Dietary nutrient balance shapes phenotypic traits of Drosophila melanogaster in interaction with gut microbiota

https://doi.org/10.1016/j.cbpa.2019.110626Get rights and content

Highlights

  • We expected interactions between diet balance and microbiota on phenotypes.

  • The physiological impact of microbiota was limited in comparison with the one of diets.

  • Yeast content in food drove most nutritional effects on development and metabolism.

  • Sugar-rich diets improved cold and starvation tolerance.

Abstract

The dietary nutrient composition can affect insects' phenotypes by modulating their physiology. Furthermore, diet can affect gut microbiota composition and abundance, with indirect consequences for the host. In this study, we reared Drosophila melanogaster on five different diets; three with balanced sugar:yeast ratio, but with increasing caloric content (2:2, 8:8, 16:16, in weight %), and two with imbalanced sugar:yeast ratio, either with low sugar and high yeast content (2:16) or vice-versa (16:2). In each of these diets, we compared flies with conventional vs. artificially altered gut microbiota with antibiotics that reduced the bacterial load. The antibiotic treatment also had the surprising effect of increasing the amount of live yeast associated with the flies. We characterized flies from these ten treatments (5 diets × 2 microbiota) in terms of development, body mass, food preference, body reserves, metabolic rate and a range of stress tolerance traits (heat, cold, starvation and desiccation tolerance). Diets, and to a lesser extent antibiotic treatment, affected development rate, weight, and cold tolerance of adult flies. Other traits such as energy reserves, metabolic rate, food preference, or starvation tolerance were affected by diet alone. When detected, the effect of antibiotic treatment was stronger in yeast-poor diets, suggesting that gut bacterial community might help to counterbalance nutritional deficiencies. These results show that changes in dietary factors lead to a global re-organization of fly's physiology and development while the manipulation of gut microorganisms had minor effects that were mainly seen in case of protein restriction.

Introduction

Most animals have a preferred dietary target in terms of macronutrient composition and amount (Simpson and Raubenheimer, 2012). In insects, the inability to meet this nutritional target represents a great challenge as nutrition affects the physiology and performance of individuals, including reproduction, development, and lifespan (Lee et al., 2008; Matzkin et al., 2011). Numerous studies addressed nutritional questions focusing on caloric restriction (Bartke et al., 2001; Masoro, 2003; Rogina et al., 2002). Food shortage may result in energy trade-off that manifests as reduced or delayed investment in development and reproduction (Edgar, 2006; Koyama et al., 2013). The shift from reproduction to survival mode is assumed to be a way to cope with harsh environment by increasing lifespan and promoting stress resistance, at the cost of a slow development (Burger et al., 2007; Carvalho et al., 2012; Kolss et al., 2009; Rion and Kawecki, 2007). Any change in nutritional supply can have considerable consequences for ecologically relevant traits of species (Raubenheimer et al., 2009), and these changes may also be driven by nutritional balance (carbohydrates:protein ratio), independent of the caloric content (Fanson et al., 2009; Mair et al., 2005; Solon-Biet et al., 2014). Experiments using artificial food with controlled nutrient compositions have helped to disentangle the contributions of specific macronutrients on insects' phenotype. For example, the “geometric framework” represents a robust method to analyze the consequences of carbohydrates:protein ratio (Lee et al., 2008; Simpson and Raubenheimer, 2012) and studies have demonstrated how low protein:carbohydrate ratios reduce development rate but increase lifespan across diverse insect species (Fanson et al., 2009; Lee et al., 2008; Simpson and Raubenheimer, 2012; Skorupa et al., 2008). Insects display various strategies to counterbalance nutritional deficiencies. Behavioral shifts represent a first response to dietary pressures, whether they manifested in food preference changes, or increased foraging (e.g.Corrales-Carvajal et al., 2016; Simpson et al., 2006). Insects may also deal with nutritional imbalance through internal physiological adjustments that can help to maintain nutritional homeostasis. For instance, nutrient sensing in the gut is regulating molecular signaling pathways such as IIS/TOR (for insulin-like growth factor signaling/target of rapamycin) that are responsible for growth (Kapahi et al., 2004; Koyama et al., 2013; Layalle et al., 2008).

Mutualistic relationships with microorganisms may provide advantageous nutritional functions to insects, including the degradation and detoxification of indigestible food and synthesis of essential nutrients (Douglas, 2009). Understanding the complex tripartite interaction among diet, microbiota and host traits represents an exciting and novel challenge in the field of nutritional ecology (Jehrke et al., 2018). In Drosophila flies, some bacterial taxa like Lactobacillus plantarum or Acetobacter sp can promote growth through the activation of insulin pathways and partially compensate for detrimental effects of protein-poor nutrition (Matos et al., 2017; Shin et al., 2011; Storelli et al., 2011). Commensal bacteria have also been found to influence behavioral decisions by limiting yeast appetite and buffer for specific amino-acid depletion (Leitão-Gonçalves et al., 2017). Furthermore, the gut microbiota can directly contribute to the nutrient supply of D. melanogaster, and thereby affect both lipid and carbohydrate metabolism (Ridley et al., 2012; Wong et al., 2014). Other roles of gut microbiota include improved peptidase activity (Erkosar et al., 2015) and provision of secondary metabolites, vitamins, and amino acids (Sannino et al., 2018; Yamada et al., 2015).

The aforementioned studies suggest that the presence of a functional microbiota could promote the host's nutritional balance and fitness. However, benefits are likely to vary according to diet, bacterial taxa, as well as host genotype (Dobson et al., 2015; Newell and Douglas, 2014; Wong et al., 2014). For instance, in D. suzukii, the presence of a functional microbiota was found to be mandatory for survival on specific poor diets, but deleterious for lifespan on balanced diets (Bing et al., 2018). Likewise, multiple studies have also reported links between stress tolerance and microbiota in insects (Ferguson et al., 2018; Henry and Colinet, 2018; Moghadam et al., 2018; Montllor et al., 2002; Russell and Moran, 2006). Since nutritional composition and microbiota may both affect insect phenotypes, including stress tolerance, it is of interest to examine how these factors compare and if one factor is more dominant than the other. As an example, it is known that carbohydrate and lipid reserves vary according to nutrition (Lee and Jang, 2014; Wong et al., 2014) but also according to gut microbiota (Huang and Douglas, 2015; Ridley et al., 2012; Wong et al., 2014), and that these reserves are important for tolerance to thermal, desiccation, and starvation stress (Arrese and Soulages, 2010; Ballard et al., 2008; Colinet et al., 2013; Colinet and Renault, 2014; Klepsatel et al., 2016). Similarly, nutritional scarcity or altered gut microbiota can both increase development duration leading to hardened phenotypes that are better able to tolerate stress (Kolss et al., 2009; Storelli et al., 2011). To our knowledge there are only a handful of studies that have investigated the impact of nutritional variables in insects harboring contrasted gut microbiota compositions or abundances (Chaston et al., 2016; Huang and Douglas, 2015; Ridley et al., 2012; Wong et al., 2014).

Here, we tested i) the effect of dietary restriction using balanced diet and ii) the effect of isocaloric but imbalanced diets with skewed sugar:yeast ratios (S:Y), on D. melanogaster. These dietary manipulations were combined (or not) with an antibiotic treatment altering the gut microbiota. This experiment aimed to disentangle the role microorganisms in the host-nutrition interaction. We first hypothesized that moderate nutrient scarcity of particular proteins (here added as yeast in diets) would decrease development rate, body weight, and metabolic rate, but provide larger energy reserves and in fine higher stress tolerance. In addition, we expected that flies could be able to show preferences for specific nutrient in two-choices experiments, with a more marked preference for the limiting nutrient in imbalanced diets. Knowing that gut microorganisms may provide nutrients for their host, we also predicted that microbial depletion would amplify phenotypical and behavioral responses in poor nutritional situations, including low-calorie diets but also imbalanced diets. As a result, we expected the importance of microbiota depletion to vary according to diet: on rich diets, microbiota alteration could be neutral or beneficial, whereas it could strengthen the nutritional stress on poor diets.

Section snippets

Fly stocks and culture medium

We conducted the experiments on an outbred laboratory population of Drosophila melanogaster derived from wild individuals collected in September 2015 in Brittany (France). Fly stocks were maintained at 25 °C under a 12:12 L:D photoperiod, on standard fly medium comprising 80 g. L−1 of inactive brewer yeast (MP Biochemicals 0290331205), 50 g. L−1 of sucrose, 10 g. L−1 of agar (Sigma-Aldrich A1296), supplemented with 8 mL. L−1 of 10% methyl 4-hydroxybenzoate (Sigma-Aldrich H5501). Wolbachia

Bacterial and yeast load

The antibiotic treatment used in the present study almost completely killed the microbial community in the treated flies (Fig. 1A; fig. S2). The bacterial load was already rather low in conventional flies from several diets, especially those with low yeast supply (2:2 and 16:2). Yeast colonization was also much dependent on diet and microbiota alteration treatment (Fig. 1B; Table 1). While the antibiotics markedly reduced bacterial load, yeasts were more abundant in bacteria-depleted flies.

Discussion

Although dietary effects have been broadly explored in Drosophila (Burger et al., 2007; Kristensen et al., 2016; Matzkin et al., 2011), it is not yet clear whether phenotypical changes result exclusively from dietary factors per se or whether they also entail some indirect influences from gut microbiota. In this study we investigated how both the caloric content of diets (sugar and yeast concentration) and the composition (sugar:yeast ratio) affected phenotypes of flies harboring conventional

Conclusions

Overall, we showed that for most of the tested traits, diet was more determining than microbiota. The antibiotic treatment interacted with diets to alter several traits, but, except for CTmin, the strength of these effects was generally minor compared to the one of diets alone, encompassing over 80% of the explained variance (Table 1). We failed to replicate the observation of hyperlipidemia, hyperglycemia and reduced metabolic rate previously detected in axenic flies (Wong et al., 2014). The

Data accessibility

Datasets are available in online repository https://doi.org/10.6084/m9.figshare.7594343

Funding

This work was supported by a grant from the Danish Council of Independent Research (to J.O.) and the international mobility grant from Rennes Metropole (to Y.H.).

This study was also supported by the International Research Project: Phenomic responses of invertebrates to changing environments and multiple stress (IRP-PRICES), funded by InEE-CNRS (to H.C. and J.O.).

Declaration of Competing Interest

Authors have no conflict of interest to declare.

Acknowledgements

Authors kindly thank Heidi MacLean as well as Torsten Kristensen for suggestions and advices on methodological aspects (desiccation and CAFE experiments respectively), and Kirsten Kromand for very welcomed technical assistance.

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