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Keywords:

  • Energy metabolism;
  • glycerol;
  • insect;
  • proteins;
  • re-feeding;
  • starvation;
  • temperature;
  • triglycerides

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Metabolic responses to prolonged food shortage (35 days) and subsequent re-feeding (14 days) were investigated in adults of an introduced beetle, Alphitobius diaperinus Panzer, as a function of temperature (12, 16, 20 and 24 °C). Various qualitative and quantitative changes that greatly vary according to the temperature experienced occurred in metabolite levels during prolonged starvation. Whereas levels of protein and ATP did not change significantly, triglycerides decreased markedly and glycogen changed little. Metabolite levels were differently affected by temperature, with triglycerides being less rapidly degraded at 20 than at 24 °C and almost completely depleted at 12 and 16 °C; in contrast to higher temperatures, glycerol is accumulated at 12 °C. Physiological adaptation to starvation and low temperatures are highly linked and energy allocation for starvation vs. temperature acclimation must be strictly regulated, both being essential for insect survival. Re-synthesis rates during recovery are probably highly temperature-dependent for all metabolites. The proteins retained during starvation and the preferential degradation of lipids allowed a rapid recovery. Above 16 °C, adult A. diaperinus regained locomotory activity rapidly and the triglyceride, glycerol and glycogen reserves were restored. This tropical species may be able to colonize other environments such as natural and/or artificial biotopes where conditions are close to those of its natural habitat.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Biological invasions and species extinction are fundamental phenomenon that govern arthropod abundance and geographical distribution (McArthur & Wilson, 1967; Cornell & Hawkins, 1993). Insect pest species introduction from tropical or subtropical regions to temperate countries is probably favoured by the current increase in international trade and thermal global changes (Howe & Freeman, 1955; Sinha & Watters, 1985). The pest beetle Alphitobius diaperinus Panzer (Coleoptera: Tenebrionidae), probably imported into temperate regions from West Africa (Salin, 1999), was introduced into animal breeding operations via contaminated foodstuffs. This invasive species has already been observed from several localities in France (Bonneau, 1988) or associated with old trees in open countryside in the U.K. (Alexander, 1998).

Pullin (1996) suggested that tropical species could have been pre-adapted to survive in colder climates, as mechanisms such as carbohydrate accumulation could have first occurred in response to drought stress in tropical regions and have been selected more recently for their cryoprotective function in cold temperate climates, making rapid colonization of these regions easier. However, climatic conditions and trophic conditions (food availability, food competition) frequently remain as limiting factors in the successful establishment of tropical insects in temperate regions where seasonal climatic fluctuations occur. In such regions, food rarely remains available throughout the year in either quality or quantity, and insects are regularly subjected to nutritional stress and concomitant cold exposures. In addition to the fact that insect metabolism is strongly temperature-dependent, studies have demonstrated that activity is significantly reduced with decreased temperatures (Renault et al., 1999; Nedved, 2000). Preliminary laboratory experiments showed that reproduction is stopped below 15–17 °C (Rueda & Axtell, 1996) and that temperatures below 11–12 °C induce chill injuries in adult Alphitobius diaperinus Panzer (Renault et al., 1999; Salin, 1999; Nedved, 2000). Lower temperatures or longer exposures to such temperatures induce chill-coma, i.e. activities are stopped, although small movements of legs and other appendages may still be observed (Block & Sømme, 1982; Renault et al., 1999), making it impossible for the insects to walk and reach food sources. In that case, energy storage during food abundance periods and metabolic energy-saving adjustments triggered by food limitation influence both geographical and temporal distributions (Hervant et al., 1999); low temperature adaptations and starvation lead to the depletion of energy reserves (David et al., 1996). Physiological and biochemical adaptations to starvation are crucial in temperate regions. An insect's capacity to withstand and recover from food shortage and temperature will contribute to determine the species invasive status, i.e. whether the population is established throughout the year in temperate regions, or if it is constrained to extinction/colonization cycles according to the seasons. A population that does not overcome stressful periods may only be maintained by immigration of individuals or may be totally eradicated.

Although most ecological and physiological studies emphasize the importance of temperature and food availability in determining species abundances and distributions (Leather et al., 1993; Schmidt-Nielsen, 1997), comparative studies of changes in metabolism tend to focus on temperature or starvation as the environmental variable of interest. Few studies seek to address the effect of both variables. The incidence of temperature intensity with concomitant food shortage may have various impacts on insect metabolism, and may provide useful information on ecological adaptations of invasive species and insect capacity to invade other biotopes. The adult beetle A. diaperinus provides a useful model to study the influence of starvation and recovery on metabolism as a function of temperature. The main question examined here is whether survival and physiological adaptations to starvation allow this species to establish yearly in temperate regions. In the present study, survival, weight parameters and water content of starved adult A. diaperinus were determined. Investigations were also performed to determine qualitative and/or quantitative changes in the adult body composition that occur during starvation and subsequent recovery by measuring several key metabolites of energy and intermediary metabolism.

Materials and methods

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Insects

Adult Alphitobius diaperinus (Coleoptera: Tenebrionidae) were originally collected from poultry house litter at Mauron (Morbihan, France). The insects were reared for 2 months at 28 °C in the laboratory and supplied with water and dry dog food ad libitum. Beetles were allocated to four groups and acclimated in air-conditioned chambers for 48 h prior to being used for the experiments. The first group was acclimated to 12 °C (n = 450), the second to 16 °C (n = 450), the third to 20 °C (n = 450) and the fourth to 24 °C (n = 450). Insects were then used either to measure the duration of survival or for metabolic assays.

Survival

To assess the effect of both starvation and temperature on survival of adult A. diaperinus, 40 acclimated beetles were kept at each studied temperature, 12, 16, 20 and 24 °C, in plastic boxes with water supplied ad libitum but without food. Survival was assessed at intervals of 3 days until the death of the beetles. The beetles were observed closely for activity or limb movement, the criterion for survival (Renault et al., 1999). At 12 °C, the insects were generally inactive and sometimes fell into short-term chill coma. To assess the survival of these beetles that showed no limb movement during observations, they were transferred to 25 °C for 30 min for a final check of movement under the microscope.

Metabolic responses to starvation and low temperatures

The effect of time, temperature and starvation on fresh mass, water content and key metabolites was studied as follow. Eight kind of batches were experienced: 200 individuals were kept at 12 °C with food and water supply ad libitum; 200 individuals were kept at 12 °C with water supply but without food; 200 individuals were kept at 16 °C with food and water supply ad libitum; 200 individuals were kept at 16 °C with water supply ad libitum but without food; 200 individuals were kept at 20 °C with food and water supply ad libitum; 200 individuals were kept at 20 °C with water supply but without food; 200 individuals were kept at 24 °C with food and water ad libitum; and 200 individuals were kept at 24 °C with water supply but without food. Eight beetles were sampled at intervals of 0 (t0, initial values), 8, 16, 25 and 35 days in every batch of fed and deprived insects maintained at 12, 16, 20 and 24 °C.

To investigate changes in fresh mass, water content and key metabolites during recovery from food deprivation, batches of beetles were starved for 35 days at 12, 16, 20 and 24 °C and then re-fed ad libitum. After verifying that a 2-day starvation period was not sufficient to induce specific metabolic responses, food was removed 2 days before sampling the insects. This ensured that the digestive tract was empty and that an overshoot in oxygen consumption due to digestive metabolism did not affect the results for both control and re-fed insects. The beetles were removed after 7 and 14 days of recovery to determine fresh weight and water content and after 14 days for metabolic assays. Water content was determined as follows: ([fresh mass – dry mass]/fresh mass × 100). Dead insects were removed regularly in all batches, and only living insects were used for analysis.

Once removed, control, starved and re-fed animals were immediately weighed (fresh mass), then frozen in liquid nitrogen before being assayed. They were then stored at −75 °C until the assay of key metabolites.

Sample preparation and metabolite assays

Whole insects were homogenized for the metabolite studies and eight insects were pooled in each extraction. The following metabolites were analysed as described in Hervant et al. (1995, 1996, 1999) using standard enzymatic methods: ATP, glucose, glycerol and glycogen. Total proteins and triglycerides were extracted according to the methods of Barclay et al. (1983), Elendt (1989) and Hervant et al. (1999), and then evaluated using specific test-combinations (Boehringer–Mannheim) (triglyceride levels were measured by determining glycerol released from triglycerides hydrolysis). All assays were performed in a recording spectrophotometer (Beckman DU-6) at 25 °C, except for the ATP assays, which were performed in a LKB 210 luminometer. Enzymes, coenzymes and substrates used for enzymatic essays were purchased from Boehringer (Mannheim, Germany) and Sigma Co. (St Louis, USA).

Statistical analysis

All statistical analyses were based on Sokal & Rohlf (1995). Differences in temperatures (four temperatures) and insect feeding status (fed, deprived) with the duration of exposure (five samples) were tested for each factor using analysis of variance (anova). Tukey's test was used to test all pair-wise differences. All statistical analyses were performed using Minitab™ software (Minitab Inc, Windows version 13, 2000). Values are presented as means ± SE.

General remarks

Preliminary biochemical and physiological analyses were performed on males and females separately. Because no sexually dimorphic response was found in either fed-controlled or starved insects, beetles were always taken randomly.

Results

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Survival, fresh mass and water content

The duration of survival greatly varied within treatments. Survival was significantly longer at 12 and 16 °C, with mean survival time during starvation of 25 and 62 days and maximum survival of 80 and 97 days. In contrast, the mean survival time during starvation ranged from 20 to 24 days at 24 and 20 °C, respectively, with maximal survival times of 49 days at 20 and 24 °C. After 35 days of starvation, 13, 14 and 13% of insects remained alive at 12, 20 and 24 °C, respectively.

Water content increased by 5–10% with time of exposure at 12 and 16 °C in fed beetles and at all temperatures in starved beetles (Fig. 1). In the four temperatures studied, fresh mass of adult A. diaperinus dropped during food deprivation when compared with initial mass but was only significantly different at 20 and 24 °C (P < 0.05) (Fig. 2). Starved and control insects showed no significant differences for fresh mass at 12 and 16 °C, suggesting that cold-exposed fed beetles also lose fresh mass.

image

Figure 1. Changes in body water content in adult Alphitobius diaperinus kept at 12, 16, 20 and 24 °C (i) during 49 days (fed beetles), (ii) during a 35-day starvation period and a subsequent 14-day re-feeding period (starved beetles). (*) indicates a value that was significantly different from the initial value (t0) (P < 0.05).

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image

Figure 2. Changes in fresh mass in adult Alphitobius diaperinus kept at 12, 16, 20 and 24 °C (i) during 49 days (fed beetles), (ii) during a 35-day starvation period and a subsequent 14-day re-feeding period (starved beetles). (*) indicates a value that was significantly different from the initial value (t0) (P < 0.05).

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Metabolites

Metabolite levels at day 8, 16 and 25 are not provided for fed beetles kept at 16 °C. No significant changes were observed in the concentration of whole animal ATP. Metabolites with absolute levels (amounts per individual) for days 0 and 35 for each treatment are presented in Table 1. Variations in metabolite levels were studied at two scales, between treatments and within treatments.

Table 1. . Metabolite levels (absolute values) at days 0 and 35 in both fed and starved insects maintained at 12, 16, 20 and 24 °C. Triglycerides (µmol triglycerides/insect); glycerol (µmol glycerol/insect); glucose (µmol glucose/insect); glycogen (µmol glycosyl/insect); proteins (g protein/insect). Non-parametric tests (Mann–Whitney: median comparison, *: P < 0.05, **: P < 0.01, ***: P < 0.001) were conducted to compare metabolite level at day 0 and day 35 in each treatment. : Table 1 . Table 1.Continued.
 12°C16°C
 FedStarvedFedStarved
 035035035035
Triglyceride80.31±16.9920.8±8.42***79.88±19.214.74±2.97***95.02±14.1476.15±20.6494.74±15.6 5.57±3.40***
Glycerol322.4±81.0345.6±86.4347.8±68.3465.5±108.0*308.2±44.9263.3±50.0306.4±42.8208.9±39.1**
Glucose100.8±17.3689.2±24.14106.46±26.2068.93±15.04**73.36±7.6568.4±15.8573.09±8.7562.09±10.63*
Glycogen1690.2±216.21348.4±257.4*1714.0±266.01211.7±260.9**1878±4431659±3231869±4591303.8±190.3**
Proteins1.925±0.361.599±0.391.93±0.251.55±0.31*2.07±0.441.85±0.462.06±0.441.62±0.25*
 20°C24°C
 FedStarvedFedStarved
 035035035035
Triglyceride82.18±21.5878.53±10.3482.80±14.4624.92±8.64***79.78±17.8286.02±17.5384.12±18.0618.27±5.54***
Glycerol281.1±66.2272.0±55.8286.2±56.7156.4±28.5***258.5±66.2232.8±79.7266.9±67.4125.7±29.8**
Glucose98.6±23.57101.6±31.0100.22±19.4588.27±15.9103.9±21.53103.3±19.08106.9±21.3792.66±16.18
Glycogen1827±4301824±3231860±3681150.0±124.4***1842±3551883±3361905±4101044.7±209.8***
Proteins1.972±0.471.98±0.542.0±0.391.45±0.31*1.88±0.361.94±0.421.94±0.421.25±0.32**

Comparison of metabolic responses among temperature treatments

Effects of starvation and temperature. anova analysis showed that the feeding status (fed/deprived), the temperature and the duration of exposure had a significant impact on triglyceride, glycerol and glycogen levels (P < 0.001, except for glycerol × feeding status: P < 0.05), whereas protein levels were significantly influenced by both feeding status and time (P < 0.01).

Tukey's pair-wise comparisons revealed significant differences in triglyceride, glycerol and glycogen levels between the fed and deprived beetles: higher values of triglycerides were observed in the fed beetles maintained at 16, 20 and 24 °C (P < 0.001), whereas no differences were found between fed and deprived beetles kept at 12 °C; higher values of glycogen were observed in fed beetles maintained at 20 and 24 °C (P < 0.05), whereas no significant differences appeared between fed and deprived beetles maintained at 12 and 16 °C; glycerol values showed significant differences for beetles kept at 24 °C, i.e. fed beetles showed higher glycerol concentrations than starved beetles (P < 0.05), but no differences were observed between fed and starved beetles kept at 12 °C.

Tukey's pair-wise comparisons also revealed significant differences in metabolite levels in starved beetles between the four temperatures studied: significantly lower values of triglycerides were observed in beetles maintained at 12 °C, triglyceride values for beetles kept at 16 °C were not significantly different from those from beetles kept at 20 and 24 °C, nor from those from starved beetles kept at 12 °C. Both temperature and starvation influenced triglyceride levels at 12 °C, whereas temperature had no significant additional influence on the levels at 20 and 24 °C. The combination of starvation and temperature significantly influenced triglyceride degradation: the more the temperature was decreased, the more the decrease in triglyceride was accelerated (P < 0.001). Glycerol values were significantly higher in starved beetles maintained at 12 °C (P < 0.001) and it is also interesting to notice that glycerol level significantly increased in starved beetles kept at 12 °C, whereas it decreased significantly in starved beetles kept at 16, 20 and 24 °C. Moreover, glycerol values were significantly higher in starved beetles kept at 16 °C when compared to those of starved beetles kept at 24 °C. Glycogen concentration had the same pattern at all four temperatures for starved beetles. No significant differences were observed for glucose concentration between all the modalities studied.

These results allowed us to distinguish the four temperatures used. Beetles maintained at 20 and 24 °C showed almost the same responses when starved but differed significantly from the responses of starved beetles kept at 12 °C. Results obtained for the 16 °C treatment indicated similarities with both higher (20 and 24 °C) and lower temperatures (12 °C). Moreover, metabolite evolution over time in fed beetles kept at 12 °C was not significantly different from those of deprived beetles kept at 12 °C.

Metabolic responses over time in fed beetles.  In fed animals kept at 16, 20 and 24 °C, no significant changes with time were observed for the concentration of whole insect glucose (data not shown), glycogen, protein, glycerol and triglyceride (Figs 3a–d). At 12 °C, triglyceride levels decreased significantly with time in fed insects (Fig. 3a) with a concomitant increase of the glycerol level. Metabolite fluctuations observed in fed animals exposed to 12 °C can be attributed to starvation resulting from the beetles' inactivity caused by this low temperature.

image

Figure 3. Changes in the levels of metabolites in fed adult A. diaperinus kept at 12, 16, 20 and 24 °C for 35 days: (a) triglycerides; (b) glycogen; (c) glycerol; (d) proteins. Values are means ± SE for n= 8 animals. (*) indicates a value that is significantly different from the initial value (t0) (P < 0.05). TGs: triglycerides.

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Metabolic responses over time in starved beetles.  Differences between times in metabolite contents for deprived beetles maintained at a given temperature were compared by one-way anova followed by a Tukey's test.

Kept at 12 °C.  The concentration of protein remained relatively stable during starvation (Fig. 4d). Glycogen content decreased to 78% of its initial value (t0) after 35 days' food deprivation, and by day 35 of starvation, glycogen level was significantly different (P < 0.05) from initial values (Fig. 4b1). The glucose level decreased and reached 72% of its initial value after 35 days' starvation. The triglycerides level decreased rapidly after the onset of fasting and the level was significantly different from the initial value after 8 days (Fig. 4a1) (P < 0.05). By day 35 of starvation, almost all the triglycerides have been used, with the remaining triglyceride level reaching 7% of the initial level (0.33 ± 0.07 µmol triglyceride/g fresh mass). In contrast, glycerol content slowly increased (Fig. 4c1) reaching 148% of its initial value at day 35, this value being significantly different from initial ones after a 25-day starvation. After 14 days' re-feeding, TG and glycerol contents remained significantly different from the initial values.

image

Figure 4. Changes in the levels of metabolites during the 35-day starvation and subsequent re-feeding (14 days) in adult A. diaperinus exposed at 12, 16, 20 and 24 °C: (a1) triglycerides (TG) of beetles kept at 12 and 24 °C; (a2) TG of beetles kept at 16 and 20 °C; (b1) glycogen of beetles kept at 12 and 24 °C; (b2) glycogen of beetles kept at 16 and 20 °C; (c1) glycerol of beetles kept at 12 and 24 °C; (c2) glycerol of beetles kept at 16 and 20 °C; (d) proteins. Values are means ± SE for n= 8 animals. (*) indicates a value that is significantly different from the initial value (t0) (P < 0.05).

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Kept at 16 °C.  Levels of proteins (Fig. 4d) and glucose did not differ significantly over time from the initial values. Glycogen decreased during starvation, reaching 76% of the initial concentration, and by day 35 of starvation, the glycogen level was significantly different (P < 0.05) from the initial concentration (Fig. 4b2). The triglycerides level decreased rapidly after the onset of starvation and the level was significantly different from the initial value after 8 days (Fig. 4a2). By day 35 of starvation, the triglycerides had almost completely disappeared (triglyceride level had reached 7% of its initial value, 0.4 ± 0.1 µmol triglyceride/g fresh mass). The glycerol content declined slowly to 74% of its initial level during 35 days of starvation (P < 0.05) (Fig. 4c2). During re-feeding, all metabolites increased sharply to initial levels.

Kept at 20 °C.  No significant changes in the concentration of whole animal proteins (Fig. 4d) or glucose were observed during starvation. The glycogen content decreased and by day 35 of starvation had reached 72% of the initial level (Fig. 4b2). The most significant change was determined for triglycerides (reaching 35% of the initial value by day 35 of starvation), with the level being significantly lower after 16 days (Fig. 4a2). Glycerol content declined slowly (Fig. 4c2) to 63% of its initial value after 35 days of food deprivation (P < 0.05). During re-feeding, all metabolites increased sharply to initial levels.

Kept at 24 °C.  No significant change was observed for glucose content over time. Glycogen decreased during starvation when compared with the initial concentrations (t0) (Fig. 4b1) and by day 25 of starvation, the level was significantly different (P < 0.05) (82% at day 16, 66% at day 35). The triglyceride level (Fig. 4a1) decreased rapidly after the onset of starvation and was significantly different from the initial value after 16 days (P < 0.05). By day 35 of starvation, the triglyceride level had reached 26% of its initial value. During starvation, the glycerol content declined slowly (Fig. 4c1), reaching 56% of the initial value by day 35 (P < 0.05). Figure 4(d) shows that the protein level was also affected, reaching 78% of its initial value at day 35. During re-feeding, all metabolites returned sharply to initial levels.

Table 2 summarizes the degradation or accumulation of metabolites in starved beetles. Rates of decrease in the levels of proteins, glycogen and glycerol declined at lower temperatures, glycerol even being accumulated at 12 °C. In contrast, glucose levels decreased less at lower temperatures, and were even stable at 20 and 24 °C. However, this does not mean that the glucose pool does not fluctuate (values are the sum of the degradation/synthesis process). Triglyceride decreased less rapidly at 20 than at 24 °C, and were almost completely depleted at 12 and 16 °C. Changes in rates of re-synthesis during recovery are probably highly temperature dependent, as demonstrated by the levels of all metabolites. In contrast, 20% of the amount of glycerol accumulated at 12 °C was degraded. Moreover, re-feeding of insects at this temperature was not as efficient as at 16, 20 and 24 °C: only 38% of the amount of triglyceride catabolized was re-synthesized vs. 87% at 24 °C, and 43% for glycogen vs. 109% at 24 °C.

Table 2.  Percent of metabolite used during a 35-day starvation period: (1 − [metabolite day 35/metabolite day 0]) × 100; and corresponding percent of re-synthesis of degraded metabolites after a 14-day recovery from food shortage: (metabolite synthesized during recovery)/(metabolite degraded during starvation)aa.
 Metabolites used during starvation (%)Metabolites re-synthesis during refeeding (%)
 12°C16°C20°C24°C12°C16°C20°C24°C
  • a

    Degradation of 20% of the amount of glycerol accumulated.

  • aa

    ([metabolite concentration at day 49–metabolite concentration at day 35]/[metabolite concentration at day 0–metabolite concentration at day 35])×.

Proteins1114172262648196
Glycogen2224283443100116109
Glucose289110100
Triglycerides9393657438707687
Glycerol14874635620a737586

Discussion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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).

Acknowledgements

  1. Top of page
  2. Abstract.
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank M. E Salvucci and M. Goto for their helpful review and amendment of the manuscript. We also thank M. Lebouvier for helpful comments on the statistical analysis.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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Accepted 25 September 2002