The metabolic profile was analyzed during constant fluctuating thermal regimes the beetles had daily pulses at higher temperatures that enabled them to recover and compared with con-stant
Trang 1tropical beetles exposed to fluctuating thermal regimes
L Lalouette1, V Kosˇta´l2, H Colinet3, D Gagneul4,5and D Renault1
1 UMR CNRS 6553, Universite´ de Rennes 1, Rennes, France
2 Institute of Entomology, Biology Centre AS CR, C ˇ eske´ Budejovice, Czech Republic
3 Unite´ d’Ecologie et de Bioge´ographie, Centre de Recherche sur la Biodiversite´, Universite´ catholique de Louvain, Louvain-la-Neuve, Belgium
4 UMR CNRS 6026, Universite´ de Rennes 1, Rennes, France
5 Department of Plant Biology, Michigan State University, East Lansing, MI, USA
Environmental stress deleteriously affects every aspect
of an ectotherm’s biological function because it
dis-rupts homeostasis, and is of sufficient magnitude to
impose geographical limits on where animal life can
occur, even if the other environmental parameters are
permissive [1,2] As arthropods’ development and
sur-vival are intimately linked to environmental
tempera-tures, these organisms have evolved a diversity of
morphological, physiological and behavioural
adapta-tions [3] Several authors have contributed to the
increased knowledge of arthropods’ cold-hardiness over the past years [4–7], but studies were usually per-formed under controlled conditions by measuring organisms’ cold-tolerance at low but constant tempera-tures Fluctuating thermal regimes (FTRs) are, how-ever, typical in natural habitats, and yearly active species may exploit intermittent periods of favourable temperatures in order to feed, develop and repair low temperature injuries (chill injuries, i.e damage caused
by low temperatures without formation of ice crystals)
Keywords
amino acid; fluctuating thermal regime;
insect; polyol; sugar
Correspondence
D Renault, UMR CNRS 6553, University of
Rennes 1 Baˆt 14A, 263 Avenue du Gal
Leclerc, CS 74205, 35042 Rennes Cedex,
France
Fax: +33 2 23235046
Tel: +33 2 23236627
E-mail: david.renault@univ-rennes1.fr
Website: http://ecobio.univ-rennes1.fr
(Received 13 November 2006, revised 26
January 2007, accepted 1 February 2007)
doi:10.1111/j.1742-4658.2007.05723.x
Environmental stress deleteriously affects every aspect of an ectotherm’s biological function Frequent exposure of terrestrial insects to temperature variation has thus led to the evolution of protective biochemical and phy-siological mechanisms However, the phyphy-siological mechanisms underlying the positive impact of fluctuating thermal regimes (FTRs) on the fitness and survival of cold-exposed insects have not been studied We have thus investigated the metabolic changes in adults of the beetle Alphitobius diaperinus in order to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds, such as free amino acids (FAAs) and polyols The metabolic profile was analyzed during constant fluctuating thermal regimes (the beetles had daily pulses at higher temperatures that enabled them to recover) and compared with con-stant cold exposure and untreated controls The increase of several essential amino acids (Lys, Iso, Leu, Phe and Trp) in cold-exposed beetles supports the conclusion that it results from the breakdown of proteins Some FAAs have been shown to have cryoprotective properties in insects, but the rela-tionship between FAAs, cold tolerance and survival has not yet been well defined Instead of considering FAAs only as a part of the osmo- and cryo-protective arsenal, they should also be regarded as main factors involved in the multiple regulatory pathways activated during cold acclimation Under FTRs, polyol accumulation probably contributes to the increased duration
of survival in A diaperinus
Abbreviations
FAA, free amino acid; FM, fresh mass; FTR, fluctuating thermal regime; HSP, heat shock protein.
Trang 2Such species may also set up physiological processes of
cold-hardening that is cued by the low temperature
but requires a stay at higher temperature for effective
expression [8]
In most species tested to date, survival rates were
con-siderably increased when insects were exposed to FTRs,
compared with those maintained under constant low
temperatures Indeed, the short bouts at a higher
tem-perature may reset the physiological state of the insects
towards the initial value [6,8–10] However, few studies
attempted to investigate the physiological and
biochemi-cal responses of insects subjected to thermal
fluctua-tions, and thus the mechanisms underlying the relative
increase of cold tolerance in insects exposed to FTRs
are poorly understood Recently, Kosˇta´l et al [11]
found that constant cold-exposed bugs of Pyrrhocoris
apterus and beetles of Alphitobius diaperinus failed to
maintain specific ion concentrations outside and inside
the cells, or across epithelia Under FTRs, however, the
primary ion pumping systems, P- and V-type ATPases,
were allowed to re-establish the ion gradients across cell
membranes and epithelia during the ‘warm’ periods [11]
The impaired function of ion pumping systems, together
with the inability to prevent⁄ restrict ion leakage down
the electrochemical gradient, led to the reduction or
unbalance of metabolites transfer This then results in
the depletion of energetic substances in cells, or in the
accumulation of potentially toxic waste substances [3]
Thermal stress strongly impacts on metabolite
con-centrations [4,12,13] Thus, metabolite changes that
occur downstream of changes in transcript or protein
levels give a good picture of the overall integrated
response of an organism [14,15] The free amino acid
(FAA) pool, which is at the centre of metabolic activity
during a variety of stress responses [16], is particularly
affected by thermal stress, and can thus indicate
chan-ges in gene and protein expression, like heat shock
pro-teins (HSPs) Indeed, it was recently found that FTRs
significantly increase the expression of HSPs during
high temperature pulses [17, L Lalouette, H Colinet,
D Siaussat & D Renault, unpublished data]
More-over, several amino acids, like Pro, Gly, Ala, and Leu,
were identified as responsive to cold stress during
con-stant cold exposure [18] They were either directly
corre-lated with stress tolerance (a causal relationship
between Pro levels and stress tolerance was found
[3,19]), or with the changes in levels of stress hormones
during thermal stress [20] However, despite their
pre-dominant role in several metabolic pathways, amino
acids were never investigated in insects exposed
to FTRs Other than amino acids, the importance of
polyols and sugars, like glycerol and trehalose, has
been emphasized regularly during insect cold
acclimation [21] However, the few studies that have attempted to investigate polyol levels in insects subjec-ted to FTRs are contradictory Indeed, cycling thermal regimes were found to increase glycerol amounts in the gall fly [22], whereas it did not differ significantly between cyclic and constant temperature regimes in the beet armyworm [23]
In the present study, we investigated the impact of cold exposure and associated metabolic changes in a year-round active beetle, A diaperinus, introduced in temperate regions from the Ivory Coast (Africa)
A diaperinus was a convenient model because it is highly chill-susceptible during cold exposures, but exhibits strong recovery capacities during the pulses at
a ‘warm’ temperature under FTRs [9] Contrasted meta-bolic responses should therefore be obtained between chilled beetles and ones that were allowed to recover daily Moreover, relatively extensive knowledge on the cold-hardiness ecology and physiology of A diaperinus has been gathered [9,11–13,24]: these studies demon-strated that its survival was progressively reduced when the temperature dropped below 8C In a more recent work, we found that beetles kept at a constant tempera-ture of 0C quickly died, whereas the effects of chilling were reversed completely when insects were kept in FTRs (5C ⁄ 22 h and 20 C ⁄ 2 h) [9] Thus, we wanted
to determine whether FTRs trigger the initiation of a metabolic response involving synthesis of protective compounds such as FAAs, polyols or HSPs The simul-taneous measurement of a large number of metabolites, relevant because the overall effect of the thermal stress
is assessed [15], was thus investigated in beetles kept under FTRs, and compared to constant cold-exposed beetles, and untreated controls
Results
Survival Lethal time for 50% of the population (Lt50) of the beetles exposed at constant 0C was 5.95 ± 0.65 days After 10 days of FTR (0C alternating with 20 C on
a 12 h basis), no mortality was observed The experi-ment was stopped after 3 weeks, and it was not possi-ble to determine the Lt50 for these beetles Longer exposures to such experimental conditions would encounter mortality unrelated to cold
Amino acids Several differences were found in metabolic profiles between insects exposed to constant temperatures and fluctuating thermal regimes
Trang 3The total FAA pool was significantly higher in control
beetles [73.19 ± 2.18 nmolÆmg)1 fresh mass (FM)]
than in constant cold-exposed (0Cc) (56.72 ± 3.31
nmolÆmg)1 FM) and 20CF0 (20C/12 hr[20 CF0]
fluctuating with 0C/12 hr[0 CF20]) ones (51.56 ±
2.35 nmolÆmg)1 FM) (P < 0.05); the total amount of
FAA was significantly higher in 0CF20 beetles
(72.58 ± 4.58 nmolÆmg)1 FM) than in 0Cc and
20CF0ones (P < 0.05)
Pro was the main amino acid found, whatever the
experimental conditions (Fig 1A,B) It is, however,
interesting to notice that it represented 35% of the
total FAA pool in beetles exposed at a constant
tem-perature of 0C, 50% of the total FAA pool at
alter-nating temperature (20CF0, 0CF20) (Fig 1A) and
>50% of the total FAA pool in control beetles Gln
and Ala were also found in high amounts in the whole
body of A diaperinus Levels of five essential amino
acids (Lys, Iso, Leu, Phe and Trp) were increased
sig-nificantly when the beetles were cold exposed (Fig 1B) No significant difference was found for Val between the four distinct thermal treatments (control,
0Cc, 0CF20and 20CF0)
Control beetles exhibited the lowest amounts of Glu and Lys, and the highest levels of Asn⁄ Ser and Arg⁄ Thr (Fig 1A,B) Gln was significantly lower in control and 0Cc beetles (P < 0.05), whereas an opposite conclusion was found with Pro (significantly higher in control and 0Ccbeetles)
Amino acid profiles in 0Ccversus 0CF20
beetles The level of several FAA differed between the 0 Cc and 0CF20 thermal treatments Ala, Gln and Pro accounted for most of the observed quantitative differ-ence; Ala and Pro being highly accumulated in the
0CF20 beetles (10.03 ± 2.14 and 37.21 ± 3.12 nmo-lÆmg)1 FM, respectively) compared with the 0Cc bee-tles (5.79 ± 1.39 and 20.78 ± 1.39 nmolÆmg)1 FM, respectively; P < 0.05) (Fig 1A) Although it was two times lower in 0Ccbeetles, Ala content was not signi-ficantly different from 0CF20 beetles (P > 0.05) Levels of Glu and Gln had opposite patterns and were significantly lower in 0CF20 than in 0Cc beetles (6.02 ± 0.66 versus 15.06 ± 1.46 nmolÆmg)1 FM, respectively, for Gln, P < 0.05) (Fig 1A) Lys was also significantly higher in 0CF20beetles (P < 0.05)
Amino acid profiles in 0CF20versus 20CF0 beetles
Several differences were observed in the amounts of amino acids between 0CF20 and 20CF0 beetles (Fig 1A,B) On the 14 amino acids detected, nine differed significantly (P < 0.05) Gln was the only amino acid that was found in significantly lower amounts in 0CF20 beetles (6.02 ± 0.66 versus 11.06 ± 0.76 nmolÆmg)1 FM) The levels of four essential amino acids (Arg⁄ Thr, Lys, Leu, Phe) and four nonessential amino acids (Asn⁄ Ser, Gly, Ala, Pro) were significantly higher in 0CF20, explaining the dif-ference observed in the total FAA pool between these two thermal treatments Again, the most important differences were recorded for Pro and Ala, which were very highly significantly reduced in 20CF0
It is interesting to notice that Gln and Ala had an opposite pattern, with the highest level in 0 Cc, the lowest level in 0CF20 and an intermediate level in
20CF0 beetles for Gln, the highest amounts in
0CF20, the lowest amounts in 20CF0and an inter-mediate situation in 0Cc beetles for Ala (Fig 1A)
Fig 1 Free amino acid body contents in A diaperinus kept at
con-stant 20 C (control), constant 0 C, and FTR (20 C ⁄ 12 h: 20 C F0 ,
and 0 C ⁄ 12 h: 0 C F20 ) (A) Nonessential amino acids, and (B)
essential amino acids Values are mean ± SE (n ¼ 7) Bars with
dif-ferent letters indicate significant differences between FAA
(P < 0.05).
Trang 4Moreover, both Ala and Pro had similar patterns in
these three thermal treatments
Sugars and polyols
Data are presented in Fig 2 Glycerol and glucose had
opposite patterns: glycerol was highly significantly
accumulated in 0CF20beetles, whereas glucose
exhib-ited the lowest amounts in these beetles (P < 0.05)
For trehalose, a trend appeared: it was detected in
lower amounts in beetles subjected to FTRs than in
control ones, whereas myo-inositol seems to be
slightly accumulated in 0CF20 beetles Mannitol was
not detected in control beetles, whereas small amounts
were found in 0CF20(0.022 ± 0.002 nmolÆmg)1FM)
and 20CF0 beetles (0.018 ± 0.003 nmolÆmg)1 FM)
Arabinitol was only detected in 0CF20beetles, but in
low amounts (0.019 ± 0.001 nmolÆmg)1 FM) No
sig-nificant differences were found for sorbitol and ribitol
(Fig 2)
Discussion
Cold survival
Though animals are regularly exposed to
thermo-vari-able environments, survival of insects subjected to
thermal fluctuating regimes had rarely been
investi-gated until some recent studies [8–10,26] When the
adults of A diaperinus were exposed to the FTRs of
0C (12 h) ⁄ 20 C (12 h), their survival was consider-ably improved (no mortality after 10 days) in compar-ison with the exposure to the constant low temperature of 0C The effect of chilling could have been mitigated simply by the significant reduction (12 h daily) of the exposure time to the low tempera-ture However, after 10 days of FTRs, the beetles had spent 5 days at 0C in total, whereas the mortality was already >30% at the same time in the constant cold-exposed beetles In earlier studies, a similar phe-nomenon was observed in several insect species [8,10,26–28], demonstrating that the positive effect of FTRs on the insect cold tolerance emerges as a general phenomenon
Literature on the physiological mechanisms underly-ing the positive impact of FTRs on the survival of cold-exposed insects is scarce Very recently, we found that the haemolymph concentrations of magnesium and sodium ions in adults of A diaperinus were either maintained relatively constant or decreased slightly during both constant cold exposure and FTRs [11] The extracellular concentration of potassium ions increased with significantly higher rates in the insects exposed to constant low temperatures than in those exposed to FTRs, and returned toward normal [K+] during the warm ‘recovery’ periods of the FTRs We speculated that this mechanism could slow down the rate of the ion homeostasis disturbance and, as a con-sequence, reduce the chill injury and delay the occur-rence of prefreeze mortality [11]
Fig 2 Polyol and sugar body contents in the adult beetle A diaperinus kept at con-stant 20 C (control), and fluctuating thermal regimes (20 C ⁄ 12 h: 20 C F0 , and
0 C ⁄ 12 h: 0 C F20 ).Values are mean ± SE (n ¼ 6) Bars with different letters indicate significant differences between FAA (P < 0.05).
Trang 5Cold exposure and associated metabolic changes
Generally, cold stress is associated with an increase in
the levels of several FAA during the first days in most
species tested to date, resulting in an increase of the
total FAA pool [13,19] Even though no significant
increase of the FAA pool was recorded in 7-day
cold-exposed beetles, our results demonstrate that protein
catabolism occurred: five essential amino acids, Lys,
Iso, Leu, Phe and Trp, were accumulated Even if
interconversions and other metabolic alterations may
occur, it was demonstrated that removal of one of
each of the essential amino acids quickly resulted in
the death of the insects [29] For instance, Tenebrionid
species supplied with a Lys- and Trp-deficient diet
were incapable to sustain growth unless it was
supple-mented with both amino acids [30] The significant
accumulation of Lys, but also Iso, Leu and Trp found
in this study, and the inability to synthesize most
essential amino acids thus supports the conclusion that
it results from the breakdown of proteins
Under FTRs, the FAA pool was significantly
reduced during warm recovery periods Indeed, energy
supplies depleted during cold exposure, as observed in
A diaperinus [12], can be regenerated during the pulse
of high temperature [31] Recent proteomic data
dem-onstrated that several proteins involved in energy
pro-duction⁄ conversion are up-regulated under FTRs
(L Lalouette, H Colinet, D Siaussat & D Renault,
unpublished data) Moreover, it has been shown in
many insects that the HSP transcripts are up-regulated
during recovery from cold shock [32], and it was
recently found that FTRs significantly increase the
expression level of HSPs [17] HSPs are synthesized
during the pulses at high temperature, consuming the
FAA pool This assumption is supported by recent
proteomic studies showing significant up-regulations
of HSPs under FTRs (L Lalouette, H Colinet,
D Siaussat & D Renault, unpublished data)
As previously shown in several other insect species,
Pro was detected in remarkably high concentrations
Causal relationships between increased proline levels
and stress tolerance were also investigated, and a
posit-ive correlation was found with the insects’ cold
accli-mation [19], i.e Pro may stabilize either membranes or
proteins [3] However, the significant decrease of Pro
amounts recorded in cold-exposed beetles demonstrates
a reduced role of this amino acid in A diaperinus cold
acclimation Pro is an important energy substrate to
maintain ATP levels [4]: the energy yield from partial
oxidation of Pro to Ala is only slightly lower in
com-parison with lipids [33] A large accumulation of Ala
that occurred after the insects were cold exposed
(0Cc and 0CF20), which prompted a search for potential sources of the amino group, is thus an inter-esting result A partial involvement of the fermentative glycolysis in cold-exposed beetles, which would have led to increased amounts of Ala, was excluded Indeed, lactate was not detected in either these beetles or the
0CF20ones (data not presented) Moreover, a partial reliance on anaerobiosis would have resulted in a quicker depletion of glucose and glycogen amounts, because this type of respiration is less efficient in gen-erating ATP [15]
Ala accumulation suggests that it is derived from the singularly large stores of free Pro In that process, Pro is first oxidized to d-pyrolline-5-carboxylate which,
in turn, can be oxidized to Glu Transamination gave rise to Ala and a-ketoglutarate Such an increase in Ala contents is necessary to shuttle the amino group derived from the conversion of Pro to alpha-Ketoglu-tarate (a-KG) in flight muscle back to body fat [34] Oxidation of the keto acid in the citric acid cycle pro-duces ATP and results in the formation of malate, which is first converted to pyruvate and then to Ala When adult A diaperinus were warm exposed, Ala was reconverted back to pyruvate in the muscle, which is then a source of carbon atoms for gluconeogenesis This result is supported by the significantly higher amount of glucose found in 20CF0 beetles More-over, Pro is a well-known precursor in glucose and gly-cogen de novo synthesis [4] The present reduced level
of Pro in 20CF0beetles and the concomitant increase
of glucose indicates a use of Pro for glucose synthesis during daily warmer periods
Gln is of interest because it has been shown to play
an important role in several physiological processes During cold exposures, proteins and amino acids can serve as an important energy source via conversion to Krebs’ cycle intermediates and subsequent oxidation to
CO2 However, an important by-product of amino acid oxidation is ammonia (here we use ammonia to refer
to both NH3 and NH4+, or a combination of the two) Ammonia can be fixed on Glu to yield Gln, which accumulates in large amounts in cold-exposed beetles A diaperinus can then utilize the nitrogen of the amide group of two Gln molecules to synthesize one uric acid molecule Indeed, most terrestrial insects are uricotelic animals (i.e they excrete uric acid), and the synthesis of Gln as a chemical compound to hide the free ammonia for posterior excretion by glutami-nase activity is the strategy used by several insects [34] Hazel et al [35] also showed that Gln levels can modulate the secretion of ions and water by isolated Malpighian tubules of Rhodnius prolixus (Hemiptera: Reduviidae) and Drosophila melanogaster (Diptera:
Trang 6Drosophilidae) Secreted fluid pH and Na+
concentra-tion increase and K+ concentration decreases in
response to Gln These findings are interesting, as Gln
levels were significantly lower in 0CF20 beetles, and
we previously demonstrated in A diaperinus that the
extracellular concentrations of potassium ions
increased during cold periods Potassium ion
concen-trations returned to normal during the pulse at high
temperature under FTRs [11]
Adult insects of different species usually respond to
environmental stresses, e.g exposure to low
tempera-tures, with a neurohormonal stress reaction involving
the metabolism of juvenile hormone, dopamine (DA),
octopamine (OA) and ecdysteroids [20,36] Tyr plays
an important role in that process, as a precursor of
several stress hormones in insects (including DA, OA
and tyramine [15,20]) It was demonstrated in
Drosophila species that heat exposures induce a rise in
the DA level [37] and a concomitant decrease of Tyr
amounts Thus, the reduced level of Tyr recorded in
stressed adults of A diaperinus might be related to an
increased hormone synthesis, like DA This hypothesis
must be tested in further studies
Other than amino acids, the importance of polyols
and sugars such as glycerol and trehalose has been
emphasized regularly during insect cold acclimation
[21] Glycerol and trehalose are usually highly
signifi-cantly accumulated during cold exposures, and have
been shown to play an important role in protecting
protein and membrane integrity during exposures to
various environmental stresses [3] However, no
signifi-cant accumulation of trehalose was observed in
cold-exposed adults of A diaperinus Trehalose that can be
converted back to glycogen may therefore relate to
energy storage functions Moreover, the decrease in
glucose levels revealed that both trehalose and glucose
are involved in the synthesis of glycerol when adults of
A diaperinus are subjected to cold stress Because a
direct correlation between the accumulation of polyols
and an increase of Lt50 in the bug P apterus has
already been observed [21], the distinct pattern
recor-ded for glycerol between constant cold exposure and
FTR may contribute to extended survival times in the
cold-exposed insects under FTR
The slight, but nonsignificant, accumulation of the
other polyols (myo-inositol, ribitol and sorbitol), and
the synthesis of arabinitol in cold-exposed beetles may
be related to their cold acclimation [18] Indeed,
relat-ively low concentrations of sugars and polyols (with
negligible colligative effects) are sufficient to enhance
survival at subzero temperatures Accumulation of
myo-inositol has been documented in a few species of
arthropods [38] In Harmonia axyridis (Coleoptera:
Coccinellidae), large amount of myo-inositol are accu-mulated during winter Its content synchronizes sea-sonally with supercooling capacity, lower lethal temperature and chilling tolerance [39], suggesting that myo-inositol may play some role in the control of cold tolerance in this beetle In Aulacophora nigripennis (Coleoptera: Chrysomelidae), a high level of chill toler-ance occurs only when myo-inositol is accumulated [40] Our data revealed changes in several specific metab-olites that are likely to be related to the thermal stress
We found that breakdown of proteins occurred within the first days of cold exposure The synthesis of Gln,
an amino acid than can hide the free ammonia for posterior excretion, and the reduced FAA pool found
in ‘warm-exposed’ beetles during FTRs, demonstrate that FAA serves as an important energy source More-over, protein synthesis, like HSPs (L Lalouette,
H Colinet, D Siaussat & D Renault, unpublished data), occurred during the warm recovery periods, con-suming the FAA pool Some FAAs have been shown
to have cryoprotective properties in insects [3], but the relationship between FAAs, cold tolerance and survival has not yet been well defined Instead of considering FAA only as a part of the osmo- and cryoprotective arsenal, they should also be regarded as main actors involved in the multiple regulatory pathways activated during cold acclimation [41] In conclusion, FTRs trig-ger the initiation of a metabolic response involving the synthesis of protective compounds such as polyols and HSPs that probably contribute to the increased dur-ation of survival in A diaperinus
Experimental procedures
Rearing and acclimation conditions
Adult A diaperinus (Coleoptera: Tenebrionidae) were origin-ally collected from poultry house litter at Mohon (Morbi-han, France, 231¢56 W, 483¢14 N; altitude: 60 m) in February 2005 The insects were then reared in darkness at
20C and supplied with water and food ad libitum, consist-ing of moistened bran and dry dog food All the insects used for this study were between 2 and 3 months old at the begin-ning of the experiment Adult beetles were then used ran-domly, either for survival experiments or biochemical assays
To investigate the duration of survival, and changes in amino acid, polyol and sugar levels, beetles were kept either
at constant or cycling low temperatures In all experiments, beetles were maintained in the darkness and supplied with water but without food It has previously been observed that beetles enter in chill-coma and are thus not able to feed [9,12] A short starvation period has a minor impact
on the survival and biochemistry of the beetles [24]
Trang 7The survival of A diaperinus has already been investigated
and discussed in previous studies [9,24], where the same
population of insects was subjected to similar constant
tem-peratures and FTRs In the present work, longer recovery
periods were used during FTRs, in order to obtain
contras-ted metabolic responses between cold- (chilled) and
warm-exposed beetles
Groups of 10 beetles were transferred to Petri dishes To
avoid potential cold-shock, the insects were exposed at
15C for 48 h before being used for the survival
experi-ments Batches of beetles were randomly assigned to each
one of the following two thermal treatments (Fig 3): (a)
Constant low temperature: 10 Petri dishes were kept at
0C; and (b) fluctuating thermal regime: the beetles were
exposed 12 h at 20C cycling with 12 h at 0 C (n ¼ 10
Petri dishes) The cycling temperature regime started at
20C
One Petri dish per treatment was removed at daily
inter-vals, and the survival was assessed as the number of beetles
that showed limb movement after 2 days of recovery at
25C
Metabolite analysis
Groups of 50 beetles were placed in Petri dishes Two series
of experiments were performed as follows (Fig 3): (a)
Con-stant low temperature: three Petri dishes were kept at 0C
(0Cc), and the Petri dishes were removed from the
incuba-tor after 7 days; and (b) fluctuating thermal regime: beetles
were exposed 12 h at 20C interrupted by a daily transfer
to 0C for 12 h The cycling temperature regime started at
20C Two Petri dishes were removed from the incubators
after 7 days The first Petri dish was removed at the end of
the 12 h)20 C cycle, just before the temperature started
dropping to 0C (20 CF0) The second Petri dish was
removed 12 h later at the end of the 12 h)0 C cycle, just
before the temperature started rising to 20C (0 CF20)
Once removed, the insects were immediately pooled (n¼
3 per sample) and weighed (FM) using a Mettler
micro-balance accurate to 0.01 mg The beetles were then frozen
in liquid nitrogen and then stored at )80 C until the amino acid assays were performed Metabolites were also analyzed in control beetles reared at 20C in darkness; food was removed 2 days before sampling the insects as described in previous studies [12,13] For each experimental condition, 6–8 samples were analyzed
Amino acids
Amino acids were extracted from fresh material as des-cribed by Renault et al [13] Groups of three animals were homogenized in 1.5 mL of 70% ethanol and Fontainebleau sand, before adding 1.5 mL of 40% ethanol The homogen-ate was centrifuged for 10 min at 4500 g and 4C (Sigma 2-16 K, angle rotor 10· 20, Sigma-Aldrich Co.), 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 4C (Sigma 2-16 K, angle rotor 10 · 20, Sigma-Aldrich Co.), and the supernatant collected The sec-ond pellet was re-suspended in 1.5 mL ultrapure water and centrifuged for 10 min at 4500 g and 4C (Sigma 2-16 K, angle rotor 10· 20, Sigma-Aldrich Co.) The combined supernatant (n¼ 3) was pooled in a balloon flask and dried
by evaporation using a rota-vapour system The insoluble residue was re-suspended in 1 mL of ultrapure water Free amino acids were assayed as described by Bouche-reau et al [25] Amino acids were characterized and quanti-fied by HPLC after precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (using a Waters Accq-Tag amino acid analysis system; Waters Cor-poration, Milford, MA, USA) and reversed-phase liquid chromatographic separation (see [25] for a full description
of the method) Aliquots (10 lL) of the crude aqueous extracts were assayed using the procedure optimized by Bouchereau et al [25]
Sugars and polyols
Once removed from the incubator, the weighed beetles (n¼ 1 per sample) were homogenized with 0.4 mL of 70% (v⁄ v) ethanol The concentration of polyols was measured
by capillary gas chromatography (Varian 3400, Palo Alto,
Fig 3 Experimental design of the protocol used to determine the impact of cold exposure and associated metabolic changes in adults of
A diaperinus Batches of beetles were exposed at constant low temperature (0 C c ) and fluctuating thermal regime (20 C ⁄ 12 h: 20 C F0 , alternating with 0 C ⁄ 12 h: 0 C F20 ).
Trang 8CA, USA) as their o-methyloxime trimethysilyl derivates.
Identity of revealed component was established against
authentic standards and by mass spectrometry (Kratos,
Manchester, UK) The protocol was fully described earlier
by Kosˇta´l & Simek [5]
Statistical analyses
Values are given as the means ± se Lethal times for 50%
of the population (Lt50) were computed using probit
analy-sis for each temperature ancovas were performed to
remove the effects of body size Tukey’s tests were used for
post hoccomparisons The data were logarithmically
trans-formed, which improved their fit to a normal distribution
When the data did not follow normal distribution,
nonpar-ametric tests (Mann–Whitney: median comparison) were
conducted These statistical analyses were performed using
minitabTM (version 13) for Windows (Minitab Inc, 2000,
Paris, France)
Acknowledgements
This paper is publication number BRC 111 of the
Bio-diversity Research Centre of the Universite´ Catholique
de Louvain
References
1 Addo-Bediako A, Chown SL & Gaston KJ (2000)
Ther-mal tolerance, climatic variability and latitude Proc R
Soc Lond B 267, 739–746
2 Chown SL, Gaston KJ & Robinson D (2004)
Macro-physiology: large-scale patterns in physiological traits
and their ecological implications Funct Ecol 18, 159–167
3 Ramløv H (2000) Aspects of natural cold tolerance in
ectothermic animals Human Reprod 15, 26–46
4 Storey KB (1983) Metabolism and bound water in
over-wintering insects Cryobiol 20, 365–379
5 Kosˇta´l V & Simek P (1996) Biochemistry and
physiol-ogy of aestivo-hibernation in the adult apple blossom
weevil, Anthonomus pomorum (Coleoptera:
Curculioni-dae) J Insect Physiol 42, 727–733
6 Bale JS (2002) Insects at low temperatures: from
molecular biology to distributions and abundance Phil
Trans Roy Soc Lond 357, 849–862
7 Danks HV (2006) Insect adaptations to cold and
chan-ging environments Can Entomol 138, 1–23
8 Hane` Z & Nedvı`d O (1999) Chill injury at alternating
temperaturesin Orchesella cincta (Collembola:
Entomo-bryidae) and Pyrrhocoris apterus (Heteroptera:
Pyrrho-coridae) Eur J Entomol 96, 165–168
9 Renault D, Nedved O, Hervant F & Vernon P (2004)
The importance of fluctuating thermal regimes for
repairing chill injuries in the tropical beetle Alphitobius
diaperinus(Coleoptera: Tenebrionidae) during exposure
to low temperature Physiol Entomol 29, 139–145
10 Colinet H, Renault D, Hance T & Vernon P (2006) The impact of fluctuating thermal regimes on the survival of
a cold-exposed parasitic wasp, Aphidius colemani Physiol Entomol 31, 234–240
11 Kosˇta´l V, Renault D, Mehrabianova´ A & Bastl J (2007) Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: role of ion homeostasis Comp Biochem Physiol A Mol Integr Physiol doi: 10.1016⁄ j.cbpa.2006.12.033
12 Renault D, Hervant F & 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 Panzer (Coleoptera: Tenebrionidae) Physiol Entomol 27, 291–301
13 Renault D, Bouchereau A, Delettre YR, Hervant F & Vernon P (2006) Changes in free amino acids in Alphito-bius diaperinus(Coleoptera: Tenebrionidae) during ther-mal and food stress Comp Biochem Physiol A Mol Integr Physiol 143, 279–285
14 Gracey AY, Fraser EJ, Li W, Fang Y, Taylor RR, Rogers J, Brass A & Cossins AR (2004) Coping with cold: an integrative, multitissue analysis of the transcrip-tome of a poikilothermic vertebrate Proc Nat Acad Sci USA 101, 16970–16975
15 Malmendal A, Overgaard J, Bundy JG, Sørensen JG, Nielsen NChr, Loeschcke V & Holmstrup M (2006) Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila Am J Physiol – Regulatory Integrative Comp Physiol 291, 205–212
16 Powell EN, Kasschaut M, Chen E, Koenig M & Pecon J (1982) Changes in the free amino acid pool during envir-onmental stress in the gill tissue of the oyster, Crassostrea virginica Comp Biochem Physiol 71A, 591–598
17 Wang HS, Zhou CS, Guo W & Kang L (2006) Thermo-periodic acclimations enhance cold hardiness of the eggs
of the migratory locust Cryobiol 53, 206–217
18 Storey KB (1997) Organic solutes in freezing tolerance Comp Biochem Physiol A 117, 319–326
19 Fields PGF, Leurat-Lassard F, Lavenseau L & Febvay
G (1998) The effect of cold acclimation and deacclima-tion on cold tolerance, trehalose and free amino acid levels in Sitophilus granarius and Cryptolestes ferrugineus (Coleoptera) J Insect Physiol 44, 955–965
20 Hirashima A, Sukhanova MJh & Rauschenbach IYu (2000) Biogenic amines in Drosophila virilis under stress conditions Biosci Biotech Biochem 64, 2625–2630
21 Kosˇta´l V, Slachta M & Simek P (2001) Cryoprotective role of polyols independent of the increase in supercool-ing capacity in diapaussupercool-ing adults of Pyrrhocoris apterus (Heteroptera: Insecta) Comp Biochem Physiol B 130, 365–374
Trang 922 Pio CJ & Baust JG (1988) Effects of temperature
cycling on cryoprotectors profiles in the goldenrod gall
fly Eurosta solidaginis (Fitch) J Insect Physiol 34,
767–771
23 Kim Y & Song W (2000) Effect of thermoperiod and
photoperiod on cold tolerance of Spodoptera exigua
(Lepidoptera: Noctuidae) Environ Entomol 29,
868–873
24 Renault D, Hervant F & Vernon P (2003) Effect of
food shortage and temperature on oxygen consumption
in the lesser mealworm, Alphitobius diaperinus (Panzer)
(Coleoptera: Tenebrionidae) Physiol Entomol 28,
261–267
25 Bouchereau A, Duhaze C, Martin-Tanguy J, Guegan
J-P & Larher F (1999) Improved analytical methods for
determination of nitrogenous stress metabolites
occurring in Limonium species J Chromatogr A 836,
209–221
26 Leopold RA, Rojas RR & Atkinson P (1998) Post
pupariation cold storage of three species of flies:
increas-ing chillincreas-ing tolerance by acclimation and recurrent
recovery periods Cryobiol 36, 213–224
27 Coulson SJ & Bale JS (1996) Supercooling and survival
of the beach leaf mining weevil Rhynchaeus fagi L
J Insect Physiol 42, 617–623
28 Nedveˇd O, Lavy D & Verhoef HA (1998) Modelling
time-temperature relationship in cold injury and effect
of high temperature interruptions on survival in a chill
sensitive collembolan Funct Ecol 12, 816–824
29 Chang CL (2004) Effect of amino acids on larvae and
adults of ceratitis capitata (wiedemann) (Diptera:
Teph-ritidae) Ann Ent Soc Am 97, 529–535
30 John AM, Davis GR & Sosulski FW (1978) Protein
nutrition of Tenebrio molitor L XIX Growth response
to levels of dietary protein and of an amino acid
mixture Arch Int Physiol Biochim 86, 761–770
31 Chen CP & Denlinger DL (1992) Reduction of cold
injury in flies using an intermittent pulse of high
temperature Cryobiol 29, 138–143
32 Rinehart JP & Denlinger DL (2000) Heat-shock protein
90 is down-regulated during pupal diapause in the flesh
fly, Sarcophaga crassipalpis, but remains responsive to thermal stress Insect Mol Biol 9, 641–645
33 Auerswald L & Ga¨de G (1999) The fate of proline in the African fruit fly beetle Pachnoda sinuata Insect Biochem Molec Biol 29, 687–700
34 Scaraffia PY & Wells MA (2003) Proline can be utilized
as an energy substrate during flight of Aedes aegypti females J Insect Physiol 49, 591–601
35 Hazel MH, Ianowski JP, Christensen RJ, Maddrell SHP
& O’Donnell MJ (2003) Amino acids modulate ion transport and fluid secretion by insect Malpighian tubules J Exp Biol 206, 79–91
36 Matsumoto H, Tanaka K, Noguchi H & Hayakawa Y (2003) Cause of mortality in insects under severe stress Eur J Biochem 270, 3469–3476
37 Rauschenbach IY, Shumnaya LV, Khlebodarova TM, Chentsova NA & Grenback LG (1995) Role of phenol oxidases and tyrosine hydroxylase in control of dopa-mine content in Drosophila virilis under normal condi-tions and heat stress J Insect Physiol 41, 279–286
38 Kosˇta´l V, Nedveˇd O & Simek P (1996) Accumulation of high concentrations of myo-inositol in the overwintering ladybird beetle Ceratomegilla undecimnotata
Cryo-Letters 17, 267–272
39 Watanabe M (2002) Cold tolerance and myo-inositol accumulation in overwintering adults of a lady beetle, Harmonia axyridis(Coleoptera: Coccinellidae) Eur
J Entomol 99, 5–9
40 Watanabe M & Tanaka K (1998) Adult diapause and cold hardiness in Aulacophora nigripennis (Coleoptera: Chrysomelidae) J Insect Physiol 44, 1103–1110
41 Colinet H, Hance T, Vernon P, Bouchereau A & Renault D (in press) Does fluctuating thermal regime trigger free amino acid production in the parasitic wasp Aphidius Colemani Hymenoptera: Aphidiinae)? Comp Biochem Physiol – Part A: Molec Integrat Physiol, in press