Dr D. Renault, Station Biologique de Paimpont, UMR 6553 CNRS, Université de Rennes-I, France. Tel: +33 0 2 99 61 81 69; Fax: +33 0 2 99 61 81 87; E-mail: email@example.com
Abstract. Temperature and food availability are limiting factors for the establishment of tropical insects in temperate countries. In the alien pest beetle, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), starvation and temperature have a significant impact on metabolic rate with oxygen consumption ranging from 0.5 µmol/g fresh mass (FM)/h at 12 °C to 3.4 µmol/g FM/h at 24 °C. At 12 °C, oxygen consumption decreased continuously during an entire period of starvation. However, at 16, 20 and 24 °C, beetles display a marked hyperactivity that leads to an increase in the oxygen consumption level during the first week of starvation, followed by a steep decrease until the end of the starvation period. Oxygen consumption either does not decline in fed beetles (observed at higher temperatures) or declines at a much shallower rate than in starved beetles (observed at cooler temperatures). During the first week of refeeding, Oxygen consumption rose steeply at 16, 20 and 24 °C before levelling off to the initial value (t0). At 12 °C, no compensation process was observed during recovery. This study reveals that an important threshold in the biology of A. diaperinus lies between 12 and 16 °C, leading to the onset of reduced locomotor activity and the promotion of survival to the detriment of reproduction. This ‘sit and wait’ behaviour is proposed as an adaptive strategy (i.e. inactivity and lower oxygen consumption coupled with low energetic requirements and high recovery abilities). Such behaviour and the observed hyperactivity were rarely described in insects before the present study. Together, the previous and present results suggest that A. diaperinus populations are likely maintained in temperate regions by immigration from warmer situations.
With the current international trade, pest species are increasingly introduced from tropical or subtropical regions to temperate ones (O'Farell & Butler, 1948; Howe & Freeman, 1955; Peters, 1977). The global spread of the introduced polyphagous beetle, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), has been facilitated by the availability of greenhouses and animal breeding facilities that obviate the need to survive external environmental conditions (Gould & Moses, 1951; Salin, 1999). In the field, A. diaperinus has already been reported in several localities in France (Bonneau, 1988) and is associated with old trees in open countryside in the U.K. (Alexander, 1998).
As metabolic rates of ectotherms are strongly dependent on temperature, food and water availability (Parsons, 1996; Gibbs et al., 1997; Davis et al., 2000), climatic conditions frequently limit the establishment of tropical insects in temperate countries. In temperate regions, insects have to cope with periods of food deprivation, which limit feeding, and the coma induced by chilling temperatures that lead to insect inactivity (Renault et al., 1999). During such periods, many insects enter a state of depressed metabolism or dormancy in which starvation is a major form of stress (Leather et al., 1993; Renault et al., 2002a). The decrease in metabolic rate mostly results from both reduced activity and reduction of the overall aerobic metabolism. The ability to withstand and rapidly recover from periods of such nutritional stress determines the survival of individuals. The capacity of an insect to overcome food shortage and cold temperatures will contribute to its invasive ability in temperate regions (i.e. whether the species is established year round, or constrained to a seasonal extinction/colonization life cycle).
Comparative studies of metabolic adaptation have often neglected the fact that more than a single variable may affect oxygen consumption (Davis et al., 2000). In many studies, temperature is considered as the environmental variable of interest, whereas the effect of food deprivation on metabolic rate is less frequently examined (Salin et al., 2000). Few studies seek to address the effect of both variables, although most ecological and physiological studies emphasize the importance of both temperature and food availability in determining species abundance and distribution (Hochachka & Somero, 1984; Schmidt-Nielsen, 1997).
Consequently, accurate analysis of these stressful conditions is a major prerequisite for an understanding of the ecological adaptations of introduced species, and to predict their capability of invading new habitats. More generally, a better knowledge of the metabolic responses to both starvation and low temperatures is required in insects to determine those environmental variables that account for the greatest variance in the data, particularly in marginal habitats. Thus, the aim of this study was to examine metabolic rate and its temperature sensitivity in both fed and starved adult A. diaperinus. In addition, Q10 values across the studied temperature range were determined.
Materials and methods
Adult A. diaperinus (Coleoptera: Tenebrionidae) were originally collected from poultry house litter at Mauron (Morbihan, France). The insects were kept for 2 months at 28 °C in the laboratory and supplied with water and dry dogfood ad libitum. Beetles were allocated to four groups and acclimated in air-conditioned chambers for 48 h before being used in 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 oxygen consumption or the duration of survival.
To assess the effect of both starvation and temperature on survival of adult A. diaperinus, 40 acclimated beetles were kept in plastic boxes at each studied temperature, 12, 16, 20 and 24 °C. Beetles were supplied with water ad libitum but were given no food. Survival was assessed at intervals of 3 days until the death of the beetles. The insects were observed closely for activity or limb movement, the criterion for survival. 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, where they were usually found to be dead.
The effect of temperature and starvation on oxygen consumption was studied using eight treatments, each comprising 200 individuals. In treatments 1–4 (12, 16, 20 and 24 °C, respectively), individuals were supplied with food and water ad libitum. In treatments 5–8 (12, 16, 20 and 24 °C, respectively), individuals were supplied with water but kept without food. Eight beetles were sampled at intervals of 0 (t0, initial values), 7, 14, 21, 28 and 35 days from every batch of fed and deprived insects maintained at 12, 16, 20 and 24 °C.
To investigate changes of oxygen consumption during recovery from food deprivation, batches of beetles were starved for 35 days at 12, 16, 20 and 24 °C and then fed ad libitum.
Measurement of oxygen consumption
Individual oxygen consumption was measured with a modified Warburg constant-volume system, as described elsewhere Hervant et al. (1997, 2000), which allowed simultaneous measurement of a large number of replicates. Fed (0, 7, 14, 21, 28, 35, 42 and 49 days), starved (0, 7, 14, 21, 28 and 35 days) or refed (7 and 14 days) beetles were transferred directly to the respiration vials. Oxygen consumption was measured every 10 min for a period of 120 min. At the end of the experiments, individuals were removed promptly from the Warburg reaction vials and then weighed (Fresh mass, FM).
All statistical analysis were carried out in accordance with Sokal and Rohlf (1995). Lethal lapse of time for 50% and 90% of the population (Lt50 and Lt90) were determined using Minitab™ (Minitab Inc., State College, PA) probit analysis to compare insect survival at each temperature studied. Analysis of covariance (ancova) was carried out to investigate the effects of insect feeding status and temperature on oxygen consumption. Dietary nutrient level (starved/fed) and temperature (four temperatures) were considered as main effect treatments. For all analyses, the duration of exposure (time) was used as the covariate. On finding significant differences between means (P < 0.05), pairwise post hoc comparisons were conducted to identify significant differences. Data were analysed using Minitab software™ (Minitab Inc., Windows, version 13). Values are presented as means ± SE.
Preliminary analyses were performed on males and females separately. Because no sexually dimorphic response was found in either fed or starved insects, beetles were always sampled randomly.
Survival curves are presented in Fig. 1. The LT50 during starvation were 17.3, 59.5, 21.8 and 16.5 days at 12, 16, 20 and 24 °C, respectively. The LT90 were 41.2, 91.2, 39.4 and 35.2 days at 12, 16, 20 and 24 °C, respectively. At 20 and 24 °C, 100% mortality occurred on day 49, whereas it occurred on day 83 and 97 at 12 and 16 °C, respectively.
Oxygen consumption of adult A. diaperinus, was significantly affected by dietary nutrient level ( d.f. = 1, F = 26.61, P < 0.000). Fed beetles exhibited significantly higher values of oxygen consumption than starved beetles at all temperatures (P < 0.001). Comparing the data for oxygen consumption of fed and starved beetles, it is evident that the oxygen consumption level declined steeply as starvation progressed and was significantly lower (P < 0.05) than that of fed insects after 21 days at 20 °C, after 28 days at 12 and 24 °C, and after 35 days at 16 °C (Fig. 2a). Oxygen consumption was also influenced significantly by temperature (d.f. = 3, F = 636.31, P < 0.000), each temperature being significantly different from the others (P < 0.001) (Fig. 2a–d). Oxygen consumption increased significantly with temperature, averaging approximately 0.65 µmol/g FM/h at 12 °C to 2.75 µmol/g FM/h at 24 °C in fed beetles. Oxygen consumption levels of fed beetles were 1.5, 2.5 and 3.5-fold less at 12 °C than at 16, 20 and 24 °C, respectively (Fig. 2a–d). A significant interaction was found between diet and temperature (d.f. = 3, F = 2.61, P < 0.05), with both reducing the level of oxygen consumption.
There was also a significant interaction between the covariate (duration of exposure) and oxygen consumption (P < 0.001). Overall, oxygen consumption level was almost constant in fed beetles kept at 16, 20 and 24 °C (P > 0.05) (Fig. 2b–d) during the experiment. At 12 °C, oxygen consumption of fed beetles appeared to be reduced, but the differences were not significant (Fig. 2a). In starved beetles kept at 16, 20 and 24 °C, oxygen consumption levels showed the same pattern (Fig. 2b–d). Oxygen consumption increased during the first week of food shortage, and was significantly different from the initial level at 24 °C (P < 0.05), with a concomitant observed hyperactivity. Following this rise, individuals displayed a decrease in oxygen consumption which was significantly lower to the initial level (t0), after 21 days at 16 °C (P < 0.01), and after 28 days at 20 and 24 °C (P < 0.01) (Fig. 2b–d). By day 35 of starvation, oxygen consumption was decreased by almost 60% of its initial value at 16, 20 and 24 °C, and was highly significantly different from the initial value (P < 0.001) at each studied temperature. Conversely, oxygen consumption of starved insects exposed at 12 °C decreased from the first to the last day of starvation (0.77–0.49 µmol/g FM/h) (Fig. 2a). Values were significantly lower from initial ones after 21 days of starvation (P < 0.05). At this temperature, insects spent most of the time in chill coma.
During refeeding, oxygen consumption rose steeply, reaching 118% of the initial value after 7 days of recovery (day 42) at 16 °C (Fig. 2b), and 126% at 20 and 24 °C (Fig. 2c,d) (P < 0.01). Oxygen consumption was twice that at the end of starvation (day 35). By day 49, the oxygen consumption of refed beetles kept at 16, 20 and 24 °C was comparable with its initial value (t0) (P < 0.05). At 12 °C, oxygen consumption increased significantly (P < 0.01) from 0.49 to 0.66 µmol/g FM/h after 7 days of refeeding (Fig. 2a).
Q10 values of fed beetles, calculated at day 35, increased with increasing temperature over the complete range of temperatures from 12 to 24 °C, and at intervals between these temperatures (Table 1). An increase of 4 °C induced a two-fold increase of reaction rates at both cold and optimal temperatures (Q10a, Q10f). The reaction rate quadrupled with an 8 °C temperature increase (Q10b, Q10e), but was three-fold higher over the complete range of temperatures from 12 to 24 °C (Q10c). An exceptionally high Q10 was found over the range 16–20 °C (Q10d).
Table 1. Q10 values of fed adult Alphitobius diaperinus kept for 35 days at 12, 16, 20 and 24 °C.
Survival of starved A. diaperinus varies significantly with temperature. The duration of survival is significantly increased when beetles are starved at 16 °C versus beetles starved at 20 and 24 °C, and is reduced when starved insects are kept at 12 °C. At this lower temperature, insects spend most of the time in chill coma. Previous work (Rueda & Axtell, 1996) has shown that inhibition of reproduction occurs between 15 and 17 °C, and that the theoretical upper threshold of chill injury (Nedved et al., 1998) and a significant decrease of activity lie between 12 and 15 °C, depending on the duration of the acclimation period (Renault et al., 1999). Above the temperature range of 15–17 °C, both metabolic rate and locomotor activity, which are strongly temperature dependent (Clarke, 1991), are higher and lead to a faster depletion of energy reserves, reducing the duration of survival. At 12 °C, starvation and accumulation of chill injury lead to a shorter duration of survival, even if metabolic rate is decreased. In these conditions, it is difficult to determine whether beetles die from an exhaustion of energy reserves rather than from other deleterious effects caused by temperature.
Highly significant different oxygen consumption levels were observed between fed beetles kept at 12, 16, 20 and 24 °C. The highly significant lower rate of oxygen demand at low temperature (12 °C) is likely related to reduced ATP demand for protein turnover, ion pump activity and other aspects of basal metabolism.
The levels of oxygen consumption of beetles starved at temperatures above 16 °C display similar features. During the beginning of starvation (days 0–7), locomotor activity is increased, and a marked hyperactivity may be even observed leading to an increase of metabolic rate, initially reflecting an increased food searching behaviour. However, during prolonged food deprivation periods (7–35 days), adult A. diaperinus behaviour exhibits a significant reduction of oxygen consumption and spontaneous activity. Such a rate of lower oxygen consumption also occurs in field-collected Collembola when feeding is ceased (Van der Woude & Joosse, 1988). The beetle's behaviour changes from active food searching to waiting, and this allows a saving of energy reserves during periods of food shortage and thus an increased duration of survival (Renault et al., 2002a). During this period, beetles are usually in a state of temporary torpor that is different from the chill coma (Renault et al., 1999; Renault et al., 2003). Karan and David (2000) also showed that this behavioural response induces a decreased metabolic rate in Drosophila melanogaster. This ‘sit-and-wait’ strategy has been demonstrated in starved amphipods (Smith & Baldwin, 1982; Hervant et al., 1997, 1999; Hervant & Renault, 2002) and in spiders (Tanaka & Itô, 1982). This period of depressed metabolism (Renault et al., 2003) during which the beetles subsist on body stores, mainly triglycerides (Renault et al., 2002a), may reflect an adaptive strategy to withstand long-term fasting.
Immediately after the onset of refeeding, A. diaperinus exhibit a large overshoot in oxygen consumption, which is the result of resuming activity and active digestive metabolism. Over-feeding phenomena (starvation compensation processes, or over-compensation) (Chown & Gaston, 1999) may explain the exceptionally high level of oxygen consumption. There is a selective advantage for an animal in such a harsh environment to optimally use and quickly restore available energy stores that were depleted during nutritional stress (Hervant et al., 1997, 2001). At 12 °C, no compensation process was observed during the recovery period. Because of temperature impact, locomotor and feeding activities remain reduced even though food is available.
The high Q10 obtained between 16 and 20 °C is ecologically relevant, indicating that temperatures below 16 °C may be considered as cold temperatures for adult A. diaperinus. It demonstrates that these organisms are able to highly alter their metabolism in response to temperature changes: a linear increase in temperature does not induce a linear increase in the rate of oxygen consumption. Such a Q10 between 16 and 20 °C was found in Scarabaeus galenus (Coleoptera: Scarabaeidae) from Southern Africa (Q10 = 5.04) (Davis et al., 2000). For this species, mean annual temperatures are approximately 22 °C (Davis et al., 2000) and are rather similar to the thermal conditions that A. diaperinus encounters in its native tropical biotope (Vuattoux, 1968). In larvae of Paractora dreuxi (Diptera: Helcomyzidae), which inhabits subantarctic regions (mean annual temperature approximately 5 °C), a high Q10 was also shown over the range 5–10 °C (Crafford & Chown, 1993). This ability to respond rapidly to a temperature increase enables species to recover quickly after starvation and cold periods, and to resume their food searching behaviour rapidly. By contrast, it may allow beetles to significantly moderate their energy expenditure during decreasing temperatures.
The observed ‘sit and wait strategy’, the possession of low energetic requirements and the high recovery abilities (rapid restoration of body reserves) have been proposed as an adaptive strategy in isopods during long starvation periods (Hervant & Renault, 2002). However, such behaviour and the observed hyperactivity were rarely described in insects before our study. During both starvation and cold exposure, adult A. diaperinus display features such as extremely low metabolic rates and relative or complete inactivity, similar to most forms of insect quiescence (Mansingh, 1971; Duman et al., 1991). The observed level of mortality of adult A. diaperinus suggests that this beetle is likely to survive November and December months outside in Paimpont (France) (mean ± SD: November 2002, 11.3 ± 2.2 °C; December 2002, 9.9 ± 3.2 °C). A higher level of mortality should be expected in January and February when temperatures are lower and beetles are subjected to sporadic food events (January 2003, 4.63 ± 4.4 °C; February 2003, 5.6 ± 3.7 °C) (Meteo France data, Station Biologique de Paimport). During these periods, A. diaperinus populations are likely maintained outside in temperate regions by immigration from warmer situations, even if the physiological ability of the beetle allows it to invade natural biotopes in mediterranean or subtropical climates. However care should be taken when drawing such conclusions. In our study, constant temperatures were used, which do not characterize cyclic variations in the field, especially in temperate regions where noticeable diurnal variations occur (Renault et al., 2002b). Longer acclimation periods may also affect significantly the duration of survival and the metabolic rate (Hanc & Nedved, 1999). Accordingly, these factors should be tested in further studies.
We thank Brent J. Sinclair for his helpful review and amendment of the manuscript.