Survival, fresh mass and water content
Temperature is an important parameter during fasting events, as it significantly influences the duration of survival. Insect activity is highly reduced below 16 °C, and as might be expected, activity stops at 12 °C, beetles falling into short-term chill coma (this study; Salin, 1999; Nedved, 2000). These insects are not able to reach food, and starve although food is available, explaining why fresh mass and metabolite changes are similar to those in starved beetles. The reduced metabolic rate described in ectotherms at low temperatures, and the concomitant reduced activity, may lead to energy savings and result in longer survival. Rearing in the laboratory showed that mean longevity in adults placed at 21–24 °C was beyond 400 days (in comparison to c. 50 days during starvation); two beetles have even lived for 703 days with food ad libitum (Preiss & Davidson, 1971).
Extensive body energy reserves may be lost as the beetles metabolize their tissues, particularly fat body tissues, to meet critical energy requirements and, as a result, fresh mass is affected during starvation. It has been demonstrated that in adult A. diaperinus food deprivation affects weight independently of sex (Salin et al., 2000). To maintain the necessary body volume (constrained by the exoskeleton in arthropods) and internal turgidity during starvation, the lost tissues must be replaced by water. Water ratio variations are the results of both water uptake, ensuring the internal cell turgidity, metabolic activity performances being linked to hydration level (Hervant et al., 1999) and dry mass decrease. The slight decrease of fresh mass in fed beetles maintained at 12 and 16 °C leads to a slight increase of percent body water. By contrast, re-feeding leads to an increase in fresh mass, resulting in a decrease in the percentage body water.
Metabolic response to food deprivation
The most pronounced changes during starvation are observed in the levels of triglycerides, these being greatly depleted at each of the four temperatures studied. The most common source of energy in insects during starvation is the oxidation of fatty acids stored in the form of triglyceride (Downer, 1985) and, usually, large amounts of lipids are accumulated in insect fat body, with fat content exceeding 50% of the total dry mass in some insects (Beenakkers et al., 1981; Beenakkers et al., 1985). Levels of stored fat significantly influence survival during food shortage, and the amounts of triglyceride are correlated positively with the duration of survival (Pullin, 1987). Complete consumption of fuel reserves may be the major reason for insect death at the four temperatures studied. In this study, levels of glycogen are little affected during starvation, in comparison with the African fruit beetle Pachnoda sinuate in which the level of carbohydrates is rapidly and almost completely depleted during food shortage (Auerswald & Gäde, 2000). In adult A. diaperinus, glycogen stores may be preserved until lipids are depleted, as shown in the spruce budworm Choristoneura fumiferana (Han & Bauce, 1998). Although trehalose is the main carbohydrate in insect heamolymph (see Becker et al., 1996), there is also a significant concentration of glucose (Friedman, 1985). Glucose levels determined in this study show no significant variations, except at the stressful 12 °C temperature. It has been suggested recently that the haemolymph glucose may play an essential role during starvation as it has a direct role in controlling carbohydrate metabolism. Its concentration might be an indicator of the status of carbohydrate metabolism in insects (Meyer-Fernandes et al., 2001). In addition, glyconeogenesis from glycerol and/or amino acids and glycogen savings are critically important for sustaining tissues and organs, the nervous system displaying an absolute requirement for glucose (Wegener, 1987). Moreover, levels of trehalose may be regulated at the expense of tissue glycogen during the first hours of starvation, but quickly level off if food shortage continues (Thompson, 1995, 1998; Meyer-Fernandes et al., 2001). The statistically significant decrease in glucose after 35 days of starvation at 12 °C may indicate a shift from lipids to carbohydrates as metabolic fuels, induced by the low levels of triglyceride.
Protein levels are not significantly affected by food shortage except at the highest temperature studied. No special forms of protein are stored for metabolic fuel, but endogenous proteins may be an important source of carbon and energy during harsh conditions. Hydrolysed proteins may be either strictly used as fuel for metabolism or converted to glucose through gluconeogenesis. Bosquet (1977) suggested that protein synthesis decreases during food deprivation, leading to a decrease in protein levels as observed here in starved beetles kept at 24 °C. However, the observed protein sparing may be an adaptation of species that sporadically endure starvation. An insect's recovery from starvation period may depend upon its ability to move about, and a compromise must be struck between the demand for carbohydrates and the preservation of muscle proteins.
The two products of the complete hydrolysis of triglyceride are free fatty acids and glycerol. During starvation, triglycerides are almost completely depleted, and glycerol levels also dropped, except at 12 °C. Glycerol may be either removed from the cell for metabolism elsewhere, or metabolized in situ (gluconeogenesis) allowing the saving of glucose and glycogen. Glycerol may also be used as an energy substrate for cell metabolism. At 12 °C, glycerol may be produced by the catabolism of a small part of glycogen reserves. It has also been suggested that the amino acids alanine and glutamate can be converted to glycerol (Raymond & Driedzic, 1997).
Because of the greater metabolic rate of insects with increased temperatures (Clarke, 1991), proteins, glycogen and glycerol are mobilized and catabolized more rapidly at 24 °C than at other temperatures. Conversely, triglycerides are almost completely depleted at 12 (even in fed beetles) and 16 °C, whereas one-third of triglycerides are used at 20–24 °C. This demonstrates the restrictive effects of temperature on adult beetles. At 12 °C, fed and starved insects both effectively starved, leading to the depletion of triglycerides. However, we may wonder why triglycerides are less used in starved insects at higher temperatures although metabolic rates and activity remained higher. During food shortage and cold exposure, energy metabolism is solely derived from body stores, and physiological adaptations to both parameters are highly energy depleting. During such periods, energy allocation for starvation (energy for metabolism) vs. temperature acclimation (membrane acclimation, production of polyols) must be strictly regulated/controlled, both being essential for insect survival. Low temperature exposures lead to lipid depletion, as suggested by David et al. (1996) in the millipede Polydesmus angustus.
Glycerol accumulation in both starved and fed insects kept at 12 °C is at first surprising. This stressful temperature, corresponding to the upper limit of the cold injury zone (Renault et al., 1999; Nedved, 2000), is well above that at which cryoprotection should be expected. Such accumulation has already been reported in Eurosta solidaginis larvae kept at 15 °C (Storey et al., 1981). Increased levels of glycerol have, at first, no colligative effects and as a result no cryoprotective function (i.e. protecting the organism against freezing) at such temperatures. However, initiation of glycerol production/accumulation that is mediated by environmental temperatures may anticipate the need for cryoprotection against sudden unseasonable frosts (Storey & Storey, 1990; Goto et al., 2001). In addition, glycerol may be implicated in temperature acclimation by increasing protein structural stability (Hochachka & Somero, 1984). Higher levels of polyols are required generally to obtain significant impact on protein stability, although it has been demonstrated that small amounts of sorbitol may have significant effects (Carpenter & Crowe, 1988; Carpenter et al., 1990). However, Yoder et al. (1992) and Pullin (1996) support the hypothesis of glycerol accumulation as a by-product of metabolic suppression. Ring & Danks (1994) have also proposed that adaptations for cryoprotection and for desiccation resistance overlapped, suggesting that tropical species such as A. diaperinus are pre-adapted to cold exposures. Recent studies (Storey & Storey, 1992; Wolfe et al., 1998; Salvucci, 2000) have shown that polyols are implicated in both cold and heat stress. This kind of pre-adaptation probably facilitates a rapid colonization of temperate regions.
Metabolic responses to subsequent re-feeding
Above 16 °C, re-feeding results in a rapid restoration of energy reserves, particularly triglycerides, and also glycerol and glycogen. The preferential degradation of lipids and also glycogen as fuel for metabolism and the protein sparing during starvation may preserve essential functions such as locomotion. This protein sparing may be of prime necessity for adult A. diaperinus to rapidly resume locomotory activity (food searching activity) when food becomes available again and may allow a rapid recovery. This may be crucial, particularly in habitats where food competition occurs: animals whose locomotory capabilities are rapidly restored may have a significant advantage for further population growth. However, during exposure at stressful (low) temperatures, insect recovery is slower because locomotory activity is highly reduced or stopped, beetles being most of the time in a temporary chill-coma state (Renault et al., 1999), and digestive metabolism is significantly depressed as shown in other species (Leather et al., 1993).