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Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster
Journal of Insect Physiology 56 (2010) 336–340
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster Laila H. Andersen a, Torsten N. Kristensen a,b,*, Volker Loeschcke a, Søren Toft a, David Mayntz a,b a b
Department of Biological Sciences, Genetics and Ecology, Aarhus University, Ny Munkegade, Building 1540, DK-8000 Aarhus C, Denmark Department of Genetics and Biotechnology, Aarhus University, Blichers Alle´ 20, Postbox 50, DK-8830 Tjele, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 September 2009 Received in revised form 9 November 2009 Accepted 10 November 2009
Larval nutrition may affect a range of different life history traits as well as responses to environmental stress in adult insects. Here we test whether raising larvae of fruit flies, Drosophila melanogaster, on two different nutritional regimes affects resistance to cold, heat and desiccation as well as egg production and egg-toadult viability. We raised larvae on a carbohydrate-enriched and a protein-enriched growth medium. We found that flies developed on the high protein medium had increased heat and desiccation tolerance compared to flies developed on the carbohydrate-enriched medium. In contrast, flies developed on the carbohydrate-enriched growth medium recovered faster from chill coma stress compared to flies developed on a protein-enriched medium. We also found gender differences in stress tolerance, with female flies being more tolerant to chill coma, heat knockdown and desiccation stress compared to males. Egg production was highest in females that had developed on the protein-enriched medium. However, there was a sex-specific effect of nutrition on egg-to-adult viability, with higher viability for males developing on the sucrose-enriched medium, while female survival was highest when developing on the protein-enriched medium. Our study indicates that larval nutrition has a strong impact on the ability to cope with stress, and that the optimal nutrient composition varies with the type of stress. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Fecundity Development Nutrition Environmental stress
1. Introduction Drosophila melanogaster is often used as a model organism in studies of physiological and evolutionary responses to various forms of stress (Hoffmann et al., 2003; Sinclair et al., 2007; Kristensen et al., 2008a). However, there is little information about how nutrition affects life history traits (Watts et al., 2006) and performance during thermal stress, and the importance of diet is often underestimated in experimental designs (Prasad et al., 2003). Hence, there is a growing need to investigate diet-related effects behind variation in traits of importance for fitness. Many organisms face a challenge of meeting their optimal nutritional requirements for somatic and reproductive growth under natural conditions (Raubenheimer and Simpson, 1999). During life, body tissues constantly require a specific quantity and proportion of nutrients, in order to attain optimal growth and performance (Bauerfeind and Fischer, 2005). Deficiency or imbalance of e.g. fat, carbohydrate or protein can affect characters such as somatic growth and reproduction. For instance, protein deficiency
* Corresponding author at: Department of Genetics and Biotechnology, Aarhus University, Blichers Alle´ 20, Postbox 50, DK-8830 Tjele, Denmark. Tel.: +45 8999 1076; fax: +45 8999 1300. E-mail address: [email protected] (T.N. Kristensen). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.11.006
reduces fecundity and growth in D. melanogaster (Wang and Clark, 1995) and in fruit-feeders protein is often a limiting macronutrient (Mattson, 1980; Adams and Gerst, 1991; Hendrichs et al., 1991; Markow et al., 2001). In contrast, diet restriction or mild starvation can increase longevity as well as tolerance to stressors such as heat stress (reviewed by Wenzel, 2006; Smith et al., 2007) demonstrating the complexity of organismal nutrient acquisition and utilization. A variety of factors may affect organismal stress tolerance. These include physiological as well as behavioural changes. Climatic changes may be met by e.g. physiological hardening processes, coma or production of metabolites making the organism tolerate temperature extremes (Sørensen et al., 2003, 2005; Lalouette et al., 2007). Also, an organism may compensate for nutritional stress and reduced body size by extending its growth period or altering its energy allocation to growth, hence postponing the reproductive period (Reichling and German, 2000; Lobe et al., 2006). Behaviourally, organisms may respond to stress by migration, use of refuges or an altered and more favourable nutrient intake to meet changed energy expenditures (Mattson, 1980). The bulk of studies on physiological and evolutionary responses to nutrient deficiencies focuses on reproduction and fecundity (Markow et al., 1999; Bauerfeind and Fischer, 2005; Naya et al., 2007; Nestel and Nemny-Lavy, 2008), while only a few studies have focused on how nutrient resources and deficiencies can affect other challenges in life such as tolerance to environmental stress
L.H. Andersen et al. / Journal of Insect Physiology 56 (2010) 336–340
(Wang and Clark, 1995; Kanazawa et al., 2003). However, Burger et al. (2007) report an increased resistance to starvation stress in D. melanogaster when reared on a sucrose-rich diet which indicates that diet composition may influence stress responses. The aim of this study was to test if larval nutrition affects the ability of adult flies to cope with a number of different stress challenges, including cold and warm temperatures and desiccation. As protein and carbohydrates are the most important macronutrients for Drosophila larvae in natural food sources (Mattson, 1980), we chose to focus on these two very different dietary conditions. In order to test how these growth media affected life history traits, we also tested for dietary effects on egg production and egg-to-adult viability. 2. Materials and methods
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were transferred individually to empty glass vials (6.0 mL) with a watertight lid and observed while submerged in a water bath set at 38.3 8C. Time until flies went into heat coma, i.e. were unable to stand, was recorded for each fly. 2.4. Desiccation resistance Males (N = 50) and females (N = 50) from each nutritional group were tested for their tolerance to an air humidity of 0–5%, obtained by means of silica gel put into a sealed fish tank, positioned at 25 8C. Each fly was separately transferred to empty glass vials (6.0 mL) which were covered with gauze to allow air to circulate through. Vials were exposed to low humidity in the fish tank and they were checked for dead flies every hour until all were dead. The tests were performed in the laboratory with 12 h light/12 h dark cycles throughout the experiment which lasted approximately 27 h.
2.1. Flies used in the experiments 2.5. Egg-to-adult viability A genetically diverse mass population of D. melanogaster reared in the laboratory since 2002 was used in the experiment (see Bubliy and Loeschcke, 2005 for further details on the population used). Prior to the experiment flies were kept on a basis medium (Sørensen et al., 2009) which is a nutritionally balanced artificial diet composed of sucrose (40 g/L water), yeast (60 g/L water), agar (16 g/L water), oatmeal (30 g/L water), nipagen (12 mL/L water) and acetic acid (1.2 mL/L water). Eggs were collected and transferred to two types of media, a carbohydrate-enriched medium and a protein-enriched medium. Both media were made by mixing either sucrose or casein (Sigma C-5890, Sigma–Aldrich) with instant Carolina basis (formula 4-24 plain) medium (Carolina Biological Supply, Burlington, NC, USA). Carolina fly medium is a nutritionally balanced artificial diet which provides the essential nutrients required by D. melanogaster larvae during development. Henceforth we refer to it as basis medium. The carbohydrate-enriched medium (20% carbohydrate) was prepared by mixing sucrose and basis medium in a 1:4 ratio before adding water. The protein-enriched medium (60% protein) was prepared by mixing casein and basis medium in a 3:2 ratio. The two nutrition types used were chosen on the background of reported variations in sugar and protein contents in the natural host choices of D. melanogaster (Markow et al., 1999; Arvanitoyannis and Mavromatis, 2009). All vials contained approximately 7 mL of medium and three to four droplets of a thin yeast suspension (ca. 0.5–1 mg/mL water) were added on top of the medium. Density of developing larvae in the vials was standardized among nutritional groups with 50 (5) eggs/vial. The eggs hatched and the larvae developed at 25 8C and 12 h light/12 h dark cycles. Hatched virgin flies were collected and sexed and transferred to standard basis medium in groups of 50 (5) flies per vial. They were left to rest after CO2anaesthesia on standard basis medium for 2–3 days and then tested as described below. 2.2. Chill coma recovery Males (N = 50) and females (N = 50) from each nutritional group were collected and tested for their ability to tolerate low temperatures. Flies were individually transferred to empty Eppendorf tubes (0.5 mL) and submerged in a water bath at 2 8C for 2 h. Immediately after exposure, they were observed separately in the laboratory at room temperature (20–23 8C) and the time it takes to regain consciousness was registered (registered as duration from exposure termination until the fly was able to stand).
Twenty female and twenty male flies from the mass population developing on basis medium were collected and sorted according to sex under CO2-anaesthesia. They were divided in four groups of 10 flies (5 males and 5 females in each group) and left to rest on standard basis medium for 2 days. After 48 h flies were transferred to a new vial for egg laying on standard basis medium during a period of 4 h. Eggs were collected and transferred in groups of 20 to vials containing 7 mL of either carbohydrate-enriched medium (N = 34) or protein-enriched medium (N = 34). Eggs were left to hatch at 25 8C with 12 h light/12 h dark cycles. We checked for newly emerged flies every 8 h and counted and sexed emerging flies. 2.6. Egg production We collected virgin males (N = 50) and virgin females (N = 50) developed on either the carbohydrate-enriched or the proteinenriched medium. One male and one female were transferred to vials containing basis medium (N = 50 pr. nutritional regime). Pairs of flies were transferred to vials with teaspoons with basis medium at age 3–4 days. Every 48 h flies were transferred to a new vial with a new teaspoon with basis medium. This procedure was repeated three times so that the number of eggs produced from day 3–4 to day 7–8 of age could be registered. 3. Statistics All statistical analyses were performed in JMP 7.0. Outliers more than three standard deviations from the mean were discarded before statistical tests were performed. In all experimental tests, except for the egg production experiment, we focused on two factors: nutritional regime (protein-enriched growth medium vs. carbohydrate-enriched growth medium) and sex. Two-way ANOVA was used to analyse egg-to-adult viability data. If the interaction term between the factors sex and nutrition was significant, we repeated the analysis for each sex separately. Egg production was analysed using one-way ANOVA, and the assumption of equal variances between groups was tested using Bartlett’s test (P > 0.05). Proportional hazard analyses were used to analyse data where the variable described the time spend until an event happened. 4. Results
2.3. Heat knockdown resistance
4.1. Chill coma recovery
Males (N = 50) and females (N = 50) from each nutritional group were tested for their ability to tolerate high temperatures. Flies
The chill coma recovery time of the flies was significantly affected by both nutritional regime and sex (Table 1). Flies
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L.H. Andersen et al. / Journal of Insect Physiology 56 (2010) 336–340
Table 1 Proportional hazards analysis on resistance to thermal stress, with focus on the two factors, nutritional regime (protein or carbohydrate-enriched medium) and sex (males or females). DF was 1 in all tests. Trait
Source
F
Chill coma recovery
Fly nutrition Fly sex Fly nutrition fly sex
13.95 25.98 0.14
0.0002 0.0001 0.71
Heat knockdown
Fly nutrition Fly sex Fly nutrition fly sex
13.09 8.56 1.49
0.0003 0.003 0.22
Fly nutrition Fly sex Fly nutrition fly sex
7.45 71.43 1.46
0.006 <0.0001 0.22
Desiccation
P
developed on the carbohydrate-enriched medium recovered on average 11% faster than flies developed on the protein-enriched medium in both males and females. In addition, females recovered faster than males on both media (Fig. 1a). We found no interaction between the two factors (Table 1); thus there was an additive effect of sex and nutrition on the ability to handle cold stress.
Table 2 Two-way ANOVA on the number of flies that developed successfully from egg-toadult from 20 eggs in either protein or carbohydrate-enriched medium. Trait/sex
Source
Df
F
P
Egg-to-adult viability
Fly nutrition Fly sex Fly nutrition fly sex Error
1 1 1 126
0.61 7.13 12.60
0.44 0.009 0.0005
Fly nutrition Fly nutrition
1,63 1,63
7.84 4.77
Contrasts Female Male
0.007 0.03
4.2. Heat knockdown resistance We found a significant influence of both sex and nutrition on heat knockdown resistance (Table 1). Male and female flies developed on the protein-enriched medium survived on average respectively 18 and 19% longer than flies developing on the carbohydrate-enriched medium. Females survived on average 10 and 8% longer than males on the protein- and carbohydrateenriched media, respectively (Fig. 1b). There was no interaction between nutritional type and sex (Table 1). 4.3. Desiccation tolerance Desiccation tolerance of flies was significantly affected by sex and nutritional regime (Table 1). Male and female flies developing on the protein-enriched medium survived on average 9 and 7% longer than male and female flies developing on the carbohydrate-enriched medium. In addition, females had
Fig. 1. Performance of adult male and female D. melanogaster in a chill coma recovery assay (a), a heat knockdown resistance assay (b) and in a desiccation tolerance assay (c). Flies were raised on either a protein- or a carbohydrate-enriched larval medium. Performance was measured by: (a) seconds it took to recover from chill coma (SE), (b) seconds until flies were knocked down by heat (SE), (c) hours before death from desiccation stress (SE).
Fig. 2. Influence of nutrient composition on (a) egg-to-adult viability in male and female D. melanogaster and on (b) egg production in female D. melanogaster. Flies were raised on a protein or a carbohydrate-enriched larval medium. Female flies in the productivity test oviposited on standard basis medium. Egg-to-adult viability was measured by calculating the average proportion of male and female adult flies emerging from eggs with an assumed 50:50 sex ratio of the eggs laid (SE). Egg production was measured as the average number of eggs produced during a period of 144 h (6 days) (SE).
L.H. Andersen et al. / Journal of Insect Physiology 56 (2010) 336–340
on average 29 and 31% higher level of desiccation tolerance than males in the protein- and carbohydrate-enriched regimes, respectively. We found no interaction between the two factors nutritional regime and sex on desiccation tolerance (Table 1, Fig. 1c). 4.4. Egg-to-adult viability Assuming that the sex ratio of the eggs collected is 50:50 we found a significant interaction between sex and nutritional regime on egg-to-adult viability (Table 2). On average 15% more females developed on the protein-enriched medium while on average 12% more males developed on the carbohydrate-enriched medium (Fig. 2a). 4.5. Egg production We found a significant difference in egg production of females reared under the two different nutritional regimes when tested on basis medium (Fig. 2b, ANOVA, df = 1,81, F = 23.9, P < 0.0001). Females developed on the protein-enriched medium produced on average 14% more eggs than females developed on the carbohydrate-enriched medium. 5. Discussion In this study we found a clear impact of nutrition on the ability to cope with stress as well as on life history traits. As larval nutrition affected tolerance to all investigated types of stress, our results indicate that stress tolerance and nutrition are tightly linked. Given different viability at the two types of medium we cannot rule out that not only plastic responses but also selection can be involved in explaining our results. 5.1. Thermal stress There are several possible physiological explanations for the faster recovery from chill coma when flies are fed a carbohydrateenriched diet. Carbohydrate is known to increase the fat content of the flies (Mayntz et al., 2005) and previous studies have detected a positive correlation between body lipid content in Drosophila spp. and resistance to cold temperatures (Hoffmann et al., 2001) and starvation (Ballard et al., 2008) and desiccation stress (Parkash et al., 2008). Hence, it is possible that the faster recovery from chill coma of the flies raised on the carbohydrate-enriched medium was due to larger lipid deposits. However, the physiological basis for how fat deposits improve chill coma recovery is not completely understood. Larger lipid deposits may affect the quality and/or quantity of the cuticular hydrocarbons influencing water loss and uptake of the cell (Kosta´l et al., 2004). Furthermore, a protein-rich diet might provide an energy source that is more complicated to utilize compared to a carbohydrate-rich diet, because the metabolizing amino acids require more complicated catabolic processes, e.g. deamination and gluconeogenesis (Thompson et al., 2003). The physiological explanation for an increased heat knockdown tolerance among flies raised on the protein-enriched medium is unknown. One possibility may be related to the induction of heat shock proteins which are known to be important for coping with several stress types (Sørensen et al., 2003; Sinclair et al., 2007; Jones and Candido, 1999; Schmidt and Paaby, 2008; Tammariello et al., 1999). Preliminary data from us suggest that Hsp70 is upregulated in flies developed on protein-enriched medium compared to in flies developed on protein deficient medium when challenged at high temperatures. However this hypothesis needs further testing.
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5.2. Desiccation stress That tolerance to desiccation was highest in flies developed on the protein-enriched medium is not what would be expected based on the results from studies by Parkash et al. (2008) where desiccation tolerance and lipid accumulation is shown to be positively correlated. It is however possible that the metabolic end-product from protein metabolization, uric acid, had a protecting effect on the increasing osmotic pressure during desiccation by reducing the water loss from cells (Smith and Smith, 1998). 5.3. Fertility and egg-to-adult viability The fact that we found a higher female developmental success on the protein-enriched larval medium while males survived better on the carbohydrate-enriched larval medium (Fig. 2a) indicates that the two sexes have different requirements during development and growth. Sex-specific responses in life history traits are well known from other studies on D. melanogaster (Matzkin et al., 2007; Sørensen et al., 2007; Yadav and Singh, 2007; Kristensen et al., 2008b). Recent data show that female D. melanogaster accumulate more lipids but less protein relative to body mass compared to males (Wilder et al., in press). While females need to build up protein for ovaries (Markow et al., 1999, 2001), males accumulate protein to build up muscle mass for activity during courtship. Our results however indicate that females suffer to a larger degree from protein deficiency than males. That egg production in protein fed females was significantly higher than in carbohydrate fed females was not surprising as studies on Drosophila and other insects have found similar results (Mattson, 1980; Cook, 1995; Markow et al., 2001; Jervis and Boggs, 2005; Nestel and Nemny-Lavy, 2008). A high protein requirement when producing eggs might reflect that synthesis of the egg-yolk protein vitellin in females is dependent on the incorporation of amino acids (Adams and Gerst, 1991; Markow et al., 1999). Females thus appear to have an advantage in being fatter than males which not only improves tolerance to chill coma, heat stress and desiccation but may also affect fecundity positively. 6. Perspectives Our finding that nutrition affects resistance towards a variety of stress types in D. melanogaster is interesting in an ecological, an evolutionary as well as in a physiological context. The diet composition of the larval medium affects how flies cope with different challenges such as reproduction, survivorship and stress resistances. This indicates that selection pressures on the ability to handle these tasks will interact with the nutritional conditions. For example, we would predict that selection on the ability to handle heat stress is stronger if the flies are experiencing a protein deficient environment (cf. Fig. 1b). Currently, there is an interest in understanding how selection for stress tolerance is correlated to the geographical variation in the expression of a given stress type. For example, numerous studies have investigated the association between thermal resistance traits and latitude and/or altitude (Hoffmann and Weeks, 2007). Clines revealing an association between latitude and/or altitude and heat or cold resistance are often taken as evidence for local adaptation to specific thermal conditions (Hoffmann and Weeks, 2007; Sarup et al., 2009). However, the nutritional composition of available food may vary with latitude and altitude too. Our study suggests that this should be taken into consideration in interpretations of results from studies on local adaptation.
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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Protein and carbohydrate composition of larval food affects tolerance to thermal stress and desiccation in adult Drosophila melanogaster Laila H. Andersen a, Torsten N. Kristensen a,b,*, Volker Loeschcke a, Søren Toft a, David Mayntz a,b a b
Department of Biological Sciences, Genetics and Ecology, Aarhus University, Ny Munkegade, Building 1540, DK-8000 Aarhus C, Denmark Department of Genetics and Biotechnology, Aarhus University, Blichers Alle´ 20, Postbox 50, DK-8830 Tjele, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 September 2009 Received in revised form 9 November 2009 Accepted 10 November 2009
Larval nutrition may affect a range of different life history traits as well as responses to environmental stress in adult insects. Here we test whether raising larvae of fruit flies, Drosophila melanogaster, on two different nutritional regimes affects resistance to cold, heat and desiccation as well as egg production and egg-toadult viability. We raised larvae on a carbohydrate-enriched and a protein-enriched growth medium. We found that flies developed on the high protein medium had increased heat and desiccation tolerance compared to flies developed on the carbohydrate-enriched medium. In contrast, flies developed on the carbohydrate-enriched growth medium recovered faster from chill coma stress compared to flies developed on a protein-enriched medium. We also found gender differences in stress tolerance, with female flies being more tolerant to chill coma, heat knockdown and desiccation stress compared to males. Egg production was highest in females that had developed on the protein-enriched medium. However, there was a sex-specific effect of nutrition on egg-to-adult viability, with higher viability for males developing on the sucrose-enriched medium, while female survival was highest when developing on the protein-enriched medium. Our study indicates that larval nutrition has a strong impact on the ability to cope with stress, and that the optimal nutrient composition varies with the type of stress. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: Fecundity Development Nutrition Environmental stress
1. Introduction Drosophila melanogaster is often used as a model organism in studies of physiological and evolutionary responses to various forms of stress (Hoffmann et al., 2003; Sinclair et al., 2007; Kristensen et al., 2008a). However, there is little information about how nutrition affects life history traits (Watts et al., 2006) and performance during thermal stress, and the importance of diet is often underestimated in experimental designs (Prasad et al., 2003). Hence, there is a growing need to investigate diet-related effects behind variation in traits of importance for fitness. Many organisms face a challenge of meeting their optimal nutritional requirements for somatic and reproductive growth under natural conditions (Raubenheimer and Simpson, 1999). During life, body tissues constantly require a specific quantity and proportion of nutrients, in order to attain optimal growth and performance (Bauerfeind and Fischer, 2005). Deficiency or imbalance of e.g. fat, carbohydrate or protein can affect characters such as somatic growth and reproduction. For instance, protein deficiency
* Corresponding author at: Department of Genetics and Biotechnology, Aarhus University, Blichers Alle´ 20, Postbox 50, DK-8830 Tjele, Denmark. Tel.: +45 8999 1076; fax: +45 8999 1300. E-mail address: [email protected] (T.N. Kristensen). 0022-1910/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2009.11.006
reduces fecundity and growth in D. melanogaster (Wang and Clark, 1995) and in fruit-feeders protein is often a limiting macronutrient (Mattson, 1980; Adams and Gerst, 1991; Hendrichs et al., 1991; Markow et al., 2001). In contrast, diet restriction or mild starvation can increase longevity as well as tolerance to stressors such as heat stress (reviewed by Wenzel, 2006; Smith et al., 2007) demonstrating the complexity of organismal nutrient acquisition and utilization. A variety of factors may affect organismal stress tolerance. These include physiological as well as behavioural changes. Climatic changes may be met by e.g. physiological hardening processes, coma or production of metabolites making the organism tolerate temperature extremes (Sørensen et al., 2003, 2005; Lalouette et al., 2007). Also, an organism may compensate for nutritional stress and reduced body size by extending its growth period or altering its energy allocation to growth, hence postponing the reproductive period (Reichling and German, 2000; Lobe et al., 2006). Behaviourally, organisms may respond to stress by migration, use of refuges or an altered and more favourable nutrient intake to meet changed energy expenditures (Mattson, 1980). The bulk of studies on physiological and evolutionary responses to nutrient deficiencies focuses on reproduction and fecundity (Markow et al., 1999; Bauerfeind and Fischer, 2005; Naya et al., 2007; Nestel and Nemny-Lavy, 2008), while only a few studies have focused on how nutrient resources and deficiencies can affect other challenges in life such as tolerance to environmental stress
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(Wang and Clark, 1995; Kanazawa et al., 2003). However, Burger et al. (2007) report an increased resistance to starvation stress in D. melanogaster when reared on a sucrose-rich diet which indicates that diet composition may influence stress responses. The aim of this study was to test if larval nutrition affects the ability of adult flies to cope with a number of different stress challenges, including cold and warm temperatures and desiccation. As protein and carbohydrates are the most important macronutrients for Drosophila larvae in natural food sources (Mattson, 1980), we chose to focus on these two very different dietary conditions. In order to test how these growth media affected life history traits, we also tested for dietary effects on egg production and egg-to-adult viability. 2. Materials and methods
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were transferred individually to empty glass vials (6.0 mL) with a watertight lid and observed while submerged in a water bath set at 38.3 8C. Time until flies went into heat coma, i.e. were unable to stand, was recorded for each fly. 2.4. Desiccation resistance Males (N = 50) and females (N = 50) from each nutritional group were tested for their tolerance to an air humidity of 0–5%, obtained by means of silica gel put into a sealed fish tank, positioned at 25 8C. Each fly was separately transferred to empty glass vials (6.0 mL) which were covered with gauze to allow air to circulate through. Vials were exposed to low humidity in the fish tank and they were checked for dead flies every hour until all were dead. The tests were performed in the laboratory with 12 h light/12 h dark cycles throughout the experiment which lasted approximately 27 h.
2.1. Flies used in the experiments 2.5. Egg-to-adult viability A genetically diverse mass population of D. melanogaster reared in the laboratory since 2002 was used in the experiment (see Bubliy and Loeschcke, 2005 for further details on the population used). Prior to the experiment flies were kept on a basis medium (Sørensen et al., 2009) which is a nutritionally balanced artificial diet composed of sucrose (40 g/L water), yeast (60 g/L water), agar (16 g/L water), oatmeal (30 g/L water), nipagen (12 mL/L water) and acetic acid (1.2 mL/L water). Eggs were collected and transferred to two types of media, a carbohydrate-enriched medium and a protein-enriched medium. Both media were made by mixing either sucrose or casein (Sigma C-5890, Sigma–Aldrich) with instant Carolina basis (formula 4-24 plain) medium (Carolina Biological Supply, Burlington, NC, USA). Carolina fly medium is a nutritionally balanced artificial diet which provides the essential nutrients required by D. melanogaster larvae during development. Henceforth we refer to it as basis medium. The carbohydrate-enriched medium (20% carbohydrate) was prepared by mixing sucrose and basis medium in a 1:4 ratio before adding water. The protein-enriched medium (60% protein) was prepared by mixing casein and basis medium in a 3:2 ratio. The two nutrition types used were chosen on the background of reported variations in sugar and protein contents in the natural host choices of D. melanogaster (Markow et al., 1999; Arvanitoyannis and Mavromatis, 2009). All vials contained approximately 7 mL of medium and three to four droplets of a thin yeast suspension (ca. 0.5–1 mg/mL water) were added on top of the medium. Density of developing larvae in the vials was standardized among nutritional groups with 50 (5) eggs/vial. The eggs hatched and the larvae developed at 25 8C and 12 h light/12 h dark cycles. Hatched virgin flies were collected and sexed and transferred to standard basis medium in groups of 50 (5) flies per vial. They were left to rest after CO2anaesthesia on standard basis medium for 2–3 days and then tested as described below. 2.2. Chill coma recovery Males (N = 50) and females (N = 50) from each nutritional group were collected and tested for their ability to tolerate low temperatures. Flies were individually transferred to empty Eppendorf tubes (0.5 mL) and submerged in a water bath at 2 8C for 2 h. Immediately after exposure, they were observed separately in the laboratory at room temperature (20–23 8C) and the time it takes to regain consciousness was registered (registered as duration from exposure termination until the fly was able to stand).
Twenty female and twenty male flies from the mass population developing on basis medium were collected and sorted according to sex under CO2-anaesthesia. They were divided in four groups of 10 flies (5 males and 5 females in each group) and left to rest on standard basis medium for 2 days. After 48 h flies were transferred to a new vial for egg laying on standard basis medium during a period of 4 h. Eggs were collected and transferred in groups of 20 to vials containing 7 mL of either carbohydrate-enriched medium (N = 34) or protein-enriched medium (N = 34). Eggs were left to hatch at 25 8C with 12 h light/12 h dark cycles. We checked for newly emerged flies every 8 h and counted and sexed emerging flies. 2.6. Egg production We collected virgin males (N = 50) and virgin females (N = 50) developed on either the carbohydrate-enriched or the proteinenriched medium. One male and one female were transferred to vials containing basis medium (N = 50 pr. nutritional regime). Pairs of flies were transferred to vials with teaspoons with basis medium at age 3–4 days. Every 48 h flies were transferred to a new vial with a new teaspoon with basis medium. This procedure was repeated three times so that the number of eggs produced from day 3–4 to day 7–8 of age could be registered. 3. Statistics All statistical analyses were performed in JMP 7.0. Outliers more than three standard deviations from the mean were discarded before statistical tests were performed. In all experimental tests, except for the egg production experiment, we focused on two factors: nutritional regime (protein-enriched growth medium vs. carbohydrate-enriched growth medium) and sex. Two-way ANOVA was used to analyse egg-to-adult viability data. If the interaction term between the factors sex and nutrition was significant, we repeated the analysis for each sex separately. Egg production was analysed using one-way ANOVA, and the assumption of equal variances between groups was tested using Bartlett’s test (P > 0.05). Proportional hazard analyses were used to analyse data where the variable described the time spend until an event happened. 4. Results
2.3. Heat knockdown resistance
4.1. Chill coma recovery
Males (N = 50) and females (N = 50) from each nutritional group were tested for their ability to tolerate high temperatures. Flies
The chill coma recovery time of the flies was significantly affected by both nutritional regime and sex (Table 1). Flies
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Table 1 Proportional hazards analysis on resistance to thermal stress, with focus on the two factors, nutritional regime (protein or carbohydrate-enriched medium) and sex (males or females). DF was 1 in all tests. Trait
Source
F
Chill coma recovery
Fly nutrition Fly sex Fly nutrition fly sex
13.95 25.98 0.14
0.0002 0.0001 0.71
Heat knockdown
Fly nutrition Fly sex Fly nutrition fly sex
13.09 8.56 1.49
0.0003 0.003 0.22
Fly nutrition Fly sex Fly nutrition fly sex
7.45 71.43 1.46
0.006 <0.0001 0.22
Desiccation
P
developed on the carbohydrate-enriched medium recovered on average 11% faster than flies developed on the protein-enriched medium in both males and females. In addition, females recovered faster than males on both media (Fig. 1a). We found no interaction between the two factors (Table 1); thus there was an additive effect of sex and nutrition on the ability to handle cold stress.
Table 2 Two-way ANOVA on the number of flies that developed successfully from egg-toadult from 20 eggs in either protein or carbohydrate-enriched medium. Trait/sex
Source
Df
F
P
Egg-to-adult viability
Fly nutrition Fly sex Fly nutrition fly sex Error
1 1 1 126
0.61 7.13 12.60
0.44 0.009 0.0005
Fly nutrition Fly nutrition
1,63 1,63
7.84 4.77
Contrasts Female Male
0.007 0.03
4.2. Heat knockdown resistance We found a significant influence of both sex and nutrition on heat knockdown resistance (Table 1). Male and female flies developed on the protein-enriched medium survived on average respectively 18 and 19% longer than flies developing on the carbohydrate-enriched medium. Females survived on average 10 and 8% longer than males on the protein- and carbohydrateenriched media, respectively (Fig. 1b). There was no interaction between nutritional type and sex (Table 1). 4.3. Desiccation tolerance Desiccation tolerance of flies was significantly affected by sex and nutritional regime (Table 1). Male and female flies developing on the protein-enriched medium survived on average 9 and 7% longer than male and female flies developing on the carbohydrate-enriched medium. In addition, females had
Fig. 1. Performance of adult male and female D. melanogaster in a chill coma recovery assay (a), a heat knockdown resistance assay (b) and in a desiccation tolerance assay (c). Flies were raised on either a protein- or a carbohydrate-enriched larval medium. Performance was measured by: (a) seconds it took to recover from chill coma (SE), (b) seconds until flies were knocked down by heat (SE), (c) hours before death from desiccation stress (SE).
Fig. 2. Influence of nutrient composition on (a) egg-to-adult viability in male and female D. melanogaster and on (b) egg production in female D. melanogaster. Flies were raised on a protein or a carbohydrate-enriched larval medium. Female flies in the productivity test oviposited on standard basis medium. Egg-to-adult viability was measured by calculating the average proportion of male and female adult flies emerging from eggs with an assumed 50:50 sex ratio of the eggs laid (SE). Egg production was measured as the average number of eggs produced during a period of 144 h (6 days) (SE).
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on average 29 and 31% higher level of desiccation tolerance than males in the protein- and carbohydrate-enriched regimes, respectively. We found no interaction between the two factors nutritional regime and sex on desiccation tolerance (Table 1, Fig. 1c). 4.4. Egg-to-adult viability Assuming that the sex ratio of the eggs collected is 50:50 we found a significant interaction between sex and nutritional regime on egg-to-adult viability (Table 2). On average 15% more females developed on the protein-enriched medium while on average 12% more males developed on the carbohydrate-enriched medium (Fig. 2a). 4.5. Egg production We found a significant difference in egg production of females reared under the two different nutritional regimes when tested on basis medium (Fig. 2b, ANOVA, df = 1,81, F = 23.9, P < 0.0001). Females developed on the protein-enriched medium produced on average 14% more eggs than females developed on the carbohydrate-enriched medium. 5. Discussion In this study we found a clear impact of nutrition on the ability to cope with stress as well as on life history traits. As larval nutrition affected tolerance to all investigated types of stress, our results indicate that stress tolerance and nutrition are tightly linked. Given different viability at the two types of medium we cannot rule out that not only plastic responses but also selection can be involved in explaining our results. 5.1. Thermal stress There are several possible physiological explanations for the faster recovery from chill coma when flies are fed a carbohydrateenriched diet. Carbohydrate is known to increase the fat content of the flies (Mayntz et al., 2005) and previous studies have detected a positive correlation between body lipid content in Drosophila spp. and resistance to cold temperatures (Hoffmann et al., 2001) and starvation (Ballard et al., 2008) and desiccation stress (Parkash et al., 2008). Hence, it is possible that the faster recovery from chill coma of the flies raised on the carbohydrate-enriched medium was due to larger lipid deposits. However, the physiological basis for how fat deposits improve chill coma recovery is not completely understood. Larger lipid deposits may affect the quality and/or quantity of the cuticular hydrocarbons influencing water loss and uptake of the cell (Kosta´l et al., 2004). Furthermore, a protein-rich diet might provide an energy source that is more complicated to utilize compared to a carbohydrate-rich diet, because the metabolizing amino acids require more complicated catabolic processes, e.g. deamination and gluconeogenesis (Thompson et al., 2003). The physiological explanation for an increased heat knockdown tolerance among flies raised on the protein-enriched medium is unknown. One possibility may be related to the induction of heat shock proteins which are known to be important for coping with several stress types (Sørensen et al., 2003; Sinclair et al., 2007; Jones and Candido, 1999; Schmidt and Paaby, 2008; Tammariello et al., 1999). Preliminary data from us suggest that Hsp70 is upregulated in flies developed on protein-enriched medium compared to in flies developed on protein deficient medium when challenged at high temperatures. However this hypothesis needs further testing.
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5.2. Desiccation stress That tolerance to desiccation was highest in flies developed on the protein-enriched medium is not what would be expected based on the results from studies by Parkash et al. (2008) where desiccation tolerance and lipid accumulation is shown to be positively correlated. It is however possible that the metabolic end-product from protein metabolization, uric acid, had a protecting effect on the increasing osmotic pressure during desiccation by reducing the water loss from cells (Smith and Smith, 1998). 5.3. Fertility and egg-to-adult viability The fact that we found a higher female developmental success on the protein-enriched larval medium while males survived better on the carbohydrate-enriched larval medium (Fig. 2a) indicates that the two sexes have different requirements during development and growth. Sex-specific responses in life history traits are well known from other studies on D. melanogaster (Matzkin et al., 2007; Sørensen et al., 2007; Yadav and Singh, 2007; Kristensen et al., 2008b). Recent data show that female D. melanogaster accumulate more lipids but less protein relative to body mass compared to males (Wilder et al., in press). While females need to build up protein for ovaries (Markow et al., 1999, 2001), males accumulate protein to build up muscle mass for activity during courtship. Our results however indicate that females suffer to a larger degree from protein deficiency than males. That egg production in protein fed females was significantly higher than in carbohydrate fed females was not surprising as studies on Drosophila and other insects have found similar results (Mattson, 1980; Cook, 1995; Markow et al., 2001; Jervis and Boggs, 2005; Nestel and Nemny-Lavy, 2008). A high protein requirement when producing eggs might reflect that synthesis of the egg-yolk protein vitellin in females is dependent on the incorporation of amino acids (Adams and Gerst, 1991; Markow et al., 1999). Females thus appear to have an advantage in being fatter than males which not only improves tolerance to chill coma, heat stress and desiccation but may also affect fecundity positively. 6. Perspectives Our finding that nutrition affects resistance towards a variety of stress types in D. melanogaster is interesting in an ecological, an evolutionary as well as in a physiological context. The diet composition of the larval medium affects how flies cope with different challenges such as reproduction, survivorship and stress resistances. This indicates that selection pressures on the ability to handle these tasks will interact with the nutritional conditions. For example, we would predict that selection on the ability to handle heat stress is stronger if the flies are experiencing a protein deficient environment (cf. Fig. 1b). Currently, there is an interest in understanding how selection for stress tolerance is correlated to the geographical variation in the expression of a given stress type. For example, numerous studies have investigated the association between thermal resistance traits and latitude and/or altitude (Hoffmann and Weeks, 2007). Clines revealing an association between latitude and/or altitude and heat or cold resistance are often taken as evidence for local adaptation to specific thermal conditions (Hoffmann and Weeks, 2007; Sarup et al., 2009). However, the nutritional composition of available food may vary with latitude and altitude too. Our study suggests that this should be taken into consideration in interpretations of results from studies on local adaptation.
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