Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes L. Lalouette 1 , V. Kos ˇ ta ´ l 2 , H. Colinet 3 , D. Gagneul 4,5 and D. Renault 1 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 Ba ˆ t. 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 physiological 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. FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS 1759 Such 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, Kos ˇ ta ´ 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 8 °C. In a more recent work, we found that beetles kept at a constant tempera- ture of 0 °C quickly died, whereas the effects of chilling were reversed completely when insects were kept in FTRs (5 °C ⁄ 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 (Lt 50 ) of the beetles exposed at constant 0 °C was 5.95 ± 0.65 days. After 10 days of FTR (0 °C alternating with 20 °Con 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 Lt 50 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. Cold exposure and associated metabolic changes L. Lalouette et al. 1760 FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS The total FAA pool was significantly higher in control beetles [73.19 ± 2.18 nmolÆmg )1 fresh mass (FM)] than in constant cold-exposed (0 °C c ) (56.72 ± 3.31 nmolÆmg )1 FM) and 20 °C F0° (20 °C/12 hr[20 °C F0° ] fluctuating with 0 °C/12 hr[0 °C F20° ]) ones (51.56 ± 2.35 nmolÆmg )1 FM) (P<0.05); the total amount of FAA was significantly higher in 0 °C F20° beetles (72.58 ± 4.58 nmolÆmg )1 FM) than in 0 °C c and 20 °C F0° ones (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 0 °C, 50% of the total FAA pool at alter- nating temperature (20 °C F0° ,0°C F20° ) (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, 0 °C c ,0°C F20° and 20 °C F0° ). 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 0 °C c beetles (P<0.05), whereas an opposite conclusion was found with Pro (significantly higher in control and 0 °C c beetles). Amino acid profiles in 0 °C c versus 0 ° C F20° beetles The level of several FAA differed between the 0 °C c and 0 °C F20° thermal treatments. Ala, Gln and Pro accounted for most of the observed quantitative differ- ence; Ala and Pro being highly accumulated in the 0 °C F20° beetles (10.03 ± 2.14 and 37.21 ± 3.12 nmo- lÆmg )1 FM, respectively) compared with the 0 °C c 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 0 °C c beetles, Ala content was not signi- ficantly different from 0 °C F20° beetles (P > 0.05). Levels of Glu and Gln had opposite patterns and were significantly lower in 0 °C F20° than in 0 °C c 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 0 °C F20° beetles (P<0.05). Amino acid profiles in 0 °C F20° versus 20 °C F0° beetles Several differences were observed in the amounts of amino acids between 0 °C F20° and 20 °C F0° 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 0 °C F20° 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 0 °C F20° , 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 20 °C F0° . It is interesting to notice that Gln and Ala had an opposite pattern, with the highest level in 0 ° C c , the lowest level in 0 °C F20° and an intermediate level in 20 °C F0° beetles for Gln, the highest amounts in 0 °C F20° , the lowest amounts in 20 °C F0° and an inter- mediate situation in 0 °C c 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). L. Lalouette et al. Cold exposure and associated metabolic changes FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS 1761 Moreover, 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 0 °C F20° beetles, 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 0 °C F20° beetles. Mannitol was not detected in control beetles, whereas small amounts were found in 0 °C F20° (0.022 ± 0.002 nmolÆmg )1 FM) and 20 °C F0° beetles (0.018 ± 0.003 nmolÆmg )1 FM). Arabinitol was only detected in 0 °C F20° beetles, 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 0 °C (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 0 °C. 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 0 °C 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). Cold exposure and associated metabolic changes L. Lalouette et al. 1762 FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS Cold 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 (0 °C c and 0 °C F20° ), 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 0 °C F20° ones (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 20 °C F0° 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 20 °C F0° beetles 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 CO 2 . However, an important by-product of amino acid oxidation is ammonia (here we use ammonia to refer to both NH 3 and NH 4 + , 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: L. Lalouette et al. Cold exposure and associated metabolic changes FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS 1763 Drosophilidae). 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 0 °C F20° 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 Lt 50 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, 2°31¢56 W, 48°3¢14 N; altitude: 60 m) in February 2005. The insects were then reared in darkness at 20 °C 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]. Cold exposure and associated metabolic changes L. Lalouette et al. 1764 FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS Survival The 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 15 °C 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 0 °C; and (b) fluctuating thermal regime: the beetles were exposed 12 h at 20 °C cycling with 12 h at 0 °C(n ¼ 10 Petri dishes). The cycling temperature regime started at 20 °C. 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 25 °C. 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 0 °C (0 °C c ), and the Petri dishes were removed from the incuba- tor after 7 days; and (b) fluctuating thermal regime: beetles were exposed 12 h at 20 °C interrupted by a daily transfer to 0 °C for 12 h. The cycling temperature regime started at 20 °C. 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 0 °C (20 °C F0° ). 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 20 °C(0°C F20° ). Once removed, the insects were immediately pooled (n ¼ 3 per sample) and weighed (FM) using a MettlerÒ 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 20 °C 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 4 °C (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 4 °C (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 4 °C (Sigma 2-16 K, angle rotor 10 · 20, Sigma-Aldrich Co.). The combined supernatant (n ¼ 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 (n ¼ 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° ). L. Lalouette et al. Cold exposure and associated metabolic changes FEBS Journal 274 (2007) 1759–1767 ª 2007 The Authors Journal compilation ª 2007 FEBS 1765 CA, 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 Kos ˇ ta ´ l & Simek [5]. Statistical analyses Values are given as the means ± se. Lethal times for 50% of the population (Lt 50 ) 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 hoc comparisons. 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Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes L. Lalouette 1 ,. polyols (myo-inositol, ribitol and sorbitol), and the synthesis of arabinitol in cold- exposed beetles may be related to their cold acclimation [18]. Indeed,