Introduction

A mild stress does not kill the animal but disturbs its homeostasis and the organism implements an adaptive response to cope with this mild stress (Minois and Rattan 2003). This adaptive response eventually improves the functional ability of the organism to resist other stresses: this phenomenon is called hormesis and is observed in various species. Mild stresses can also have positive effects on aging and longevity (review in Le Bourg and Rattan 2008). For instance, exposure to a mild stress, such as hypergravity (HG: gravity levels higher than 1 g, the Earth gravity level, e.g., Le Bourg and Minois 1997), cold shock (Le Bourg 2007), or heat shock (e.g., in the nematode Caenorhabditis elegans: Cypser and Johnson 2002; in Drosophila melanogaster: Khazaeli et al. 1997; Le Bourg et al. 2001; Hercus et al. 2003), can slightly increase longevity. Other stresses also increase longevity in D. melanogaster (X-irradiation: Sacher 1963; Vaiserman et al. 2003) or in C. elegans (hyperbaric oxygen: Cypser and Johnson 2002). However, some mild stresses do not increase longevity as, for instance, UV or gamma-irradiations in C. elegans (Cypser and Johnson, 2002). Furthermore, a mild stress can increase longevity of D. melanogaster males but not of females (HG: Le Bourg and Minois 1997; Le Bourg et al. 2000; X-irradiation: Vaiserman et al. 2003). Mild stresses can delay behavioral aging, but this is not always observed (compare Le Bourg and Minois 1999 and Le Bourg 2007 on the one hand and Le Bourg et al. 2001 on the other hand).

Exposure to a mild stress can increase resistance to a strong stress. For instance, survival time at 37°C is increased after pretreatment with heat, cold or HG (Le Bourg 2007; Le Bourg et al. 2001; Minois and Le Bourg 1999). Exposure to HG at young age for two weeks also increases survival time of males exposed at 4 weeks of age to a single or several non-lethal heat shocks, but no positive effect is observed in males heat-shocked at later ages and in females heat-shocked at 4, 5, or 6 weeks of age (Le Bourg et al. 2004; Le Bourg 2005). Similar positive effects have been reported in both sexes exposed at 4 weeks of age to several non-lethal heat shocks, if flies were pretreated with cold at a young age (Le Bourg 2007). A treatment applied at young age can thus partially protect flies at middle age from a non-lethal heat shock. Otherwise, HG has no effect on resistance to desiccation or cold but decreases resistance to starvation (Minois and Le Bourg 1999), while a cold pretreatment increases resistance to cold but also decreases resistance to starvation (Le Bourg 2007). Finally, neither heat, cold or HG had positive effects on resistance to hydrogen peroxide (Le Bourg, 2008a).

Beyond the study of resistance to abiotic stresses, such as heat shocks, it would be useful to know whether a mild stress could also help to resist infection, because flies encounter fungi and bacteria in the wild, like other species. This article has thus studied the resistance of flies to infection by spores of the fungus Beauveria bassiana, which kill the flies in a few days (e.g., Lemaître et al. 1997; Gobert et al. 2003).

Material and methods

Flies of the wild-type strain Meyzieu routinely used in mild stress experiments in our laboratory were subjected or not to one of three stresses known to have positive effects on longevity, i.e., heat, cold, and HG, and were subsequently infected: their longevity after infection was recorded. As one of these mild stresses had positive effects on resistance to infection (see below), the effect of infection on resistance to lethal heat and on climbing scores were assessed in flies subjected or not subjected to this mild stress.

Flies

The experimental flies were adult males and females of the wild strain Meyzieu caught at the end of the seventies in France, near the city of Lyon. The strain is maintained by mass mating (several bottles containing hundreds of flies mixed every three weeks) on the standard medium (agar, sugar, corn meal and killed yeast) containing a mold inhibitor (para-hydroxymethyl-benzoic acid) and enriched with live yeast at the surface of the medium.

In order to obtain the parents of the experimental flies, flies were allowed to lay eggs for one night in a bottle containing the medium described above. About 50 pairs emerging from this bottle 9–10 days after egg-laying were transferred to bottles (ca. 25 pairs in a bottle) containing the medium previously described: these flies are the parents of the experimental flies. Experimental flies were obtained as follows: eggs laid by 5 day-old parents during a 15 h period on a petri dish containing the usual medium colored with charcoal and a drop of live yeast were transferred by batches of 25 into 80 ml glass vials containing the medium described above. At emergence, virgin flies with a duration of preimaginal development of 9–10 days were transferred under ether anesthesia in groups of 15 flies of the same sex to 20 ml polystyrene vials containing the standard medium with a drop of live yeast.

Flies were transferred to new vials twice a week. Flies spent their life in an incubator (except if they were subjected to HG at young age, see below); the rearing temperature was 25 ± 0.5°C; light was on from 08.00 to 20.00 h (fluorescent lamp).

Pretreatment of flies by a mild stress

The procedures used to provide cold and heat shocks, and to rear flies in HG have been described at length, respectively, in Le Bourg (2007), Le Bourg et al. (2001) and in all previous papers on HG (e.g., Le Bourg et al. 2002).

Cold pretreatment

Flies were exposed from 5 days of age to 0°C for 60 min a day during two periods of 5 days separated by 2 days with no cold shock. Flies were transferred without anesthesia in early morning to empty polystyrene vials (diameter: 17 mm, length: 63 mm) closed by a polypropylene plug. These vials were kept for 1 h in ice (0°C) and afterwards at room temperature for at least 20 min and, after that, flies were transferred back to their rearing vials.

The vials used for the cold shock did not contain food to avoid any delay of the temperature fall and prevent flies from being stuck to food when asleep. Therefore, control flies were kept in their rearing vials, because, as these flies are not knocked down by cold, transferring them to empty vials would be a period of starvation.

Hypergravity pretreatment

HG is obtained by putting flies in a continuously rotating centrifuge (for a picture, see Le Bourg 2008b). Flies were subjected to HG (3 or 5 g) for 2 weeks from the second day of adult life (if we except 20 min stops twice a week to change the rearing vials) and after that transferred to 1 g: these flies are called the HG-flies. Flies never subjected to HG were placed near the rotating centrifuge during the first two weeks of adult life: these flies are called the 1 g-flies. After the 2 weeks of centrifugation, all flies were transferred into the incubator described above.

Heat pretreatment

Flies were transferred without anesthesia just before shock from their rearing vials to empty polystyrene vials (diameter: 17 mm, length: 63 mm) closed by a polypropylene plug. This plug contained absorbent cotton that was soaked with distilled water. The vials were placed 5 min daily in a 37°C water-bath during five successive days, the first shock being applied at 5 days of age. A group of flies was not subjected to heat shock. After each heat shock, flies were transferred back to their rearing vials.

Infection procedure

The spores of the fungus Beauveria bassiana kept at −80°C in 20% glycerol were incubated at 25°C in 90 mm Petri dishes containing the appropriate medium (for one liter of distilled water, the autoclaved medium contained: peptone (Sigma P463): 1 g, glucose (Fluka 49159): 20 g, malt extract (Fluka 70167): 20 g, agar: 15 g). After sporulation, which occurs ca. 4 weeks after spreading spores on the medium, flies were infected.

The day of infection, flies were transferred to new vials before to be very slightly anaesthetized with ether and then shaken for ca. 1 min in a Petri dish containing a sporulating fungal culture. After having checked under stereomicroscope that all flies were well covered with spores, flies were transferred back to their vials. The main difference between this infection procedure and that of Lemaître et al. (1997) is the fungus incubation temperature and flies rearing temperature after infection, which is 25°C here and 29°C in Lemaître et al. (1997). We reasoned that transferring flies from 25 to 29°C would be an extra-stress and that using the same temperature throughout experiment allows to observe solely the effects of infection. Preliminary experiments (data not shown) showed that, even at 25°C, the fungus killed flies; therefore, the fungus was virulent at 25°C.

Longevity experiments

In all experiments, longevity was recorded daily from the day following infection until the death of the last fly. Day 0 is the day of infection. The longevity results of each experiment were analyzed with factorial ANOVAs testing for the effect of sex, infection, pretreatment by a mild stress and their interactions.

Experiments testing the effect of cold stress were done at several ages. In a first experiment, flies were infected at 19 days of age, i.e., 3 days after the last cold shock. Three or four vials of 15 flies were used for each level of sex, cold pretreatment and infection factors (total n = 395). It was impossible to record longevities during the last four week-ends of the experiment and the 38 flies dying during these week-ends were considered to have died on Saturdays (and not on Sundays or Mondays). Since the cold pretreatment increased longevity (see below), this procedure minimizes the positive effect of mild stress on the longevity of non-infected flies. In a second experiment, flies were infected at 26 days of age, i.e., 10 days after the last cold shock. Five or six vials of 15 flies were used for each level of sex, cold pretreatment and infection factors (total n = 561). In a third experiment, flies were infected at 33 days of age, i.e., 17 days after the last cold shock. Six or seven vials of 15 flies were used for each level of sex, cold pretreatment and infection factors (total n = 608). In a fourth and last experiment, flies were infected at 40 days of age, i.e., 24 days after the last cold shock. Five to seven vials of 15 flies were used for each level of sex, cold pretreatment and infection factors (total n = 585).

Flies subjected to the HG pretreatment were infected at 18 days of age, i.e., 3 days after HG-flies were removed from the centrifuge. Three or four vials of 15 flies were used for each level of sex, gravity and infection factors (total n = 627).

Flies subjected to the heat pretreatment were infected at 12 days of age, i.e., 3 days after the last heat shock. Four or five vials of 15 flies were used for each level of sex, heat pretreatment and infection factors (total n = 483).

Resistance to heat

Resistance to heat was observed in control and cold-pretreated flies, which have been infected with B. bassiana at 20 days of age or have not been infected. The survival time in a water-bath set at 37°C was observed at 3 or 4 weeks of age, i.e., 3 or 7 days after infection. Flies were transferred just before shock into empty polystyrene vials (diameter: 17 mm, length: 63 mm), the plug containing absorbent cotton with distilled water to prevent desiccation. Flies were observed every 5 min and flies totally immobile were considered to be dead. About 30 flies were observed in each group of sex, cold pretreatment, age, and infection condition.

Climbing activity

In the climbing activity test, flies are individually placed in a vertical vial subjected to a mechanical stimulation and the highest height reached within 20 s after cessation of the stimulation is recorded. Climbing scores decrease with age and this test has been routinely used in aging research for many years (e.g., Miquel et al. 1972; Feany and Bender 2000). The procedure has been described in detail (Le Bourg and Minois 1999); the stimulation intensity used by Le Bourg et al. (2004, rotating speed of the vial: 130 rpm) was used in the present experiment. Climbing activity was observed in control and cold-pretreated flies, which were infected with B. bassiana at 20 days of age or not infected. Experiments were carried out in the morning at 3 or 4 weeks of age, i.e., 3 or 7 days after infection. Twenty flies with intact legs were observed in each group of sex, cold pretreatment, age, and infection condition.

A square-root transformation was applied to the climbing scores before they were analyzed with an ANOVA testing for the effect of sex, cold pretreatment, age, infection and interactions.

Results

Effect of cold stress on longevity and resistance to fungi

When flies were infected at 19 days of age, the cold pretreatment increased longevity (Figs. 1, 2a, 3a, F(1, 387) = 56.57, P < 0.0001). Infection decreased longevity, as expected (F(1, 387) = 518.04, P < 0.0001), and the cold pretreatment by infection interaction showed that the positive effect of cold pretreatment was more important in non-infected flies than in infected ones (F(1, 387) = 29.54, P < 0.0001: compare Fig. 1a and b). The sex factor and the other interactions were not significant, if we except the second-order interaction between sex, infection, and cold pretreatment (F(1, 387) = 9.12, P = 0.0027). This interaction shows that the effect of cold pretreatment was more important in non-infected males (ca. 15 days) than in non-infected females (ca. 7 days), while the contrary was observed in infected flies (ca. 0.5 day in males and 3 days in females), a positive effect being nearly absent in males. In other words, this interaction shows that the effect of the cold pretreatment on resistance to infection is not only explained by a higher longevity of non-infected cold pretreated flies. If the ANOVA is restricted to infected flies, the positive effect of the cold pretreatment was still significant (data not shown).

Fig. 1
figure 1

Mean longevity ± SEM as a function of sex, exposure to cold and infection. Flies were cold-pretreated daily (0°C for 60 min) or not during two periods of 5 days separated by 2 days, starting at 5 days of age. The day of infection one half of flies were infected with the fungus Beauveria bassiana. Day 0 is the day of infection. a Longevity of infected flies, b longevity of non-infected flies. Each point is the mean of 41–86 flies

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Fig. 2
figure 2

Survival curves of males as a function of exposure to cold and infection. a Infection at 19 days of age. b Infection at 26 days of age. c Infection at 33 days of age. d Infection at 40 days of age

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Fig. 3
figure 3

Survival curves of females as a function of exposure to cold and infection. a Infection at 19 days of age. b Infection at 26 days of age. c Infection at 33 days of age. d Infection at 40 days of age

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When flies were infected at 26 days of age, the cold pretreatment increased longevity (Figs. 1, 2b, 3b, F(1, 553) = 7.66, P = 0.0058). Infection decreased longevity (F(1, 553) = 548.31, P < 0.0001) but the cold pretreatment by infection interaction (F(1, 553) = 2.93, P = 0.0873) failed to reach significance, contrarily to what was observed when infection was done at 19 days of age. Males lived longer than females (F(1, 553) = 8.71, P = 0.0033). The sex by infection interaction (F(1, 553) = 8.91, P = 0.0030) showed that the deleterious effect of infection was more important in females (ca. −20 days) than in males (ca. −15 days). The other interactions were not significant. If the ANOVA is restricted to infected flies, the positive effect of the cold pretreatment was not significant, but the sex by infection interaction (F(1, 264) = 5.73, P = 0.0173) showed that the cold shock very slightly decreased longevity of females and increased that of males.

When flies were infected at 33 days of age, the cold pretreatment increased longevity (Figs. 1, 2c, 3c, F(1, 600) = 55.78, P < 0.0001). Infection decreased longevity (F(1, 600) = 615.16, P < 0.0001) and the cold pretreatment by infection interaction (F(1, 600) = 9.45, P = 0.0022) showed that the positive effect of the cold pretreatment was more important in non-infected flies (ca. 6 days) than in infected ones (ca. 2 days). Males lived longer than females (F(1, 600) = 5.63, P = 0.0180). The sex by cold pretreatment interaction (F(1, 600) = 16.87, P < 0.0001) showed that the positive effect of a cold pretreatment was more important in males (ca. 6 days) than in females (ca. 1 day) and the sex by infection interaction (F(1, 600) = 16.71, P < 0.0001) showed that the deleterious effect of infection was more important in females (ca. −15 days) than in males (ca. −11 days). The second-order interaction between sex, cold pretreatment and infection was not significant. If the ANOVA is restricted to infected flies, the positive effect of the cold pretreatment was still significant (data not shown).

Finally when flies were infected at 40 days of age, the cold pretreatment also increased longevity (Figs. 1, 2d, 3d, F(1, 577) = 19.43, P < 0.0001). Infection decreased longevity (F(1, 577) = 539.20, P < 0.0001) and the cold pretreatment by infection interaction (F(1, 577) = 8.12, P = 0.0045) showed that the positive effect of the cold pretreatment was more important in non-infected flies (ca. 4 days) than in infected ones (ca. 1 day). Males lived longer than females (F(1, 577) = 10.56, P = 0.0012). The other interactions were not significant. If the ANOVA is restricted to infected flies, the positive effect of the cold pretreatment was still significant (data not shown).

Therefore, these experiments confirmed that a cold pretreatment increases longevity of non-infected flies and showed that infected flies survive for a longer time if they are subjected to a cold pretreatment at a young age. This positive effect was observed if flies were infected early after the end of the cold pretreatment (19 days of age) or after a long delay (26, 33 or 40 days of age). However, the positive effect of cold was more important in females than in males if flies were infected at 19 days of age, while it was more important in males if flies were infected at later ages.

Remarkably, the survival curves (Figs. 2, 3) show that, in males, some flies have a similar lifespan as non-infected flies, while this is not observed in females, except in flies infected at 19 days of age. Therefore, it seems that some males are able to escape infection.

Effect of hypergravity on longevity and resistance to fungi

Infection decreased longevity (Fig. 4, F(1, 615) = 1351.54, P < 0.0001). Both the gravity factor and its interaction with infection were not significant (Fs close to 1). Males lived slightly longer than females (F(1, 615) = 5.65, P = 0.0177) and the sex by infection interaction showed that the deleterious effect of infection was more important in females than in males (F(1, 615) = 7.49, P = 0.0064). The interaction between sex and gravity factors was also significant (F(2, 615) = 6.54, P = 0.0015), showing that HG increased longevity of males and decreased that of females. However, the second-order interaction between sex, gravity and infection (F(2, 615) = 3.73, P = 0.0245) showed that HG increased longevity of non-infected males and decreased that of non-infected females, while it had no effect in infected flies, with the exception of a very slight longevity increase in 5 g males when compared to 1 g ones (less than 1 day). When the ANOVA was restricted to infected flies, the effect of HG was not significant and, if the ANOVA was restricted to non-infected flies, the significant interaction between the sex and gravity factors showed that HG decreased longevity in females and increased it in males (data not shown).

Fig. 4
figure 4

Mean longevity ± SEM as a function of sex, exposure to HG and infection. Flies were kept or not in HG for 2 weeks from the second day of adult life (1, 3, or 5 g). At 18 days of age, one half of these flies were infected with the fungus Beauveria bassiana. Day 0 is the day of infection, i.e., 18 days of adult age. Each bar is the mean of 38–61 flies

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Therefore, these results show that while the usual effects of HG on non-infected flies are observed, i.e., a positive effect in males and a negative one in females, HG does not protect flies against infection.

Effect of heat stress on longevity and resistance to fungi

Infection decreased longevity (Fig. 5, F(1, 475) = 450.55, P < 0.0001), but neither heat shocks nor sex had a significant effect on longevity (Fs < 1). The sex by infection interaction showed that the deleterious effect of infection was more important in females than in males (F(1, 475) = 56.42, P < 0.0001). The interaction between heat shock and sex factors was also significant (F(2, 475) = 6.48, P = 0.0112), showing that heat shocks slightly increased longevity of females and decreased that of males. The other interactions were not significant. When the ANOVA was restricted to infected flies, the effect of heat was not significant and, if the ANOVA was restricted to non-infected flies, the significant interaction between the sex and heat factors showed that heat decreased longevity in males and increased it in females (data not shown).

Fig. 5
figure 5

Mean longevity ± SEM as a function of sex, exposure to heat and infection. Flies were heat-pretreated daily (37°C for 5 min) or not during 5 days from 5 days of age. At 12 days of age, one half of these flies were infected with the fungus Beauveria bassiana. Day 0 is the day of infection, i.e., 12 days of adult age. Each bar is the mean of 49–68 flies

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This experiment was replicated (five vials of 15 flies for each level of sex, heat pretreatment and infection factors: total n = 566) and provided very similar results. However, the interaction between heat shock and sex factors was not significant and, if the ANOVA was restricted to non-infected flies, neither sex, heat shock and their interaction had a significant effect on longevity (data not shown).

In this experiment, non-infected flies lived slightly longer than in the first one (mean difference, ca. 2 days) and infected flies lived shorter (mean difference, ca. 4 days), which shows that infection was more deleterious to flies.

To sum up, these experiments show that heat shocks do not increase longevity of non-infected flies, contrarily to a previous study (Le Bourg et al. 2001), and that heat shocks do not increase resistance to infection.

Resistance to heat after infection of cold-pretreated flies

In addition to an increased survival time, it could be that a cold pretreatment has other positive effects in infected flies. The present experiment tested whether infection decreases resistance to a lethal heat stress and if a cold pretreatment could minimize this possible effect.

In each sex, survival time data were analyzed with a factorial ANOVA testing for the effect of infection, cold pretreatment, age and all interactions. Sexes were analyzed separately because an ANOVA testing both sexes showed a strong effect of sex, a significant interaction between infection and sex, and another one between cold pretreatment, age, and sex (data not shown).

In males, the cold pretreatment increased survival time (Fig. 6a, F(1, 232) = 12.01, P = 0.0006, means ± SEM of control and pretreated males, respectively: 108.54 ± 1.71 min, 116 ± 1.28 min), as previously shown (Le Bourg 2007). Age and infection factors, and all interactions were not significant. Therefore, infection did not decrease survival time of 3 or 4 week-old males, i.e., 3 or 7 days after infection.

Fig. 6
figure 6

Mean survival time at 37°C ± SEM as a function of sex, age, exposure to cold, and infection. Flies were cold-pretreated or not (60 min daily during two periods of 5 days separated by two days, starting at 5 days of age) and survival time was observed at 23 or 27 days of age, i.e., 3 or 7 days after infection. Each bar is the mean of 28–30 flies. a Males, b females

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In females, cold had no effect on survival time (Fig. 6b, F < 1), while a slight positive effect has been previously reported (Le Bourg 2007). Infection decreased survival time (F(1, 229) = 5.69, P = 0.0178, means ± SEM of control and infected females, respectively: 135.87 ± 2.73 min, 126.15 ± 3.17 min), and the age by infection interaction showed that this effect was only observed at 4 weeks of age, i.e., 7 days after infection (F(1, 229): 5.89, P = 0.0160). The age factor and the other interactions were not significant. Therefore, infection decreased survival time late after infection, when most females have begun to die due to infection, and a cold pretreatment did not protect flies against it. As most of deaths occurred soon after this time, it could be that infected females were less resistant to heat simply because many of them were currently dying. However, this negative effect of infection on survival time could also be explained by a stronger virulence of the fungus at 37°C in these fragile females.

This experiment thus shows that fungal infection decreases resistance to heat in females but not in males. This lower resistance is observed in females only when mortality due to infection is strongly rising: it could be that heat only accelerates this dying process. In any case, cold does not protect flies against infection if they are subjected to a lethal heat stress.

Climbing activity after infection of cold-pretreated flies

The present experiment tested whether infection decreases climbing score and if a cold pretreatment could minimize this possible effect.

The cold pretreatment increased climbing scores (Fig. 7, F(1, 304) = 16.98, P < 0.0001, means ± SEM of control and pretreated flies, respectively: 0.87 ± 0.15, 1.58 ± 0.16 cm), as previously observed (Le Bourg 2007). Climbing scores decreased with age (F(1, 304) = 10.59, P = 0.0013, means ± SEM of 3 and 4 week-old flies, respectively: 1.61 ± 0.18, 0.85 ± 0.13 cm). Sex and infection had no significant effect on climbing scores, as well as all interactions. However, a nearly significant interaction between infection and cold pretreatment showed that infected flies had higher climbing scores if they have been cold-pretreated (F(1, 304) = 3.82, P = 0.0516). Figure 7 shows that this not significant effect is mainly due to females.

Fig. 7
figure 7

Mean climbing score ± SEM as a function of sex, age, exposure to cold, and infection. Flies were cold-pretreated or not (60 min daily during two periods of 5 days separated by 2 days, starting at 5 days of age). The climbing score is the height (cm) reached by a fly in 20 s after the cessation of a standardized mechanical stimulation. Each bar is the mean of 20 flies; (a) males, (b) females

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Therefore, this experiment confirms that a cold pretreatment increases climbing scores and shows that infection did not decrease these scores. However, there was a tendency in females for a decreased score in infected flies, this decrease being erased in cold-pretreated females.

Discussion

Mild stresses can have positive effects on longevity, aging and resistance to some strong stresses in D. melanogaster. Particularly, both HG and cold pretreatments increase survival time after repeated non-lethal heat shocks (Le Bourg et al. 2004; Le Bourg 2005, 2007), i.e., these mild stresses improve resistance to a surrogate of “summer heatwave” in humans, and it would be of interest if a mild stress could also protect against infection, i.e., a surrogate of “winter pathologies” in humans.

The present results show that heat and HG pretreatments failed to protect flies against fungi. In addition, we failed to replicate the previously observed slight positive effect of heat on longevity (Le Bourg et al. 2001); however, several hundreds of flies were necessary to observe this effect, which shows that it was of a low magnitude. Therefore, it is not surprising that we failed to confirm this slight positive effect of a heat stress on longevity.

A cold pretreatment at young age increased survival to infection and this effect was observed throughout life, i.e., a long time after the cold pretreatment was applied. Figure 1a shows that infection soon after the cold pretreatment had a stronger positive effect in females than in males. At later ages, no effect of a cold pretreatment was observed in females and cold-pretreated males survived longer than control ones. On the whole, the positive effect of cold was thus more important in males than in females. This sex effect may be explained by a higher sensitivity of females to the daily cold shocks, since nearly no males died when these shocks were applied, whereas about 20 cold-shocked females died when infection occurred at 26 or 40 days of age. Thus, it seems that, depending on the experiment, females can suffer or not from repeated cold shocks. A more important positive effect of a mild stress in males has already been observed. For instance, HG or irradiation increase longevity of males, but not of females (Le Bourg et al. 2000; Vaiserman et al. 2003; see also Sørensen et al. 2007). Burger and Promislow (2004) have discussed possible causes of the sex effect in flies subjected to an environmental intervention which increases lifespan. Nevertheless, for the time being, no author has been able to propose a satisfactory explanation of this sex effect.

Cold is thus a mild stress with various positive effects, since it delays behavioral aging, increases longevity, resistance to lethal heat, non-lethal heat shocks, cold shock (Le Bourg 2007), and fungal infection (this work). However, infection did not clearly impair resistance to heat and climbing activity and, thus, it was not possible to test whether cold could minimize deleterious effects of infection upon these traits.

Therefore, while other mild stresses can increase resistance to lethal heat shock and/or longevity, cold is the most efficient mild stress currently described in flies, since it has beneficial effects on various traits. In such conditions, it would be of interest to reassess the effect of a cold mild stress in rodents, because it seems that only one study has been done. Holloszy and Smith (1986) exposed rats to a cold shock by immerging them 4 h a day, 5 days a week from 9 to 32 months of age, in a 23°C water after a 3-months period of adaptation to this cold shock. This 23-months exposure to cold shocks had a not significant positive effect on longevity but seemed to protect against several malignancies (neoplasias, sarcomas). Nevertheless, cold exposure also increased cardiovascular pathologies. This experiment thus showed that a cold shock can have some protective effects at old age and one may wonder whether similar or even better effects would be obtained with shorter exposures.

Flies express antimicrobial peptide genes when they are infected. The Toll pathway is activated by fungal infection and directs the expression of the drosomycin and metchnikowin antimicrobial peptide genes (Lemaître et al. 1997). When activated by fungi, the Toll pathway induces the translocation into the nucleus of the transcription factor DIF (review in Martinelli and Reichhardt 2005). Dif mutants are more sensitive to fungal infection than wild-type flies and do not synthetize Drosomycin after infection with B. bassiana (Rutschmann et al. 2000). Using this mutant could allow to know whether the Toll pathway explains the increased resistance to infection after a cold pretreatment: a cold pretreatment should not protect Dif mutants against infection. Daibo et al. (2001) have reported the existence in D. triauraria of a gene called drosomycin-like, which is upregulated in diapausing flies: this gene shares some homology with the drosomycin gene of D. melanogaster. Could it be that cold-pretreated flies synthetize more antimicrobial peptides when infected, and particularly Drosomycin, which could explain their better resistance to fungal infection? However, Drosomycin and the other antimicrobial peptides are not sufficient to protect against fungal infection, because infected flies die after infection despite the synthesis of these peptides. Besides, even if the expression levels of the genes coding for these peptides is higher in aged flies than in young ones (Pletcher et al. 2002; Landis et al. 2004; Ren et al. 2007), the present results show that aged flies do not resist infection longer than young ones. Moreover, Libert et al. (2008) reported that the increased survival to bacterial infection of long-lived D. melanogaster mutants was decoupled from the expression level of antimicrobial peptides such as Drosomycin.

It is of interest to note that some flies, mainly males, were able to escape infection because their longevity was similar to that of non-infected flies (see the tail of the survival curves in Figs. 2 and 3). Therefore, it could be that some flies are better protected than most of flies, for unknown reasons. Could it be that these flies have a higher expression of antimicrobial peptide genes or rely on other, unknown, mechanisms?

Experiments testing the Dif mutant and measuring the synthesis of antimicrobial peptides in flies subjected to a cold pretreatment remain to be done. Gaining some knowledge of the mechanism explaining the positive effects of mild stress is of concern because previous experiments discarded superoxide dismutase and catalase, two antioxidant enzymes, as possible causes of these positive effects (Le Bourg and Fournier 2004). Moreover, the 70 kDa heat-shock protein (HSP70) was shown to explain, at least partly, the increased resistance to lethal heat in flies previously subjected to HG, but not any other positive effect, since HSP70 is not synthetized at 25°C (Le Bourg et al. 2002). However, while a mild heat stress increases survival time at 37 or 38°C, it does not clearly induce HSP70 (Le Bourg et al. 2001; Sørensen et al. 2007). Therefore, mild stress has positive effects on various traits but, up to now, there is no explanation for these effects.

Our studies performed in flies show that a mild stress has positive effects, particularly at middle or old age, on resistance to lethal or non-lethal heat shocks and to infection. Therefore, an environmental intervention is able to modulate aging and longevity and to partially protect flies against life-threatening stresses. In such conditions, performing more studies on the effects of mild stress in mammals (e.g., Abete and Rengo 2008; Ji 2008) is of interest. Such studies could maybe open the way to clinical research (Abete et al. 2008).