Late-born intermittently fasted juvenile garden dormice use torpor to grow and fatten prior to hibernation: consequences for ageing processes

Torpor is thought to slow age-related processes and to sustain growth and fattening of young individuals. Energy allocation into these processes represents a challenge for juveniles, especially for those born late in the season. We tested the hypothesis that late-born juvenile garden dormice (Eliomys quercinus) fed ad libitum (‘AL’, n = 9) or intermittently fasted (‘IF’, n = 9) use short torpor bouts to enhance growth and fat accumulation to survive winter. IF juveniles displayed more frequent and longer torpor bouts, compared with AL individuals before hibernation. Torpor frequency correlated negatively with energy expenditure and water turnover. Hence, IF juveniles gained mass at the same rate, reached similar pre-hibernation fattening and displayed identical hibernating patterns and mass losses as AL animals. We found no group differences in relative telomere length (RTL), an indicator of ageing, during the period of highest summer mass gain, despite greater torpor use by IF juveniles. Percentage change in RTL was negatively associated with mean and total euthermic durations among all individuals during hibernation. We conclude that torpor use promotes fattening in late-born juvenile dormice prior to hibernation. Furthermore, we provided the first evidence for a functional link between time spent in euthermy and ageing processes over winter.     

Body temperature patterns and use of torpor in an alpine glirid species,woolly dormouse

Woolly dormice, Dryomys laniger Felten and Storch (Senckenbergiana Biol 49(6):429–435, 1968), are a small (20–30 g), omnivorous (mainly insectivorous), nocturnal glirid species endemic to Turkey. Although woolly dormice have been assumed to hibernate during winter, no information exists on body temperature patterns and use of torpor in the species. In the present study, we aimed to determine body temperature patterns and use of torpor in woolly dormice under controlled laboratory conditions. Accordingly, body temperature (Tb) of woolly dormice was recorded using surgically implanted Thermochron iButtons, small and inexpensive temperature-sensitive data loggers. Woolly dormice exhibited robust, unimodal daily Tb rhythmicity during the euthermic stage before the beginning of hibernation. They displayed short torpor before they began hibernation, although the tendency to enter short torpor was different among individuals. Woolly dormice began hibernation within 1–3 days after exposure to cold and darkness, i.e., on October 22–24, and ended hibernation in the first half of April. Hibernation consisted of a sequence of multiday torpor bouts, interrupted by euthermic intervals. Thus, the patterns of hibernation in woolly dormice were similar to those observed in classical hibernating mammals.     

Group hibernation does not reduce energetic costs of young yellow-bellied marmots

We investigated mechanisms of energy conservation during hibernation. The amount of time torpid was significantly less for groups of three young marmots than for marmots hibernating singly. Mean daily mass loss (DML; as mg d(-1) g(-1) immergence mass) averaged 1.33 for single marmots and 1.46 for grouped young. Animals were active 17.3% of the time, which used 82.4% of the energy, and were torpid 82.7% of the time, which used 17.6% of the energy expenditure. During longer torpor bouts, more time was spent in deep torpor, which decreased the hourly cost of a complete bout. Bout oxygen consumption V dot o2, percent time in deep torpor, and body temperature (T(B)) during deep torpor changed seasonally and were curvilinearly related to when in the hibernation period the measurements were made and probably represent a stage in the circannual metabolic cycle. The decrease of environmental temperature (T(E)) to 2 degrees C significantly increased metabolism. Potential costs of low T(E) were reduced by allowing T(B) to decrease, thereby reducing the T(B) to T(E) gradient. Average monthly metabolic rate was high early and late in the hibernation period when time spent euthermic was greater and when VO2 was higher. Over the hibernation period, energy saved averaged 77.1% and 88.0% of the costs for winter and summer euthermic metabolism, respectively. Hibernation costs were reduced by the seasonal changes, the high percentage of time in torpor, the rapid decline in V dot o2 following arousal, and allowing T(B) to decline at lower T(E). Asynchrony in the torpor cycles increased energy expenditures in group hibernators, which negated possible beneficial effects of group hibernation.     

Daily torpor in free-ranging common dormice (Muscardinus avellanarius) in Lithuania

Torpid common dormice (Muscardinus avellanarius) were found in nestboxes during all the activity period from early April until late October. Prevalence of torpor among dormice was highest in spring, decreased considerably in summer and increased again in October. The proportion of adult dormice that were torpid was inversely related to mean monthly air temperature, except in September, when dormice prepare for hibernation by accumulating fat reserves. In spring and summer, torpid dormice were found when ambient temperature was up to 14–15 °C and in the arousal phase of torpor – up to 19–20 °C. In autumn, dormice were active at much lower ambient temperatures compared to spring and summer, and only dormice that were fat enough and ready for hibernation were torpid. Torpor bouts usually lasted only until noon, but also in the afternoon during persistently low ambient temperatures in April and October. In April–July, torpor was more frequent among adult males than females (75% vs. 46%), and pregnant females were never found in deep torpor. Prevalence of torpor among young-of-the-year was lower compared to adult dormice at the same time. In October, the average weight of torpid young-of-the-year dormice was significantly higher compared to the weight of active dormice (22.3 vs. 17.5 g).

Zusammenfassung

Tagestorpor bei frei lebenden Haselmäusen (Muscardinus avellanarius) in LitauenHaselmäuse (Muscardinus avellanarius) im Torpor wurden während der gesamten aktiven Saison zwischen zeitigem April und Ende Oktober in Nistkästen gefunden. Im Frühjahr war die Torporfrequenz am höchsten, während des Sommers ging sie beträchtlich zurück, und im Oktober stieg sie wieder deutlich. Der Anteil an Haselmäusen im Torpor war umgekehrt abhängig von der mittleren Monatslufttemperatur, mit Ausnahme des Septembers, wo sich die Haselmäuse Fettreserven in Vorbereitung des Winterschlafes anlegen. Während des Frühlings und des Sommers konnten Haselmäuse im Torpor gefunden werden, wenn die Umgebungstemperatur bis 14–15 °C war, und in der Aufwachphase bei Temperaturen bis 19–20 °C. Im Herbst waren die Haselmäuse bei viel tieferen Temperaturen als im Sommer oder Frühjahr aktiv und nur die Haselmäuse die fett genug waren, um in den Winterschlaf gehen zu können, waren lethargisch. Die Torporphasen dauerten in der Regel nur bis zum Mittag, aber im April und Oktober während lang anhaltenden tiefen Umgebungstemperaturen auch bis in den Nachmittag hinein. Zwischen April und Juli konnten mehr adulte Männchen als Weibchen im Torpor nachgewiesen werden (75% vs 46%). Tragende Weibchen waren niemals im tiefen Torpor zu finden. Die Torporfrequenz war bei Haselmäusen in ihrem ersten Lebensjahr geringer als bei adulten im selben Zeitraum. Im Oktober war bei den lethargischen Jungtieren des Jahres das Durchschnittsgewicht höher als bei den aktiven Haselmäusen (22.3 vs. 17.5 g).     

Summer torpor in a free-ranging bat from subtropical Australia

It is widely believed that torpor is mainly an adaptation of endotherms for cold stress and food limitation. We studied torpor use in the wild by a small tree-roosting microbat from a sub-tropical area during summer when food was abundant. Surprisingly, two torpor bouts per day were employed on each roost-day observed. The first bout occurred in the early morning and the second bout in the late afternoon, whilst a period of normothermia was maintained over the warmest part of the day. Torpor likely reduced energy expenditure substantially, and may be common in small microbats whose day-roosts are poorly insulated, even in sub-tropical climates.     

The role of energy availability in Mammalian hibernation: a cost-benefit approach

Hibernation is widely regarded as an adaptation to seasonal energy shortage, but the actual influence of energy availability on hibernation patterns is rarely considered. Here we review literature on the costs and benefits of torpor expression to examine the influence that energy may have on hibernation patterns. We first establish that the dichotomy between food- and fat-storing hibernators coincides with differences in diet rather than body size and show that small or large species pursuing either strategy have considerable potential scope in the amount of torpor needed to survive winter. Torpor expression provides substantial energy savings, which increase the chance of surviving a period of food shortage and emerging with residual energy for early spring reproduction. However, all hibernating mammals periodically arouse to normal body temperatures during hibernation. The function of these arousals has long been speculated to involve recovery from physiological costs accumulated during metabolic depression, and recent physiological studies indicate these costs may include oxidative stress, reduced immunocompetence, and perhaps neuronal tissue damage. Using an optimality approach, we suggest that trade-offs between the benefits of energy conservation and the physiological costs of metabolic depression can explain both why hibernators periodically arouse from torpor and why they should use available energy to minimize the depth and duration of their torpor bouts. On the basis of these trade-offs, we derive a series of testable predictions concerning the relationship between energy availability and torpor expression. We conclude by reviewing the empirical support for these predictions and suggesting new avenues for research on the role of energy availability in mammalian hibernation.     

Allometries of the durations of torpid and euthermic intervals during mammalian hibernation: A test of the theory of metabolic control of the timing of changes in body temperature

Summary The durations of the intervals of torpor and euthermia during mammalian hibernation were found to be dependent on body mass. These relationships support the concept that the timing of body temperature changes is controlled by some metabolic process. Data were obtained from species spanning nearly three orders of magnitude in size, that were able to hibernate for over six months without food at 5°C. The timing of body temperature changes was determined from the records of copper-constantan thermocouples placed directly underneath each animal. Because all species underwent seasonal changes in their patterns of hibernation, animals were compared in midwinter when the duration of euthermic intervals was short and relatively constant and when the duration of torpid intervals was at its longest. Large hibernators remained euthermic longer than small hibernators (Fig. 2). This was true among and within species. The duration of euthermic intervals increased with mass at the same rate (mass^0.38) that mass-specific rates of euthermic metabolism decrease, suggesting that hibernators remain at high body temperatures until a fixed amount of metabolism has been completed. These data are consistent with the theory that each interval of euthermia is necessary to restore some metabolic imbalance that developed during the previous bout of torpor. In addition, small species remained torpid for longer intervals, than large species (Fig. 3). The absolute differences between different-sized species were large, but, on a proportional basis, they were comparatively slight. Mass-specific rates of metabolism during torpor also appear to be much less dependent on body mass than those during euthermia, but the precision of these metabolic measurements is insufficient for them to provide a conclusive test of the metabolic theory. Finally, small species with high mass-specific rates of euthermic metabolism are under tighter energetic constraints during dormancy than large species. The data presented here show that, in midwinter, small species compensate both by spending less time at high body temperatures following each arousal episode and by arousing less frequently, although the former is far more important energetically than the latter.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.     

Seasonal torpor and normothermic energy metabolism in the Eastern chipmunk (Tamias striatus)

To assess the changes in thermoregulatory characteristics that accompany the seasonal expression of torpor we measured seasonal differences in body mass adjustments, body temperature (T _b) and metabolic rate (MR) in both summer- and winter-acclimated individuals from a species of food-storing hibernator, the Eastern chipmunk (Tamias striatus). Torpor occurred only in the winter and was associated with lower normothermic T _b, during inter-bout arousal periods than in the summer. Chipmunks increased body mass before the initiation of torpor in winter, and steadily lost mass as the hibernation season progressed. Torpor expression was correlated to initial mass gain, with the individuals who showed the largest mass increase in the fall showing the highest degree of torpor. Acclimation to winter-like conditions produced a decline in normothermic MR at all ambient temperatures examined. The findings indicate that torpor expression is accompanied by a decrease in T _b and MR during normothermy, indicating that a conservation of energy metabolism occurs, not only in torpor, but also during the inter-bout arousal periods.     

Summer and winter torpor use by a free-ranging marsupial

Torpor is usually associated with low ambient temperatures (T(a)) in winter, but in some species it is also used in summer, often in response to limited food availability. Since the seasonal expression of torpor of both placental and marsupial hibernators in the wild is poorly documented by quantitative data, we investigated torpor and activity patterns of the eastern pygmy-possum Cercartetus nanus (17.4 g) over two seasons. We used radio telemetry to track animals during winter (n=4) and summer (n=5) in a warm-temperate habitat and found that torpor was used in both seasons. In winter all animals entered periods of short-term hibernation (from 5 to 20 days) containing individual torpor bouts of up to 5.9 days. In summer, torpor bouts were always <1 day in duration, only used by males and were not related to daily mean T(a). Pygmy-possums entered torpor at night as T(a) cooled, and rewarmed during the afternoon as T(a) increased. Individuals interspersed torpor bouts with nocturnal activity and the percentage of the night animals were active was the same in summer and winter. Our study provides the first information on torpor patterns in free-ranging C. nanus, and shows that the use of torpor throughout the year is important for energy management in this species.     

Daily torpor in free-ranging rock elephant shrews, Elephantulus myurus: a year-long study

Under laboratory conditions, rock elephant shrews, Elephantulus myurus, use daily torpor under both short and long photoperiod acclimation. However, use of heterothermy often differs under field and laboratory conditions. We investigated the use of torpor in free-ranging elephant shrews from May 2001 to May 2002. The elephant shrews were capable of daily torpor throughout the year, with torpor most prevalent during winter. We recorded two torpor bouts during early summer (November). We recorded a total of 467 torpor bouts during the year. The mean torpor minimum body temperature (Tbmin) for the whole year was 15.3 degrees +/-4.4 degrees C, and the mean bout length was 8.6+/-3.5 h. These values were in the range expected for daily heterotherms. However, there was some marginal overlap with hibernation characteristics; a few torpor bouts were longer than 24 h in duration, and Tbmin decreased below 10 degrees C. Torpor was highly correlated with low ambient temperature and photoperiod. Torpor was also correlated with invertebrate abundance after controlling for photoperiod effects. During the year in which this study was conducted, the rainfall was 14% below long-term average. Historical rainfall records show that summer rainfall during strong El Nino years is up to 40% below the long-term average. During these drought years, the frequency of summer torpor may be higher, highlighting the need for long-term physiological data in free-ranging animals.     

The role of energy availability in Mammalian hibernation: an experimental test in free-ranging eastern chipmunks

Reduced torpor expression by hibernating mammals is often attributed to physiological constraints that limit their hibernation ability but may instead reflect adaptive, plastic responses to surplus energy availability. We evaluated this hypothesis by supplementing the food hoards of free-ranging eastern chipmunks (Tamias striatus) before hibernation and then documenting their use of torpor during the subsequent winter. In both years of study, chipmunks that received additional food were euthermic more than twice as frequently as nonsupplemented individuals. Furthermore, when food-supplemented individuals did express torpor, their minimum collar temperature was 5 degrees -10 degrees C warmer than nonsupplemented animals. These results indicate that reduced torpor expression by hibernators can result from an absence of energetic necessity rather than a lack of physiological capability and suggest that even endotherms sequestered in a hibernaculum may benefit from maintaining an elevated body temperature whenever possible.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Survival rates in a small hibernator,the edible dormouse: a comparison across Europe

Understanding how local environmental factors lead to temporal variability of vital rates and to plasticity of life history tactics is one of the central questions in population ecology. We used long‐term capture‐recapture data from five populations of a small hibernating rodent, the edible dormouse Glis glis, collected over a large geographical range across Europe, to determine and analyze both seasonal patterns of local survival and their relation to reproductive activity. In all populations studied, survival was lowest in early summer, higher in late summer and highest during hibernation in winter. In reproductive years survival was always lower than in non‐reproductive years, and females had higher survival rates than males. Very high survival rates during winter indicate that edible dormice rarely die from starvation due to insufficient energy reserves during the hibernation period. Increased mortality in early summer was most likely caused by high predation risk and unmet energy demands. Those effects have probably an even stronger impact in reproductive years, in which dormice were more active. Although these patterns could be found in all areas, there were also considerable differences in average survival rates, with resulting differences in mean lifetime reproductive success between populations. Our results suggest that edible dormice have adapted their life history strategies to maximize lifetime reproductive success depending on the area specific frequency of seeding events of trees producing energy‐rich seeds.     

Hibernating patterns of free-ranging Columbian ground squirrels

Summary The patterns of torpor and euthermy during hibernation was documented for 28 free-ranging Columbian ground squirrels (Spermophilus columbianus) fitted with temperature-sensitive radio transmitter collars. Adult males began hibernation earlier, were euthermic for a greater proportion of the hibernating season and emerged earlier than other age and sex classes. The patterns of hibernation of adult females did not differ significantly from those of juveniles. Emergence from the hibernaculum was preceded by a long (3–12 d) euthermic interval in adult males but not in adult females or juveniles. Changes in soil temperature did not appear to initiate emergence. The greater time spent euthermic by adult males is interpreted as a significantly greater energy cost of hibernation for adult males than for other age and sex classes. The benefits offsetting these costs may be increased reproductive potential in spring and avoidance of predation in late summer.     

Nucleoli undergo structural and molecular modifications during hibernation

The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.     

Hormonal changes and energy substrate availability during the hibernation cycle of Syrian hamsters

Animals have to adapt to seasonal variations in food resources and temperature. Hibernation is one of the most efficient means used by animals to cope with harsh winter conditions, wherein survival is achieved through a significant decrease in energy expenditure. The hibernation period is constituted by a succession of torpor bouts (hypometabolism and decrease in body temperature) and periodic arousals (eumetabolism and euthermia). Some species feed during these periodic arousals, and thus show different metabolic adaptations to fat-storing species that fast throughout the hibernation period. Our study aims to define these metabolic adaptations, including hormone (insulin, glucagon, leptin, adiponectin, GLP-1, GiP) and metabolite (glucose, free fatty acids, triglycerides, urea) profiles together with body composition adjustments. Syrian hamsters were exposed to varied photoperiod and temperature conditions mimicking different phases of the hibernation cycle: a long photoperiod at 20 °C (LP20 group), a short photoperiod at 20 °C (SP20 group), and a short photoperiod at 8 °C (SP8). SP8 animals were sampled either at the beginning of a torpor bout (Torpor group) or at the beginning of a periodic arousal (Arousal group). We show that fat store mobilization in hamsters during torpor bouts is associated with decreased circulating levels of glucagon, insulin, leptin, and an increase in adiponectin. Refeeding during periodic arousals results in a decreased free fatty acid plasma concentration and an increase in glycemia and plasma incretin concentrations. Reduced incretin and increased adiponectin levels are therefore in accordance with the changes in nutrient availability and feeding behavior observed during the hibernation cycle of Syrian hamsters.     

The role of succinate dehydrogenase and oxaloacetate in metabolic suppression during hibernation and arousal

Hibernation elicits a major reduction in whole-animal O₂ consumption that corresponds with active suppression of liver mitochondrial electron transport capacity at, or downstream of, succinate dehydrogenase (SDH). During arousal from the torpor phase of hibernation this suppression is reversed and metabolic rates rise dramatically. In this study, we used the 13-lined ground squirrel (Ictidomys tridecemlineatus) to assess isolated liver mitochondrial respiration during the torpor phase of hibernation and various stages of arousal to elucidate a potential role of SDH in metabolic suppression. State 3 and state 4 respiration rates were seven- and threefold lower in torpor compared with the summer-active and interbout euthermic states. Respiration rates increased during arousal so that when body temperature reached 30°C in late arousal, state 3 and state 4 respiration were 3.3- and 1.8-fold greater than during torpor, respectively. SDH activity was 72% higher in interbout euthermia than in torpor. Pre-incubating with isocitrate [to alleviate oxaloacetate (OAA) inhibition] increased state 3 respiration rate during torpor by 91%, but this rate was still fourfold lower than that measured in interbout euthermia. Isocitrate pre-incubation also eliminated differences in SDH activity among hibernation bout stages. OAA concentration correlated negatively with both respiration rates and SDH activity. These data suggest that OAA reversibly inhibits SDH in torpor, but cannot fully account for the drastic metabolic suppression observed during this hibernation phase.