) and was the highest (up to 151%) for the Cormoran population (+110±26%, mean of increase ± s.d.).
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-alanine. Total free amino
acid content (FAA) increased when temperature decreased from 10°C to
3°C but it was only significant for four of the seven studied populations
(Fig. 4): Cormoran (FAA
increased by 55%, P=0.000139***), Alex (FAA increased by
57%, P=0.000143***), Pissoir (FAA increased by 147%,
P=0.000138***) and Volognat (FAA increased by 243%,
P=0.000138***). Glutamine, alanine and lysine predominated
over the free amino acid pool and, together, their levels presented a
statistically significant increase during cold exposure for five of the seven
populations (P-values were equal to 0.000138*** or
0.000192***). In the Cormoran population, these three amino acids
accounted for 46% and 50% of the total FAA pool at 10°C and 3°C,
respectively, 40% and 53% in the Pissoir population, 44% and 45% in the
Volognat population (all amino acids increased drastically at 3°C for this
population), 37% and 45% in the Alex population, and 28% and 42% in the
Froidières population (Fig.
5; supplementary material Fig. S1).
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The variability observed between populations at 3°C (Fig. 2A) seemed to be predominantly linked to three amino acids: lysine and glutamine on the one hand and arginine on the other hand (Fig. 2B). The most notable effect is the differential accumulation between the Charabotte 2 population situated on the right end of the first axis and the Cormoran population situated on the left end of this axis (Fig. 2A). In the Charabotte 2 population, arginine was found in high amounts at 3°C, and overall this amino acid presented very heterogeneous fluctuations between populations (supplementary material Fig. S1). Arginine concentrations increased for six populations between 10°C and 3°C but it was significant for only two of them (the Volognat and Charabotte 1 populations) (supplementary material Fig. S1). It is interesting to note that for both the Charabotte populations, arginine was the predominant amino acid at 3°C whereas lysine body content was very low compared with all the other populations.
To summarize, the Cormoran population was characterized by the lowest decrease of glycerol and by the highest increase of trehalose during cold acclimation. Volognat exhibited one of the highest Q10, the lowest reduction of ventilatory activity and the highest increases of FAA, arginine, alanine and lysine amounts. Except for glycerol and trehalose, which were highly significantly decreased and increased, respectively, the Charabotte 1 population tended to have similar characteristics between the cold-exposed organisms (3°C) and the control organisms (10°C).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Farrell et al., 2008).
However, in the present study, we demonstrated that such a conclusion does not
apply to populations of the subterranean crustacean Niphargus
rhenorhodanensis. Moreover, we also proved that the thermal response is
not restricted to subterranean populations living in the phreatic system
(Issartel et al., 2005a;
Issartel et al., 2005b) but is
more widely distributed, including in the most typical and extremely thermally
buffered karstic environments.
We characterized cold-hardiness in seven populations of N.
rhenorhodanensis at three levels: behaviour, metabolism and biochemistry.
Most studies have analyzed only one or two of these aspects and, to date, we
have only found two studies dealing with these three components of the
response to cold in crustacean species
(Issartel et al., 2005a;
Issartel et al., 2005b).
In the seven populations we studied, locomotory activity was drastically
reduced at 3°C, but no organisms were motionless during cold exposure. By
contrast, Issartel et al. showed that individuals of another subterranean
crustacean, Niphargus virei, were motionless at 3°C
(Issartel et al., 2005a).
Thus, the ability to maintain a minimal locomotory activity at cold
temperature is not a general trend for subterranean organisms. As these
organisms lack visual organs, they need to move permanently to find trophic
resources. The minimal activity maintained even at cold temperatures probably
enables N. rhenorhodanensis species to continue to feed, which
represents an important functional advantage. Interestingly, Issartel et al.
found a similar response in a surface-dwelling crustacean adapted to large
thermal variations, Gammarus fossarum
(Issartel et al., 2005a).
Further experiments should examine whether the non-zero locomotory activity
displayed by N. rhenorhodanensis at 3°C is adaptive or reflects
nonadaptive physicochemical effects of the decrease in temperature.
Most biological processes are slowed down when temperature decreases.
Oxygen consumption follows this rule, and expected Q10
values range between 2 and 3
(Schmidt-Nielsen, 1997;
Daoud et al., 2007;
Irwin et al., 2007;
Jimenez and Bennett, 2007).
For all populations, except Alex and Volognat, N. rhenorhodanensis
populations responded as expected (Q10=2.09±0.12,
mean ± s.d.) (Table 2).
The very high reduction of oxygen consumption (and the associated high
Q10 value) measured in the Alex population highlighted
that the metabolism was more highly depressed in those crustaceans than in the
other populations. It leads us to think that this atypical decrease in oxygen
consumption may be the expression of physiological changes other than standard
thermal-induced changes occurring on physicochemical processes. As for the
Volognat population, our results are rather surprising. Indeed, the Volognat
population displayed a highly depressed oxygen consumption
(Q10=3.93) (Table
2) but the weakest decrease in ventilatory activity. While further
experiments are needed, this suggests that a decoupling exists between the
ventilatory activity and the metabolic rate in these organisms. Keeping in
mind that all the populations studied inhabit similar environments where the
temperature is highly buffered all year long, these heterogeneous responses
are particularly interesting. We can thus hypothesize that the current
environmental conditions experienced by the animals are not the only
parameters driving the amplitude of the stress response of the organisms we
studied.
Despite the large decrease in oxygen consumption observed during cold
exposure, crustaceans did not switch to anaerobic metabolism. Indeed, no
lactate was detected, this compound being a major end-product of anaerobic
metabolism in N. rhenorhodanensis
(Hervant et al., 1995;
Hervant et al., 1996). Cold
exposure is well known to induce aerobic metabolism perturbations
(Hochachka and Somero, 2002),
resulting in a decrease in ATP production. Thus, some organisms have to use
anaerobic means for some portion of their energy needs
(Costanzo et al., 2004;
Hartley et al., 2000;
Packard and Packard, 2004).
However, the energetic yield of aerobic metabolism is largely superior to that
of anaerobic metabolism (Hochachka and
Somero, 2002). Consequently, organisms such as N.
rhenorhodanensis, which are able to maintain their whole metabolism as
aerobic, present a functional advantage that enhances their survival time
during cold exposure.
From a biochemical point of view, several molecules were accumulated or
degraded at 3°C in the seven populations we examined. Organisms were not
able to maintain their glucose body contents. Since stabilising the glucose
level in the haemolymph is essential for the regular functioning of the
nervous, muscle and reproductive systems of the organisms
(Buckup et al., 2008), one
might expect that, even if glucose body content decreased, haemolymph glucose
content would remain more stable. Unfortunately, our analytical procedure did
not allow us to differentiate glucose in the haemolymph from cellular glucose.
Glycerol amounts were also reduced during cold exposure, indicating that this
metabolite, well known as a colligative antifreeze compound accumulated in
large amounts in insects during cold acclimation
(Bennett et al., 2005;
Kostal et al., 2001;
Michaud and Denlinger, 2007;
Renault et al., 2002), did not
play any cold acclimation role in the seven studied populations of N.
rhenorhodanensis. As far as we are aware, only two studies have dealt
with cold responses in Crustacea. Issartel et al. studied three species
– Gammarus fossarum, Niphargus virei and Niphargus
rhenorhodanensis – (Issartel et
al., 2005b), and Karanova and Gakhova studied a cryotolerant
crustacean, Gammarus lacustris
(Karanova and Gakhova, 2002).
These works showed that glycerol is not accumulated in response to cold in
these crustacean species. Thus, the absence of glycerol accumulation in the
populations studied here is not surprising and cannot be considered as
evidence for nonadaptive features to cold. Glycerol in N.
rhenorhodanensis can be used either as a glucose precursor to maintain a
stable haemolymph glucose level using the neoglucogenesis pathway or as an
intermediate to generate trehalose. This idea is supported by the significant
rise in trehalose concentration during cold exposure, in particular in the
Cormoran and Pissoir populations. Similar results have been reported in the
overwintering isopod Porcellio scaber
(Tanaka and Udagawa, 1993) and
in a population of N. rhenorhodanensis living in a porous aquifer
(Issartel et al., 2005a;
Issartel et al., 2005b).
Accumulation of trehalose probably helped in protecting protein and membrane
integrity during the exposure at 3°C
(Fields et al., 1998;
Ramløv, 2000;
Ring and Danks, 1998) and
suggests that this response could be adaptive.
Cold stress was very often found to be associated with an increase in the
level of several free amino acids (FAA) during the first days in most species
tested to date, resulting in an increase in the total FAA pool
(Fields et al., 1998;
Renault et al., 2006). In the
present study, the FAA pool of the subterranean crustacean N.
rhenorhodanensis was significantly altered by thermal stress, and the
highest increase was found in the Volognat population. This cold-induced
accumulation of several amino acids possibly resulted from a nonadaptive
change in gene and protein expression
(Colinet et al., 2007;
Lalouette et al., 2007), i.e.
a change in the balance between protein anabolism/catabolism. And yet, we
would like to stress that four amino acids were significantly accumulated:
alanine, glutamine, lysine and arginine, representing more than 50% of the
total amino acid body content for each population. This point is particularly
interesting because these amino acids are known to play an important role
during cold exposure, conferring cryoprotection. In vitro experiments
have demonstrated that alanine acts as a cryoprotectant, maintaining enzyme
activities at low temperatures (Carpenter
and Crowe, 1988; Carpenter et
al., 1990), and several studies have observed an alanine
upregulation in insects during cold acclimation
(Fields et al., 1998;
Goto et al., 2001;
Michaud and Denlinger, 2007).
Alanine seems to have the same colligative effect as glycerol when it is used
as a cryoprotectant in ectothermic organisms
(Michaud and Denlinger, 2007);
it protects proteins and membrane integrity and promotes supercooling of body
fluids (Lee, 1989;
Ramløv, 2000).
Glutamine variations are of interest because this amino acid is involved in
several physiological processes. Catabolism of both amino acids and proteins
during exposure to low temperatures leads to the production of ammonia (here
we use ammonia to refer to NH3 or NH4+, or a
combination of the two). Ammonia can be fixed on glutamate to yield glutamine,
which is accumulated in large amounts in cold-exposed subterranean
crustaceans. Moreover, as glutamine contains positively charged amine groups,
Anchordoguy et al. suggested that it could minimise membrane disruption at
cold temperatures by interacting with phospholipids
(Anchordoguy et al., 1988).
Numerous studies dealing with cold-acclimated insects
(Fields et al., 1998;
Hanzal and Jegorov, 1991;
Michaud and Denlinger, 2007;
Renault et al., 2006) have
shown an increase in the glutamine content similar to what we found here in
five populations of N. rhenorhodanensis.
The third amino acid to be significantly accumulated during cold exposure
by four populations is lysine, an essential amino acid in arthropods
(Ramsay and Houston, 2003).
Such an accumulation of lysine was previously found in two species of
Coleoptera acclimated to cold temperatures
(Fields et al., 1998) and in
larvae of the wax moth Galleria mellonella during cold acclimation
(Hanzal and Jegorov, 1991). As
lysine is an essential amino acid, the increase in lysine concentration is
probably due to protein catabolism.
Arginine, another essential amino acid, presented highly heterogeneous
variations among the seven populations of N. rhenorhodanensis. This
highly variable tendency could reflect (1) protein catabolism at 3°C
(which is highly individual and population dependent) or (2) the breakdown of
arginine phosphate, a phosphagen largely accumulated by hypogean crustaceans
(Hervant et al., 1995;
Hervant et al., 1996), to
produce and therefore maintain their ATP body content. We can point out that
the Volognat population presented the highest increase of arginine at 3°C
so it seems that this population displayed the highest disturbance in ATP
production during cold exposure, which would be consistent with
Q10 measurements.
Proline has very often been found to be accumulated in response to cold
exposure in a wide range of arthropod species
(Storey, 1997;
Ramløv, 1999;
Ramløv, 2000) and is
usually the most accumulated amino acid in insects
(Hanzal and Jegorov, 1991;
Fields et al., 1998;
Ramløv, 1999). N.
rhenorhodanensis exhibited very low amounts of proline whatever the
experimental conditions and, even if it was accumulated in four cold-exposed
populations, its concentration remained at low levels. As previously suggested
in other studies, this amino acid (and this holds true even if it had been
accumulated) may not play a cold acclimation role in all arthropod species
(Issartel et al., 2005b;
Lalouette et al., 2007).
Since temperature in subterranean ecosystems is very stable throughout the
year, the physiological responses and adaptations to cold exhibited by these
crustaceans cannot be linked to the thermal variations they undergo in their
living environment. Some of the responses we found, which typically occur in
regularly cold-exposed arthropods
(Lalouette et al., 2007;
Ramløv, 2000), are also
intriguing in view of the thermal characteristics of the habitats of N.
rhenorhodanensis. In a recent study, Issartel et al. questioned the
ecological relevance of the cold-hardiness of N. rhenorhodanensis and
compared it to the response of another subterranean karstic species, N.
virei (Issartel et al.,
2005a). As expected, N. virei exhibited a typical
stenothermal profile. Issartel et al. thus hypothesised that the observed
responses may result from the past life history of these two species
(Issartel et al., 2005a), i.e.
this cold-hardiness would be a relict adaptation that enabled the survival of
N. rhenorhodanensis within glaciers during the Pleistocene
(Issartel et al., 2005b).
Lefébure et al. supplied DNA evidence that several populations of
N. rhenorhodanensis survived within the area covered by glaciers,
possibly in small areas called nunataks (locations free of ice surrounded by
glaciers), during the last glacial maximum (LGM)
(Lefébure et al.,
2007), whereas Foulquier et al. showed that all populations of
N. virei survived the LGM outside of the glacier area
(Foulquier et al., 2008).
These two studies are thus in agreement with the hypothesis of a relict
adaptation for the cold-hardiness found in N. rhenorhodanensis.
Keeping in mind that the seven populations of N. rhenorhodanensis live in very similar biotopes and have endured the same evolution process, another interesting point of this study is the distinct amplitudes of the response we found between cold-exposed populations (Fig. 2). Cormoran, Pissoir and Volognat organisms appeared to be the `most cold-hardy' (i.e. exhibited the most pronounced metabolic and biochemical responses), whereas the `least cold-hardy' individuals were found in both the Charabotte populations. Three hypotheses can be put forward to explain the heterogeneity observed here in the responses to cold.
). Knowing whether or not the seven populations we used belong
to distinct lineages would be helpful in testing the possible relationship
between historical factors and the cold-hardiness heterogeneity we found
here. To conclude, we found that the overall relationship that can be established between the amplitude of thermal variations in the biotope and cold-hardiness abilities of the species may be more complex in subterranean crustaceans than in other arthropods. Indeed, we found that populations of N. rhenorhodanensis surprisingly displayed likely adaptive strategies to the cold even if they live in strongly thermally buffered environments: they accumulated cryoprotective molecules, they maintained a locomotory activity and managed to conserve an aerobic metabolism even at cold temperatures. Thus, subterranean environments seem to constitute a counter-example to the theory generally accepted. Moreover, we found a high heterogeneity in the cold responses between populations inhabiting similar and geographically close biotopes. The thermal plasticity of theses amphipods may result from (1) their past life history or (2) the intensity of the stress to which they are subjected. Thus, present environmental conditions (approximately 10°C) cannot explain the cold-adaptation abilities of the subterranean crustacean N. rhenorhodanensis but could partly explain the heterogeneity found in physiological and metabolic responses to cold exposure among different populations.
|
Footnotes |
|---|
This research was supported by funds from Universities Claude Bernard – Lyon 1 and Beaulieu – Rennes 1 and from the IFR41 of the National Centre of French Scientific Research (CNRS). The authors thank Dr F. Malard for his valuable assistance in collecting individuals of Niphargus rhenorhodanensis and his helpful suggestions for this manuscript. We are also grateful to M. Laparie, who helped us with the statistical analysis. We also wish to thank Christina Richardson and two anonymous reviewers, who helped us to improve this article.
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Addo-Bediako, A., Chown, S. L. and Gaston, K. J.
(2000). Thermal tolerance, climatic variability and latitude.
Proc. Biol. Sci. 267,739
-745.
Anchordoguy, T., Carpenter, J. F., Loomis, S. H. and Crowe, J. H. (1988). Mechanisms of interaction of amino acids with phospholipid bilayers during freezing. Biochim. Biophys. Acta 946,299 -306.[Medline]
Bennett, V. A., Sformo, T., Walters, K., Toienn, O., Jeannet,
K., Hochstrasser, R., Pan, Q., Serianni, A. S., Barnes, B. M. and Duman, J.
G. (2005). Comparative overwintering physiology of Alaska and
Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of
antifreeze proteins, polyols, dehydration and diapause. J. Exp.
Biol. 208,4467
-4477.
Botosaneanu, L. (1986). Stygofauna Mundi: A Faunistic, Distributional, and Ecological Synthesis of the World Fauna Inhabiting Subterranean Waters (ed. L. Botosaneanu), pp.740 . Leiden: Brill Academic Publishers.
Bouchereau, A., Duhazé, C., Martin-Tanguy, J., Guégan, J. P. and Larher, F. (1999). Improved analytical methods for determination of nitrogenous stress metabolites occurring in Limonium species. J. Chromatogr. A 836,209 -221.[CrossRef]
Buckup, L., Dutra, B. K., Ribarcki, F. P., Fernandes, F. A., Noro, C. K., Oliveira, G. T. and Vinagre, A. S. (2008). Seasonal variations in the biochemical composition of the crayfish Parastacus defossus (Crustacea, Decapoda) in its natural environment. Comp. Biochem. Physiol. 149A,59 -67.
Carpenter, J. F. and Crowe, J. H. (1988). The mecanism of cryoprotection of proteins by solutes. Cryobiology 25,244 -255.[CrossRef][Medline]
Carpenter, J. F., Crowe, J. H. and Arakawa, T. (1990). Comparison of solute-induced protein stabilization in aqueous solution in the frozen and dried states. J. Dairy Sci. 73,3627 -3636.[Abstract]
Castany, G. (1998). Hydrogéologie: Principes et Methodes, pp.236 . Paris: Dunod.
Chown, S. L. (2001). Physiological variation in insects: hierarchical levels and implications. J. Insect Physiol. 47,649 -660.[CrossRef][Medline]
Colinet, H., Vernon, P. and Hance, T. (2007). Does thermal-related plasticity in size and fat reserves influence supercooling abilities and cold-tolerance in Aphidius colemani (Hymenoptera: Aphidiinae) mummies? J. Therm. Biol. 32,374 -382.[CrossRef]
Costanzo, J. P., Dinkelacker, S. A., Iverson, J. B. and Lee, R. E., Jr (2004). Physiological ecology of overwintering in the hatchling painted turtle: multiple-scale variation in response to environmental stress. Physiol. Biochem. Zool. 77, 74-99.[CrossRef][Medline]
Culver, D. C., Christman, M. C., Sket, B. and Trontelj, P. (2004). Sampling adequacy in an extreme environment: species richness patterns in Slovenian caves. Biodivers. Conserv. 13,1209 -1229.[CrossRef]
Daoud, D., Chabot, D., Audet, C. and Lambert, Y. (2007). Temperature induced variation in oxygen consumption of juvenile and adult stages of the northern shrimp, Pandalus borealis.J. Exp. Mar. Biol. Ecol. 347, 30-40.[CrossRef]
Farrell, A. P., Hinch, S. G., Cooke, S. J., Patterson, D. A., Crossin, G. T., Lapointe, M. and Mathes, M. T. (2008). Pacific Salmon in hot water: applying aerobic scope models and biotelemetry to predict the success of spawning migrations. Physiol. Biochem. Zool. 81,697 -709.[CrossRef][Medline]
Fields, P. G., Fleurat-Lessart, F., Lavenseau, L., Febvay, G., Peypelut, L. and Bonnot, G. (1998). The effect of cold acclimation and deacclimation on cold tolerance, trehalose and free amino acids levels in Sitophilus granaries and Cryptolestes ferrugineus (Coleoptera). J. Insect Physiol. 44,955 -965.[CrossRef][Medline]
Foulquier, A., Malard, F., Lefébure, T., Douady, C. J. and Gibert, J. (2008). The imprint of Quaternary glaciers on the present-day distribution of the obligate groundwater amphipod Niphargus virei (Niphargidae). J. Biogeogr. 35,552 -564.[CrossRef]
Freeze, R. A. and Cherry, J. A. (1979). Groundwater, pp. 604. Upper Saddle River, NJ: Prentice-Hall.
Gaston, K. J. and Chown, S. L. (1999). Elevation and climatic tolerance: a test using dung beetles. Oikos 86,584 -590.[CrossRef]
Ghalambor, C. K., Huey, R. B., Martin, P. R., Tewksbury, J. J.
and Wang, G. (2006). Are mountain passes higher in the
tropics? Janzen's hypothesis revisited. Integr. Comp.
Biol. 46,5
-17.
Gibert, J. (2001). Basic attributes of groundwater ecosystems. In Groundwater Ecology: A Tool For Management of Water Resources (ed. C. Griebler, D. L. Danielopol, J. Gibert, H. P. Nachtnebel and J. Notenboom), pp.39 -52. Luxembourg: Office for Official Publications of the European Communities.
Gibert, J. and Deharveng, L. (2002). Subterranean ecosystems: a truncated functional biodiversity. Biosciences 52,473 -481.[CrossRef]
Gibert, J., Stanford, J. A., Dole-Olivier, M. J. and Ward, J. V. (1994). Basic attributes of groundwater ecosystems and prospects for research. In Groundwater Ecology (ed. J. Gibert, D. L. Danielopol and J. A. Stanford), pp.7 -40. San Diego, CA: Academic Press.
Ginet, R. and Decou, V. (1977). Initiation à la Biologie et à l'Écologie Souterraines, pp. 345. Paris: Delarge.
Ginet, R. and Mathieu, J. (1968). Comparaison des temperatures létales supérieures de Niphargus longicaudatus (Crust. Amphipodes) hypogés et épigés. Ann. Spéléo. 23,425 -440.
Goto, M., Sekine, Y., Outa, H., Hujikura, M. and Suzuki, K. (2001). Relationships between cold hardiness and diapause, and between glycerol and free amino acid contents in overwintering larvae of the oriental corn borer, Ostrinia furnacalis. J. Insect Physiol. 47,157 -165.[CrossRef][Medline]
Hanzal, R. and Jegorov, A. (1991). Changes in free amino acid composition in haemolymph of larvae of the wax moth, Galleria mellon ella L., during cold acclimation. Comp. Biochem. Physiol. 100A,957 -962.
Hartley, L. M., Packard, M. J. and Packard, G. C. (2000). Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival. J. Comp. Physiol. B 170,45 -50.[CrossRef][Medline]
Hervant, F., Mathieu, J., Garin, D. and Freminet, A. (1995). Behavioral, ventilatory and metabolic responses to severe hypoxia and subsequent recovery of the hypogean Niphargus rhenorhodanensis and the epigean Gammarus fossarum (Crustacea: Amphipoda). Physiol. Biochem. Zool. 68,223 -244.
Hervant, F., Mathieu, J., Garin, D. and Freminet, A. (1996). Behavioral, ventilatory and metabolic responses of the hypogean amphipod Niphargus virei and the epigean isopod Asellus aquaticus to severe hypoxia and subsequent recovery. Physiol. Biochem. Zool. 69,1277 -1300.
Hervant, F., Mathieu, J., Barré, H., Simon, K. and Pinon, C. (1997). Comparative study on the behavioural, ventilatory and respiratory responses of hypogean and epigean crustacean to long-term starvation and subsequent feeding. Comp. Biochem. Physiol. 118A,1277 -1283.
Hochachka, P. and Somero, G. (2002). Biochemical Adaptation, Mechanism and Physiological Evolution, pp. 480. New York: Oxford University Press.
Huey, R. B. and Kingsolver, J. G. (1989). Evolution of ectotherm performance. Trends Ecol. Evol. 4, 131-135.[CrossRef]
Humphreys, W. F. (2000). Relict faunas and their derivation. In Subterranean Ecosystems (ed. H. Wilkens, D. C. Culver and W. F. Humphreys), pp.417 -432. Amsterdam: Elsevier.
Irwin, S., Wall, V. and Davenport, J. (2007). Measurement of temperature and salinity effects on oxygen consumption of Artemia franciscana K., measured using fibre-optic oxygen microsensors. Hydrobiologia 575,109 -115.[CrossRef]
Issartel, J., Hervant, F., Voituron, Y., Renault, D. and Vernon, P. (2005a). Behavioural, ventilatory and respiratory responses of epigean and hypogean crustaceans to different temperatures. Comp. Biochem. Physiol. 141A, 1-7.[CrossRef]
Issartel, J., Renault, D., Voituron, Y., Bouchereau, A., Vernon,
P. and Hervant, F. (2005b). Metabolic responses to cold in
subterranean crustaceans. J. Exp. Biol.
208,2923
-2929.
Issartel, J., Voituron, Y. and Hervant, F. (2007). Impact of temperature on the survival, the activity and the metabolism of the cave-dwelling Niphargus virei, the ubiquitous stygobiotic N. rhenorhodanensis and the surface-dwelling Gammarus fossarum (Crustacea, Amphipoda). Subterr. Biol. 5, 9-14.
Jimenez, A. G. and Bennett, W. A. (2007). Metabolic responses of sand fiddler crabs, Uca pugilator, in northwest Florida to seasonal temperature change. J. Therm. Biol. 32,308 -313.[CrossRef]
Juberthie, C. and Decu, V. (ed.) (1994a). Encyclopaedia Biospeologica I. Moulis-Bucarest: Société de Biospéléologie.
Juberthie, C. and Decu, V. (1994b). Structure et diversité du domaine souterrain: particularités des habitats et adaptations des espèces. In Encyclopaedia Biospeologica I (ed. C. Juberthie and V. Decu), pp.5 -22. Moulis-Bucarest: Société de Biospéléologie.
Juberthie, C. and Decu, V. (ed.) (1998). Encyclopaedia Biospeologica II. Moulis-Bucarest: Société de Biospéléologie.
Juberthie, C. and Decu, V. (ed.) (2001). Encyclopaedia Biospeologica III. Moulis-Bucarest: Société de Biospéléologie.
Karanova, M. V. and Gakhova, E. N. (2002). Molecular nature of the cryotolerant lake amphipod Gammarus lacustris.Biofizika 47,116 -124.[Medline]
Kostal, V., Slachta, M. and Simek, P. (2001). Cryoprotective role of polyols independent of the increase in supercooling capacity in diapausing adults of Pyrrhocoris apterusi (Heteroptera: Insecta). Comp. Biochem. Physiol. 130B,365 -374.[CrossRef][Medline]
Lalouette, L., Kostal, V., Colinet, H., Gagneul, D. and Renault, D. (2007). Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes. FEBS J. 274,1759 -1767.[CrossRef][Medline]
Lee, R. E. (1989). Insect cold-hardiness: to freeze or not to freeze? BioScience 39,308 -313.[CrossRef]
Lefébure, T., Douady, C. J., Malard, F. and Gibert, J. (2007). Testing dispersal and cryptic diversity in a widely distributed groundwater amphipod (Niphargus rhenorhodanensis). Mol. Phylogenet. Evol. 42,676 -686.[CrossRef][Medline]
Malard, F. and Hervant, F. (1999). Oxygen supply and the adaptations of animals in groundwater. Freshw. Biol. 41,1 -30.[CrossRef]
Michaud, R. M. and Denlinger, D. L. (2007). Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison. J. Comp. Physiol. B 177,753 -763.[CrossRef][Medline]
Packard, G. C. and Packard, M. J. (2004). To
freeze or not to freeze: adaptations for overwintering by hatchlings of the
North American painted turtle. J. Exp. Biol.
207,2897
-2906.
Peck, L. S., Webb, K. E. and Bailey, D. M. (2004). Extreme sensitivity of biological function to temperature in Antarctic marine species. Funct. Ecol. 18,625 -630.[CrossRef]
Pörtner, H. O. (2006). Climate-dependent evolution of Antarctic ectotherms: an integrative analysis. Deep Sea Res. Part II Top. Stud. Oceanogr. 53,1071 -1104.[CrossRef]
Rakotomalala, R. (2005). TANAGRA: a free software for research and academic purposes. In Proceeding of EGC'2005, RNTI-E-3, vol. 2, pp.697 -702. Paris: Cépaduès-Editions. (In French.)
Ramløv, H. (1999). Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae). J. Comp. Physiol. B 169,224 -235.[CrossRef]
Ramløv, H. (2000). Aspects of natural
cold tolerance in ectothermic animals. Hum. Reprod.
15, 26-46.
Ramsay, S. L. and Houston, D. C. (2003). Amino acid composition of some woodland arthropods and its implications for breeding tits and other passerines. Ibis 145,227 -232.[CrossRef]
Renault, D., Hervant, F. and Vernon, P. (2002). Comparative study of the metabolic responses during food shortage and subsequent recovery at different temperatures in the adult lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Physiol. Entomol. 27,291 -301.[CrossRef]
Renault, D., Bouchereau, A., Delettre, Y., Hervant, F. and Vernon, P. (2006). Changes in free amino acids amounts in Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae) during thermal and food stress. Comp. Biochem. Physiol. 143A,279 -285.[CrossRef][Medline]
Ring, R. A. and Danks, H. V. (1998). The role of trehalose in cold-hardiness and dessication. Cryo Letters 19,275 -282.
Schmidt-Nielsen, K. (1997). Animal Physiology: Adaptation and Environment, 5th edn, pp.217 -238. Cambridge: Cambridge University Press.
Stevens, G. C. (1989). The latitudinal gradient in geographical range: how so many species coexist in the tropics. Am. Nat. 133,240 -256.[CrossRef]
Storey, K. B. (1997). Organic solutes in freezing tolerance. Comp. Biochem. Physiol. A 117,319 -326.[Medline]
Tanaka, K. and Udagawa, T. (1993). Cold adaptation of the terrestrial isopod, Porcellio scaber, to subnivean environments. J. Comp. Physiol. B 163,439 -444.
Thioulouse, J., Chessel, D., Dolédec, S. and Olivier, J. M. (1997). ADE-4: a multivariate analysis and graphical display software. Stat. Comput. 7, 75-83.[CrossRef]
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