Drosophila melanogaster as a Versatile Model Organism in Food

Review Cite This: J. Agric. Food Chem. 2018, 66, 3737−3753

pubs.acs.org/JAFC

Drosophila melanogaster as a Versatile Model Organism in Food and Nutrition Research Stefanie Staats,*,† Kai Lüersen,† Anika E. Wagner,‡ and Gerald Rimbach† †

Institute of Human Nutrition and Food Science, University of Kiel, Hermann-Rodewald-Strasse 6, D-24118 Kiel, Germany Institute of Nutritional Medicine, University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany

‡

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. KEYWORDS: Drosophila melanogaster, model organism, nutrition, longevity, metabolism

1. INTRODUCTION Drosophila melanogaster is a proven model organism in genetic research. The fruit fly has been further established as an emerging and valuable model in experimental food and nutrition research in the past few decades. D. melanogaster is suitable to be used in nutritional intervention studies as it exhibits many similarities with mammalian species. Although the insect body plan is simpler than that of mammals, the anatomy of fruit flies includes organ systems with equivalent functions of the mammalian heart, lung, kidney, liver, and gonads. Moreover, the fruit fly holds a complex and dynamic gut similar in structure and organization of the mammalian gut. Also, the tissues, physiology, and anatomy of mammalian and D. melanogaster intestines exhibit similar properties. Moreover, the fruit fly possesses a central and peripheral nervous system, produces gastrointestinal and sex hormones like insulin-like peptides, juvenile hormone, and ecdysone that affect the fly’s metabolism and development, and occupies genes and proteins displaying 50−60% orthology to mammalian ones. Within this Review article we describe fly anatomy and physiology including nutrient sensing and endocrine signaling. Since feed intake and the composition of experimental diets are important determinants in nutritional studies, we introduce different methods as far as the quantification of feed intake and also the examination of food preferences are concerned. Furthermore, we discuss the pros and cons of complex versus so-called holidic diets. We then compile different Drosophila studies concerning the impact of diet on various outcome measurements including life span and longevity-associated gene expression, locomotor activity, intestinal barrier function, and gut microbiota as well as fertility. Finally, we make suggestions © 2018 American Chemical Society

in terms of future directions of research applying D. melanogaster as a model organism in food and nutritional sciences.

2. FLY ANATOMY 2.1. Morphology and Gut Anatomy. Drosophila melanogaster is a holometabolous insect. Hence, its life cycle encompasses the four developmental stages embryo, larvae (first, second, and third instar larvae separated by molts), pupa, and adult, of which only the larvae and the adult flies are active feeders. In accordance with a typical insect morphology, the segmented body of an adult D. melanogaster can be divided into three parts: the head, the thorax, and the abdomen. The head carries sucking mouthparts and sensory organs, including a pair of antennae and compound eyes. Each of the three thoracic segments (pro-, meso-, and metathorax) bears a pair of limbs. In addition, a pair of wings is attached to the mesothorax, and a pair of halters is attached to the metathoracic segment. The abdominal segments lack extremities but contain the male and female genitalia (Figure 1A).1 The worm-like body of larvae is also segmented but lacks compound eyes, limbs, and wings. Adult flies ingest mainly liquid food via their proboscis, whereas the mouth hooks of larvae allows the ingestion of solid food.2 Although the insect body plan is simpler than that of mammals, the anatomy of the fruit fly includes organ systems with functions equivalent to the mammalian brain and Received: Revised: Accepted: Published: 3737

December 18, 2017 March 21, 2018 March 22, 2018 April 5, 2018 DOI: 10.1021/acs.jafc.7b05900 J. Agric. Food Chem. 2018, 66, 3737−3753

Review

Journal of Agricultural and Food Chemistry

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.

zones along its anterior−posterior axis.12 In particular, a stomach-like acidic compartment of the Drosophila midgut has been determined. Here, copper cells secret acid in a manner similar to mammalian gastric parietal cells, which leads to a local pH < 3.8,9 Malpighian tubules, which play a role analogous to the kidneys in vertebrates, are connected to the gut at the midgut−hindgut junction. The hindgut functions in water and ion reabsorption prior to fecal excretion, similar to the mammalian large intestine. The hindgut ends in the anus, which is located at the last abdominal segment.2,8 The overall organization of the larval gastrointestinal tract is similar to that of the adult fly. However, larvae lack a crop but contain four gastric ceca, blind-ending pouches attached to the anterior midgut, that do not exist in the adult alimentary tract.2,8 Comparable to the situation in adults, the larval midgut can be functionally subdivided into specific segments.8,13,14 During metamorphosis, the larval gut is replaced by a de novo generated adult intestinal tract.2 2.2. Size, Body Weight, and Body Composition. Size is determined in terms of thorax length in Drosophila and is affected by ambient temperature15,16 and by genetic17 and dietary factors18 which affect mating behavior and survival.19 Nutrition may affect larval development and the adult body weight of fruit flies either by modifying the fly’s metabolism or by direct caloric restriction due to a reduced feed intake. Effects of nutritional intervention on body weight development can be easily assessed by live weighing of a defined pool of flies.20 Male flies weigh approximately 700 μg, while female flies weigh 1000−1200 μg. Body composition can be defined by means of lean body mass, fat, and protein content and depends on the delicate adjustment of catabolic and anabolic pathways in the fruit fly. Thus, body composition can be modified by nutritional compounds and dietary supplements that affect the metabolism of Drosophila. Changes in fruit fly body composition can be determined by measuring whole body protein and triglyceride

peripheral nervous system, heart, lung (trachea system in the fly), kidney (Malpighian tubules in the fly), liver (fat body in the fly), gut, and gonads.3−7 For instance, like the mammalian gastro-intestinal system, the digestive tract of the fruit fly is responsible for the digestion and uptake of nutrients. The gut tube that traverses the entire body of adult animals is lined by a simple epithelium of columnar or cuboidal cells called enterocytes that secrete digestive enzymes and absorb nutrients. As in mammals, the basal side of this epithelial monolayer is aligned to the basement membrane, an extracellular collagenous matrix. Visceral muscles that surround the epithelial tube elicit peristaltic movement. They are innervated by the central nervous system and oxygenated by trachea.2,8 Associated enteroendocrine cells are responsible for humoral signaling, and stem cells enable regeneration processes; e.g., the Drosophila midgut epithelium is completely renewed every 1−2 weeks.9,10 The alimentary canal of Drosophila consists of three main parts: the foregut, the midgut, and the hindgut. As in mammals, the different parts of the Drosophila digestive tract are highly specialized in anatomy, organization, and function.2,8 The foregut starts with the oral cavity (pharynx) followed by the esophagus, which is connected to the crop, a food storage organ. The foregut and midgut are separated by a valve-like organ, the cardia, whose foregut portion is the proventriculus. The cardia secretes the peritrophic matrix, a non-cellular semipermeable structure that is composed of chitin and glycoproteins, and surrounds the food bolus in the midgut. Analogous to the role of mucous secretions of the vertebrate digestive tract, the peritrophic matrix forms a physical barrier that is thought to protect the Drosophila midgut epithelium from harmful particles and pathogens. 11 According to morphometric parameters the midgut consists of six regions. However, molecular and transcript analyses revealed that the midgut region can be further subdivided into 14 functional 3738

DOI: 10.1021/acs.jafc.7b05900 J. Agric. Food Chem. 2018, 66, 3737−3753

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Journal of Agricultural and Food Chemistry levels following fly homogenization and lysis using commercially available kits, e.g., the bicinchoninic acid colorimetric assay and the lipase/glycerol kinase/peroxidase-based colorimetric assay.20 Similarly, glucose and trehalose levels, the most important energy resources and storage carbohydrates in Drosophila,21,22 are quantifiable using a commercial glucose oxidase/peroxidase-based colorimetric assay optionally combined with trehalose digestion.20,22 Importantly, glucose and triglyceride levels determined in Drosophila should be normalized to live body weight rather than to total protein content. In in vitro studies, protein normalization is often used as an internal control, but in the fruit fly, nutritional intervention may affect the total protein content, thus normalization to the protein content may lead to false results. In addition, the water content can be determined by calculating the difference between a fly’s live and dry (following drying with the help of heat or CaCl2) weights.23−25 Both body weight and body composition may vary, dependent on the fly strain and the age of the flies (Figure 1B). The application of the above-mentioned methods and kits to determine macronutrients in the fruit fly itself is functional and allows the analysis of small sample pools of flies or isolated tissue and organs with an adequate repetition number to minimize quantitation errors between different measurement days. In order to determine macronutrient composition in the fly feed, standard AOAC analytical methods are recommended. 2.3. Enzymatic Machinery. The digestion and absorption of food occur predominantly in the midgut of fruit flies. The distinct enzymatic infrastructure of the different midgut zones is an important prerequisite for an ordered digestion and absorption process within the Drosophila gut lumen.2,8 Like mammals, dietary macronutrients have to be broken down prior to absorption by the enterocytes of D. melanogaster. According to an in silico search by Lemaitre and Miguel-Aliaga in 2013, 349 putative digestive enzymes have been predicted in the fruit fly genome.2 The vast majority of the hydrolases involved in these digestive processes are homologous to the respective mammalian enzymes found in the intestinal tract. As in humans, dietary polysaccharides, such as starch, are broken down, usually into monosaccharides, in the Drosophila gut lumen by the consecutive action of α-amylases (Amy-p, Amy-d, and Amyrel) and several α-glucosidases (eight putative αglucosidase genes have been identified, e.g., glucosidase 1, glucosidase 2 α and β).26−28 To date, there are no indications for the subsistence of enzymes with dietary β-glucosidase activity in the fruit fly. Nevertheless, D. melanogaster exhibits the lysosomal-expressed glucocerebrosidase 1b (Gba1b) cleaving glucosylceramide and playing a major role in Parkinson’s disease.29 Dietary lipids, such as triacylglycerols, phospholipids, and other acylglycerols, are digested by the action of neutral lipases that are secreted into the midgut lumen, similar to their human counterparts.30−32 Moreover, the fruit fly is equipped with a broad range of endo- and exopeptidases, including, e.g., trypsin, that cleave dietary proteins and peptides into dipeptides and amino acids.2,33 In humans, the end products of luminal digestion (monosaccharides and dipeptides/amino acids) are then taken up by specific transport systems located in the apical membrane of the small intestine. Given that many genes homologous to known mammalian transporter family members are present in the genome of D. melanogaster, one can assume similar uptake mechanisms for the midgut epithelium in fruit flies.2 Nevertheless, it is remarkable that these transport processes and the

participating transporter proteins are not yet well characterized. An exception is the dipeptide transporter Opt1, a homolog of the human SLC15 dipeptide transporters pepT1 and pepT2. Drosophila Opt1 is expressed on the apical membrane of the midgut epithelium and exhibits proton-dependent, high-affinity dipeptide transport activity.34 To be dispersed within the entire body, the absorbed nutrients have to be released into the fly’s open circulatory system, which is filled with hemolymph, a blood-like fluid. Similar to humans, the transfer of lipids across the gut epithelium and their further transport in the hemolymph require lipoproteins. Following absorption into midgut enterocytes, dietary fatty acids are incorporated into diacylglycerols, which together with dietary sterols, are bound to the apolipoprotein B (ApoB)-family lipoprotein lipid transfer particle (LTP). For hemolymph transport, the lipids are shifted onto lipophorin (Lpp), another ApoB-family lipoprotein.35 The Drosophila intestinal tract is also a good model to study the transporter-mediated uptake processes of micronutrients such as zinc (via the SLC39A zinc transporters dZIP) and copper (via the copper transporters CtrA and CtrB).36,37 2.4. Nutrient Sensing and Endocrine Signaling. Similar to mammalian species, D. melanogaster has a complex neuroendocrine system that produces peptide and steroid hormones, such as insulin-like peptides, juvenile hormone, and ecdysone,38−42 to control the fly’s metabolism and development. The actual levels of certain nutrients affect the release of hormones and control nutrient- and energy-sensing signaling pathways, which then modulate metabolism to maintain homeostasis at the cellular and organismic levels. In humans, the sensing of ingested food starts with the enteroendocrine system in the intestinal tract, which leads to the secretion of gastrointestinal peptide hormones such as cholecystokinin (CCK), glucagon-like peptide (GLP1), peptide YY, and glucose-dependent insulinotropic peptide (GIP).43 These hormones affect peristaltic movement, digestive enzyme secretion, glucose homeostasis, and appetite. The enteroendocrine cells of the D. melanogaster midgut have been shown to express a battery of potential regulatory peptides. It is very likely that most of these peptides are involved in nutrient sensing, the control of gut function and metabolic homeostasis.44,45 However, up to now a physiological function could be ascribed to only a limited number of these peptides. For example, activin-β (Actβ) expression is upregulated in enteroendocrine cells by a chronically high sugar diet and counteracts hyperglycemia by eliciting the action of adipokinetic hormone (AKH, a glucagon-like peptide; see below) in the fat body.46,47 The best-known example of nutrient-induced hormone regulation is the impact of the serum glucose level on the release of the antagonistic peptide hormones insulin and glucagon from mammalian pancreatic β- and α-cells, respectively. Insulin is an indicator for high glucose and high energy levels, whereas its counterpart, glucagon, is secreted under low glucose and low energy conditions. The interplay between insulin and glucagon is also crucial for systemic glucose homeostasis in D. melanogaster. The fly possesses eight homologous insulin-like peptides (dILP1−8) and the glucagonlike peptide AKH. A glucose-rich diet induces the secretion of dILP2, dILP3, and dILP5 that are mainly produced in 14 β-celllike insulin producing cells (IPCs) located in the central nervous system of the fly.48,49 Similar to the mechanism reported for mammalian β-cells, dILP release from IPCs is regulated autonomously by glucose sensing performed by 3739


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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 500 min (right panel) leading to an accumulation of the food dye in the fly’s fat body.

glucose transporters, KATP channels, voltage-sensitive Ca2+ channels, and dietary amino acids, in particular leucine.48,50 Moreover, as in mammals, several neuronal and humoral interorgan communication networks have been identified that link the systemic nutrition and/or energy status sensed in peripheral organs to IPC activity.48,51−54 The fat body, the functional analog of the mammalian liver/adipose tissue, and the intestine are metabolic key integrators in these processes. The SirtUPD2-IIS axis, for example, which connects the nutrition/ energy status of the fat body to IPC activity, has been deciphered.51 The evolutionarily conserved NAD+-dependent metabolic sensor Sir2/Sirt1 stimulates the release of UPD2, the functional homolog of mammalian leptin, from the fat body into the hemolymph, specifically in response to glucose or lipid feeding. As a result, the JAK/STAT ligand UPD2 increases dILP release from IPCs most probably by inhibition of GABAergic brain neurons that tonically inhibit IPC.48,51 As in mammals, binding of dILP to the insulin/IGF-like tyrosine kinase receptor (INR) activates phosphoinositide 3kinase (PI3K)-dependent signaling pathways via the Drosophila insulin receptor substrate (IRS) Chico, thereby promoting the activation of the serine/threonine-specific protein kinase AKT. Owing to its phosphorylating activity, AKT prevents the translocation of the forkhead transcription factor FOXO from the cytosol into the nucleus. In target tissues, dILP signaling has anabolic effects, increases nutrient storage, and supports growth.48,49,55 The glucagon-like peptide AKH is synthesized in α-cell-like corpus cardiacum (CC) cells that are part of the ring gland directly connected to the fly’s aorta. Similar to human α-cells, CC cells respond to low sugar levels in the hemolymph by activating the internal low energy status sensor adenosine monophosphate-activated kinase (AMPK) which results in Ca 2+ -dependent AKH release. In accordance with its antagonistic function to insulin, AKH elicits the mobilization of glycogen and lipid stores in target tissues via binding to the G protein-coupled AKH receptor.56 In addition to this insulin mechanism, the abundance of certain amino acids is also sensed by the target of rapamycin (TOR) pathway. As in humans, high amino acid levels activate the Drosophila dTOR pathway leading to the phosphorylation of kinase S6K and eIF-4E binding protein (4E-BP), which promote the initiation of translation and elongation as well as ribosome biogenesis.49 Similar to mammals, dTOR is also controlled by cellular and systemic nutrient statuses. A high intracellular adenosine monophosphate-to-adenosine triphosphate ratio inhibits dTOR activity through AMPK. Indicative of a high systemic nutrient and energy status, AKT, a key integration factor and downstream target of IIS signaling (see

above), directly inhibits tuberous sclerosis tumor suppressor (TSC1/2), which in turn inhibits the small GTPase Ras homolog enriched in brain (Rheb). Since Rheb functions as a negative dTOR regulator, IIS/AKT promotes dTOR signaling.48,49 Hence, similar to humans, in fruit flies, the IIS and dTOR pathways converge to regulate protein synthesis and growth.

3. MEASUREMENT OF FEED INTAKE AND COMPOSITION OF EXPERIMENTAL DIETS 3.1. Quantification of Food Ingestion and Examination of Marked Feed Preferences. Administration of various dietary factors such as secondary plant compounds to the Drosophila standard medium may affect the taste of the food due to sweetness, bitterness, or saltiness and thus might result in reduced food uptake by the supplemented flies. As calorie restriction extends the life span of the fruit fly,57−59 alters body weight,57 may affect molecular signaling pathways and locomotor activity,60 and might mask plant bioactive-dependent effects on the fly’s metabolism, equal food uptake has to be ascertained by determining the quantity of ingested food. Moreover, the general acceptance of administered plant metabolites can be evaluated through the use of choice assays. 3.1.1. Ingestion Quantity. To quantify food uptake in Drosophila, e.g., to exclude dietary restriction-dependent effects following administration of an ill-tasting secondary plant compound, methods such as administration of food colorings, radioisotope labeling with 32P, observation of proboscis extension (PE), and application of the capillary feeding (CAFE) assay are applicable. Estimating food uptake by dye usage is easy and inexpensive while it can be rapidly assessed by visual inspection of the flies. FCF Brilliant Blue and Sulforhodamine B (Figure 2, left panel) represent suitable food dyes that are added to the fly’s standard food medium at concentrations of 2.5% and 0.2% (w/v) and do not affect food intake per se.58,61,62 Flies that ingest the dyes display colored thoraxes and abdomens, particularly tinted fat bodies (Figure 2, right panel). Food uptake can be calculated by scoring the intensity of body coloring following visual evaluation and by using photometric and fluorometric measurements of the processed flies at dye-specific wavelengths adjusted to a standard curve. Quantification of food uptake by a dye-based method is rather imprecise and challenging in term of its reproducibility.62 However, dye-based methods can be further improved by using radioactive tracers, as radioisotopes are effectively incorporated and retained in the flies’ bodies. Thus, administration of 32P at concentrations of 0.5−4 μCi/mL [α-32P] dCTP allows for consistent and time-dependent 3740


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dye is correlated to the amount of food ingested and can be fluorometrically determined in whole flies or whole body homogenates using a microplate reader.72 In Drosophila larvae food preferences can be similarly estimated performing choice assays with supplemented agarose-filled Petri dishes, e.g., with sweet or bitter-tasting macro- and micronutrients and secondary plant compounds. Larvae in the third instar feeding-stage are washed and put on the prepared agarose plates prior to larvae counting following a defined time lapse.73−76 Similarly, Petri dishes containing filter paper soaked with the experimental liquid diet can be applied.69 The distribution of larvae then points to the preferred food. 3.2. Complex versus Chemically Defined Diets. There are different methods available for the preparation of solid or semi-solid media for optimal Drosophila larval growth, maintenance of adults, and the performance of nutrition experiments. Most commonly, solid media are prepared with varying amounts of sugars, agar, and yeast serving as the protein and micronutrient sources, while sometimes corn meal is also added and inactive dry yeast is replaced by brewer’s yeast.20,77,78 Although food ingredients are specifically manufactured for Drosophila husbandry, these complex media do not have defined protein, vitamin, and mineral concentrations. Even more undefined media, based on bananas, jaggery, yeast, and barley are occasionally used.79,80 However, many studies investigating the effect of nutritional compounds and plant metabolites on life span and health performance parameters of the fruit fly use a modified Caltech medium81 consisting of 5−10.5% 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 medium of 10% sucrose, 10% yeast, and 2% agar is commonly used.86,87 Methylparaben and propionic acid are commonly used as preservatives in Drosophila food preparations. The advantage of all of the above-mentioned media is that the preparation is simple, provides flies with all the essential nutrients, and reveals supplement-dependent effects on fly development and growth parameters if they have a significant impact. In addition to ambient temperature and virginity,88 nutrient density might affect the fly’s metabolism88−91 and may alter its response to nutritional interventions, leading to falsepositive amplification or false-negative attenuation of detectable effects.85 In this particular case, the application of a chemically defined food preparation, a holidic medium, is applicable. Piper et al. introduced this fully synthetic diet to the Drosophila experimental portfolio in 2014.92 In addition to sucrose and agar that ensure the solidity of the medium, only isolated dietary factors are added. Therefore, defined concentrations of cholesterol, minerals (Na, K, Ca, Cl, Cu, Fe, Mg, Mn, Zn and SO4, CO3, PO4), essential and non-essential amino acids (isoleucine, leucine, arginine, histidine, lysine, phenylalanine, threonine, tryptophan, valine and tyrosine, alanine, asparagine, aspartic acid, cysteine, glutamine, glycine, proline, serine), glutamate, vitamins (B1, B2, B6, biotin, folic acid, niacin, Capantothenate), choline, myo-inositol, inosine, and uridine are added. The pH value is adjusted using a buffer. There are additional recipes available for the preparation of chemically defined media that represent modifications of the holidic diet. Compared with holidic medium, 400 kcal/L chemically defined food (CDF400K) additionally contains vitamins A, E, D3, and K as well as the metal ion Cr and lactose, glucose, and trehalose sugars.93 In contrast, Sang’s medium contains casein instead of isolated amino acids and is supplemented with fructose and lecithin but lacks Cu, Zn, myo-inositol, inosine, and uridine

measurement of food uptake while quantification is carried out by a scintillation counter.59,62−64 The CAFE assay is more complex but more precise and highly reproducible, and it allows for the continuous real-time quantitation of food ingestion in individual fruit flies. This method is based on the feeding of a liquid food preparation instead of the solid fly medium.55 Flies are maintained in experimental vials closed with a specific lid that is pierced with a microcapillary containing the liquid food. Food uptake is calculated with the help of a graduated glass microcapillary with a holding capacity of 5 μL.65 Food color is dispensable but can be used for better visualization.65 Another possibility to quantify food uptake in Drosophila is to observe the proboscis extension (PE) of flies maintained on the experimental food medium. The proboscis extension reflex is a congenital behavior of wild-type flies ensuring food uptake.66,67 Food uptake is defined as the event when a fly extends its proboscis, touches the medium surface, and performs a bobbing motion. Events are scored by an observer repeatedly monitoring the flies for 3 s every 2−5 min for a total of 90 min.68 However, the PE assay is not recommended as the sole feeding assay as it hardly detects reproducible differences and shows considerable variability in feed intake in the fruit fly, thus increasing the risk of obtaining false negative results.62 Similarly, food intake can be determined in Drosophila larvae. Preferably, food-dye based methods using FCF Brilliant blue (2.5% w/v) are used. Following a defined feeding period, food intake is photometrically quantified in tinted larvae following homogenization of whole larvae or isolated guts.69,70 3.1.2. Food Choice. In addition to the above-mentioned food intake assays, there are some methods available that allow for the examination of food preferences. The fly’s proclivity for a specific macronutrient composition and aversion to some secondary plant compounds due to its bitterness can be evaluated using the two-choice preference test. Therefore, two different liquid food preparations containing, e.g., specific plant bioactives or micronutrients, are stained with red and blue food dyes, respectively. Food is applied to filter discs and put into an enclosed box, and the flies are allowed to ingest for a defined interval. The food dyes accumulate in the flies as they eat, and food uptake can be evaluated visually (red-, blue-, and purplecolored flies) and photometrically or fluorometrically.67 The choice assay can also be performed with the help of the CAFE assay setup while providing different food preparations in a number of capillaries.65 Further, a modified version of the PE assay, the manual feeding assay (MAFE)71 combined with the proboscis extension reflex assay (PER)66,67 can be applied to test Drosophila for its ingestion readiness of selected nutrients and substances. Therefore, fixed flies manually receive a liquid food preparation repeatedly delivered by a pipet tip or a fine graduated capillary. A feeding (PE) event is counted if flies fully extend their proboscises and start drinking the liquid food. Food preference and food quantity can be simultaneously determined by calculating the number of PE events and by reading out the ingestion volume from the capillary.71 Applying the FRAPPÉ assay is a specific opportunity to evaluate ethanol consumption in the fruit fly. This assay allows disclosing alcohol preferences in Drosophila by fast and highthroughput measurements of consumption in individual ethanol-primed flies using a fluorescence plate reader. Different liquid food preparations are stained with fluorescent dyes, such as rhodamine B and fluorescein, and the flies are allowed to ingest the food for a defined period of time. The intensity of the 3741


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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 maximum life span (longest-lived 10%) than males [maximum 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.] (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, based on our own unpublished work.

compared with the holidic diet.94 Importantly, varying food preparations differ in their protein, carbohydrate, lipid, vitamin, mineral, and gelling agent content and, therefore, affect larval development, time lapse until adult eclosion, maximum life span, and fecundity differently.92,95 For instance, a reduction or depletion of sterols and single amino acids or vitamins in the diet markedly reduces fertility of the flies,92 while increasing the carbohydrate content may significantly prolong life span.95 Moreover, considering the impact of the nutritional geometry on life span and fitness parameters, not only micronutrients and secondary plant compounds but also changes in macronutrient availability and energy content of the food may determine the health and survival of the fly.59,96−98 There is evidence that the protein: carbohydrate ratio of the diet significantly determines lifetime egg production and life expectancy rather than dietary restriction.99,100 For instance, the egg production rate remains constant until the carbohydrate content of the diet exceeds the protein content by 2-fold. Similarly, there is a continuous decline in life span when the protein: carbohydrate ratio rises but a marked increase in life expectancy if protein: carbohydrate ratio is low (about 1/10 or lower).98,99,101,102 Interestingly, similar effects of macronutrient balance, so-called “nutritional geometry”, on life span and reproductive capability are reported in mice.103 Therefore, researchers are advised to carefully consider the aims and anticipated outcomes of their dietary intervention studies in D. melanogaster and make use of the Geometric Framework approach102 so that they choose the most suitable food preparation for life span and reproduction studies.

4. BIOMARKERS TO BE MONITORED IN RESPONSE TO DIETARY FACTORS 4.1. Life Span. Drosophila has been used in genetic studies for decades but has only become interesting for nutritiondependent intervention studies recently. The number of publications investigating the effects of dietary factors on life span, physical fitness, fecundity, and development has markedly increased within the past 10 years. Therefore, the fruit fly is an excellent model organism to study putative changes to life expectancy caused by dietary factors. The fruit fly features easy husbandry, is relatively cheap, allows for a high sample number for optimal power of experiments, and exhibits a rapid reproduction and lifecycle with a comparatively short lifetime. Furthermore, there are lots of mutant strains for the mechanistic validation of initial findings. Similar to humans, where on average females live longer than males,104 female flies often exhibit a prolonged life span compared with their male counterparts (Figure 3A,B).105 Life expectancy depends on genotype, female fecundity and mating status,106 and, remarkably, sex-dependent differences in intestinal stem cell activity and background systemic inflammation also determine longevity differences.107 Moreover, male and female D. melanogaster from the same strain show diverging effects on life span that are dependent on dietary interventions. Various isolated plant compounds, complex plant extracts, and macronutrients have recently been investigated for their life span-modulating abilities and have revealed both beneficial and detrimental effects or have 3742


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Table 1. Longevity-Associated Genes of Drosophila melanogaster Whose Expression May Be Controlled by Dietary Factors gene

full name

biological function

AMPK Atg8a Chico

AMP-activated protein kinase α subunit autophagy-related 8a chico

dILP6 Foxo

insulin-like peptide 6 forkhead box, subgroup O

Indy Mth

I’m not dead yet methuselah

starvation response, lipid metabolism, regulation of digestive system processes, TOR signaling regulation of autophagy, life span determination regulation of immune response, organ/organism growth, metabolic processes, aging growth regulation carbohydrate metabolism, circadian rhythm, regulation of proliferation and growth, hormone synthesis triglyceride metabolism, citrate cycle stress response, aging

Sir2 Sod2

sirtuin 1 superoxide dismutase 2 (Mn)

Srl Teq

spargel tequila

regulation of protein/histone acetylation regulation of metabolic processes, autophagy and life span, hemocyte proliferation, heart morphogenesis energy homeostasis glucose homeostatis, memory function

life-extending if...

reference(s)

up-regulation/activation

193, 194

up-regulation down-regulation

195, 196 197−199

overexpression overexpression

200 114, 115, 201 202, 203 119, 204

down-regulation down-regulation/partial loss of function overexpression up-regulation/activation overexpression down-regulation

20, 82 83, 205−207 110 208

the mRNA expression of both srl and sir2 can be induced by dietary administration of plant bioactives, e.g., the green tea catechin epigallocatechin gallate78 and the isoflavone prunetin20 that contribute to life span extension in fruit flies. The Drosophila foxo, an ortholog of mammalian Foxo3a, prolongs life span when its expression is exclusively activated in the pericerebral fat body resulting in a reduction of insulin-like peptide 2 transcription in the neuronal insulin-producing cells.114 Similarly, life span is elongated when foxo is overexpressed in the fly’s abdominal fat body.115,116 Foxo overexpression contributes to a reduction of the insulin/insulin growth factor-like signaling pathway, leading to life span extension. Therefore, the induction of foxo during early adulthood in flies is most effective extending life span compared to foxo upregulation at older ages.116 By contrast, the loss of chico expression, an insulin receptor substrate in Drosophila mediating insulin/insulin-like growth factor signaling, leads to longevity.117 This effect is putatively associated with the chicodependent olfactory associative learning ability and cognitive function of the fruit fly118 and may be related to the fly’s nutritional behavior. Likewise, the life span of Drosophila increases when flies carry a truncated version of mth119 or when mth expression is impaired by the use of specific mth inhibitors.120 The RNAi-mediated knockdown of mth expression in insulin-producing cells in the fly’s brain is sufficient to extend its life span through modification of the expression and release of insulin-like peptides and reduction of insulin/ IGF signaling.121 As mth is assumed to act as a class B (secretinlike) G protein-coupled receptor involved in the drug response and transduction of odorant stimuli, among other functions,119 the dietary modulation of mth expression and function and the induction of longevity are conceivable. Notably, the effects of mth are foxo-dependent and interactive, extending the life span of Drosophila.121 Besides the above-mentioned longevityassociated genes, many other genes may affect the life expectancy of Drosophila as ascertained in mutant fly experiments.122 Table 1 gives a short overview on genes encoding proteins that are putatively involved in life span regulation in the fruit fly. 4.3. Locomotor Activity. Ingestion of plant bioactives may alter the metabolism and general health status of Drosophila as reflected by changes in the movement behavior of the flies. Locomotor activity can be easily assessed by performing the rapid iterative negative geotaxis (RING) assay. This method

even been ineffective. Researchers have to take into account that different fly strains exhibit variances in their genetically determined life expectancy and in their susceptibility toward dietary interventions. Therefore, the use of various substance concentrations and the inclusion of diverse fly strains in nutritional intervention studies are recommended. Importantly, both Drosophila adults and larva display learning processes and associate foods to their nutritional values and tasting resulting in utilization or avoidance of a given food. As this ability can cause confounding effects, especially in longterm feeding experiments like lifespan determination, the performance of gustatory and choice assays (as described above) is highly recommended. Moreover, larval feeding habits may affect the adult phenotype of D. melanogaster. It has been shown, that administration of high contents of short carbohydrates, e.g., sucrose and fructose, can cause an obese phenotype in mature fruit flies.108,109 Researchers should consider the most suitable dietary composition for their nutritional studies in respect of the desired phenotype and research question. 4.2. Expression of Longevity-Associated Genes. The fruit fly possesses several longevity-associated genes that are related to nutrient-dependent signaling pathways and may be affected by caloric restriction. The expression of these longevity-associated genes can further be affected by dietary secondary plant metabolites and nutrients, resulting in life span modification. Some promising candidate genes are spargel (srl), sirtuin 2 (sir2), chico, methuselah (mth), and forkhead box, sub-group O (foxo). Thereby, both induction and downregulation of the expression of these genes may change the life span of the fruit fly. On the one hand, upregulation of srl and sir2 transcript levels extends life span, while downregulation of chico and mth or solely targeted overexpression of foxo at an adult age contributes to an extended life span. Overexpression of srl, the Drosophila ortholog to mammalian PGC1, is associated with an increased life span in Drosophila. The induction of srl transcription in intestinal stem and progenitor cells is especially correlated with a significant extension in life span110 while reduced srl expression causes a decrease in life span.111 Similarly, sir2, a sirtuin 1 ortholog, is necessarily involved in life span regulation in Drosophila as decreased expression of sir2 causes early lethality112 while moderate overexpression of sir2, especially in the fat body and neurons, prolongs the life span of fruit flies.82,113 Importantly, 3743


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Table 2. Intestinal Integrity and Stem Cell Proliferation Can Be Visualized Using the Smurf Assaya and Phospho-histone H3 (PH3) Immunofluorescence Staining To Indicate Stem Cell Proliferationb

a 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. bStem 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

was introduced by Gargano et al.123 and takes advantage of the negative geotaxis of Drosophila. The assay is based on the successive induction of climbing by tapping the flies onto the bottom of clear experimental vials (Figure 3C) and letting them climb up the walls for a defined period of time. The higher the flies climb within that interval the better their estimated overall health status, quantified by calculating the average climbing score. The assay is useful for estimating the physical condition of fruit flies dependent on age, genetic, and dietary factors.20,124−128 The advantages of this procedure are its sensitivity and the ability to examine a large number of flies at the same time. Another way to quantify spontaneous locomotor activity in Drosophila is the use of a PC-based locomotor activity monitoring system. This method is based on using the interruption of an infrared light beam gate by the fly to record the locomotor activity of individual flies and is costlier in terms of equipment. However, this method possesses the advantage of being able to monitor the fly’s locomotor behavior over longer periods of time under standardized housing conditions, and allows for the examination of both circadian rhythm and sleep/rest parameters.129−131 4.4. Intestinal Barrier Function. Gut barrier function is closely correlated with the overall life expectancy and health status of Drosophila.12,123,132,133 The epithelial surface of the gut serves as a first line of defense against microorganisms by producing anti-microbial peptides (AMPs). Moreover, gut health and intestinal epithelial integrity depend on appropriate stem cell proliferation and tissue homeostasis,134 which warrant gut integrity.110 Adherence and septate junction proteins, e.g., armadillo, catenins, coracle, paxillin, polychaetoid, shotgun, and

spectrins among others, are important for ensuring a strong gut barrier function in the fruit fly that is comparable to that of mammals.135 Importantly, the loss of intestinal integrity causes a shortening of the medium and maximum life spans of D. melanogaster,58,136 while intestinal barrier dysfunction increases with age136 and predicts age-onset mortality.58 Premature mortality is also associated with increased AMP expression58 related to changes in the intestinal immune response. Furthermore, alterations of the gut microbiome and development of midgut dysplasia that is reported for some mutant fly strains are associated with increased gut dysfunction and barrier loss137 and lead to a shortened life span compared with wildtype flies.138 As dietary factors are able to affect the fly’s metabolism, gene expression, and microbiome composition,20,78,139−141 they may in turn modulate health and life span in Drosophila via modification of gut integrity. 4.4.1. Visualization of Gut Integrity/Leakage Using Food Dyes. The smurf assay is a non-invasive method of evaluating gut integrity in the fruit fly58,142 in response to dietary factors. Therefore, flies are fed a specific diet for at least 30 days followed by concurrent dietary administration of the food medium administered with a food dye, e.g., Brilliant Blue FCF (E133) or Fluorescein (2.5% w/v) that normally do not traverse the gut for 9 h to 7 d.58,110 Flies are visually categorized into the smurf phenotype, by displaying intense body coloring indicating a leaky gut, or the non-smurf phenotype with sole staining of the proboscis, anus, and the gut itself, representing flies with an intact intestinal barrier (Table 2, upper panel). Defined populations are scored according to these phenotypes, and the percentage change of the ratio of healthy flies to flies with impaired gut integrity is calculated, highlighting the 3744


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amplicons.152 The most dominating species of the Firmicutes and the Proteobacteria belong to Lactobacillus and Acetobacter spp., respectively. The main species detected in the midguts of flies are A. pomorum, A. tropicalis, L. f rucitvorans, L. brevis, and L. plantarum.152,153 Importantly, the composition of the microbiome depends on both the laboratory fly strain, sex, and age of the flies.151 In particular, frequently used strains differ in major bacterial species as Canton S flies hosted higher numbers of Leuconostoc species whereas w1118 flies hosted higher numbers of Enterococcus species. Furthermore, Lactobacillus species were more prominent in aged than in young flies and in Canton S more than in w1118 flies. Age-dependent changes across fly strains and sex have also been demonstrated for the bacterial species A. pasteurianus, L. plantarum and L. f ructivorans.151 In yeast-deprived flies being mono-associated with L. plantarum an increased larval growth as well as a reduction in developmental timing has been documented being associated with an earlier metamorphosis.154 This L. plantaruminduced accelerated growth is not detrimental to the flies as increased survival was detected even in mono-associated male food-deprived flies.155 This study revealed that commensal L. plantarum seems to be a beneficial member of the fly’s microbiome mediating an earlier hatching and an increased life span without affecting fitness and reproduction. These results support the important role of the intestinal microbiota in essential evolutionary processes. It has also been demonstrated that the microbiota in Drosophila exert beneficial effects on the fly gut including the immune response, intestinal physiology, gut function, and gut homeostasis.137,156−159 Due to the presence of only a limited number of commensal aerotolerant bacteria in the gut, the fruit fly offers an ideal research model to systematically unravel effects on the host− microbiota interaction.151,154,160 As the fruit fly’s microbiota composition is mainly shaped by the host’s diet, the effect on the microbiota of different compounds, nutrients and/or drugs can easily be investigated.161 Additionally, the relatively cheap maintenance and the simple generation of germ-free and gnotobiotic animals make the fruit fly an important model organism to elucidate interactions between the host and its intestinal microbiota.162 However, it should be taken into account that the microbiota of the fly depends on permanent ingestion from the diet rather than on intestinal growth.153 Therefore, the total numbers of flies kept per vial as well as the flipping procedure, should be carefully reconsidered for each experiment. In addition to the environmental influence on the composition of microbiota in the fruit fly, the stomach-like copper cell region in the midgut of flies also contributes to the colonization as well as to the composition of the intestinal microbiota.163 In addition to the mentioned properties, the fruit fly also offers an ideal model organism to deeply investigate the effects on a potential host−microbiota interaction as it combines both genetic and experimental tractability. 4.4.3.2. Infection Studies. The fruit fly is an appropriate model organism for investigating the preventive effects of nutritional interventions on the severity of targeted infections, e.g., plant bioactives with putative anti-inflammatory or antibacterial activity. Several Drosophila pathogens induce either local or systemic infections with mild or severe disease progression.164 Pathogens that induce mild infections in Drosophila are Pectobacterium carotovorum subsp. carotovorum 15165 and Serratia marcescens Db11,144 while Pseudomonas entomophila L48 induces severe disease.145,166,167 All of these pathogens can either be introduced systemically or orally in

potential increase in health status by the dietary compound of interest. 4.4.2. Determination of Intestinal Immune Function by Quantification of Anti-microbial Peptide (AMP) Expression and Stem Cell Proliferation. Whether plant bioactives and nutrient composition affect the health and life span of Drosophila, and to what extent, potentially by their antibacterial or immuno-modulatory activities, can be evaluated by various methods that give some indication of alterations in the immune status and function of the fly. Therefore, flies are generally pre-fed with the experimental diet for a defined time and analyzed for AMP expression, stem cell proliferation, and bacterial load in the intestine. Unchallenged, compound-treated and pathogen-infected flies can be generated for AMPs and stem cell proliferation targeted experiments. Oral infection of fruit flies with Drosophila pathogens significantly induces AMP expression, e.g., Metchnikowin, Diptericin, Attacins, and Drosocin, in the gut that can be quantified by qRT-PCR.107,135,143−145 Accordingly, the repair of the epithelium is an important trait for survival following exposure to intestinal stressors. Similar to mammals, the fruit fly possesses multipotent intestinal stem cells (ISCs) in the gut. ISC responsiveness to gut damage maintains intestinal homeostasis and promotes survival in young flies, while dysregulated intestinal stem ISC division may be detrimental at older ages.143,146,147 Phospho-Histone H3 (PH3) staining of the gut is an appropriate method for depicting intestinal stem cell proliferation.135,143,145,148 Mild infections with a low infection dose of Drosophila pathogens, e.g., Pectobacterium carotovorum subsp. carotovorum 15 and Pseudomonas entomophila L48, may induce stem cell proliferation (Table 2, lower panel), while severe infections with high infection doses irreparably damage the gut, resulting in distinctly decreased stem cell proliferation.143,145 Both gut-derived AMP expression and intestinal epithelial mitosis increase with age in the fruit fly107 underlining the substantial importance of intestinal stem cell activity and background systemic inflammation on the determination of Drosophila’s life expectancy.107 The treatment of flies with secondary plant metabolites may affect the inflammatory response, AMP expression, and stem cell proliferation, making these methods suitable tools for monitoring the effects of dietary supplements on the health status of Drosophila. Notably, the indigenous gut microbiota activate a basal level of ISC activity in the fruit fly,148 emphasizing the importance the gut microbiome for the immune and health statuses of Drosophila. 4.4.3. Impact of Gut Microbiota and Infection Studies. 4.4.3.1. The Fly Microbiome. The Drosophila gastrointestinal tract is colonized by various commensal microorganisms similar to the mammalian gut. However, the fly gut contains a limited number of microorganisms from approximately 30 species compared with more than 500 different bacterial species in mammals; thus the fly gut displays a lower bacterial diversity.149,150 Several human diseases including inflammatory bowel disease, obesity, and cardiovascular diseases have been connected to a detrimental change in the gut microbiota.151 Therefore, model organisms hosting a rather simple microbiota are needed to elucidate the underlying pathogenic mechanisms to establish preventive and therapeutic strategies in the treatment of microbiota-associated diseases. Most of the bacteria detected in the gut of fruit flies belong to the phyla Firmicutes and Proteobacteria. Furthermore, Actinobacteria, Bacteroides, and Cyanobacteria have been detected by 454 pyrosequencing of 16S rRNA gene 3745


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Journal of Agricultural and Food Chemistry high or low sub-lethal bacterial concentrations.145,147,167 Simple experimental settings require the pre-feeding of flies for a defined time period before the flies are orally or systemically exposed to pathogenic bacteria. Oral infections are then carried out by application of a bacteria−sucrose solution (OD600 = 100−200) following starvation for 2h. For systemic infections flies are infected by pricking into the thorax with a thin needle inoculated with a concentrated bacterial pellet (4 × 1011 cells/ mL at OD600 = 200)164,165 or by microinjection of a defined volume of the bacterial pellet using Nanoject and a pulled glass capillary.164 When performing nutritional intervention studies targeting gut health in the fruit fly, researchers have to be aware that male individuals display a higher level of systemic inflammation and are more susceptible to intestinal infection than females, in general.107 Moreover, adequate bacterial uptake has to be ensured, which can be ascertained by quantifying the bacterial load. Bacterial load assays are performed to estimate the number of ingested pathogenic bacteria. Flies are starved for 2 h prior to the application of a bacterial-sucrose suspension, e.g., using GFP-labeled Erwinia carotovora carotovora 15 or Serratia marcescens Db11 containing 0.5% FCF blue, for another 2 h. For quantification of the ingested amounts of bacteria, flies are visually inspected under a fluorescent dissecting microscope when GFP-labeled bacteria are used.164 Otherwise, flies are homogenized, and their OD600 is measured spectrophotometrically. Homogenized flies are plated onto lysogenic broth agar plates with selective antibiotics (if applicable), and colony-forming units are counted following incubation at 29−30 °C overnight.107,144 Both wild-type and mutant fly strains, e.g., reporter flies carrying immune-inducible promoters of AMPs ligated to green and red fluorescent proteins or LacZ-derived β-galactosidase activity,164 are suitable infection models. Sensible and available read-outs are survivorship, AMPs expression, gut integrity, stem cell proliferation, and bacterial load/bacterial clearance. 4.5. Fertility. The sexual activity of the fruit fly affects its life expectancy and vice versa.168,169 Fertility is a fitness component and is correlated with life expectancy as germ cell ablation results in increased life expectancy in D. melanogaster.170 Moreover, the expression of distinct hormone receptors affects life span in a sex-specific manner.133 A reduction in fecundity is putatively associated with longevity in the fruit fly, possibly mediated by dietary restriction-dependent effects.59,171,172 Interestingly, the nutritional status of females affects their mating response.173,174 Therefore, nutritional interventions, e.g., administration of secondary plant bioactives that may act as CR mimetics, potentially affect the fertility of female Drosophila. Male flies are able to transfer nutrients with their ejaculate175 and thus may affect female mating behavior with pheromone effective compounds.176 Testis development, accessory gland protein synthesis, and mating success are controlled by the Drosophila sex hormones 20-OH-ecdysone and juvenile hormone.177,178 Thus, plant bioactives exhibiting hormone-like structures might exhibit estrogenic activity and affect fertility in the fruit fly.

Table 3. Comparison of the Invertebrate Models Drosophila melanogaster and Caenorhabditis elegansa D. melanogaster

C. elegans

phylum sexes

Arthropoda females and males

chromosomes

three pairs of autosomes (2−4) + an X/Y pair

genome size and gene number orthologs in human genome lifetime fecundity

165 Mb, approx. 17 000

Nematoda hermaphrodites and males five pairs of autosomes (I−V) + one or two sex chromosomes 100 Mb, approx. 20 000

50%

38%

approximately 400 progeny

egg-to-adult development average lifespan

egg, instar larvae 1−3, pupa, adult 10 d at 25 °C approximately 50−60 d at 25 °C

laboratory diet

• complex diets

• approximately 300 self-progeny; • up to 1000 progeny after mating egg, L1 to L4 larvae, adult 3 d at 20 °C approximately 15−20 d at 20 °C • E. coli lawn on agar plates • E. coli in complex liquid media • axenic semidefined medium mouth, pharynx, intestine, rectum 20 polyploid epithelial cells

• defined holidic medium

anatomy of intestinal tract intestinal cell types microbiota

cell culture a

mouth, pharynx, esophagus, crop, cardia, midgut, hindgut, anus • enterocytes, enteroendocrine cells • stem cells species of the phyla Firmicutes, Proteobacteria, Actinobacteria, Bacteroides, and Cyanobacteria available

lacking

not available

Data from refs 164, 191, and 209.

and gene−environment interactions and often deliver fast answers to conserved fundamental biological problems. Both fly and worm are easy and cost-effective to grow in the laboratory. Their genome has been sequenced, and powerful forward and reverse genetics including RNAi and CRISPR are feasible. For both models, a huge collection of mutants is available from community projects. Moreover, when employing D. melanogaster or C. elegans usually one has to consider few ethical and regulatory restrictions. Nevertheless, we have the opinion that the fruit fly provide some key features that favor it as model with respect to nutritional research questions. (1) Despite its small body size, the fruit fly has a relatively complex gastrointestinal system that, as in mammals, is highly specialized in anatomy, organization, and function. It consists of different cell types that enable, e.g., frequent regeneration processes. Compared to that, the gastrointestinal tract of C. elegans is very simple. It encompasses 20 polyploid enterocytes and lacks enteroendocrine and stem cells. (2) As in mammals, the alimentary tract of the fruit fly harbors various commensal microorganisms enabling the investigation of host−microbiota interaction. C. elegans lacks a microbiota. (3) The recent introduction of chemically defined holidic media for D. melanogaster offers a broad range of opportunities to examine, e.g., micronutrient function and nutritional geometry.98,101 A comparable chemically defined medium for C. elegans has not yet been developed. (4) For nutritional studies it is often crucial to know how much food has been ingested by an organism. However, appropriate methods for food uptake quantification

5. D. MELANOGASTER OR C. ELEGANS? PROS AND CONS Invertebrate model organisms have become a cornerstone of various fields of biological and biomedical research. Most prominent are the fruit fly D. melanogaster and the nematode Caenorhabditis elegans that share many advantages listed in Table 3 that permit systematic deciphering of the gene−gene 3746


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Journal of Agricultural and Food Chemistry have been established only for D. melanogaster, but are rather lacking for C. elegans. (5) Furthermore, Drosophila cell lines can be deemed as an extension of the fly model.179−181 Their culturing is easy and inexpensive, and a broad range of genetic, molecular, and biochemical methodology has been established. Drosophila cell cultures can be used to examine, e.g., cellular and biochemical response to environmental cues including nutrients, bioactives, and hormones. Until now, no C. elegans cell culture lines exist. (6) Finally, D. melanogaster contains a higher number of genes with orthologs in the human genome than C. elegans.

(Indiana, USA), the Drosophila Species Stock Center (California, USA), the Vienna Drosophila Resource Center (Austria), and the Drosophila Genomics and Genetic Resources: Kyoto Stock Center (Japan). Further, distinct target genes and signaling pathways can be specifically examined using RNAi and the CRISPR/Cas system for genome engineering, as they can be used in the fruit fly. The fruit fly is an appropriate model organism to target many aspects of basic nutritional research as central metabolic pathways are evolutionarily well conserved. Moreover, D. melanogaster is an excellent model organism to evaluate the impact of nutritional factors and diet composition on health and life span among many other aspects in both wild-type and mutant fly strains and in various disease models, e.g., fly strains developing metabolic disorders like obesity, diabetes, and immunological and motoric diseases.188−191 Results may be partly extrapolated to mammalian species. However, when the transfer of findings made in the fruit fly to mammals is intended, verification in mammalian models, e.g., laboratory rodents, is still required.

6. OUTLOOK: FUTURE DIRECTIONS The fruit fly D. melanogaster is an emerging and valuable model organism for food and nutrition research, as there are numerous experimental methods, tools for analysis, and genetic models available. Nevertheless, specific aspects should be considered when designing future fruit fly nutrition studies to improve experimental outcomes and study comparability. First, diet has a huge impact on many biochemical, physiological, and biological processes. A multitude of different, mainly undefined, fly food recipes have been used to cultivate Drosophila. We encourage the Drosophila community to develop highly standardized diets to ensure good reproducibility and comparability of the results between nutritional intervention studies. Defined and purified experimental diets used for laboratory rodents (including rats and mice) are already designed by the American Institute of Nutrition (AIN).182,183 In particular, it may be useful to include a standardized defined holidic food medium. Recent progress in the formulation of holidic fly media is very promising.92,95 However, flies reared on these holidic food preparations display a delay in larval development and a reduction of fecundity rates, indicating that these diets still have to be improved or adapted to the different life stages of D. melanogaster. The exact nutrient requirements of the fruit fly still remain unsolved and must be elucidated in detail. The use of holidic food preparations offers an excellent opportunity to prove the essentialness of micronutrients in D. melanogaster development and physiology. Furthermore, the absorption of nutrients in the gastrointestinal tract and the cellular uptake of metabolites, which represent key steps in alimentation, as well as nutrient-dependent transporters in the Drosophila gut are scarcely known and have to be identified and characterized. Importantly, researchers have to take into account that the impact of nutrients on biochemical, physiological, and biological processes is affected by the genetic background of an organism. As different fly strains show distinct inherent life expectancies and responsiveness to dietary interventions, we recommend including at least two different wild-type fly strains in each key experiment, where the effects of nutrients on D. melanogaster are examined. Additionally, it is essential to ensure a sufficient replicate number and an adequate cohort size in certain experiments, such as life-span assays that are laborious, long, and interference-prone, to create robust and solid data. Compared with vertebrate models, D. melanogaster as a model organism offers a broad range of methodologies (especially the versatile genetic toolbox) with which to approach basic problems of nutrition research in a cost- and time-efficient way. The fruit fly occupies genes and proteins that are 50−60% orthologous to mammalian ones184−186 and that can be easily explored using FlyBase.187 Numerous mutant fly strains are already commercially available at places such as the Bloomington Drosophila Stock Center

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 431 880 5313. Fax +49 431 880 2628. E-mail: [email protected]. ORCID

Stefanie Staats: 0000-0001-9642-9204 Funding

University of Kiel (federate state Schleswig-Holstein, Germany). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED foxo, forkhead box, sub-group O; IPC, insulin-producing cell; LCL, lower control limit; mth, methuselah; sir2, sirtuin 2; srl, spargel; UCL, upper control limit

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REFERENCES

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Drosophila melanogaster as a Versatile Model Organism in Food

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