Drosophila melanogaster as a Versatile Model Organism in Food and Nutrition Research

Abstract

Drosophila melanogaster has been widely used in the biological sciences as a model organism. Drosophila has a relatively short life span of 60–80 days, which makes it attractive for life span studies. Moreover, approximately 60% of the fruit fly genes are orthologs to mammals. Thus, metabolic and signal transduction pathways are highly conserved. Maintenance and reproduction of Drosophila do not require sophisticated equipment and are rather cheap. Furthermore, there are fewer ethical issues involved in experimental Drosophila research compared with studies in laboratory rodents, such as rats and mice. Drosophila is increasingly recognized as a model organism in food and nutrition research. Drosophila is often fed complex solid diets based on yeast, corn, and agar. There are also so-called holidic diets available that are defined in terms of their amino acid, fatty acid, carbohydrate, vitamin, mineral, and trace element compositions. Feed intake, body composition, locomotor activity, intestinal barrier function, microbiota, cognition, fertility, aging, and life span can be systematically determined in Drosophila in response to dietary factors. Furthermore, diet-induced pathophysiological mechanisms including inflammation and stress responses may be evaluated in the fly under defined experimental conditions. Here, we critically evaluate Drosophila melanogaster as a versatile model organism in experimental food and nutrition research, review the corresponding data in the literature, and make suggestions for future directions of research.

Full text

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Drosophila melanogaster as a versatile model organism in food and 1
nutrition research 2
Stefanie Staatsa*, Kai Lüersena, Anika E. Wagnerb, Gerald Rimbacha 3
a Institute of Human Nutrition and Food Science, Christian-Albrechts-University Kiel, Hermann-4
Rodewald-Strasse 6, D-24118 Kiel, Germany 5
b Institute of Nutritional Medicine, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, 6
Germany 7
* Corresponding author: Tel +49 431 880 5313. Fax +49 431 880 2628. E-mail address: 8
staats@foodsci.uni-kiel.de 9

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Abstract 10
Drosophila melanogaster has been widely used in the biological sciences as a model organism. 11
Drosophila has a relatively short life span of 60 to 80 days, which makes it attractive for life span studies. 12
Moreover, approximately 60% of the fruit fly genes are orthologs to mammals. Thus, metabolic and 13
signal transduction pathways are highly conserved. Maintenance and reproduction of Drosophila do not 14
require sophisticated equipment and are rather cheap. Furthermore, there are fewer ethical issues 15
involved in experimental Drosophila research compared with studies in laboratory rodents, such as rats 16
and mice. Drosophila is increasingly recognized as a model organism in food and nutrition research. 17
Drosophila is often fed complex solid diets based on yeast, corn and agar. There are also so-called 18
holidic diets available that are defined in terms of their amino acid, fatty acid, carbohydrate, vitamin, 19
mineral and trace element compositions. Feed intake, body composition, locomotor activity, intestinal 20
barrier function, microbiota, cognition, fertility, ageing and life span can be systematically determined 21
in Drosophila in response to dietary factors. Furthermore, diet-induced pathophysiological mechanisms 22
including inflammation and stress responses may be evaluated in the fly under defined experimental 23
conditions. Here, we critically evaluate Drosophila melanogaster as a versatile model organism in 24
experimental food and nutrition research, review the corresponding data in the literature and make 25
suggestions for future directions of research. 26
Key words: Drosophila melanogaster, model organism, nutrition, longevity, metabolism
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Introduction 28
Drosophila melanogaster is a proven model organism in genetic research. The fruit fly has been further 29
established as an emerging and valuable model in experimental food and nutrition research in the past 30
few decades. D. melanogaster is suitable to be used in nutritional intervention studies as it exhibits many 31
similarities with mammalian species. Although the insect body plan is simpler than that of mammals, 32
the anatomy of fruit flies includes organ systems with equivalent functions of the mammalian heart, 33
lung, kidney, liver and gonads. Moreover, the fruit fly holds a complex and dynamic gut exhibiting a 34
similar structure and organization of the mammalian gut. Also, the tissues, physiology and anatomy of 35
mammalian and D. melanogaster intestines exhibit similar properties. Moreover, the fruit fly possesses 36
a central and peripheral nervous system, produces gastrointestinal and sex hormones like insulin-like 37
peptides, juvenile hormone and ecdysone that affect the fly’s metabolism and development, as well as 38
occupies genes and proteins displaying 50-60% orthology to mammalian ones. Within this review article 39
we describe fly anatomy and physiology including nutrient sensing and endocrine signaling. Since feed 40
intake and the composition of experimental diets are important determinants in nutritional studies, we 41
introduce different methods as far as the quantification of feed intake and also the examination of food 42
preferences are concerned. Furthermore, we discuss the pros and cons of complex versus so-called 43
holidic diets. We then compile different Drosophila studies concerning the impact of diet on various 44
outcome measurements including life span and longevity-associated gene expression, locomotor 45
activity, intestinal barrier function and gut microbiota as well as fertility. Finally, we make suggestions 46
in terms of future directions of research applying D. melanogaster as a model organism in food and 47
nutritional sciences. 48
Fly anatomy 49
a. Morphology and gut anatomy 50
Drosophila melanogaster is a holometabolous insect. Hence, its life cycle encompasses the four 51
developmental stages embryo, larvae (first, second and third instar larvae separated by molts), pupa and 52
adult, of which only the larvae and the adult flies are active feeders. In accordance with a typical insect 53

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morphology, the segmented body of an adult D. melanogaster can be divided into three parts, the head, 54
the thorax and the abdomen. The head carries sucking mouthparts and sensory organs, including a pairs 55
of antennae and compound eyes. Each of the three thoracic segments (pro-, meso- and metathorax) bears 56
a pair of limbs. In addition, a pair of wings is attached to the mesothorax and a pair of halters is attached 57
to the metathoracic segment. The abdominal segments lack extremities but contain the male and female 58
genitalia (Fig. 1A) 1. The worm-like body of larvae is also segmented but lacks compound eyes, limbs 59
and wings. Adult flies ingest mainly liquid food via their proboscis, whereas the mouth hooks of larvae 60
allows the ingestion of solid food 2. 61
Although the insect body plan is simpler than that of mammals, the anatomy of the fruit fly includes 62
organ systems with functions equivalent to the mammalian brain and peripheral nervous system, heart, 63
lung (trachea system in the fly), kidney (Malpighian tubules in the fly), liver (fat body in the fly), gut 64
and gonads 3-7. For instance, like the mammalian gastro-intestinal system, the digestive tract of the fruit 65
fly is responsible for the digestion and uptake of nutrients. The gut tube that traverses the entire body of 66
adult animals is lined by a simple epithelium of columnar or cuboidal cells called enterocytes that secrete 67
digestive enzymes and absorb nutrients. As in mammals, the basal side of this epithelial monolayer is 68
aligned to the basement membrane, an extracellular collagenous matrix. Visceral muscles that surround 69
the epithelial tube elicit peristaltic movement. They are innervated by the central nervous system and 70
oxygenated by trachea 2, 8. Associated enteroendocrine cells are responsible for humoral signaling and 71
stem cells enable regeneration processes e.g. the Drosophila midgut epithelium is completely renewed 72
every 1-2 weeks 9, 10. 73
The alimentary canal of Drosophila consists of three main parts, the foregut, the midgut and the hindgut. 74
As in mammals, the different parts of the Drosophila digestive tract are highly specialized in anatomy, 75
organization and function 2, 8. The foregut starts with the oral cavity (pharynx) followed by the 76
esophagus, which is connected to the crop, a food storage organ. The foregut and midgut are separated 77
by a valve-like organ, the cardia, whose foregut portion is the proventriculus. The cardia secretes the 78
peritrophic matrix, a non-cellular semi-permeable structure that is composed of chitin and glycoproteins, 79
and surrounds the food bolus in the midgut. Analogous to the role of mucous secretions of the vertebrate 80

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digestive tract, the peritrophic matrix forms a physical barrier that is thought to protect the Drosophila 81
midgut epithelium from harmful particles and pathogens 11. According to morphometric parameters the 82
midgut consists of six regions. However, molecular and transcript analyses revealed that the midgut 83
region can be further subdivided into 14 functional zones along its anterior–posterior axis 12. In 84
particular, a stomach-like acidic compartment of the Drosophila midgut has been determined. Here, 85
copper cells secret acid in a manner similar to mammalian gastric parietal cells, which leads to a local 86
pH < 3 8, 9. Malpighian tubules, which play a role analogous to the kidneys in vertebrates, are connected 87
to the gut at the midgut-hindgut junction. The hindgut functions in water and ion reabsorption prior to 88
fecal excretion, similar to the mammalian large intestine. The hindgut ends in the anus, which is located 89
at the last abdominal segment 2, 8. 90
The overall organization of the larval gastrointestinal tract is similar to that of the adult fly. However, 91
larvae lack a crop but contain four gastric ceca, blind-ending pouches attached to the anterior midgut, 92
that do not exist in the adult alimentary tract 2, 8. Comparable to the situation in adults, the larval midgut 93
can be functionally subdivided into specific segments 8, 13, 14. During metamorphosis, the larval gut is 94
replaced by a de novo generated adult intestinal tract 2. 95
b. Size, body weight, body composition 96
Size is determined in terms of thorax length in Drosophila and is affected by ambient temperature 15, 16 97
and by genetic 17 and dietary factors 18 which affect mating behavior and survival 19. Nutrition may affect 98
larval development and the adult body weight of fruit flies either by modifying the fly’s metabolism or 99
by direct caloric restriction due to a reduced feed intake. Effects of nutritional intervention on body 100
weight development can be easily assessed by live weighing of a defined pool of flies 20. Male flies 101
weigh approximately 700 µg while female flies weigh 1000-1200 µg. Body composition can be defined 102
by means of lean body mass, fat and protein content and depends on the delicate adjustment of catabolic 103
and anabolic pathways in the fruit fly. Thus, body composition can be modified by nutritional 104
compounds and dietary supplements that affect the metabolism of Drosophila. Changes in fruit fly body 105
composition can be determined by measuring whole body protein and triglyceride levels following fly 106
homogenization and lysis using commercially available kits e.g., the bicinchoninic acid colorimetric 107

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assay and the lipase/glycerol kinase/peroxidase-based colorimetric assay 20. Similarly, glucose and 108
trehalose levels, the most important energy resources and storage carbohydrates in Drosophila 21, 22, are 109
quantifiable using a commercial glucose oxidase/peroxidase-based colorimetric assay optionally 110
combined with trehalose digestion 20, 22. Importantly, glucose and triglyceride levels determined in 111
Drosophila should be normalized to live body weight rather than to total protein content. In in vitro 112
studies, protein normalization is often used as an internal control, but in the fruit fly, nutritional 113
intervention may affect the total protein content, thus normalization to the protein content may lead to 114
false results. In addition, the water content can be determined by calculating the difference between a 115
fly’s live and dry (following drying with the help of heat or CaCl2) weights 23-25. Both body weight and 116
body composition may vary dependent on the fly strain and the age of the flies (Abb. 1B). The 117
application of the abovementioned methods and kits to determine macronutrients in the fruit fly itself is 118
functional and allows the analysis of small sample pools of flies or isolated tissue and organs with an 119
adequate repetition number to minimize quantitation errors between different measurement days. In 120
order to determine macronutrient composition in the fly feed, standard AOAC analytical methods are 121
recommended. 122
c. Enzymatic machinery 123
The digestion and absorption of food occurs predominantly in the midgut of fruit flies. The distinct 124
enzymatic infrastructure of the different midgut zones is an important prerequisite for an ordered 125
digestion and absorption process within the Drosophila gut lumen 2, 8. Like mammals, dietary 126
macronutrients have to be broken down prior to absorption by the enterocytes of D. melanogaster. 127
According to an in silico search by Lemaitre and Miguel-Aliaga (2013), 349 putative digestive enzymes 128
have been predicted in the fruit fly genome 2. The vast majority of the hydrolases involved in these 129
digestive processes are homolog to the respective mammalian enzymes found in the intestinal tract. As 130
in humans, dietary polysaccharides, such as starch, are broken down, usually into monosaccharides, in 131
the Drosophila gut lumen by the consecutive action of alpha-amylases (Amy-p, Amy-d, and Amyrel) 132
and several alpha-glucosidases (eight putative alpha-glucosidase genes have been identified, e.g. 133
glucosidase 1, glucosidase 2 α and β) 26-28. Up to date, there are no indications for the subsistence of 134

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enzymes with dietary beta-glucosidase activity in the fruit fly. Nevertheless, D. melanogaster exhibits 135
the lysosomal-expressed glucocerebrosidase 1b (Gba1b) cleaving glucosylceramide and playing a major 136
role in Parkinson’s disease 29. Dietary lipids, such as triacylglycerols, phospholipids and other 137
acylglycerols, are digested by the action of neutral lipases that are secreted into the midgut lumen, similar 138
to their human counterparts 30-32. Moreover, the fruit fly is equipped with a broad range of endo- and 139
exopeptidases, including e.g., trypsin that cleave dietary proteins and peptides into dipeptides and amino 140
acids 2, 33. In humans, the end products of luminal digestion (monosaccharides and dipeptides/amino 141
acids) are then taken up by specific transport systems located in the apical membrane of the small 142
intestine. Given that many genes homologous to known mammalian transporter family members are 143
present in the genome of D. melanogaster, one can assume similar uptake mechanisms for the midgut 144
epithelium in fruit flies 2. Nevertheless, it is remarkable that these transport processes and the 145
participating transporter proteins are not yet well characterized. An exception is the dipeptide transporter 146
Opt1, a homolog of the human SLC15 dipeptide transporters pepT1 and pepT2. Drosophila Opt1 is 147
expressed on the apical membrane of the midgut epithelium and exhibits proton-dependent, high-affinity 148
dipeptide transport activity 34. To be dispersed within the entire body, the absorbed nutrients have to be 149
released into the fly’s open circulatory system, which is filled with hemolymph, a blood-like fluid. 150
Similar to humans, the transfer of lipids across the gut epithelium and their further transport in the 151
hemolymph requires lipoproteins. Following absorption into midgut enterocytes, dietary fatty acids are 152
incorporated into diacylglycerols, which together with dietary sterols, are bound to the apolipoprotein B 153
(ApoB)-family lipoprotein lipid transfer particle (LTP). For hemolymph transport, the lipids are shifted 154
onto lipophorin (Lpp), another ApoB-family lipoprotein 35. The Drosophila intestinal tract is also a good 155
model to study the transporter-mediated uptake processes of micronutrients such as zinc (via the 156
SLC39A zinc transporters dZIP) and copper (via the copper transporters CtrA and CtrB) 36, 37. 157
d. Nutrient sensing and endocrine signaling 158
Similar to mammalian species, D. melanogaster has a complex neuroendocrine system that produces 159
peptide and steroid hormones, such as insulin-like peptides, juvenile hormone and ecdysone 38-42, to 160
control the fly’s metabolism and development. The actual levels of certain nutrients affect the release of 161

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hormones and control nutrient- and energy-sensing signaling pathways, which then modulate 162
metabolism to maintain homeostasis at the cellular and organismic level. In humans, the sensing of 163
ingested food starts with the enteroendocrine system in the intestinal tract, which leads to the secretion 164
of gastrointestinal peptide hormones such as cholecystokinin (CCK), glucagon-like peptide (GLP1), 165
peptide YY and glucose-dependent insulinotropic peptide (GIP) 43. These hormones affect peristaltic 166
movement, digestive enzyme secretion, glucose homeostasis and appetite. The enteroendocrine cells of 167
the D. melanogaster midgut have been shown to express a battery of potential regulatory peptides. It is 168
very likely that most of these peptides are involved in nutrient sensing, the control of gut function and 169
metabolic homeostasis 44, 45. However, up to now a physiological function could be ascribed to only a 170
limited number of these peptides. For example, activin-β (Actβ) expression is upregulated in 171
enteroendocrine cells by a chronically high sugar diet and counteracts hyperglycemia by eliciting the 172
action of adipokinetic hormone (AKH, a glucagon-like peptide; see below) in the fat body 46, 47. 173
The best-known example of nutrient-induced hormone regulation is the impact of the serum glucose 174
level on the release of the antagonistic peptide hormones insulin and glucagon from mammalian 175
pancreatic β- and α-cells, respectively. Insulin is an indicator for high glucose and high energy levels, 176
whereas its counterpart, glucagon, is secreted under low glucose and low energy conditions. The 177
interplay between insulin and glucagon is also crucial for systemic glucose homeostasis in D. 178
melanogaster. The fly possesses eight homologous insulin-like peptides (dILP1-8) and the glucagon-179
like peptide AKH. A glucose-rich diet induces the secretion of dILP2, dILP3 and dILP5 that are mainly 180
produced in 14 β-cell-like insulin producing cells (IPCs) located in the central nervous system of the fly 181
48, 49. Similar to the mechanism reported for mammalian β-cells, dILP release from IPCs is regulated 182
autonomously by glucose sensing performed by glucose transporters, KATP channels, voltage sensitive 183
Ca2+ channels and dietary amino acids, in particular leucine 48, 50. Moreover, as in mammals, several 184
neuronal and humoral inter-organ communication networks have been identified that link the systemic 185
nutrition and/or energy status sensed in peripheral organs to IPC activity 48, 51-54. The fat body, the 186
functional analog of the mammalian liver/adipose tissue, and the intestine are metabolic key integrators 187
in these processes. The Sirt-UPD2-IIS axis, e.g., which connects the nutrition/energy status of the fat 188

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body to IPC activity has been deciphered 51. The evolutionarily conserved NAD+-dependent metabolic 189
sensor Sir2/Sirt1 stimulates the release of UPD2, the functional homolog of mammalian leptin, from the 190
fat body into the hemolymph, specifically in response to glucose or lipid feeding. As a result, the 191
JAK/STAT ligand UPD2 increases dILP release from IPCs most probably by inhibition of GABAergic 192
brain neurons that tonically inhibit IPC 48, 51. 193
As in mammals, binding of dILP to the insulin/IGF-like tyrosine kinase receptor (INR) activates 194
phosphoinositide 3-kinase (PI3K)-dependent signaling pathways via the Drosophila insulin receptor 195
substrate (IRS) Chico, thereby promoting the activation of the serine/threonine-specific protein kinase 196
AKT. Owing to its phosphorylating activity, AKT prevents the translocation of the forkhead 197
transcription factor FOXO from the cytosol into the nucleus. In target tissues, dILP signaling has 198
anabolic effects, increases nutrient storages and supports growth 48, 49, 55. 199
The glucagon-like peptide AKH is synthesized in α-cell-like corpus cardiacum (CC) cells that are part 200
of the ring gland directly connected to the fly’s aorta. Similar to human α-cells, CC cells respond to low 201
sugar levels in the hemolymph by activating the internal low energy status sensor adenosine 202
monophosphate-activated kinase (AMPK) which results in Ca2+ dependent AKH release. In accordance 203
with its antagonistic function to insulin, AKH elicits the mobilization of glycogen and lipid stores in 204
target tissues via binding to the G protein-coupled AKH receptor 56. 205
In addition to this insulin mechanism, the abundance of certain amino acids is also sensed by the target 206
of rapamycin (TOR) pathway. As in humans, high amino acid levels activate the Drosophila dTOR 207
pathway leading to the phosphorylation of kinase S6K and eIF-4E binding protein (4E-BP), which 208
promote the initiation of translation and elongation as well as ribosome biogenesis (Oldham, 2011). 209
Similar to mammals, dTOR is also controlled by cellular and systemic nutrient statuses. A high 210
intracellular adenosine monophosphate ÷ adenosine triphosphate ratio inhibits dTOR activity through 211
AMPK. Indicative of a high systemic nutrient and energy status, AKT, a key integration factor and 212
downstream target of IIS signaling (see above), directly inhibits tuberous sclerosis tumor suppressor 213
(TSC1/2), which in turn inhibits the small GTPase Ras homolog enriched in brain (Rheb). Since Rheb 214

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functions as a negative dTOR regulator, IIS/AKT promotes dTOR signaling 48, 49. Hence, similar to 215
humans, in fruit flies, the IIS and dTOR pathways converge to regulate protein synthesis and growth. 216
Measurement of feed intake and composition of experimental diets 217
a. Quantification of food ingestion and examination of marked feed preferences 218
Administration of various dietary factors such as secondary plant compounds to the Drosophila standard 219
medium may affect the taste of the food due to sweetness, bitterness or saltiness and thus might result in 220
reduced food uptake by the supplemented flies. As calorie restriction extends the life span of the fruit 221
fly 57-59, alters body weight 57, may affect molecular signaling pathways and locomotor activity 60 and 222
might mask plant bioactive-dependent effects on the fly’s metabolism, equal food uptake has to be 223
ascertained by determining the quantity of ingested food. Moreover, the general acceptance of 224
administered plant metabolites can be evaluated through the use of choice assays. 225
Ingestion quantity. To quantify food uptake in Drosophila, e.g., to exclude dietary restriction-226
dependent effects following administration of an ill-tasting secondary plant compound, both 227
administration of food colorings, radioisotope-labelling with 32P, observation of proboscis extension 228
(PE) and application of the capillary feeding (CAFE) assay are applicable. 229
Estimating food uptake by dye usage is easy and inexpensive while it can be rapidly assessed by visual 230
inspection of the flies. FCF Brilliant Blue and Sulforhodamine B (Fig. 2, left panel) represent suitable 231
food dyes that are added to the fly’s standard food medium at concentrations of 2.5% and 0.2% (w/v) 232
and do not affect food intake per se 58, 61, 62. Flies that ingest the dyes display colored thoraxes and 233
abdomens, particularly tinted fat bodies (Fig. 2, right panel). Food uptake can be calculated by scoring 234
the intensity of body coloring following visual evaluation and by using photometric and fluorometric 235
measurements of the processed flies at dye-specific wavelengths adjusted to a standard curve. 236
Quantification of food uptake by a dye-based method is rather imprecise and challenging in term of its 237
reproducibility 62. However, dye-based methods can be further improved by using radioactive tracers, as 238
radioisotopes are effectively incorporated and retained in the flies bodies. Thus, administration of 32P at 239

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concentrations of 0.5–4 μCi/ml [α-32P] dCTP allows for consistent and time-dependent measurement 240
of food uptake while quantification is carried out by a scintillation counter 59, 62-64. 241
The CAFE assay is more complex but more precise, highly reproducible and allows for the continuous 242
real-time quantitation of food ingestion in individual fruit flies. This method is based on the feeding of 243
a liquid food preparation instead of the solid fly medium 55. Flies are maintained in experimental vials 244
closed with a specific lid that is pierced with a microcapillary containing the liquid food. Food uptake is 245
calculated with the help of a graduated glass microcapillary with a holding capacity of 5 µl 65. Food 246
color is dispensable but can be used for better visualization 65. 247
Another possibility to quantify food uptake in Drosophila is to observe the proboscis extension (PE) of 248
flies maintained on the experimental food medium. The proboscis extension reflex is a congenital 249
behavior of wild-type flies ensuring food uptake 66, 67. Food uptake is defined as the event when a fly 250
extends its proboscis, touches the medium surface and performs a bobbing motion. Events are scored by 251
an observer repeatedly monitoring the flies for 3 sec every 2-5 min for a total of 90 min 68. However, the 252
PE assay is not recommended as the sole feeding assay as it hardly detects reproducible differences and 253
shows considerable variability in feed intake in the fruit fly thus increasing the risk of obtaining false 254
negative results 62. 255
Similarly, food intake can be determined in Drosophila larvae. Preferably, food-dye based methods 256
using FCF Brilliant blue (2.5% w/v) are used. Following a defined feeding period, food intake is 257
photometrically quantified in tinted larvae following homogenization of whole larvae or isolated guts 69,
258
70. 259
Food choice. In addition to the abovementioned food intake assays, there are some methods available 260
that allow for the examination of food preferences. The fly’s proclivity for a specific macronutrient 261
composition and aversion to some secondary plant compounds due to its bitterness can be evaluated 262
using the two-choice preference test. Therefore, two different liquid food preparations containing e.g., 263
specific plant bioactives or micronutrients, are stained with red and blue food dyes, respectively. Food 264
is applied to filter discs, put into an enclosed box and the flies are allowed to ingest for a defined interval. 265
The food dyes accumulate in the flies as they eat and food uptake can be evaluated visually (red-, blue- 266

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and purple-colored flies) and photometrically or fluorometrically 67. The choice assay can also be 267
performed with the help of the CAFE assay setup while providing different food preparations in a 268
number of capillaries 65. Further, a modified version of the PE assay, the manual feeding assay (MAFE) 269
71 combined with the proboscis extension reflex assay (PER) 66, 67 can be applied to test Drosophila for 270
its ingestion readiness of selected nutrients and substances. Therefore, fixed flies manually receive a 271
liquid food preparation repeatedly delivered by a pipette tip or a fine graduated capillary. A feeding (PE) 272
event is counted if flies fully extend their proboscises and start drinking the liquid food. Food preference 273
and food quantity can be simultaneously determined by calculating the number of PE events and by 274
reading out the ingestion volume from the capillary 71. 275
Applying the FRAPPÉ assay is a specific opportunity to evaluate ethanol consumption in the fruit fly. 276
This assay allows disclosing alcohol preferences in Drosophila by fast and high throughput 277
measurements of consumption in individual ethanol-primed flies using a fluorescence plate reader. 278
Different liquid food preparations are stained with fluorescent dyes, such as rhodamine B and 279
fluorescein, and the flies are allowed to ingest the food for a defined period of time. The intensity of the 280
dye is correlated to the amount of food ingested and can be fluorometrically determined in whole flies 281
or whole body homogenates using a microplate reader 72. 282
In Drosophila larvae food preferences can be similarly estimated performing choice assays with 283
supplemented agarose-filled petri dishes, e.g., with sweet or bitter tasting macro- and micronutrients and 284
secondary plant compounds. Larvae in the third instar feeding-stage are washed and put on the prepared 285
agarose plates prior larvae counting following a defined time lapse 73-76. Similarly, petri dishes 286
containing filter paper soaked with the experimental liquid diet can be applied 69. The distribution of 287
larvae then points to the preferred food. 288
b. Complex versus chemically-defined diets 289
There are different methods available for the preparation of solid or semi-solid media for optimal 290
Drosophila larval growth, maintenance of adults and the performance of nutrition experiments. Most 291
common, solid media are prepared with varying amounts of sugars, agar and yeast serving as the protein 292
and micronutrient source, while sometimes corn meal is also added and inactive dry yeast is replaced by 293

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brewer’s yeast 20, 77, 78. Although food ingredients are specifically manufactured for Drosophila 294
husbandry, these complex media do not have defined protein, vitamin and mineral concentrations. Even 295
more undefined media, based on bananas, jaggery, yeast and barley are occasionally used 79, 80. However, 296
many studies investigating the effect of nutritional compounds and plant metabolites on life span and 297
health performance parameters of the fruit fly use a modified Caltech medium 81 consisting of 5-10.5% 298
sucrose, 2.1-5% yeast, 8.6-10.5% corn meal and 0.5-1.3% agar 20, 78, 82-85. Accordingly, a sugar-yeast 299
medium of 10% sucrose, 10% yeast and 2% agar is commonly used 86, 87. Methylparaben and propionic 300
acid are commonly used as preservatives in Drosophila food preparations. The advantage of all of the 301
abovementioned media is that the preparation is simple, provides flies with all the essential nutrients and 302
reveals supplement-dependent effects on fly development and growth parameters if they have a 303
significant impact. In addition to ambient temperature and virginity 88, nutrient density might affect the 304
fly’s metabolism 88-91 and may alter its response to nutritional interventions leading to false-positive 305
amplification or false-negative attenuation of detectable effects 85. In this particular case, the application 306
of a chemically defined food preparation, a holidic medium, is applicable. Piper et al. introduced this 307
fully synthetic diet to the Drosophila experimental portfolio in 2014 92. In addition to sucrose and agar 308
that ensure the solidity of the medium, only isolated dietary factors are added. Therefore, defined 309
concentrations of cholesterol, minerals (Na, K, Ca, Cl, Cu, Fe, Mg, Mn, Zn and SO4, CO3, PO4), essential 310
and non-essential amino acids (isoleucine, leucine, arginine, histidine, lysine, phenylalanine, threonine, 311
tryptophan, valine and tyrosine, alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, proline, 312
serine), glutamate, vitamins (B1, B2, B6, biotin, folic acid, niacin, Ca-pantothenate), choline, myo-313
inositol, inosine and uridine are added. The pH value is adjusted using a buffer. There are additional 314
recipes available for the preparation of chemically defined media that represent modifications of the 315
holidic diet. Compared with holidic medium, 400 Kcal/L chemically defined food (CDF400K) 316
additionally contains vitamins A, E, D3 and K as well as the metal ion Cr and lactose, glucose and 317
trehalose sugars 93. In contrast, Sang’s medium contains casein instead of isolated amino acids and is 318
supplemented with fructose and lecithin but lacks Cu, Zn, myo-inositol, inosine and uridine compared 319
with the holidic diet 94. Importantly, varying food preparations differ in their protein, carbohydrate, lipid, 320

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vitamin, mineral and gelling agent content and, therefore, affect larval development, time lapse until 321
adult eclosion, maximum life span and fecundity differently 92, 95. For instance, a reduction or depletion 322
of sterols and single amino acids or vitamins in the diet markedly reduces fertility of the flies 92 while 323
increasing the carbohydrate content may significantly prolong life span 95. Moreover, considering the 324
impact of the nutritional geometry on life span and fitness parameters, not only micronutrients and 325
secondary plant compounds but also changes in macronutrient availability and energy content of the 326
food may determine the health and survival of the fly 59, 96-98. There is evidence that the protein: 327
carbohydrate ratio of the diet significantly determines lifetime egg production and life expectancy rather 328
than dietary restriction 99, 100. For instance, the egg production rate remains constant until the 329
carbohydrate content of the diet exceeds the protein content by 2-fold. Similarly, there is a continuous 330
decline in life span when the protein: carbohydrate ratio rises but a marked increase in life expectancy 331
if protein: carbohydrate ratio is low (about 1/10 or lower) 98, 99, 101, 102. Interestingly, similar effects of 332
macronutrient balance, so called “nutritional geometry”, on life span and reproductive capability are 333
reported in mice 103. Therefore, researchers are advised to carefully consider the aims and anticipated 334
outcomes of their dietary intervention studies in D. melanogaster and make use of the Geometric 335
Framework approach 102 so that they choose the most suitable food preparation for life span and 336
reproduction studies. 337
Biomarkers to be monitored in response to dietary factors 338
a. Life span 339
Drosophila has been used in genetic studies for decades but has only become interesting for nutrition-340
dependent intervention studies recently. The number of publications investigating the effects of dietary 341
factors on life span, physical fitness, fecundity and development has markedly increased within the last 342
ten years. Therefore, the fruit fly is an excellent model organism to study putative changes to life 343
expectancy caused by dietary factors. The fruit fly features easy husbandry, is relatively cheap, allows 344
for a high sample number for optimal power of experiments and exhibits a rapid reproduction and 345
lifecycle with a comparatively short lifetime. Furthermore, there are lots of mutant strains for the 346

15
mechanistic validation of initial findings. Similar to humans, where on average females live longer than 347
males 104, female flies often exhibit a prolonged life span compared with their male counterparts (Fig. 348
3A,B; 105). Life expectancy depends on genotype, female fecundity and mating status 106 and, 349
remarkably, sex-dependent differences in intestinal stem cell activity and background systemic 350
inflammation also determine longevity differences 107. 351
Moreover, male and female D. melanogaster from the same strain show diverging effects on life span 352
that are dependent on dietary interventions. Various isolated plant compounds, complex plant extracts 353
and macronutrients have recently been investigated for their life span-modulating abilities and have 354
revealed both beneficial and detrimental effects or have even been ineffective. Researchers have to take 355
into account that different fly strains exhibit variances in their genetically determined life expectancy 356
and in their susceptibility towards dietary interventions. Therefore, the use of various substance 357
concentrations and the inclusion of diverse fly strains in nutritional intervention studies are 358
recommended. 359
Importantly, both Drosophila adults and larva display learning processes and associate foods to their 360
nutritional values and tasting resulting in utilization or avoidance of a given food. As this ability can 361
cause confounding effects, especially in long-term feeding experiments like lifespan determination, the 362
performance of gustatory and choice assays (as described above) is highly recommended. Moreover, 363
larval feeding habits may affect the adult phenotype of D. melanogaster. It has been shown, that 364
administration of high contents of short carbohydrates, e.g., sucrose and fructose, can cause an obese 365
phenotype in mature fruit flies 108, 109. Researchers should consider the most suitable dietary composition 366
for their nutritional studies in respect of the desired phenotype and research question. 367
b. Expression of longevity-associated genes 368
The fruit fly possesses several longevity-associated genes that are related to nutrient dependent signaling 369
pathways and may be affected by caloric restriction. The expression of these longevity-associated genes 370
can further be affected by dietary secondary plant metabolites and nutrients resulting in life span 371
modification. Some promising candidate genes are spargel (srl), sirtuin 2 (sir2), chico, methuselah (mth) 372
and forkhead box, sub-group O (foxo). Thereby, both induction and down-regulation of the expression 373

16
of these genes may change the life span of the fruit fly. On the one hand, upregulation of srl and sir2 374
transcript levels extend life span while downregulation of chico and mth or solely targeted 375
overexpression of foxo at an adult age contributes to an extended life span. 376
Overexpression of srl, the Drosophila ortholog to mammalian PGC1, is associated with an increased 377
life span in Drosophila. The induction of srl transcription in intestinal stem and progenitor cells is 378
especially correlated with a significant extension in life span 110 while reduced srl expression causes a 379
decrease in life span 111. Similarly, sir2, a sirtuin 1 ortholog, is necessarily involved in life span 380
regulation in Drosophila as decreased expression of sir2 causes early lethality 112 while moderate 381
overexpression of sir2, especially in the fat body and neurons, prolongs the life span of fruit flies 82, 113. 382
Importantly, the mRNA expression of both srl and sir2 can be induced by dietary administration of plant 383
bioactives, e.g., the green tea catechin epigallocatechin gallate 78 and the isoflavone prunetin 20 that 384
contribute to life span extension in fruit flies. The Drosophila foxo, an ortholog of mammalian Foxo3a, 385
prolongs life span when its expression is exclusively activated in the pericerebral fat body resulting in a 386
reduction of insulin-like peptide 2 transcription in the neuronal insulin-producing cells 114. Similarly, life 387
span is elongated when foxo is overexpressed in the fly’s abdominal fat body 115, 116. Foxo overexpression 388
contributes to a reduction of the insulin/insulin growth factor-like signaling pathway, leading to life span 389
extension. Therefore, the induction of foxo during early adulthood in flies is most effective extending 390
life span compared to foxo upregulation at older ages 116. By contrast, the loss of chico expression, an 391
insulin receptor substrate in Drosophila mediating insulin/insulin-like growth factor signaling, leads to 392
longevity 117. This effect is putatively associated with the chico-dependent olfactory associative learning 393
ability and cognitive function of the fruit fly 118 and may be related to the fly’s nutritional behavior. 394
Likewise, the life span of Drosophila increases when flies carry a truncated version of mth 119 or when 395
mth expression is impaired by the use of specific mth inhibitors 120. The RNAi-mediated knockdown of 396
mth expression in insulin-producing cells in the fly’s brain is sufficient to extend its life span through 397
modification of the expression and release of insulin-like peptides and reduction of insulin/IGF signaling 398
121. As mth is assumed to act as a class B (secretin-like) G protein–coupled receptor involved in the drug 399
response and transduction of odorant stimuli, among other functions 119, the dietary modulation of mth 400

17
expression and function and the induction of longevity is conceivable. Notably, the effects of mth are 401
foxo-dependent and interactive, extending the life span of Drosophila 121. Besides the abovementioned 402
longevity-associated genes, many other genes may affect the life expectancy of Drosophila as 403
ascertained in mutant fly experiments 122. Tab. 1 gives a short overview on genes encoding proteins that 404
are putatively involved in life span regulation in the fruit fly. 405
c. Locomotor activity 406
Ingestion of plant bioactives may alter the metabolism and general health status of Drosophila as 407
reflected by changes in the movement behavior of the flies. Locomotor activity can be easily assessed 408
by performing the rapid iterative negative geotaxis (RING) assay. This method was introduced by 409
Gargano et al. 123 and takes advantage of the negative geotaxis of Drosophila. The assay is based on the 410
successive induction of climbing by tapping the flies onto the bottom of clear experimental vials (Fig. 411
3C) and letting them climb up the walls for a defined period of time. The higher the flies climb within 412
that interval the better their estimated overall health status, quantified by calculating the average 413
climbing score. The assay is useful for estimating the physical condition of fruit flies dependent on age, 414
genetic and dietary factors 20, 124-128. The advantages of this procedure are its sensitivity and the ability 415
to examine a large number of flies at the same time. 416
Another way to quantify spontaneous locomotor activity in Drosophila is the use of a PC-based 417
locomotor activity monitoring system. This method is based on using the interruption of an infrared light 418
beam gate by the fly to record the locomotor activity of individual flies and is costlier in terms of 419
equipment. However, this method possesses the advantage of being able to monitor the fly’s locomotor 420
behavior over longer periods of time under standardized housing conditions, and allows for the 421
examination of both circadian rhythm and sleep/rest parameters 129-131. 422
d. Intestinal barrier function 423
Gut barrier function is closely correlated with the overall life expectancy and health status of Drosophila 424
12, 123, 132, 133. The epithelial surface of the gut serves as a first-line of defense against microorganisms by 425
producing anti-microbial peptides (AMPs). Moreover, gut health and intestinal epithelial integrity 426
depend on appropriate stem cell proliferation and tissue homeostasis 134, which warrant gut integrity 110. 427

18
Adherence and septate junction proteins, e.g., armadillo, catenins, coracle, paxillin, polychaetoid, 428
shotgun and spectrins among others, are important for ensuring a strong gut barrier function in the fruit 429
fly that is comparable to that of mammals 135. Importantly, the loss of intestinal integrity causes a 430
shortening of the medium and maximum life spans of D. melanogaster 58, 136 while intestinal barrier 431
dysfunction increases with age 136 and predicts age-onset mortality 58. Premature mortality is also 432
associated with increased AMP expression 58 related to changes in the intestinal immune response. 433
Furthermore, alterations of the gut microbiome and development of midgut dysplasia that is reported for 434
some mutant fly strains are associated with increased gut dysfunction and barrier loss 137 and lead to a 435
shortened life span compared with wild-type flies 138. As dietary factors are able to affect the fly’s 436
metabolism, gene expression and microbiome composition 20, 78, 139-141, they may in turn modulate health 437
and life span in Drosophila via modification of gut integrity. 438
Visualization of gut integrity/leakage using food dyes 439
The Smurf assay is a non-invasive method of evaluating gut integrity in the fruit fly 58, 142 in response to 440
dietary factors. Therefore, flies are fed a specific diet for at least 30 days followed by concurrent dietary 441
administration of the food medium administered with a food dye, e.g., Brilliant Blue FCF (E133) or 442
Fluorescein (2.5% w/v) that normally do not traverse the gut for 9h to 7d 58, 110. Flies are visually 443
categorized into the smurf phenotype, by displaying intense body coloring indicating a leaky gut, or the 444
non-smurf phenotype with sole staining of the proboscis, anus and the gut itself, representing flies with 445
an intact intestinal barrier (Tab. 2, upper panel). Defined populations are scored according to these 446
phenotypes and the percentage change of the ratio of healthy flies to flies with impaired gut integrity is 447
calculated, highlighting the potential increase in health status by the dietary compound of interest. 448
Determination of intestinal immune function by quantification of anti-microbial peptide (AMP) 449
expression and stem cell proliferation 450
Whether plant bioactives and nutrient composition affect the health and life span of Drosophila, and to 451
what extent, potentially by their anti-bacterial or immuno-modulatory activities, can be evaluated by 452
various methods that give some indication of alterations in the immune status and function of the fly. 453
Therefore, flies are generally pre-fed with the experimental diet for a defined time and analyzed for AMP 454

19
expression, stem cell proliferation and bacterial load in the intestine. Unchallenged, compound-treated 455
and pathogen-infected flies can be generated for AMPs and stem cell proliferation targeted experiments. 456
Oral infection of fruit flies with Drosophila pathogens significantly induces AMP expression, e.g., 457
Metchnikowin, Diptericin, Attacins and Drosocin, in the gut that can be quantified by qRT-PCR 107, 135,
458
143-145. Accordingly, the repair of the epithelium is an important trait for survival following exposure to 459
intestinal stressors. Similar to mammals, the fruit fly possesses multipotent intestinal stem cells (ISC) in 460
the gut. ISC responsiveness to gut damage maintains intestinal homeostasis and promotes survival in 461
young flies while dysregulated intestinal stem ISC division may be detrimental at older ages 143, 146, 147. 462
Phospho-Histone H3 (PH3) staining of the gut is an appropriate method for depicting intestinal stem cell 463
proliferation 135, 143, 145, 148. Mild infections with a low infection dose of Drosophila pathogens, e.g., 464
Pectobacterium carotovorum subsp. carotovorum 15 and Pseudomonas entomophila L48, may induce 465
stem cell proliferation (Tab. 2, lower panel) while severe infections with high infection doses irreparably 466
damage the gut, resulting in distinctly decreased stem cell proliferation 143, 145. 467
Both gut-derived AMP expression and intestinal epithelial mitosis increase with age in the fruit fly 107 468
underlining the substantial importance of intestinal stem cell activity and background systemic 469
inflammation on the determination of Drosophila’s life expectancy 107. The treatment of flies with 470
secondary plant metabolites may affect the inflammatory response, AMP expression and stem cell 471
proliferation making these methods suitable tools for monitoring the effects of dietary supplements on 472
the health status of Drosophila. Notably, the indigenous gut microbiota activate a basal level of ISC 473
activity in the fruit fly 148, emphasizing the importance the gut microbiome for the immune and health 474
statuses of Drosophila. 475
Impact of gut microbiota and infection studies 476
The fly microbiome. The Drosophila gastrointestinal tract is colonized by various commensal 477
microorganisms similar to the mammalian gut. However, the fly gut contains a limited number of 478
microorganisms from approximately 30 species compared with more than 500 different bacterial species 479
in mammals; thus the fly gut displays a lower bacterial diversity 149, 150. Several human diseases including 480
inflammatory bowel disease, obesity and cardiovascular diseases have been connected to a detrimental 481

20
change in the gut microbiota 151. Therefore, model organisms hosting a rather simple microbiota are 482
needed to elucidate the underlying pathogenic mechanisms to establish preventive and therapeutic 483
strategies in the treatment of microbiota-associated diseases. 484
Most of the bacteria detected in the gut of fruit flies belong to the phyla Firmicutes and Proteobacteria. 485
Furthermore, Actinobacteria, Bacteroides and Cyanobacteria have been detected by 454 pyrosequencing 486
of 16S rRNA gene amplicons 152. The most dominating species of the Firmicutes and the Proteobacteria 487
belong to Lactobacillus and Acetobacter spp., respectively. The main species detected in the midguts of 488
flies are A. pomorum, A. tropicalis, L. frucitvorans, L. brevis and L. plantarum 152, 153. Importantly, the 489
composition of the microbiome depends on both the laboratory fly strain, sex and age of the flies 151. In 490
particular, frequently used strains differ in major bacterial species as Canton S flies hosted higher 491
numbers of Leuconostoc species whereas w1118 flies hosted higher numbers of Enterococcus species. 492
Furthermore, Lactobacillus species were more prominent in aged than in young flies and in Canton S 493
more than in w1118 flies. Age-dependent changes across fly strains and sex have also been demonstrated 494
for the bacterial species A. pasteurianus, L. plantarum and L. fructivorans 151. In yeast-deprived flies 495
being mono-associated with L. plantarum an increased larval growth as well as a reduction in 496
developmental timing has been documented being associated with an earlier metamorphosis 154. This L. 497
plantarum-induced accelerated growth is not detrimental to the flies as increased survival was detected 498
even in mono-associated male food-deprived flies 155. This study revealed that commensal L. plantarum 499
seems to be a beneficial member of the fly’s microbiome mediating an earlier hatching and an increased 500
life span without affecting fitness and reproduction. These results support the important role of the 501
intestinal microbiota in essential evolutionary processes. It has also been demonstrated that the 502
microbiota in Drosophila exert beneficial effects on the fly gut including the immune response, intestinal 503
physiology, gut function and gut homeostasis 137, 156-159. 504
Due to the presence of only a limited number of commensal aerotolerant bacteria in the gut, the fruit fly 505
offers an ideal research model to systematically unravel effects on the host-microbiota-interaction 151,
506
154, 160. As the fruit fly’s microbiota composition is mainly shaped by the host’s diet, the effect on the 507
microbiota of different compounds, nutrients and/or drugs can easily be investigated 161. Additionally, 508

21
the relatively cheap maintenance and the simple generation of germ-free and gnotobiotic animals makes 509
the fruit fly an important model organism to elucidate interactions between the host and its intestinal 510
microbiota 162. However, it should be taken into account that the microbiota of the fly depends on 511
permanent ingestion from the diet rather than on intestinal growth 153. Therefore, the total numbers of 512
flies kept per vial as well as the flipping procedure, should be carefully reconsidered for each experiment. 513
In addition to the environmental influence on the composition of microbiota in the fruit fly, the stomach-514
like copper cell region in the midgut of flies also contributes to the colonization as well as to the 515
composition of the intestinal microbiota 163. In addition to the mentioned properties, the fruit fly also 516
offers an ideal model organism to deeply investigate the effects on a potential host-microbiota-517
interaction as it combines both, genetic and experimental tractability. 518
Infection studies. The fruit fly is an appropriate model organism for investigating the preventive effects 519
of nutritional interventions on the severity of targeted infections, e.g., plant bioactives with putative anti-520
inflammatory or anti-bacterial activity. Several Drosophila pathogens induce either local or systemic 521
infections with mild or severe disease progression 164. Pathogens that induce mild infections in 522
Drosophila are Pectobacterium carotovorum subsp. carotovorum 15 165 and Serratia marcescens Db11 523
144 while Pseudomonas entomophila L48 induce severe disease 145, 166, 167. All of these pathogens can 524
either be introduced systemically or orally in high or low sub-lethal bacterial concentrations 145, 147, 167. 525
Simple experimental settings require the pre-feeding of flies for a defined time period before the flies 526
are orally or systemically exposed to pathogenic bacteria. Oral infections are then carried out by 527
application of a bacteria-sucrose-solution (OD600 =100 to 200) following starvation for 2h. For systemic 528
infections flies are infected by pricking into the thorax with a thin needle inoculated with a concentrated 529
bacterial pellet (4×1011 cells/ml at OD600 =200) 164, 165 or by microinjection of a defined volume of the 530
bacterial pellet using Nanoject™ and a pulled glass capillary 164. When performing nutritional 531
intervention studies targeting gut health in the fruit fly, researchers have to be aware that male 532
individuals display a higher level of systemic inflammation and are more susceptible to intestinal 533
infection than females, in general 107. Moreover, adequate bacterial uptake has to be ensured, which can 534
be ascertained by quantifying the bacterial load. Bacterial load assays are performed to estimate the 535

22
number of ingested pathogenic bacteria. Flies are starved for 2 h prior to the application of a bacterial-536
sucrose suspension, e.g., using GFP-labeled Erwinia carotovora carotovora 15 or Serratia marcescens 537
Db11 containing 0.5% FCF blue, for another 2 h. For quantification of the ingested amounts of bacteria, 538
flies are visually inspected under a fluorescent dissecting microscope when GFP-labeled bacteria are 539
used 164. Otherwise, flies are homogenized and their OD600 is measured spectrophotometrically. 540
Homogenized flies are plated onto lysogenic broth agar plates with selective antibiotics (if applicable) 541
and colony-forming units are counted following incubation at 29°C-30 °C overnight 107, 144. 542
Both wild-type and mutant fly strains, e.g., reporter flies carrying immune-inducible promoters of AMPs 543
ligated to green and red fluorescent proteins or LacZ-derived β-galactosidase activity 164, are suitable 544
infection models. Sensible and available read-outs are survivorship, AMPs expression, gut integrity, 545
stem cell proliferation and bacterial load/bacterial clearance. 546
e. Fertility 547
The sexual activity of the fruit fly affects its life expectancy and vice versa 168, 169. Fertility is a fitness 548
component and is correlated with life expectancy as germ cell ablation results in increased life 549
expectancy in D. melanogaster 170. Moreover, the expression of distinct hormone receptors affects life 550
span in a sex-specific manner 133. A reduction in fecundity is putatively associated with longevity in the 551
fruit fly, possibly mediated by dietary restriction-dependent effects 59, 171, 172. Interestingly, the nutritional 552
status of females affects their mating response 173, 174. Therefore, nutritional interventions e.g., 553
administration of secondary plant bioactives that may act as CR mimetics potentially affect the fertility 554
of female Drosophila. Male flies are able to transfer nutrients with their ejaculate 175 and thus may affect 555
female mating behavior with pheromone effective compounds 176. Testis development, accessory gland 556
protein synthesis and mating success are controlled by the Drosophila sex hormones 20-OH-ecdysone 557
and juvenile hormone 177, 178. Thus, plant bioactives exhibiting hormone-like structures might exhibit 558
estrogenic activity and affect fertility in the fruit fly. 559
Using D. melanogaster or C. elegans? – pros and cons 560

23
Invertebrate model organisms have become a cornerstone of various fields of biological and biomedical 561
research. Most prominent are the fruit fly D. melanogaster and the nematode Caenorhabditis elegans 562
that share many advantages listed in tab. 3 that permit systematic deciphering of the gene-gene and gene-563
environment interactions and often deliver fast answers to conserved fundamental biological problems. 564
Both, fly and worm are easy and cost-effective to grow in the laboratory. Their genome has been 565
sequenced and powerful forward and reverse genetics including RNAi and CRISPR are feasible. For 566
both models, a huge collection of mutants is available from community projects. Moreover, when 567
employing D. melanogaster or C. elegans usually one has to consider few ethical and regulatory 568
restrictions. Nevertheless, we have the opinion that the fruit fly provide some key features that favor it 569
as model with respect to nutritional research questions. (1) Despite its small body size, the fruit fly has 570
a relatively complex gastrointestinal system that, as in mammals, is highly specialized in anatomy, 571
organization and function. It consists of different cell types that enable e.g. frequent regeneration 572
processes. Compared to that, the gastrointestinal tract of C. elegans is very simple. It encompasses 573
twenty polyploid enterocytes and lacks enteroendocrine and stem cells. (2) As in mammals, the 574
alimentary tract of the fruit fly harbors various commensal microorganisms enabling the investigation 575
of host-microbiota-interaction. C. elegans lacks a microbiota. (3) The recent introduction of chemically 576
defined holidic media for D. melanogaster offers a broad range of opportunities to examine e.g. 577
micronutrient function and nutritional geometry 98,101. A comparable chemically defined medium for C. 578
elegans has not yet developed. (4) For nutritional studies it is often crucial to know how much food has 579
been ingested by an organism. However, appropriate methods for food uptake quantification have been 580
established only for D. melanogaster, but are rather lacking for C. elegans. (5) Furthermore, Drosophila 581
cell lines can be deemed as an extension of the fly model 179-181. Their culturing is easy and inexpensive 582
and a broad range of genetic, molecular and biochemical methodology has been established. Drosophila 583
cell cultures can be used to examine, e.g., cellular and biochemical response to environmental cues 584
including nutrients, bioactives and hormones. Up to now, no C. elegans cell culture lines exist. (6) 585
Finally, D. melanogaster contains a higher number of genes with orthologs in the human genome than 586
C. elegans. 587

24
Outlook – future directions 588
The fruit fly D. melanogaster is an emerging and valuable model organism for food and nutrition 589
research as there are numerous experimental methods, tools for analysis and genetic models available. 590
Nevertheless, specific aspects should be considered when designing future fruit fly nutrition studies to 591
improve experimental outcomes and study comparability. First, diet has a huge impact on many 592
biochemical, physiological and biological processes. A multitude of different, mainly undefined, fly 593
food recipes have been used to cultivate Drosophila. We encourage the Drosophila community to 594
develop standardized diets to ensure good reproducibility and comparability of the results between 595
nutritional intervention studies. Defined and purified experimental diets used for laboratory rodents 596
(including rats and mice) are already designed by the American Institute of Nutrition (AIN) 182, 183. In 597
particular, it may be useful to include a standardized defined holidic food medium. Recent progress in 598
the formulation of holidic fly media is very promising 92, 95. However, flies reared on these holidic food 599
preparations display a delay in larval development and a reduction of fecundity rates indicating that these 600
diets still have to be improved or adapted to the different life stages of D. melanogaster. The exact 601
nutrient requirements of the fruit fly still remain unsolved and must be elucidated in detail. The use of 602
holidic food preparations offers an excellent opportunity to prove the essentialness of micronutrients in 603
D. melanogaster development and physiology. Furthermore, the absorption of nutrients in the 604
gastrointestinal tract and the cellular uptake of metabolites, which represent key steps in alimentation, 605
as well as nutrient dependent transporters in the Drosophila gut are scarcely known and have to be 606
identified and characterized. Importantly, researchers have to take into account that the impact of 607
nutrients on biochemical, physiological and biological processes is affected by the genetic background 608
of an organism. As different fly strains show distinct inherent life expectancies and responsiveness to 609
dietary interventions, we recommend including at least two different wild-type fly strains in each key 610
experiment, where the effects of nutrients on D. melanogaster are examined. Additionally, it is essential 611
to ensure a sufficient replicate number and an adequate cohort size in certain experiments, such as life-612
span assays that are laborious, long and interference-prone, to create robust and solid data. Compared 613
with vertebrate models, D. melanogaster as a model organism offers a broad range of methodologies 614

25
(especially the versatile genetic toolbox) with which to approach basic problems of nutrition research in 615
a cost- and time-efficient way. The fruit fly occupies genes and proteins that are 50-60% orthologous to 616
mammalian ones 184-186 and that can be easily explored using FlyBase 187. Numerous mutant fly strains 617
are already commercially available at places such as the Bloomington Drosophila Stock Center (Indiana, 618
USA), the Drosophila Species Stock Center (California, USA), the Vienna Drosophila Resource Center 619
(Austria) and the Drosophila Genomics and Genetic Resources: Kyoto Stock Center (Japan). Further, 620
distinct target genes and signaling pathways can be specifically examined using RNAi and the 621
CRISPR/Cas system for genome engineering, as they can be used in the fruit fly. The fruit fly is an 622
appropriate model organism to target many aspects of basic nutritional research as central metabolic 623
pathways are evolutionarily well conserved. Moreover, D. melanogaster is an excellent model organism 624
to evaluate the impact of nutritional factors and diet composition on health and life span amongst many 625
other aspects in both wild type and mutant fly strains and in various disease models, e.g. fly strains 626
developing metabolic disorders like obesity, diabetes, immunological and motoric diseases188-191. 627
Results may be partly extrapolated to mammalian species. However, when the transfer of findings made 628
in the fruit fly to mammals is intended, verification in mammalian models, e.g., laboratory rodents, is 629
still required. 630

26
Abbreviations 631
foxo: forkhead box, sub-group O 632
IPC: insulin-producing cell 633
LCL: lower control limit 634
mth: methuselah 635
sir2: sirtuin 2 636
srl: spargel 637
UCL: upper control limit 638
Conflicts of interest 639
The authors declare no competing financial interest. 640
Funding sources 641
Source of funding: University of Kiel (federate state Schleswig-Holstein, Germany).
642

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Figure captions
Figure 1. (A) Anatomy of Drosophila melanogaster with a focus on the digestive tract (schematic).
Graph made according to Prokop and Hartenstein 3, 192. (B) Body weight and composition of the w1118
D. melanogaster wild type strain.
IPCs: insulin-producing cells; crop: represents the stomach; midgut: small intestine; hindgut: large
intestine and rectum; Malpighian tubules: kidney
Figure 2. Chemical structure of sulforhodamine B (left panel) and white light pictures of male and
female w1118 D. melanogaster that ingested sulforhodamine B with the fly food medium (0.2% w/v) for
500min (right panel) leading to an accumulation of the food dye in the fly’s fat body. Own unpublished
work.
Figure 3. Life span and locomotor activity in w1118 D. melanogaster reared on a standard sugar-yeast-
corn meal diet dependent on sex. (A) Representative life span curves of male and female w1118 D.
melanogaster that received a life-long standard diet. (B) Females had a longer mean, median and
maximum1 life span (longest-lived 10%) than males. (C) The rapid iterative negative geotaxis (RING)
assay is a common method used for the evaluation of the locomotor activity of D. melanogaster
dependent on age and nutritional interventions. Flies are repeatedly tapped to the bottom of clear vials
and allowed to climb up for a defined time period before a photograph is taken. Climbing activity is
quantified by scoring the distance that is overcome by the flies. The higher the climbing distance the
better the overall health status of the flies.
1maximum refers to the longest-lived 10%. Median life span -lower control limit: 42 days for males and
58 days for females; median life span -upper control limit: 47 days for males and 63 days for females.
Own unpublished work.

39
Figures
Figure 1
Figure 1. (A) Anatomy of Drosophila melanogaster with a focus on the digestive tract (schematic).
Graph made according to Prokop and Hartenstein 3, 192. (B) Body weight and composition of the w1118
D. melanogaster wild type strain.
IPCs: insulin-producing cells; crop: represents the stomach; midgut: small intestine; hindgut: large
intestine and rectum; Malpighian tubules: kidney

40
Figure 2
Figure 2. Chemical structure of sulforhodamine B (left panel) and white light pictures of male and
female w1118 D. melanogaster that ingested sulforhodamine B with the fly food medium (0.2% w/v) for
500min (right panel) leading to an accumulation of the food dye in the fly’s fat body. Own unpublished
work.

41
Figure 3
Figure 3. Life span and locomotor activity in w1118 D. melanogaster reared on a standard sugar-yeast-
corn meal diet dependent on sex. (A) Representative life span curves of male and female w1118 D.
melanogaster that received a life-long standard diet. (B) Females had a longer mean, median and
maximum1 life span (longest-lived 10%) than males. (C) The rapid iterative negative geotaxis (RING)
assay is a common method used for the evaluation of the locomotor activity of D. melanogaster
dependent on age and nutritional interventions. Flies are repeatedly tapped to the bottom of clear vials
and allowed to climb up for a defined time period before a photograph is taken. Climbing activity is

42
quantified by scoring the distance that is overcome by the flies. The higher the climbing distance the
better the overall health status of the flies.
1maximum refers to the longest-lived 10%. Median life span -lower control limit: 42 days for males and
58 days for females; median life span -upper control limit: 47 days for males and 63 days for females.
Own unpublished work.

43
Tables
Table 1
Table 1: Longevity-associated genes of Drosophila melanogaster whose expression may is controlled
by dietary factors.
Gene Full name Biological function Life-extending if Reference
AMPK
AMP-activated
protein kinase α
subunit
starvation response, lipid
metabolism, regulation of
digestive system processes, TOR
signaling
up-regulation/
activation 193, 194
Atg8a Autophagy-
related 8a
regulation of autophagy, life span
determination up-regulation 195, 196
Chico chico
regulation of immune response,
organ/organism growth,
metabolic processes, ageing
down-regulation 197-199
dILP6 Insulin-like
peptide 6 growth regulation overexpression 200
Foxo forkhead box,
sub-group O
carbohydrate metabolism,
circadian rhythm, regulation of
proliferation and growth,
hormone synthesis
overexpression 114, 115, 201
Indy I'm not dead yet triglyceride metabolism, citrate
cycle down-regulation 202, 203
Mth methuselah stress response, ageing
down-regulation/
partial loss-of-
function
119, 204
Sir2 Sirtuin 1 regulation of protein/histone
acetylation overexpression 20, 82
Sod2
Superoxide
dismutase 2
(Mn)
regulation of metabolic
processes, autophagy and life
span, hemocyte proliferation,
heart morphogenesis
up-regulation/
activation 83, 205-207
Srl spargel energy homeostasis overexpression 110
Teq Tequila glucose homeostatis, memory
function down-regulation 208

44
Table 2
Table 2. Intestinal integrity and stem cell proliferation can be visualized using the smurf assay and
phospho-histone H3 (PH3) immunofluorescence staining to indicate stem cell proliferation.
Strain/Sex/Age
Smurf assay Non-smurf smurf phenotype Dye used for
visualization
w1118 ♂
30 days
Brilliant Blue FCF
(E133)
Stem cell
proliferation Healthy (non-infected control)
Infected with Pseudomonas
entomophila at a low infectious
dose of OD600=5
w1118 ♂
10 days
Smurf assay: The flies were fed an experimental diet for 30 days prior to the dietary administration of an E133
Brilliant Blue FCF-supplemented diet (2.5% w/v) for 7 days. Subsequently, flies were anaesthetized with carbon
dioxide and the number of flies with or without the Smurf phenotype were counted.
Stem cell proliferation: The flies were fed an experimental diet for 10 days. The midguts were dissected and fixed
using a 4% paraformaldehyde solution (pH=7.4). Samples were incubated with an anti-phospho-histone H3
antibody (PH3) prior to incubation with an AlexaFluor594-conjugated secondary antibody and counterstaining
with DAPI. Images were acquired with a fluorescence microscope using TexasRed and DAPI filter systems as
previously described 135.
Pictures: Own unpublished work.

45
Table 3
Table 3: Comparison of the invertebrate models Drosophila melanogaster and Caenorhabditis elegans.
D. melanogaster C. elegans
Phylum Arthropoda Nematoda
Sexes females and males hermaphrodites and males
Chromosomes 3 pairs of autosomes (2-4)
+ an X/Y pair
5 pairs of autosomes (I-V)
+ 1 or 2 sex chromosomes
Genome size & gene
number 165 Mb, approx.. 17.000 100 Mb, approx.. 20,000
Orthologs in human
genome 50% 38%
Lifetime fecundity approx. 400 progeny
- approx. 300 self-progeny;
- up to 1000 progeny after
mating
Egg to adult
development
egg, instar larvae 1-3, pupa, adult
10 d at 25 °C
egg, L1 to L4 larvae, adult
3 d at 20 °C
Average lifespan approx. 50-60 d at 25 °C approx. 15-20 d at 20 °C
Laboratory diet
 complex diets
 defined holidic medium
 E. coli lawn on agar plates
 E. coli in complex liquid media
 Axenic semi-defined medium
Anatomy of intestinal
tract
mouth, pharynx, esophagus, crop,
cardia, midgut, hindgut, anus mouth, pharynx, intestine, rectum
Intestinal cell-types
 enterocytes, enteroendocrine
cells
 Stem cells
20 polyploid epithelial cells
Microbiota
species of the phyla Firmicutes,
Proteobacteria, Actinobacteria,
Bacteroides and Cyanobacteria
lacking
Cell culture Available Not available
164, 191, 209

46
TOC Graphic
--- for table of contents only ---

47
Table of contents
ABSTRACT ............................................................................................................................................. 2
INTRODUCTION ................................................................................................................................... 3
FLY ANATOMY..................................................................................................................................... 3
A. MORPHOLOGY AND GUT ANATOMY ................................................................................................ 3
B. SIZE, BODY WEIGHT, BODY COMPOSITION ...................................................................................... 5
C. ENZYMATIC MACHINERY................................................................................................................ 6
D. NUTRIENT SENSING AND ENDOCRINE SIGNALING ........................................................................... 7
MEASUREMENT OF FEED INTAKE AND COMPOSITION OF EXPERIMENTAL DIETS 10
A. QUANTIFICATION OF FOOD INGESTION AND EXAMINATION OF MARKED FEED PREFERENCES ........ 10
B. COMPLEX VERSUS CHEMICALLY-DEFINED DIETS .......................................................................... 12
BIOMARKERS TO BE MONITORED IN RESPONSE TO DIETARY FACTORS ................... 14
A. LIFE SPAN ..................................................................................................................................... 14
B. EXPRESSION OF LONGEVITY-ASSOCIATED GENES ......................................................................... 15
C. LOCOMOTOR ACTIVITY ................................................................................................................ 17
D. INTESTINAL BARRIER FUNCTION ................................................................................................... 17
E. FERTILITY .................................................................................................................................... 22
USING D. MELANOGASTER OR C. ELEGANS? – PROS AND CONS ........................................ 22
OUTLOOK – FUTURE DIRECTIONS ............................................................................................. 24
REFERENCES ...................................................................................................................................... 27
FIGURE CAPTIONS ........................................................................................................................... 38
FIGURES ............................................................................................................................................... 39
TABLES ................................................................................................................................................. 43
TOC GRAPHIC .................................................................................................................................... 46