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Fluctuations in nutrient composition affect male reproductive output in Drosophila melanogaster
Journal of Insect Physiology 118 (2019) 103940
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Fluctuations in nutrient composition affect male reproductive output in Drosophila melanogaster
T
⁎
Lucy Rebecca Daviesa, , Mads F. Schoua, Torsten N. Kristensena,b, Volker Loeschckea a b
Department of Bioscience, Aarhus University, DK-8000 Aarhus C, Denmark Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg East, Denmark
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluctuating environments Protein to carbohydrate ratios Sexual dimorphism Nutrient availability Unpredictable environments Starvation resistance
Insects are known to selectively balance their intake of protein and carbohydrate to optimize reproduction and survival. For insects who feed on decomposing fruit, fluctuations in macronutrient composition occur as fruits ripe and decomposition progresses which may challenge optimal resource allocation. Using Drosophila melanogaster, we tested the effect of macronutrient fluctuations and the variability of these fluctuations on starvation resistance and components of reproductive output; traits known to be sensitive to different protein to carbohydrate (P:C) ratios in the diet. For 8 days, flies were fed the same protein to carbohydrate (P:C) ratio (constant feeding), or fed diets with fluctuations in P:C ratio on each day; these fluctuations being regular (predictably fluctuating) or irregular (unpredictably fluctuating). The three feeding regimes yielded the same average P:C ratio across the duration of the experiment. We found no difference in starvation resistance across the feeding regimes. Interestingly, there was a sexual dimorphism in the effect on reproductive output with males performing worst in the unpredictable feeding regime, and with no effect of feeding regime on female performance. Our study provides evidence for means of adapting to fluctuating macronutrient composition and suggests females are more tactful than males in storing and allocating resources for reproduction.
1. Introduction Changes in light, temperature and rainfall affect plant and microbial growth and force organisms such as insects to deal with changes in nutrient availability throughout their life time (Wolda, 1978; Morais et al., 1995). As well as overall abundance of nutrient availability, the ratio of different macronutrients, such as protein and carbohydrate, can also fluctuate within an organism’s environment. For example, for insects feeding on decomposing fruit, such as most Drosophila spp., natural fluctuations in macronutrient composition of the diet can occur due to yeast growth and metabolism which modifies the fruit tissues during different stages of fruit decay (Morais et al., 1995). Protein and carbohydrate are critical macronutrients, but as fruit ripens the availability of these macronutrients varies and will not always be synchronized with the protein to carbohydrate (P:C) ratio ideal for consumers. For insects, surpluses or deficiencies of certain macronutrients can be detrimental or beneficial (Lee et al., 2008; Simpson and Raubenheimer, 2009; Dussutour et al., 2012). Over intake of protein can be detrimental for lifespan in many insects, while insufficient intake can have negative effects on reproduction and larval development (Lee et al., 2008; Fanson et al., 2009; Clark et al., 2015; Rodrigues et al.,
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2015). The opposite, however, is found for carbohydrate intake (Lee et al., 2008; Fanson et al., 2009; Clark et al., 2015; Rodrigues et al., 2015). Insects, therefore, selectively balance the intake of macronutrients in order to optimize a particular trait (Simpson and Raubenheimer, 2012). For example, once female Drosophila melanogaster have mated, they increase their protein intake and lower their carbohydrate intake to optimize reproduction (Lee et al., 2013). Conversely, virgin females and males choose a diet that is rich in carbohydrate, benefitting lifespan and starvation resistance (Lee et al., 2013; Davies et al., 2018). In times of starvation D. melanogaster survive by utilizing stored lipids; lipids that have come from carbohydrate (Lee and Jang, 2014). During nutrient fluctuations insects are unable to choose their desired diet and nutrient utilization and resource allocation to a specific trait may be problematic. Despite the biological importance of this intriguing ecological and physiological challenge, which we must assume insects have adapted to, few studies have been performed attempting to understand this adaptation. Here we investigated the effects of macronutrient fluctuations and the predictability of these fluctuations on resource allocation and reproductive potential of D. melanogaster. Previous experiments investigating food abundance fluctuations show that the tephritid fly,
Corresponding author at: Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus C, Denmark. E-mail address: [email protected] (L.R. Davies).
https://doi.org/10.1016/j.jinsphys.2019.103940 Received 20 May 2019; Received in revised form 13 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0022-1910/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 118 (2019) 103940
L.R. Davies, et al.
Fig. 1. The protein to carbohydrate ratios in the three feeding regimes; constant, predictable and unpredictable.
resistance and measures of reproduction were set up within 3 h after the completion of the feeding.
Anastrepha ludens, and the medfly, Ceratitis capitate, minimize the allocation of body lipid, glycogen and protein stores to reproduction and metabolism in times of food shortages and maximize storage levels in times of high food availability (Aluja et al., 2011; Carey et al., 2002). In our experiment, we tested protein and carbohydrate allocation efficiency when both macronutrients were available ad libitum. To do this we tested the impact of three feeding regimes, constant, predictably and unpredictably fluctuating regimes, on investment in starvation resistance and several components of reproductive output; traits known to be sensitive to different P:C ratios (Lee et al., 2008; Fricke et al., 2015). In the constant regime the P:C ratio was kept the same whereas in the predictable regime, the P:C ratio changed between high and low each day in a regular manner. The unpredictable regime changes were irregular and varied in big and small changes (see Fig. 1 for a timeline of the P:C ratio in each regime). In order to evaluate whether it is the delivery of P:C ratio that is important or the overall P:C ratio, the average P:C ratio was the same in the three regimes (Fig. 1).
2.3. Protein to carbohydrate ratios Instant dry yeast (instant yeast, LeSaffre, Marq-en-Baroeul, France) and sucrose were used as sources of protein and carbohydrate. P:C ratios were calculated based on the yeast containing 98.5% Saccharomyces cerevisiae and 1.5% sorbitan monostearate, with nutrient values of 50% protein, 6% digestible carbohydrate, 27% indigestible fibre, 6% fats and 11% sodium, vitamin C, calcium and iron. Sorbitan monostearate does not affect the P:C ratio. The yeast and sucrose were combined with 16 g/L agar, 12 mL/L nipagen and 1.2 mL/L acetic acid mixed in water. To avoid the growth of yeast and the increase of protein in the diets, the yeast was killed in boiling water before the addition of the other components. Yeast to sucrose ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1: 3.5 and 1:4 were used to make the P:C ratios 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8 respectively.
2. Methods 2.4. Starvation resistance 2.1. Fly stock After exposure to the different feeding regimes for 8 days, flies were mixed within feeding regimes and randomly placed in individual vials containing 3.5 mL of a 2% agar solution (for constant, predictable and unpredictable regimes: females n = 15, n = 14, n = 15; males n = 17, n = 17, n = 18, respectively) i.e. without access to nutrients. Starvation resistance was measured as the survival time in these vials. The number of dead flies was counted every 8 h until all flies had died.
All D. melanogaster used for this study were from a laboratory stock established from the offspring of approximately 200 inseminated females caught near Odder (55°56′42.46″N, 10°12′45.31″E), Denmark, in 2012. The flies were maintained at 19 °C on a 12 h:12 h light:dark cycle on a standard laboratory medium containing 60 g/L yeast, 40 g/L sucrose, 30 g/L oatmeal, 16 g/L agar, 12 mL/L nipagin and 1.2 mL/L acetic acid mixed with water. 200 flies were taken from the stock as the experimental base population. This base population was maintained at 25 °C for two generations to allow for acclimation to the temperature of the feeding regimes. To obtain flies for the experiment, eggs from the second generation flies were collected and developed at a density of 30 eggs per vial on 7 mL of standard medium.
2.5. Females’ offspring viability We measured egg-to-adult viability of eggs laid after exposing the females to the feeding regimes, and will refer to this component of female reproductive output as females’ offspring viability. After exposure to the feeding regimes, 30 females from each regime were grouped in units of 10 and added to vials containing standard laboratory medium. 10 virgin control males were also put in each vial. The control males were of the same age, from the same original population and had been maintained on the standard laboratory medium in a density controlled environment. After 3 days (to allow for mating and egg production), the females were transferred to vials containing a spoon with the standard laboratory medium for egg laying. After 12 h, eggs were transferred to vials containing the standard laboratory medium such that exactly 20 eggs were in each vial (number of vials for constant, predictable and unpredictable feeding regimes: n = 13, n = 13 and n = 14, respectively). Every other day, the emerged flies were counted and removed.
2.2. Feeding regimes Virgin flies that had emerged within 6 h were separated by sex and placed in 60 vials in groups of five. Flies were exposed to one of three feeding regimes for 8 days; constant, predictably or unpredictably fluctuating (Fig. 1). Flies exposed to the constant regime were fed P:C ratio 1:5 every day (Fig. 1). The predictably fluctuating regime provided flies with a P:C ratio diet alternating between 1:2 and 1:8 each day, whereas the flies in the unpredictably fluctuating regime were provided with ratios that changed irregularly over the 8 days (Fig. 1). The average P:C ratio was 1:5 for all feeding regimes. Starvation 2
Journal of Insect Physiology 118 (2019) 103940
L.R. Davies, et al.
predictable-unpredictable P < 0.001) (Fig. 2C). There was a significant effect of feeding regime on the hatchability of the eggs laid by control females that had mated with males from the different feeding regimes (χ21,36 = 54.0, P < 0.001). The post hoc test showed the hatchability of eggs sired by males from the unpredictably fluctuating regime was significantly lower than that from the predictable regime (unpredictable-predictable P < 0.01; constant-predictable P = 0.55; constant-unpredictable P = 0.08) (Fig. 2D). Pearson’s correlation test revealed that overall there was a significant positive correlation between mating time and the hatchability of the eggs (r = 0.42, d.f. = 43, P < 0.01).
2.6. Male reproductive output Male reproductive output was measured by mating time and the hatchability of the eggs laid by the mated female. After exposure to the feeding regimes, male flies were individually placed in wells with one control virgin female in a fly arena (number of males for constant, predictable and unpredictable regimes: n = 14, n = 16 and n = 15 respectively). The fly arena (dimensions 12 cm × 12 cm) contained 6 × 6 wells with diameter of 1.6 cm. The control females were of the same age, from the same original population and had been maintained on the standard laboratory medium in a density controlled environment. A transparent plastic lid was placed over the top such that the flies were trapped in their wells. A camera was left for 8 h above the wells to video mating time. The mated females were transferred to individual vials containing a spoon with the standard laboratory medium for egg laying. Fresh vials and spoons were supplied every other day for 12 days. Egg hatchability was calculated from counts of laid eggs and hatched larvae.
4. Discussion This study aimed to investigate the consequences of macronutrient fluctuations and the predictability of these fluctuations on traits known to be affected by different protein to carbohydrate (P:C) ratios (Lee et al., 2008; Fricke et al., 2015). We show that predictability in the nutritional fluctuations had an effect on male reproductive output, but not on starvation resistance and females’ offspring viability. Male reproductive output was measured by the mating time and hatchability of the eggs produced by females exposed to standard laboratory medium that had mated with males from the different feeding regimes. Previous studies have shown mating time to be a good indicator of male investment into reproduction and hatchability to be affected by male diet (Wigby et al., 2009; Moatt et al., 2014; Fricke et al., 2015). In this study, male mating time was reduced when nutrients were provided in an unpredictable fluctuating way (Fig. 2C). In a previous experiment on D. simulans, unpredictable fluctuations in temperature also resulted in reduced performance in life-history related traits compared to flies experiencing predictable thermal fluctuations (Manenti et al., 2014). The authors suggested that this was due to energy expenditures in monitoring a changing environment and in attempting to produce an optimal response to the changes. This explanation is likely in our set up as well, with the males frequently adjusting their metabolism and resource allocation. If the adjustment is costly, or if it fails to be in synchrony with the unpredictable change in macronutrient levels, it will cause a non-optimal resource allocation and therefore suboptimal nutritional state for successful reproduction. When given a choice, males prefer carbohydrate-rich diets (Lee et al., 2013) in order to invest in the energy demands for courtship behaviors, the mating itself and for the production of accessory gland proteins (Droney, 1998; Maklakov et al., 2008). On day 7 of the unpredictably fluctuating feeding regime, however, flies were provided with a relatively low carbohydrate diet which could have had negative effects on male reproductive success. Though if this was the case the low carbohydrate diet would likely also have had negative effects on starvation resistance and this was not seen (Fig. 2A). An experiment subjecting flies to multiple unpredictable regimes with different sequences of fluctuations would provide further evidence as to whether the reduced reproductive fitness is due to the particular sequence of the diets or indeed the unpredictability of the fluctuations. As for the male reproductive output, we also expected a reduced performance in starvation resistance and females’ offspring viability in the fluctuating environments. Previous experiments have also shown suboptimal nutritional environments for female adults to have negative effects on the development of her offspring (Kyneb & Toft, 2006; Geister et al., 2008). This, however, was not found in our study (Fig. 1A, B). For many insects, it has been found that different amounts of protein and carbohydrate optimize or weaken particular traits (Geister et al., 2008; Lee et al., 2008; Lee et al., 2013; Simpson and Raubenheimer, 2009; Dussutour et al., 2012; Sisodia et al., 2015). Geister et al. (2008) and Sisodia et al. (2015) for example, found higher amounts of protein led to lower offspring viability compared to diets higher in carbohydrate. Lee et al. (2008) also found diets higher in carbohydrate enhanced lifespan. In our study, the three regimes had an average P:C ratio of 1:5.
2.7. Statistical analysis A logistic regression in a generalized linear model was used to investigate the effect of feeding regime on females’ offspring viability and egg hatchability for male productive output. The full model contained feeding regime as the predictor variable which was then compared with a reduced model from which feeding regime was removed, using a likelihood ratio test to obtain a P-value. We detected no over-dispersion. The effect of feeding regime on starvation resistance was tested by comparing linear models. The model contained starvation resistance as the response variable and feeding regime and sex as the predictor variables and the interaction between feeding regime and sex. To obtain P-values of the model components, we performed sequential model reduction and compared models using F-tests. The effect of feeding regime on mating time was tested by comparing linear models containing mating time as the response variable and feeding regime as the predictor variable with an identical model in which feeding regime was removed. The models were compared using an F-test to obtain the P-value. When F-tests showed a significant effect of feeding regime, we used Tukey’s post hoc comparisons to identify which regimes were significantly different from each other. All statistical analyses were carried out in R version 3.5.2 (R Core Team, 2018). Assumptions for parametric analysis were fulfilled in all models. 3. Results 3.1. Starvation resistance Females survived starvation significantly longer than males (F1,93 = 90.99, P < 0.001), but we found no significant interaction between feeding regime and sex (F1,92 = 2.04, P = 0.13). Starvation resistance did not differ significantly between females from the different feeding regimes (F1,94 = 2.87, P = 0.06) (Fig. 2A). 3.2. Females’ offspring viability and male reproductive output Feeding regime had no impact on the egg-to-adult viability of eggs from female flies mated after the 8 days of exposure to the feeding regime (χ21,39 = 37.8, P = 0.40) (Fig. 2B). Male reproductive fitness was measured by mating time and the number of eggs hatched from the female they had mated with. There was a significant effect of feeding regime on mating time (F1,44 = 19.67, P < 0.001) with the post hoc test revealing no difference in performance between the constant and predictable regimes (P = 0.54), but males from the unpredictable regime mated for the shortest time (constant-unpredictable P < 0.001; 3
Journal of Insect Physiology 118 (2019) 103940
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Fig. 2. Mean ( ± s.e.m.) starvation resistance and female and male reproductive output for each feeding regime; constant, predictable and unpredictable. A. Starvation resistance. B. Percentage of adult D. melanogaster that emerged from 20 eggs. C. Mating time, in seconds, of males mated with a control female. D. Hatchability of eggs from control females that had mated with males from the 3 feeding regimes. Same letters indicate no significant difference.
ratio of the regimes affects the phenotype.
Here, it could therefore be the average ratio that was affecting starvation resistance and females’ offspring viability and not the way in which the nutrients were consumed. In our female reproduction analysis, female flies from the three feeding regimes were tested after being on the same laboratory standard medium for 3 days. This was necessary to allow time for mating, fertilization of eggs and egg laying. We argue that these 3 days had little influence on our result as previous experiments show the nutritional environment before mating can determine allocation in to reproduction (Carey et al., 2002; Gorter et al., 2016). This mechanism was proposed as a means of adapting to fluctuating environments, and our study provides evidence for that. The ingestion of the same P:C ratio over the 8 days would give the flies from the three feeding regimes equal resources to invest in reproductive output, which is confirmed by equal female reproductive output across feeding regimes. The same is probably happening for starvation resistance in both the males and females. A study focusing on nutritional environment and starvation resistance found starvation resistance was dependent on lipid reserves which were determined by different P:C ratios (Lee and Jang, 2014). Although Lee & Jang (2014) did not vary the diets, the assimilation of the average 1:5 P:C ratio would explain why we do not see a difference across the three feeding regimes. Overall females survived starvation resistance longer than males in our study. Lee and Jang (2014) also observed this and proposed that this was due to the larger lipid content of females compared to males. Further research could investigate effects of lowering or increasing the average P:C ratio across the three regimes. By raising the average ratio to levels between 1:1 and 1:4, we would expect to observe lower starvation resistance (Lee et al., 2008; Davies et al., 2018) and offspring viability (Geister et al., 2008; Sisodia et al., 2015) in all regimes and vice versa if the average ratio was to be lowered compared to the 1:5 ratio. The addition of egg count will also give another component of female reproductive output and would be expected to be optimized at higher P:C ratios. This would provide further insight in how the average
5. Conclusion Consequences of fluctuating macronutrients on reproductive output differed between males and females, and our results likely reflect a better ability of females to store and allocate nutrients in fluctuating environments. We previously found females to make the same foraging choices across a range of different P:C ratios in the developmental media (Davies et al., 2018). Both these studies, in addition to findings by Gorter et al. (2016) and Carey et al., (2002), lead us to conclude that females have developed strategies for nutrient uptake and resource investment that allow for optimal egg production across fluctuating nutritional conditions.
Acknowledgements We are grateful to Trine Bech Søgaard and Annemarie Højmark for help in the laboratory.
Funding This research was supported by funding from the Graduate School of Science and Technology (GSST) at Aarhus University and by grants from the Danish Natural Research Council to V.L. (large frame grant DFF-4002-00113) and T.N.K. (DFF-8021-00014B).
Accessibility Data available from figshare: https://figshare.com/projects/ Fluctuations_in_nutrient_availability_affect_male_reproductive_output_ in_Drosophila_melanogaster/60551. 4
Journal of Insect Physiology 118 (2019) 103940
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Aluja, M., Birke, A., Guillén, L., Díaz-Fleischer, F., Nestel, D., 2011. Coping with an unpredictable and stressful environment: the life history and metabolic response to variable food and host availability in a polyphagous tephritid fly. J. Insect Physiol. 57, 1592–1601. https://doi.org/10.1016/j.jinsphys.2011.08.001. Carey, J.R., Liedo, P., Harshman, L., Liu, X., Müller, H.-G., Partridge, L., Wang, J.L., 2002. Food pulses increase longevity and induce cyclical egg in Mediterranean fruit flies production. Funct. Ecol. 16, 313–325. https://doi.org/10.1046/j.1365-2435.2002. 00633.x. Clark, R.M., Zera, A.J., Behmer, S.T., 2015. Nutritional physiology of life-history tradeoffs: how food protein–carbohydrate content influences life-history traits in the wingpolymorphic cricket Gryllus firmus. J. Exp. Biol. 218, 298–308. https://doi.org/10. 1242/jeb.112888. Davies, L.R., Schou, M.F., Kristensen, T.N., Loeschcke, V., 2018. Linking developmental diet to adult foraging choice in Drosophila melanogaster. J. Exp. Biol. 221, e9. https:// doi.org/10.1242/jeb.175554. Droney, D.C., 1998. The influence of the nutritional content of the adult male diet on testis mass, body condition and courtship vigour in a Hawaiian Drosophila. Funct. Ecol. 12, 920–928. https://doi.org/10.1046/j.1365-2435.1998.00266.x. Dussutour, A., Simpson, S.J., Sabatier, P., 2012. Ant workers die young and colonies collapse when fed a high-protein diet. Proc. R. Soc. B: Biol. Sci. 279, 2402–2408. https://doi.org/10.1098/rspb.2012.0051. Fanson, B.G., Weldon, C.W., Perez-Staples, D., Simpson, S.J., Taylor, P.W., 2009. Nutrients, not caloric restriction, extend lifespan in Queensland. Agin Cell 8, 514–523. https://doi.org/10.1111/j.1474-9726.2009.00497.x. Fricke, C., Adler, M.I., Brooks, R.C., Bonduriansky, R., 2015. The complexity of male reproductive success: effects of nutrition, morphology, and experience. Behav. Ecol. 26, 617–624. https://doi.org/10.1093/behaco/aru240. Geister, T.L., Lorenz, M.W., Hoffmann, K.H., Fischer, K., 2008. Adult nutrition and butterfly fitness: effects of diet quality on reproductive output, egg composition, and egg hatching success. Front. Zool. 5, 1–13. https://doi.org/10.1186/1742-9994-5-10. Gorter, J.A., Jagadeesh, S., Gahr, C., Boonekamp, J.J., Levine, J.D., Billeter, J., 2016. The nutritional and hedonic value of food modulate sexual receptivity in Drosophila melanogaster females. Sci. Rep. 6, e19441. https://doi.org/10.1038/srep19441. Kyneb, A., Toft, S., 2006. Effects of maternal diet quality on offspring performance in the rove beetle Tachyporus hypnorum. Ecol. Entomol. 31 (4), 322–330. https://doi.org/ 10.1111/j.1365-2311.2006.00775.x. Lee, K.P., Jang, T., 2014. Exploring the nutritional basis of starvation resistance in Drosophila melanogaster. Funct. Ecol. 28, 1144–1155. https://doi.org/10.1111/13652435.12247.
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Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Fluctuations in nutrient composition affect male reproductive output in Drosophila melanogaster
T
⁎
Lucy Rebecca Daviesa, , Mads F. Schoua, Torsten N. Kristensena,b, Volker Loeschckea a b
Department of Bioscience, Aarhus University, DK-8000 Aarhus C, Denmark Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg East, Denmark
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluctuating environments Protein to carbohydrate ratios Sexual dimorphism Nutrient availability Unpredictable environments Starvation resistance
Insects are known to selectively balance their intake of protein and carbohydrate to optimize reproduction and survival. For insects who feed on decomposing fruit, fluctuations in macronutrient composition occur as fruits ripe and decomposition progresses which may challenge optimal resource allocation. Using Drosophila melanogaster, we tested the effect of macronutrient fluctuations and the variability of these fluctuations on starvation resistance and components of reproductive output; traits known to be sensitive to different protein to carbohydrate (P:C) ratios in the diet. For 8 days, flies were fed the same protein to carbohydrate (P:C) ratio (constant feeding), or fed diets with fluctuations in P:C ratio on each day; these fluctuations being regular (predictably fluctuating) or irregular (unpredictably fluctuating). The three feeding regimes yielded the same average P:C ratio across the duration of the experiment. We found no difference in starvation resistance across the feeding regimes. Interestingly, there was a sexual dimorphism in the effect on reproductive output with males performing worst in the unpredictable feeding regime, and with no effect of feeding regime on female performance. Our study provides evidence for means of adapting to fluctuating macronutrient composition and suggests females are more tactful than males in storing and allocating resources for reproduction.
1. Introduction Changes in light, temperature and rainfall affect plant and microbial growth and force organisms such as insects to deal with changes in nutrient availability throughout their life time (Wolda, 1978; Morais et al., 1995). As well as overall abundance of nutrient availability, the ratio of different macronutrients, such as protein and carbohydrate, can also fluctuate within an organism’s environment. For example, for insects feeding on decomposing fruit, such as most Drosophila spp., natural fluctuations in macronutrient composition of the diet can occur due to yeast growth and metabolism which modifies the fruit tissues during different stages of fruit decay (Morais et al., 1995). Protein and carbohydrate are critical macronutrients, but as fruit ripens the availability of these macronutrients varies and will not always be synchronized with the protein to carbohydrate (P:C) ratio ideal for consumers. For insects, surpluses or deficiencies of certain macronutrients can be detrimental or beneficial (Lee et al., 2008; Simpson and Raubenheimer, 2009; Dussutour et al., 2012). Over intake of protein can be detrimental for lifespan in many insects, while insufficient intake can have negative effects on reproduction and larval development (Lee et al., 2008; Fanson et al., 2009; Clark et al., 2015; Rodrigues et al.,
⁎
2015). The opposite, however, is found for carbohydrate intake (Lee et al., 2008; Fanson et al., 2009; Clark et al., 2015; Rodrigues et al., 2015). Insects, therefore, selectively balance the intake of macronutrients in order to optimize a particular trait (Simpson and Raubenheimer, 2012). For example, once female Drosophila melanogaster have mated, they increase their protein intake and lower their carbohydrate intake to optimize reproduction (Lee et al., 2013). Conversely, virgin females and males choose a diet that is rich in carbohydrate, benefitting lifespan and starvation resistance (Lee et al., 2013; Davies et al., 2018). In times of starvation D. melanogaster survive by utilizing stored lipids; lipids that have come from carbohydrate (Lee and Jang, 2014). During nutrient fluctuations insects are unable to choose their desired diet and nutrient utilization and resource allocation to a specific trait may be problematic. Despite the biological importance of this intriguing ecological and physiological challenge, which we must assume insects have adapted to, few studies have been performed attempting to understand this adaptation. Here we investigated the effects of macronutrient fluctuations and the predictability of these fluctuations on resource allocation and reproductive potential of D. melanogaster. Previous experiments investigating food abundance fluctuations show that the tephritid fly,
Corresponding author at: Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus C, Denmark. E-mail address: [email protected] (L.R. Davies).
https://doi.org/10.1016/j.jinsphys.2019.103940 Received 20 May 2019; Received in revised form 13 August 2019; Accepted 3 September 2019 Available online 04 September 2019 0022-1910/ © 2019 Elsevier Ltd. All rights reserved.
Journal of Insect Physiology 118 (2019) 103940
L.R. Davies, et al.
Fig. 1. The protein to carbohydrate ratios in the three feeding regimes; constant, predictable and unpredictable.
resistance and measures of reproduction were set up within 3 h after the completion of the feeding.
Anastrepha ludens, and the medfly, Ceratitis capitate, minimize the allocation of body lipid, glycogen and protein stores to reproduction and metabolism in times of food shortages and maximize storage levels in times of high food availability (Aluja et al., 2011; Carey et al., 2002). In our experiment, we tested protein and carbohydrate allocation efficiency when both macronutrients were available ad libitum. To do this we tested the impact of three feeding regimes, constant, predictably and unpredictably fluctuating regimes, on investment in starvation resistance and several components of reproductive output; traits known to be sensitive to different P:C ratios (Lee et al., 2008; Fricke et al., 2015). In the constant regime the P:C ratio was kept the same whereas in the predictable regime, the P:C ratio changed between high and low each day in a regular manner. The unpredictable regime changes were irregular and varied in big and small changes (see Fig. 1 for a timeline of the P:C ratio in each regime). In order to evaluate whether it is the delivery of P:C ratio that is important or the overall P:C ratio, the average P:C ratio was the same in the three regimes (Fig. 1).
2.3. Protein to carbohydrate ratios Instant dry yeast (instant yeast, LeSaffre, Marq-en-Baroeul, France) and sucrose were used as sources of protein and carbohydrate. P:C ratios were calculated based on the yeast containing 98.5% Saccharomyces cerevisiae and 1.5% sorbitan monostearate, with nutrient values of 50% protein, 6% digestible carbohydrate, 27% indigestible fibre, 6% fats and 11% sodium, vitamin C, calcium and iron. Sorbitan monostearate does not affect the P:C ratio. The yeast and sucrose were combined with 16 g/L agar, 12 mL/L nipagen and 1.2 mL/L acetic acid mixed in water. To avoid the growth of yeast and the increase of protein in the diets, the yeast was killed in boiling water before the addition of the other components. Yeast to sucrose ratios of 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1: 3.5 and 1:4 were used to make the P:C ratios 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8 respectively.
2. Methods 2.4. Starvation resistance 2.1. Fly stock After exposure to the different feeding regimes for 8 days, flies were mixed within feeding regimes and randomly placed in individual vials containing 3.5 mL of a 2% agar solution (for constant, predictable and unpredictable regimes: females n = 15, n = 14, n = 15; males n = 17, n = 17, n = 18, respectively) i.e. without access to nutrients. Starvation resistance was measured as the survival time in these vials. The number of dead flies was counted every 8 h until all flies had died.
All D. melanogaster used for this study were from a laboratory stock established from the offspring of approximately 200 inseminated females caught near Odder (55°56′42.46″N, 10°12′45.31″E), Denmark, in 2012. The flies were maintained at 19 °C on a 12 h:12 h light:dark cycle on a standard laboratory medium containing 60 g/L yeast, 40 g/L sucrose, 30 g/L oatmeal, 16 g/L agar, 12 mL/L nipagin and 1.2 mL/L acetic acid mixed with water. 200 flies were taken from the stock as the experimental base population. This base population was maintained at 25 °C for two generations to allow for acclimation to the temperature of the feeding regimes. To obtain flies for the experiment, eggs from the second generation flies were collected and developed at a density of 30 eggs per vial on 7 mL of standard medium.
2.5. Females’ offspring viability We measured egg-to-adult viability of eggs laid after exposing the females to the feeding regimes, and will refer to this component of female reproductive output as females’ offspring viability. After exposure to the feeding regimes, 30 females from each regime were grouped in units of 10 and added to vials containing standard laboratory medium. 10 virgin control males were also put in each vial. The control males were of the same age, from the same original population and had been maintained on the standard laboratory medium in a density controlled environment. After 3 days (to allow for mating and egg production), the females were transferred to vials containing a spoon with the standard laboratory medium for egg laying. After 12 h, eggs were transferred to vials containing the standard laboratory medium such that exactly 20 eggs were in each vial (number of vials for constant, predictable and unpredictable feeding regimes: n = 13, n = 13 and n = 14, respectively). Every other day, the emerged flies were counted and removed.
2.2. Feeding regimes Virgin flies that had emerged within 6 h were separated by sex and placed in 60 vials in groups of five. Flies were exposed to one of three feeding regimes for 8 days; constant, predictably or unpredictably fluctuating (Fig. 1). Flies exposed to the constant regime were fed P:C ratio 1:5 every day (Fig. 1). The predictably fluctuating regime provided flies with a P:C ratio diet alternating between 1:2 and 1:8 each day, whereas the flies in the unpredictably fluctuating regime were provided with ratios that changed irregularly over the 8 days (Fig. 1). The average P:C ratio was 1:5 for all feeding regimes. Starvation 2
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predictable-unpredictable P < 0.001) (Fig. 2C). There was a significant effect of feeding regime on the hatchability of the eggs laid by control females that had mated with males from the different feeding regimes (χ21,36 = 54.0, P < 0.001). The post hoc test showed the hatchability of eggs sired by males from the unpredictably fluctuating regime was significantly lower than that from the predictable regime (unpredictable-predictable P < 0.01; constant-predictable P = 0.55; constant-unpredictable P = 0.08) (Fig. 2D). Pearson’s correlation test revealed that overall there was a significant positive correlation between mating time and the hatchability of the eggs (r = 0.42, d.f. = 43, P < 0.01).
2.6. Male reproductive output Male reproductive output was measured by mating time and the hatchability of the eggs laid by the mated female. After exposure to the feeding regimes, male flies were individually placed in wells with one control virgin female in a fly arena (number of males for constant, predictable and unpredictable regimes: n = 14, n = 16 and n = 15 respectively). The fly arena (dimensions 12 cm × 12 cm) contained 6 × 6 wells with diameter of 1.6 cm. The control females were of the same age, from the same original population and had been maintained on the standard laboratory medium in a density controlled environment. A transparent plastic lid was placed over the top such that the flies were trapped in their wells. A camera was left for 8 h above the wells to video mating time. The mated females were transferred to individual vials containing a spoon with the standard laboratory medium for egg laying. Fresh vials and spoons were supplied every other day for 12 days. Egg hatchability was calculated from counts of laid eggs and hatched larvae.
4. Discussion This study aimed to investigate the consequences of macronutrient fluctuations and the predictability of these fluctuations on traits known to be affected by different protein to carbohydrate (P:C) ratios (Lee et al., 2008; Fricke et al., 2015). We show that predictability in the nutritional fluctuations had an effect on male reproductive output, but not on starvation resistance and females’ offspring viability. Male reproductive output was measured by the mating time and hatchability of the eggs produced by females exposed to standard laboratory medium that had mated with males from the different feeding regimes. Previous studies have shown mating time to be a good indicator of male investment into reproduction and hatchability to be affected by male diet (Wigby et al., 2009; Moatt et al., 2014; Fricke et al., 2015). In this study, male mating time was reduced when nutrients were provided in an unpredictable fluctuating way (Fig. 2C). In a previous experiment on D. simulans, unpredictable fluctuations in temperature also resulted in reduced performance in life-history related traits compared to flies experiencing predictable thermal fluctuations (Manenti et al., 2014). The authors suggested that this was due to energy expenditures in monitoring a changing environment and in attempting to produce an optimal response to the changes. This explanation is likely in our set up as well, with the males frequently adjusting their metabolism and resource allocation. If the adjustment is costly, or if it fails to be in synchrony with the unpredictable change in macronutrient levels, it will cause a non-optimal resource allocation and therefore suboptimal nutritional state for successful reproduction. When given a choice, males prefer carbohydrate-rich diets (Lee et al., 2013) in order to invest in the energy demands for courtship behaviors, the mating itself and for the production of accessory gland proteins (Droney, 1998; Maklakov et al., 2008). On day 7 of the unpredictably fluctuating feeding regime, however, flies were provided with a relatively low carbohydrate diet which could have had negative effects on male reproductive success. Though if this was the case the low carbohydrate diet would likely also have had negative effects on starvation resistance and this was not seen (Fig. 2A). An experiment subjecting flies to multiple unpredictable regimes with different sequences of fluctuations would provide further evidence as to whether the reduced reproductive fitness is due to the particular sequence of the diets or indeed the unpredictability of the fluctuations. As for the male reproductive output, we also expected a reduced performance in starvation resistance and females’ offspring viability in the fluctuating environments. Previous experiments have also shown suboptimal nutritional environments for female adults to have negative effects on the development of her offspring (Kyneb & Toft, 2006; Geister et al., 2008). This, however, was not found in our study (Fig. 1A, B). For many insects, it has been found that different amounts of protein and carbohydrate optimize or weaken particular traits (Geister et al., 2008; Lee et al., 2008; Lee et al., 2013; Simpson and Raubenheimer, 2009; Dussutour et al., 2012; Sisodia et al., 2015). Geister et al. (2008) and Sisodia et al. (2015) for example, found higher amounts of protein led to lower offspring viability compared to diets higher in carbohydrate. Lee et al. (2008) also found diets higher in carbohydrate enhanced lifespan. In our study, the three regimes had an average P:C ratio of 1:5.
2.7. Statistical analysis A logistic regression in a generalized linear model was used to investigate the effect of feeding regime on females’ offspring viability and egg hatchability for male productive output. The full model contained feeding regime as the predictor variable which was then compared with a reduced model from which feeding regime was removed, using a likelihood ratio test to obtain a P-value. We detected no over-dispersion. The effect of feeding regime on starvation resistance was tested by comparing linear models. The model contained starvation resistance as the response variable and feeding regime and sex as the predictor variables and the interaction between feeding regime and sex. To obtain P-values of the model components, we performed sequential model reduction and compared models using F-tests. The effect of feeding regime on mating time was tested by comparing linear models containing mating time as the response variable and feeding regime as the predictor variable with an identical model in which feeding regime was removed. The models were compared using an F-test to obtain the P-value. When F-tests showed a significant effect of feeding regime, we used Tukey’s post hoc comparisons to identify which regimes were significantly different from each other. All statistical analyses were carried out in R version 3.5.2 (R Core Team, 2018). Assumptions for parametric analysis were fulfilled in all models. 3. Results 3.1. Starvation resistance Females survived starvation significantly longer than males (F1,93 = 90.99, P < 0.001), but we found no significant interaction between feeding regime and sex (F1,92 = 2.04, P = 0.13). Starvation resistance did not differ significantly between females from the different feeding regimes (F1,94 = 2.87, P = 0.06) (Fig. 2A). 3.2. Females’ offspring viability and male reproductive output Feeding regime had no impact on the egg-to-adult viability of eggs from female flies mated after the 8 days of exposure to the feeding regime (χ21,39 = 37.8, P = 0.40) (Fig. 2B). Male reproductive fitness was measured by mating time and the number of eggs hatched from the female they had mated with. There was a significant effect of feeding regime on mating time (F1,44 = 19.67, P < 0.001) with the post hoc test revealing no difference in performance between the constant and predictable regimes (P = 0.54), but males from the unpredictable regime mated for the shortest time (constant-unpredictable P < 0.001; 3
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Fig. 2. Mean ( ± s.e.m.) starvation resistance and female and male reproductive output for each feeding regime; constant, predictable and unpredictable. A. Starvation resistance. B. Percentage of adult D. melanogaster that emerged from 20 eggs. C. Mating time, in seconds, of males mated with a control female. D. Hatchability of eggs from control females that had mated with males from the 3 feeding regimes. Same letters indicate no significant difference.
ratio of the regimes affects the phenotype.
Here, it could therefore be the average ratio that was affecting starvation resistance and females’ offspring viability and not the way in which the nutrients were consumed. In our female reproduction analysis, female flies from the three feeding regimes were tested after being on the same laboratory standard medium for 3 days. This was necessary to allow time for mating, fertilization of eggs and egg laying. We argue that these 3 days had little influence on our result as previous experiments show the nutritional environment before mating can determine allocation in to reproduction (Carey et al., 2002; Gorter et al., 2016). This mechanism was proposed as a means of adapting to fluctuating environments, and our study provides evidence for that. The ingestion of the same P:C ratio over the 8 days would give the flies from the three feeding regimes equal resources to invest in reproductive output, which is confirmed by equal female reproductive output across feeding regimes. The same is probably happening for starvation resistance in both the males and females. A study focusing on nutritional environment and starvation resistance found starvation resistance was dependent on lipid reserves which were determined by different P:C ratios (Lee and Jang, 2014). Although Lee & Jang (2014) did not vary the diets, the assimilation of the average 1:5 P:C ratio would explain why we do not see a difference across the three feeding regimes. Overall females survived starvation resistance longer than males in our study. Lee and Jang (2014) also observed this and proposed that this was due to the larger lipid content of females compared to males. Further research could investigate effects of lowering or increasing the average P:C ratio across the three regimes. By raising the average ratio to levels between 1:1 and 1:4, we would expect to observe lower starvation resistance (Lee et al., 2008; Davies et al., 2018) and offspring viability (Geister et al., 2008; Sisodia et al., 2015) in all regimes and vice versa if the average ratio was to be lowered compared to the 1:5 ratio. The addition of egg count will also give another component of female reproductive output and would be expected to be optimized at higher P:C ratios. This would provide further insight in how the average
5. Conclusion Consequences of fluctuating macronutrients on reproductive output differed between males and females, and our results likely reflect a better ability of females to store and allocate nutrients in fluctuating environments. We previously found females to make the same foraging choices across a range of different P:C ratios in the developmental media (Davies et al., 2018). Both these studies, in addition to findings by Gorter et al. (2016) and Carey et al., (2002), lead us to conclude that females have developed strategies for nutrient uptake and resource investment that allow for optimal egg production across fluctuating nutritional conditions.
Acknowledgements We are grateful to Trine Bech Søgaard and Annemarie Højmark for help in the laboratory.
Funding This research was supported by funding from the Graduate School of Science and Technology (GSST) at Aarhus University and by grants from the Danish Natural Research Council to V.L. (large frame grant DFF-4002-00113) and T.N.K. (DFF-8021-00014B).
Accessibility Data available from figshare: https://figshare.com/projects/ Fluctuations_in_nutrient_availability_affect_male_reproductive_output_ in_Drosophila_melanogaster/60551. 4
Journal of Insect Physiology 118 (2019) 103940
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