David Renault
email: david.renault@univ-rennes1.fr

Hesham Yousef

Amr A Mohamed

The multilevel antibiotic-induced perturbations to biological systems: early-life exposure induces long-lasting damages to muscle structure and mitochondrial metabolism in flies

Keywords: Antibiotics, eukaryote, insect flight muscles, mitochondrial dysfunction, metabolomics, energy metabolism 4

Antibiotics have been increasingly used over the past decades for human medicine, foodanimal agriculture, aquaculture, and plant production. A significant part of the active molecules of antibiotics can be released into the environment, in turn affecting ecosystem functioning and biogeochemical processes. At lower organizational scales, these substances affect bacterial symbionts of insects, with negative consequences on growth and development of juveniles, and population dynamics. Yet, the multiple alterations of cellular physiology and metabolic processes have remained insufficiently explored in insects. We evaluated the effects of five antibiotics with different mode of action, i.e. ampicillin, cefradine, chloramphenicol, cycloheximide, and tetracycline, on the survival and ultrastructural organization of the flight muscles of newly emerged blow flies Chrysomya albiceps. Then, we examined the effects of different concentrations of antibiotics on mitochondrial protein content, efficiency of oxidative phosphorylation, and activity of transaminases (Glutamate oxaloacetate transaminase and glutamate pyruvate transaminase) and described the cellular metabolic perturbations of flies treated with antibiotics. All antibiotics affected the survival of the insects and decreased the total mitochondrial protein content in a dose-dependent manner.

Ultrastructural organization of flight muscles in treated flies differs dramatically compared to the control groups and severe pathological damages/structures disorganization of mitochondria appeared. The activities of mitochondrial transaminases significantly increased with increased antibiotic concentrations. The oxidation rate of pyruvate+proline from isolated mitochondria of the flight muscles of 1-day-old flies was significantly reduced at high doses of antibiotics. In parallel, the level of several metabolites, including TCA cycle intermediates, was reduced in antibiotics-treated flies. Overall, antibiotics provoked a system-wide alteration of the structure and physiology of flight muscles of the blow fly Ch. albiceps, and may have fitness consequences at the organism level. Environmental antibiotic pollution is likely to have unwanted cascading ecological effects of insect population dynamics and community structure.

INTRODUCTION

The use of pharmaceuticals has been consistently developed over the past decades [START_REF] Aminov | History of antimicrobial drug discovery: Major classes and health impact[END_REF], and antibiotics have revolutioned human medicine. Antibiotics are also highly employed for food-animal agriculture, aquaculture and plant production [START_REF] Witte | Selective pressure by antibiotic use in livestock[END_REF][START_REF] Le | Antibiotic resistance in bacteria from shrimp farming in mangrove areas[END_REF][START_REF] Cabello | Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment[END_REF], for disease prevention, or for promoting growth and feed efficiency of livestock animals [START_REF] Gaskins | Antibiotics as growth promotants: mode of action[END_REF]. However, the use of antibiotics is not without posing significant problems [START_REF] Finley | The scourge of antibiotic resistance: the important role of the environment[END_REF][START_REF] Larsson | Antibiotics in the environment[END_REF][START_REF] Rico | Use, fate and ecological risks of antibiotics applied in tilapia cage farming in Thailand[END_REF], and alternatives to these growth promotors are increasingly discussed [START_REF] Seal | Alternatives to antibiotics: a symposium on the challenges and solutions for animal production[END_REF]. Besides antibiotic resistance [START_REF] Neu | The crisis in antibiotic resistance[END_REF][START_REF] Laxminarayan | Antibiotic resistance-the need for global solutions[END_REF], the adverse effects of antibiotics on environmental ecosystems is a rapidly emerging concern. [START_REF] Andersson | Microbiological effects of sublethal levels of antibiotics[END_REF] suggested that up to 80% of the active substances of antibiotics can be released into the environment. Several antibiotics are not degraded within the gut of livestock animals, and the several pathways that permit the wide-scale dissemination of these drugs [START_REF] Sarmah | A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (VAs) in the environment[END_REF][START_REF] Chang | Antibiotics in agriculture and the risk to human health: how worried should we be?[END_REF] rapidly expand the antibiotic load in the environment [START_REF] Perry | The antibiotic resistome: what's new?[END_REF][START_REF] Amos | Validated predictive modelling of the environmental resistome[END_REF]. The effects of antibiotics on ecosystems and biogeochemical processes have thus become a major research topic [START_REF] Roose-Amsaleg | Do antibiotics have environmental side-effects? Impact of synthetic antibiotics on biogeochemical processes[END_REF].

Antibiotics are present in most ecosystems [START_REF] Daghrir | Tetracycline antibiotics in the environment: a review[END_REF], often as mixtures.

Several antibiotics (tetracyclines) are widely used as crop protectants [START_REF] Mcmanus | Antibiotic use in plant agriculture[END_REF][START_REF] Chang | Antibiotics in agriculture and the risk to human health: how worried should we be?[END_REF], but their exact ecological impacts remain insufficiently explored.

Antibiotics have different modes of actions to kill or limit growth of bacteria: they can inhibit/regulate metabolism of nucleic acids [START_REF] Floss | Rifamycin-mode of action, resistance, and biosynthesis[END_REF], protein biosynthesis [START_REF] Walsh | Molecular mechanisms that confer antibacterial drug resistance[END_REF][START_REF] Siibak | Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins[END_REF][START_REF] Wilson | Ribosome-targeting antibiotics and mechanisms of bacterial resistance[END_REF], activities of certain enzymes [START_REF] Kohanski | How antibiotics kill bacteria: from targets to networks[END_REF], or induce toxic metabolic perturbations [START_REF] Belenky | Bactericidal antibiotics induce toxic metabolic perturbations that lead to cellular damage[END_REF]. Antibiotics are most efficient when cells of the microorganisms are multiplying [START_REF] Stewart | Antibiotic resistance of bacteria in biofilms[END_REF], and can particularly affect bacterial symbionts of organisms, with negative consequences on growth and development of juveniles [START_REF] Kafil | Role of symbiotic bacteria in the growth and development of the Sunn pest, Eurygaster integriceps[END_REF].

Insects are likely to be exposed to environmentally released antibiotics, and more particularly tetracyclines. Tetracyclines are highly persistent into the environment, and can reach very high amounts into winter manure, up to 43 and 183 mg kg-1 for tetracycline and oxytetracycline, respectively [START_REF] Hu | Occurrence and source analysis of typical veterinary antibiotics in manure, soil, vegetables and groundwater from organic vegetable bases, northern China[END_REF], and up to 200 µg kg-1 for tetracycline in the first layers of the soil [START_REF] Hamscher | Determination of persistent tetracycline residues in soil fertilized with liquid manure by high-performance liquid chromatography with electrospray ionization tandem mass spectrometry[END_REF]. Surprisingly, most of the studies have focused on the fitness consequences of adding of these drugs to the food of laboratory cultured insects [START_REF] Büyükgüzel | Galleria mellonella (L.) survivorship, development and protein content in response to dietary antibiotics[END_REF][START_REF] Ridley | Microbe-dependent and nonspecific effects of procedures to eliminate the resident microbiota from Drosophila melanogaster[END_REF][START_REF] Xu | Impacts of antibiotic and bacteriophage treatments on the gut-symbiont-associated Blissus insularis (Hemiptera: Blissidae)[END_REF]. At low concentrations of ciprofloxacin (3.33 to 13.33 µg/mL), the development of the fly Chrysomia putoria was not altered [START_REF] Ferraz | Evaluation of the influence of the antibiotic ciprofloxacin in the development of an Old World screwworm fly, Chrysomya putoria[END_REF], while addition of streptomycin to the food at concentrations of 0.03 to 0.15% increased the growth of the larvae of the butterfly Spodoptera litura, without affecting its survival [START_REF] Thakur | Effect of antibiotic on survival and development of Spodoptera litura (Lepidoptera: Noctuidae) and its gut microbial diversity[END_REF]. [START_REF] Dickel | Long-term prophylactic antibiotic treatment: effects on survival, immunocompetence and reproduction success of Parasemia plantaginis (Lepidoptera: Erebidae)[END_REF] reported that long-term exposure to prophylactic treatments (fumagillin) negatively affects the number of eggs laid by females of the wood tiger moth Parasemia plantaginis. Interestingly, metabolic perturbations provoked by antibiotics to insects, and their end-target consequences, have remained unexplored.

Bacteria and mitochondria share many similarities in terms of ribosomal machinery [START_REF] Zhang | Antibiotic susceptibility of mammalian mitochondrial translation[END_REF], and there are growing evidence that antibiotics can affect mitochondrial protein synthesis and mitochondrial functioning in a wide range of non-bacterial taxa [START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF][START_REF] Wang | Antibiotic use and abuse: A threat to mitochondria and chloroplasts with impact on research, health, and environment[END_REF]. For instance, doxycycline (tetracycline family) has been demonstrated to affect proteostasis in both invertebrate and vertebrate cells [START_REF] Houtkooper | Mitonuclear protein imbalance as a conserved longevity mechanism[END_REF]. [START_REF] Ballard | Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila[END_REF] demonstrated that tetracycline reduces ATP production in Drosophila simulans and increases mtDNA density in insects free of Wolbachia. In parallel, several works have demonstrated that rapid metabolic changes occur after antibiotic treatments in bacteria [START_REF] Belenky | Bactericidal antibiotics induce toxic metabolic perturbations that lead to cellular damage[END_REF][START_REF] Hoerr | Characterization and prediction of the mechanism of action of antibiotics through NMR metabolomics[END_REF][START_REF] Zampieri | Metabolic constraints on the evolution of antibiotic resistance[END_REF], but there are many unknowns regarding the mechanisms of action of antibiotics, and their impacts, at the intra-organismal level in insects. Hence, the use of extracellular footprints and physiological intracellular fingerprints should improve our understanding of the effects of antibiotics on insects.

Juveniles of dipterans are poorly mobile insects which often thrive in microhabitats near the surroundings of human settlements, including agricultural lands and farms. The probability of exposure of fly larvae and pupae to antibiotics is thus very high, and should greatly affect the biochemistry, physiology and morpho-anatomy of these developing individuals, with potential long-lasting effects. At the larval stage, wings and flight muscles are absent from the thorax [START_REF] Paululat | Determination and development of the larval muscle pattern in Drosophila melanogaster[END_REF][START_REF] Roy | Muscle pattern diversification in Drosophila: the story of imaginal myogenesis[END_REF], and muscle cells appear and progressively arrange and differentiate at the pupal stage [START_REF] Roy | Muscles in the Drosophila second thoracic segment are patterned independently of autonomous homeotic gene function[END_REF].

Also, in a number of fly species, the maturation of flight muscle mitochondria only occurs after the emergence of adults [START_REF] Marden | Variability in the size, composition, and function of insect flight muscles[END_REF], and this process is associated with a high biosynthetic activity of mitochondrial proteins from flight muscles [START_REF] Lennie | Changes in the nucleic acid content and structure of the thoracic mitochondria during development of the blowfly Lucilia cuprina[END_REF][START_REF] Tribe | Biochemical and structural variations in the flight muscle mitochondria of aging blowflies Calliphora erythrocephala[END_REF]. Because several antibiotics are inhibitor of protein biosynthesis, the exposure of juvenile flies to these drugs should affect the mitochondrial efficiency and mitochondrial DNA density, as shown in Drosophila flies [START_REF] Ballard | Tetracycline treatment influences mitochondrial metabolism and mtDNA density two generations after treatment in Drosophila[END_REF].

In this work, we investigated the effects of different antibiotics inhibiting (i) cell wall synthesis (ampicillin and cefradine) and (ii) protein synthesis (chloramphenicol, cycloheximide, and tetracycline) (Table S1) on the survival and ultrastructural organization of the flight muscles of newly emerged blow fly, Chrysomya albiceps. Having these phenotypic data, we further examined the effects of these drugs on the physiology and metabolism of the individuals. Considering the prominent importance of mitochondria for flight muscles, we examined the effects of different concentrations of antibiotics on mitochondrial protein content, efficiency of oxidative phosphorylation, and activity of transaminases. Among transaminases that are localized in mitochondria, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) are the most active in insects [START_REF] Gäde | Anaerobic energy metabolism[END_REF].

These enzymes are crucial for amino acid metabolism, but the effects of antibiotics on their activity have received little attention. Finally, we were interested in describing the cellular metabolic perturbations of flies treated with antibiotics, and run metabolomics assays to obtain a system-wide picture of biochemical networks altered by antibiotics.

We expected that moderate to high concentrations of protein synthesis inhibitors would be detrimental to the survival of newly emerged adult Ch. albiceps, whose mitochondriogenesis and biosynthetic processes should be affected. Chloramphenicol and tetracycline bind on large and small subunits of the mitochondrial ribosomes, and can inhibit mitochondrial protein synthesis and biogenesis in mammals [START_REF] Brodersen | The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit[END_REF][START_REF] Houtkooper | Mitonuclear protein imbalance as a conserved longevity mechanism[END_REF], while cycloheximide provokes a complete inhibition of cytoplasmic protein synthesis of eukaryotic cells. We assumed that mitochondrial structures and protein amounts would be altered in muscle tissues of flies injected with these three drugs. Ampicillin and cefradine are cell wall synthesis inhibitors, and these bactericidal antibiotics can strongly reduce the activity of the electron transfer chain of mitochondria [START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF]. At low to moderate concentrations, mitochondrial metabolism of adult Ch. albiceps treated with ampicillin, cefradine, chloramphenicol, cycloheximide and tetracycline should be reduced, rendering cells' energy deficient (reduced oxidative phosphorylation). Finally, we hypothesized that metabolic alterations would occur after the flies would be injected by the different antibiotics, and that the effects of the metabolic profiles would mirror the mode of actions of these different drugs.

MATERIALS AND METHODS

Insect rearing

Adults of the hairy maggot blow fly (GenBank common name: taxid:81419) Chrysomya albiceps (Wiedemann, 1819) (Diptera: Calliphoridae) were field-collected from Giza, Egypt (30°01'34.4"N 31°12'21.6"E). Colonies were established at the insectary of the Faculty of Science (Cairo University, Giza, Egypt) as described in [START_REF] Queiroz | Temperature requirements of Chrysomya albiceps (Wiedemann, 1819) (Diptera, Calliphoridae) under laboratory conditions[END_REF]. Adult flies were placed into wooden cages (50X50X80 cm, LxLxH) with sides made of fine mesh wire-gauze; cages containing flies were maintained under controlled conditions (27±1 °C, 60-70% Relative Humidity (R.H.), 14:10 h light:dark), and reared for five generations before being used for the experiments. Newly emerged adults were supplied with sucrose and water. Two to three days after emergence, adults were offered fresh meat (about 250 mg) as food and oviposition media. Females oviposit batches of up to 250-300 eggs, which were periodically transferred to larval rearing dishes (38 cm diameter, 7 cm height) containing a rearing medium (moistened sawdust and fresh meat blocks). These dishes were covered with muslin cloth fastened with rubber bands. Larval populations of Ch. albiceps are cannibals; hence, 2 nd instars were regularly sampled from the semi-dried meat, and transferred to new rearing dishes. Pupation took place in the sawdust, or within dried meat chunks, and the pupae were collected and transferred to adult cages for emergence.

Experimental treatments: injection of antibiotics and monitoring of the survival of the flies

The effects of antibiotics with bacteriostatic or bactericidal activity (Table S1) on the biology and physiology of the blow flies were assessed by injection of different concentrations of: ampicillin (A9518 Sigma-Aldrich), cefradine (CAT # C0690000 Sigma-Aldrich), chloramphenicol (CAT # C3787 Sigma-Aldrich), cycloheximide (CAT # C7698 Sigma-Aldrich), and tetracycline (CAT # T7660 Sigma-Aldrich). To standardize the amount of drug given to each insect, antibiotics were injected into the adults. Before injections, newly emerged adults were allowed to harden for 1-2 hour, and they were then briefly cooled on ice prior to injection. A 10-µL Hamilton microsyringe was used for injections. Different concentrations of antimicrobial solutions were freshly prepared into distilled water and protected from light by wrapping tubes in foil. A volume of 2 µL of antimicrobial drugs was injected to each fly, meaning that we had different independent groups of flies that received distinct antibiotics at different concentrations (ampicillin: 100, 200, 300, 400 and 500 µg/fly; cefradine: 100, 200, 300, 400, 500, 600 and 700 µg/fly; chloramphenicol: 100, 150, 200, 250 and 300 µg/fly; cycloheximide: 2.5, 5, 10, 20, 30 and 40 µg/fly; tetracycline: 10, 20, 30 and 40 µg/fly). The concentrations of the antibiotics were determined by running a preliminary test that measured the mortality dose-response of the flies (each experimental group started with an initial number of 50 flies). Two groups of control flies were also prepared: flies pricked with the syringe (no injection, control), and flies that were injected 2 µL of saline solution (control 'saline'). All injections were made in the flight muscles, by entering the syringe beneath the wings, between the thorax and the abdomen.

For this part of the experiment, flies were individually injected one of the abovementioned antimicrobial concentration. Three independent survival assays (N = 50 flies per survival assay) were done for each experimental condition, including control flies. After injection, flies were maintained into rearing cages on sugar and water diet at 27±1 °C, 60-70% R.H, and 14:10 h (light: dark) photoperiod. Flies from each experimental condition were examined daily for mortality for up to four days. The results from this experiment were used for selecting the antibiotic concentrations used for the second part of the study, i.e. for the characterization of extracellular footprints and physiological intracellular fingerprints of antimicrobial and antifungal treatments.

Extracellular footprints of the effects of antibiotic treatments on flight muscles

For this part of the experiment, antibiotic quantities generating 50% mortality after one day (LD 50 values obtained from the survival experiment) were injected to the flies (cefradine: 500 µg/fly; chloramphenicol: 200 µg/fly; cycloheximide: 20 µg/fly; Tetracycline: 30 µg/fly), except for ampicillin (300 µg/fly) for which lower quantities were injected to have flies surviving up to the fourth day post injection.

Heads, legs and abdomens of flies collected 1 and 4 days after injection of antibiotics were carefully removed after the flies were immobilized at 4 °C. The mid line of the dorsal surface of the thorax was cut with a sharp razor, so that the flight muscles were exposed on each side of the insect. A droplet of 2.5% glutaraldehyde fixative was put onto the flight muscles, which were then left for in situ fixation for 30 min. Small strips of about 1 mm of muscles were collected and immersed again in glutaraldehyde solution at 4 °C for 2 hours.

Then, muscles were washed in several baths of 0.05 M cacodylate buffer (pH 7.3) and let overnight at 4 °C. Postfixation was carried out in 1% osmium tetroxide for 45 min at room temperature. Muscles were then washed by cacodylate buffer, and dehydrated in graded ethanol concentrations, from 60 to 100%. A final dehydration step was carried by washes with toluene or propylene oxide. The tissues were then left overnight in 1:1 toluene and embedding medium (Epon) for infiltration. After this step, muscles were transferred to closed plastic capsules containing fresh epon. Epon capsules were polymerized at 60 °C for 48 hours.

Ultrathin sections were cut with glass knives by an LKB 2088-Ultramicrotome V (Cambridge Instruments Ltd., Cambridge, UK). Sections were mounted on uncoated copper grids.

Mounted sections were stained with uranyl acetate, followed by lead citrate and ultra automatic stainer (Leica-Reichert). The sections were examined with a Jeol 120 kV high resolution TEM (JEOL Ltd. Tokyo, Japan), and structural elements were identified based on [START_REF] Smith | The structure of insect muscles[END_REF].

Physiological intracellular fingerprints of the effects of antibiotics

Here, antibiotic quantities generating around 50% mortality after one day (according to LD 50 values obtained from the survival experiment), and one lower and one higher dose, were injected to the flies for each drug (cefradine: 200, 400 or 600 µg/fly; chloramphenicol: 100, 200 or 250 µg/fly; cycloheximide: 10, 20 or 30 µg/fly; tetracycline: 20, 30 or 40 µg/fly), except for ampicillin for which we used an intermediate dose lower than LD 50 (100, 300 or 500 µg/fly). Flies were collected for physiological analyses 24 hours after the injections.

Isolation of mitochondria

Twenty-four hours after the injection of antimicrobial solutions or distilled water, adult blow flies were collected from cages and immobilized by transferring them to -20 °C for 5 min.

Flies were subsequently kept on ice, and heads, wings, legs and abdomens were quickly removed. Thoraces were conserved for isolation of flight muscle mitochondria as described by [START_REF] Tribe | Changes taking place in the respiratory efficiency of isolated flight muscle sarcosomes, associated with the age of the blowfly, Calliphora erythrocephala[END_REF]. Thoraces from 25-50 flies were homogenized in a Dounce homogenizer containing pre-cooled isolation medium [(0.32 M sucrose, 0.01 M Tris buffer, pH 7.4 and 0.01 M EDTA (ethylenediaminetetraacetic acid disodium salt)]. Homogenates were filtered through four layers of muslin, previously soaked in ice-cold sucrose medium, and filtrates were centrifuged at 750 g for 10 min at 4 °C. The pellet was discarded, and the supernatant that contained mitochondria was centrifuged at 12,000 g for 20 min at 4 °C. The supernatant was discarded, and the mitochondrial pellet was resuspended gently in isolation solution using a pre-cooled fire-polished glass rod, and centrifuged again at 12,000 g for 20 min at 4 °C. The samples were then used for analyses of the protein content of the mitochondria.

Protein content of the mitochondria and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separation

The mitochondrial pellet was suspended into 5 mL of ice-cold 10% TCA. After 10 min at 4 °C, the samples were centrifuged at 12,000 g for 10 min, and the pellet was washed twice with ice-cold 10% TCA. The last mitochondrial TCA insoluble protein pellet was dissolved into 0.5 mL of NaOH (1 N). Aliquots of 100 µL were used for measuring protein amounts with the Folin-phenol method of [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF]. Bovine serum albumin (BSA; Sigma-Aldrich, fraction V) was used as the standard for the protein calibration curve. Absorbance of the samples was read at 750 nm. SDS-PAGE gels were performed according to the method of [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]

in a

Tris-glycine discontinuous gel system under denaturing conditions. Electrophoresis was performed on either 10 or 12% separating gel, overlaid with 5% stacking gel, at a constant current of 30 mA for 4 hours. The following reference proteins were used as molecular weight markers (Sigma-Aldrich), and ran concomitantly in SDS-PAGE: α-lactalbumin (14,200 Da), trypsin inhibitor (21,500 Da), bovine carbonic anhydrase (31,000 Da), egg albumin (42,699 daltons), bovine serum albumin (66,200 Da) and phosphorylase b (97,400 Da). Gel-Pro Analyzer 3.1 software (Media Cybernetics, Washington -MD 20850, USA) was used for estimating the relative molecular weight (Mwt) and relative mobility (Rf) values.

Measurement of the effects of antibiotics on energy production of the mitochondria

Oxygen consumption of mitochondria was determined with the Warburg manometric technique at 25 °C [START_REF] Tribe | Changes taking place in the respiratory efficiency of isolated flight muscle sarcosomes, associated with the age of the blowfly, Calliphora erythrocephala[END_REF], and expressed in terms of oxygen consumed per hour per milligram dry mass of mitochondrial flight muscles (µL O 2 /mg/h or QO 2 ). We focused on the mitochondria of the flies injected with antibiotics at the LD 50 . For each replicate, a volume of 100 µL of the mitochondrial extract was homogenized into a standard reaction mixture containing 130 mM KCl, 10 mM Tris HCl (pH 7.4), 30 mM potassium phosphate, 30 mM glucose, 1 mg BSA/mL, and 10 µL 50 mM ADP. For this assay, pyruvate (10 mM) and proline (4 mM) were the supplied substrates. Then, a volume of 100 µL of sarcosomal suspension (containing 0.2-0.4 mg protein) was added. The center of the flasks was filled with 200 µL of 20% KOH, and an acid washed filter paper wick was added. An incubation period of 25 min at 25 °C was conducted, with air as the gas phase. The reaction was stopped by the addition of 1 mL of ice-cold 25% TCA, and the flasks were placed immediately on ice in a refrigerator at 0 °C to prevent degradation of ATP. The contents of the flasks were centrifuged (12,000 g for 5 min at 4 °C). Then, inorganic phosphate of the supernatant was determined by mixing 100 µL from each sample with 3 mL of copper acetate buffer, 0.5 mL of ammonium molybdate and 0.5 mL of reducing agent. After 10 min at room temperature, absorbance of the samples was read at 880 nm. Corrections for endogenous oxidation and phosphorylation were not done, since these amounts are small when mitochondria are isolated from intact tissues.

Measurement of the glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT)

Enzymatic assays were carried out as described in [START_REF] Reitman | A colorimetric method for the determination of serum glutamic oxaloacetic acid and glutamic pyruvic transaminases[END_REF], with minor modifications. Oxaloacetate and pyruvate formed by the transaminase enzymes GOT and GPT, respectively, react with dinitrophenylhydrazine (DNPH) to form hydrazone. The absorbance of hydrazone produced in the alkaline medium was measured at 546 nm. For each test, 100 µL of the mitochondrial suspension was pipetted and transferred to 500 µL of phosphate buffer (GOT substrate: phosphate buffer (pH 7.2) 100 mM/L, L-aspartate 80 mM/L, α-ketoglutarate 4 mM/L; GPT: phosphate buffer (pH 7.2) 100 mM/L, D.L-alanine 80 mM/L, α-ketoglutarate 4 mM/L). After incubation for 30 min at 37 °C, a volume of 500 µL of the color reagent (DNPH) was added. The samples were mixed for 20 min at room temperature before adding 5 mL of 0.4 M NaOH. The enzyme activity was expressed in standard international units (U) per g of mitochondrial protein. One U is defined as the amount of enzyme that catalyzes the conversion of 1 µmole of substrate per min at 25 °C.

Metabolomic fingerprinting of flight muscles treated with antibiotics

In the last part of the work, we focused on antibiotics with bacteriostatic activity (chloramphenicol, cycloheximide, and tetracycline). We decided to collect the flies two days after the injections to ensure that we passed the peak of mortality of day 1. Based on the results of the survival assays, we selected one mild and one moderate antibiotic concentration for the metabolomic assays: 100 or 200 µg of chloramphenicol per fly (Chloramph C2 and C1, respectively), 2.5 or 10 µg of cycloheximide per fly (Cyclohex C2 and C1, respectively), 10 or 30 µg of tetracycline per fly (Tetracl C2 and C1, respectively). Untreated flies were injected saline solution (2 µL, hereafter refer to as 'control saline'), and a second group of flies was just pricked with the syringe (no injection, hereafter refer to as 'control'). After injections (or prick), all flies were returned to optimal growth conditions, and were collected for subsequent analyses when they were aged of 2 days. Each replicate consisted of flight muscles from at least nine flies. Four to six independent replicates were run for each experimental condition. The replicates were vacuum-dried and stored at -20 °C prior to analyses.

Gas chromatography coupled with mass spectrometry (GC-MS) was used detect the 62 metabolites of our database, encompassing amino acids, organic acids, polyamines, polyols, and sugars. We used the method described in [START_REF] Khodayari | Deciphering the metabolic changes associated with diapause syndrome and cold acclimation in the two -spotted spider mite Tetranychus urticae[END_REF]. Each sample was homogenized in 900 µL of a solution of ice-cold methanol: chloroform (2:1, v:v). Then, a volume of 600 µL of ultra-pure water was added. Samples were homogenized and centrifuged for 10 min at 4,000 g (4 °C). Ninety µL of the upper phase containing metabolites was transferred to new glass vials and samples were vacuum-dried (Speed Vac Concentrator, MiVac, Genevac Ltd., Ipswich, England) at 34 °C. Then, we used the derivatization process and GC-MS settings described by [START_REF] Khodayari | Deciphering the metabolic changes associated with diapause syndrome and cold acclimation in the two -spotted spider mite Tetranychus urticae[END_REF]. Randomized sample sequences were established for sample injection, chromatograms were analyzed using XCalibur v2.0.7.

Standard samples, consisting of 62 pure reference compounds at 1,2,5,10,20,50,100,200,500,750,1000, and 1500 µM were run and metabolite levels were quantified using the quadratic calibration curves for each reference compound.

Statistical analysis

The Kaplan-Meier estimates and Mantel-Cox two-sided log-rank tests were used to calculate the mean cumulative mortality of the flies after antibiotic treatments (± the standard error of mean, SEM), and draw the effects of doses of antibiotics on survival probabilities. For estimating LC 50 and comparing the different antibiotic doses, probit analysis was applied to fit multiple regression using SAS 9.2 (SAS Institute Inc., NC, USA). LC 50 were obtained by drawing a cumulative 96 hours mortality curve (dependent variable).

Biochemical data were analyzed with independent t-tests to assess for differences among the treated groups and controls. All experiments were performed in four replicates, unless otherwise indicated. The results are presented as the mean ± SEM. Data were analyzed with IBM SPSS Statistics V22.0 (IBM Corp. Armonk, NY, USA).

For metabolomics, the concentration of the metabolites was log-transformed, and data were mean-centered. The effects of antibiotics on the metabolic signatures of the flight muscles were investigated using Principal Component Analysis (PCA). A heatmap was constructed using Ward's method as the clustering method, and the Pearson correlation as the distance measure. The dendrogram was constructed according to Ward's method, and Euclidean correlation as the distance measure. Variations in the quantities of individual metabolites were then examined among the experimental conditions using an ANOVA with a Tukey post-hoc procedure. Analyses of metabolomic data were conducted with MetaboAnalyst 3.0 (http://www.metaboanalyst.ca; [START_REF] Xia | Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis[END_REF].

RESULTS

Effect of different concentrations of antibiotics on the survival of the flies

All injected antibiotics affected the survival of the flies (Fig. 1, Table 1), while little mortality was observed in flies injected with the saline solution, or in flies that were just pricked. Logrank tests demonstrated that increased antibiotic doses significantly increased mortality of the flies (p<0.001, Chi-square value (χ2) were: ampicillin χ2 = 67.98, df=4; cefradine χ2 = 93.15, df=5; chloramphenicol χ2 = 101.35, df=4; cycloheximide χ2 = 168.85, df=5; tetracycline χ2 = 107.56, df=3 (Fig. 1). LD 50 values for each antibiotic are presented in Table 1. At the lowest concentration, the injected antibiotics had small effects on the survival of Ch. albiceps. The number of dead individuals was the highest 1-day after injection for cefradine, chloramphenicol and tetracycline, whatever the concentration of the drug. As a result, LD 50 values remained stable for these three drugs when calculated 1 or 4 days after the injection (Table 1). Conversely, mortality progressed constantly over the four days post-injection for the flies that were treated with ampicillin or cycloheximide, and LD 50 values greatly varied.

For instance, LD 50 was 21.76 (19.63-23.89) µg /fly for cycloheximide when calculated at day 1, and dropped to 7.09 (6.47-7.71) µg/fly when LD 50 was calculated at day 4.

Extracellular footprints of the effects of antimicrobial treatments on flight muscles

Electron micrographs of flight muscles of flies treated with antibiotics showed a degenerative alteration of the fine structure of flight muscles and mitochondria; alterations were observed 1 (Fig. S1) and 4 (Fig. S2) days after the injection of the antibiotics. Except for flies treated with cefradine, mitochondria were swollen, cristae were distorted, and glycogen granules were dense (Fig. S2). In 4-day-old flies, some organelles appeared, without any internal compartment; moreover, mitochondrial and flight muscles interspaces were relatively large. Fewer effects were observed with cefradine (Fig. S2). Ultrastructural sections of ampicillintreated flies showed few irregularities in mitochondrial shapes but were overall very similar to controls (data not shown).

Physiological intracellular fingerprints of the effects of antibiotics

Effects of antimicrobial on the protein content of the mitochondria of the cells from flight muscles

A consistent pattern was observed across the experimental treatments, with the amount of proteins from flight muscles being decreased when the concentration of the drugs was increased (Fig. 2). At the highest antibiotic concentration, a larger decrease of the protein amount was observed for chloramphenicol and cycloheximide (reduction of about 29%), while the lowest decrease was observed for cefradine and tetracycline (reduction of about 14%).

Mitochondrial proteins of the flight muscles were characterized by SDS-PAGE, which allowed the separation of 41 bands (Fig. S3, Table S2). The 10% SDS-PAGE separated 27 protein subunits, ranging from 11,000 to 120,500 daltons, while the 12.5% SDS-PAGE separated 24 protein bands, ranging from 28,500 to 97,000 daltons. Proteins with molecular weights of 50, 42.5, 38 and 22 kilodaltons for the 10% gel, and 34.5, 33 and 17.5 kDa for the 12.5% gel were the most concentrated protein subunits. The highest changes were observed for flies injected with cycloheximide and chloramphenicol, whose gels were characterized by a lower number of bands of fainter colors, as compared with control flies (Fig. S3).

Oxidative phosphorylation of flight muscle mitochondria

The oxygen consumption (oxidation rate of pyruvate and proline) was 102 ± 0.2 µL O 2 /h/mg mitochondrial proteins in 1-day-old control flies (Fig. 3a). It was two times lower in flies that were injected with antibiotics, with values ranging from 39.5 ± 0.9 µL O 2 /h/mg for chloramphenicol at 200 µg/fly to 55.7 ± 1.5 µL O 2 /h/mg for cycloheximide at 20 µg/fly.

The considerable inhibition of the rate of oxygen uptake following the treatment with high doses of antibiotics altered P/O ratio, which oscillated around 2 across in the experimental treatments (Fig. 3b).

Effects of antimicrobial on the transaminase activity

Flight muscle mitochondria from untreated newly emerged flies were characterized by similar transaminase (GOT & GPT) activities, of about 14 U/g mitochondrial protein (Fig. 4). Except for the GOT activity of the flies injected with 100 µg/fly ampicillin, all antibiotic treatments significantly affected the activity of GOT and GPT. In three groups, all from the cefradine treatment, activity of GPT was reduced significantly as compared with control flies.

Conversely, activity of GPT and GOT was significantly increased in all of the other experimental treatments. Activity of mitochondrial GPT was increased, relative to control, in flies injected with moderate and high doses of ampicillin (2.4-and 2.7-fold, respectively), chloramphenicol (3.6-and 3.4-fold, respectively), cycloheximide (1.6-and 3.0-fold, respectively), and tetracycline (1.23-and 1.20-fold, respectively). Mitochondrial GOT activity was increased significantly in all flies that were treated with antibiotics as compared with control flies (Fig. 4).

Metabolomics

Forty-nine metabolites were quantified from our samples, with alanine, glycerol and lactic acid being the most abundant metabolites whatever the experimental condition. The first and second principal components (PC1, PC2) of the PCA accounted for 71.4% and 9.3% of the total inertia (Fig. 5a). The metabolic phenotypes of the samples injected with two different doses of antibiotics remained very similar, i.e. largely overlapped. PC1 strongly separated the flies treated with cycloheximide from the other experimental groups. Concentrations of ethanolamine, pipecolic acid, putrescine, glutamic acid and ribose (ranked by contribution to the axis) were positively correlated to PC1, and were more abundant in flies that were injected cycloheximide (Fig. 5b). Lactic acid, trehalose, fructose-6-P, glucose, fructose, glucose-6-P, spermine, sorbitol and alanine were positively correlated to PC2, and were in lower amounts in flies treated with cycloheximide.

The second axis of the PCA (PC2) separated control flies (pricked flies or flies injected with the saline solution) from flies treated with tetracycline or chloramphenicol. Lactic acid, glycerol-3-P, saccharose and gluconolactone were measured in higher amounts in pricked and saline-injected flies, whose metabolic phenotypes did not significantly differ. Glucose, fructose-6-P, free phosphate, phenylalanine, spermine, glycerol, glucose-6-P and trehalose were quantified in higher concentrations in flies that were injected with chloramphenicol.

Hierarchical clustering of the samples and heatmap (Fig. S4 andS5) were drawn to facilitate pattern recognition, i.e. visualize similarities / differences among replicates without a priori on the experimental conditions. The hierarchical cluster analysis confirmed the separation of the experimental groups observed in the PCA, with samples from a given experimental condition clustering together for all experimental treatments (Fig. S4). A subset of amino acids, polyamines and organic acids were decreased significantly in flies treated with chloramphenicol and tetracycline, with 21 metabolites having decreased amounts in these two conditions as compared with controls (Fig. S5). The concentrations of most metabolites from the flies injected with cycloheximide were altered as compared with controls, while only few metabolites (i.e. most of those positively correlated to PC1 in the PCA, putrescine, fumaric acid, ethanolamine, pipecolic acid, aspartic acid, and glutamic acid) being accumulated (Table S6.

DISCUSSION

Effect of antibiotics on the survival of the flies

While the lowest doses of ampicillin, cefradine, chloramphenicol, cycloheximide and tetracycline induced little mortality to the flies, survival was very quickly impaired as soon as the quantity of drugs was increased. For cefradine, chloramphenicol, and tetracycline, most of the mortality of the flies was recorded one day after injection, and survival remained relatively stable afterwards. Contrariwise, mortality progressed over the course of the experiment when the flies were injected ampicillin or cycloheximide, as depicted by the decrease of LD 50 from day 1 to 4; yet, the highest mortalities were observed after a day at the highest doses for these two antibiotics. Our findings suggest that cefradine, chloramphenicol, and tetracycline may have diffused and reached their targets more quickly within the flies after the injection than the two other antibiotics. Moreover, the time-dependent sequence of antibiotic-associated effects that inhibit various vital cellular functions, such as transcription, translation, or DNA supercoiling, or alter the microbiome of the flies, must be drug-specific.

At the highest antibiotic doses, the little to null activity of the ribosomes into the cells of the bacteria and flies likely explains the death of our insects.

The toxic effects of chloramphenicol were particularly striking, with survival of the flies dropping very quickly when the injected amount was only slightly increased (1.5 or 2 times as compared with the lowest dose), and no survival was measured at 300 µg chloramphenicol/fly. These results are consistent with the findings of [START_REF] Ashour | Chloramphenicol and ethidium bromide effects on flight-muscle mitochondria of the blowfly Calliphora erythrocephala[END_REF]Ashour et al. ( , 1980a) ) who reported low mortality in newly emerged flies of Calliphora erythrocephala injected with 100 µg chloramphenicol/fly, whereas all flies died when doses of this drug were higher than 400 µg/fly. There are now growing suspicions that chloramphenicol could induce aplastic anemia to human, and that it could be carcinogenic for vertebrates [START_REF] Baynes | Health concerns and management of select veterinary drug residues[END_REF], including ectothermic vertebrates [START_REF] El-Mofty | Induction of leukaemia in chloramphenicol-treated toads[END_REF]. However, the main physiological causes that provoked the death of our blow flies at moderate to high concentrations of chloramphenicol remain uncertain.

Tetracycline, which is often used for clearing insects infected by Wolbachia, had very low effects on the survival of the flies when injected at low amounts. However, a 3-or 4-fold increase of the injected dose greatly reduced survival, which reached 0% after 4 days at the highest concentration of tetracycline. A similar finding was found with cycloheximide.

Working with cycloheximide and other protein DNA synthesis inhibitors, Campbel & Birt (1975) reported in the sheep blow fly Lucilia cuprina that doses of 1 µg of cycloheximide were quickly detoxified by the individuals, whereas longer durations were required for higher concentrations, like for instance puromycin (mitochondrial protein inhibitor) at 5 µg. At moderate to high antibiotic concentrations, the detoxifying capacities of the blow flies may have been exceeded, resulting in the death of the individuals.

Our present survival data were used to calibrate the doses of antibiotics that were further injected to the insects to investigate the physiological effects of drugs on developing/maturing flies. We speculated that the different target / mode of action of each drug, and the differential rates of detoxification of each of these molecules by the insect, would explain the different survival kinetics we measured.

Effects of antibiotics on mitochondrial structure and oxidative rate

Most of the mitochondria of 1-day-old flies were not fully differentiated, i.e. cristae were not densely packed, which is consistent with the findings of [START_REF] Gregory | An electron-microscopic study of flight muscle development in the blowfly Lucilia cuprina[END_REF] on the blow fly Lucilia cuprina. The cytoplasmic and mitochondrial protein inhibitors we used induced marked changes to the mitochondrial ultrastructure of the flies, with chloramphenicol, cycloheximide and tetracyline having the most severe effects, by provoking swelling, disorganization of cristae, vacuolation and whorl shape formation. Consistently, the mitochondrial protein inhibitors chloramphenicol and ethidium bromide caused irregular alignment of cristae in C. erythrocephala (Ashour et al., 1980b); higher doses inflicted considerable swelling to whole mitochondria, most likely because cristae maintenance and maturation post emergence require considerable amounts of ATP production from mitochondria. Similar changes, i.e. swollen, short and fragmented mitochondria, have also been reported from mammalian cells [START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF], demonstrating the genericity of these antibiotic-induced changes across taxa.

Whorl-like shapes of the mitochondria were particularly significant for the blow fly Ch. albiceps treated with cycloheximide. [START_REF] Bulos | Bioenergetic properties of mitochondria from flight muscle of aging blowflies[END_REF][START_REF] Webb | Are there major degenerative changes in the flight muscle of ageing Diptera?[END_REF][START_REF] Webb | Are there major degenerative changes in the flight muscle of ageing Diptera?[END_REF] suggested that whorls are associated with the absence of cytochrome oxidase band, and possibly of other key enzymes. In the absence of a repair mechanism, configuration of the membranes could change towards an organization observed in myelin sheath i.e. concentric lipoprotein bilayers which seem more stable configuration in these circumstances.

Three out of the five injected antibiotics used in this study are inhibitors of protein synthesis. Consistently, the amount of proteins of mitochondria was decreased for up to ca.

25% in flies treated with chloramphenicol, cycloheximide and tetracycline. These results confirmed that the drugs reached their targets and affected the physiology of the flies. [START_REF] Sebald | Incorporation of amino acids into mitochondrial protein of the flight muscle of Locusta migratoria in vitro and in vivo in the presence of cycloheximide[END_REF], in Locusta migratoria, and Bartelink & DeKort (1973), in the Colorado beetle, found that the mitochondrial protein synthesized in the presence of cycloheximide was about 15% lower than in controls. Ashour et al. (1980a) found that the highest rate of protein synthesis occurred 3 to 4 days after emergence in C. erythrocephala, and the highest values of flight muscle mitochondrial proteins were recorded in 6-10 days old flies. Consistent with this observation, Ashour et al. (1980a) found that flies displayed the greatest susceptibility to drug treatment when the protein synthesis was the most intense (in 3 to 4 days old flies), while older flies (6-10 days old flies) displayed the greatest resistance to drug treatment. As we worked with newly emerged flies, we may have minimized the effects of drugs on protein synthesis, which could have been greater some few days later.

An injection of chloramphenicol (100 or 200 µg/fly) was expected to limit protein synthesis (50S ribosomal subunits) by inhibiting the activity of the peptidyl transferase and cytochrome oxidase enzymes [START_REF] Houtkooper | Mitonuclear protein imbalance as a conserved longevity mechanism[END_REF]. The earlier investigations conducted on this drug reported a 92% inhibition of the protein synthesis in Saccharomyces cereviciae [START_REF] Schweyen | Protein synthesis by yeast mitochondria in vivo. Quantitative estimate of mitochondrially governed synthesis of mitochondrial proteins[END_REF]; in the Colorado beetle, doses of chloramphenicol of 50, 100 and 150 µg induced an inhibition of the mitochondrial protein ranging from 40 to 70% (Bartelink & DeKort, 1973). In the present work, the amount of mitochondrial proteins of the flight muscles were progressively reduced when the dose of chloramphenicol was increased, and the highest dose of this antibiotic resulted in a 30% reduction of the protein content. The electrophoretical differentiation of the proteins, as compared with control flies, revealed that chloramphenicol, but also cycloheximide, rapidly decreased mitochondrial protein synthesis, and had the highest effect on protein subunits differentiation. Cycloheximide alters ribosomes when translation is ongoing [START_REF] De Loubresse | Structural basis for the inhibition of the eukaryotic ribosome[END_REF]. As the turnover of the synthesis of protein subunits from blow fly flight muscle mitochondria is relatively low over time (Jeffreys & Craig, 1976a;Jeffreys & Craig, 1976b;[START_REF] Ashour | Differentiation between mitochondrial and cytoplasmic protein subunits of blowfly flight muscle mitochondria synthesized in vivo[END_REF], the observed changes are maintained over several days.

In mammalian cells, antibiotics have been demonstrated to induce mitochondria dysfunction [START_REF] Houtkooper | Mitonuclear protein imbalance as a conserved longevity mechanism[END_REF][START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF][START_REF] Moullan | Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research[END_REF]. Damages and swelling of mitochondria may lead to reduction to suppression of ATP production, because antibiotics alter the inner membrane of mitochondria, in turn affecting the coupling of oxygen consumption to ADP phosphorylation [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. In this part of the study, all antibiotics were injected at doses that cause ca. 50% mortality after one day (except for ampicillin), and we reported a considerable decrease of the oxidative rate of surviving flies as compared with controls. There are few data on the mode of action of antibiotics in insects; in mammals, antibiotics have been proven to alter the electron transfer chain, thus decreasing the mitochondrial membrane potential and ATP levels [START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF]. The leakage of electrons from the electron transfer chain also contributes to increase the production of reactive oxygen species. For instance, treatments of mammalian cells with ampicillin increased the amount of superoxide ions, which potentially induces increased DNA and protein damages [START_REF] Kalghatgi | Bactericidal antibiotics induce mitochondrial dysfunction and oxidative damage in mammalian cells[END_REF]. While electron leaks are expected to affect mitochondrial coupling efficiency, the P/O ratios were similar among the different experimental conditions. This result suggests that the electrochemical gradient was not affected by the drugs, and revealed that ATP production versus O 2 consumption was reduced in antibiotic-treated flies.

The putative mitochondrial dysfunctions post-antibiotic treatments can be explored by ratiometric analysis of O 2 consumption (QO 2 ) after addition of pyruvate and proline (state 2 of mitochondrial respiration), followed by the addition of ADP to reach state 3, and returning to basal oxygen mitochondrial consumption when all ADP had been phosphorylated (state 4).

Respiratory control ratio of state 3/state 4 is a measure of the level of mitochondrial coupling and phosphorylation efficiency [START_REF] Brand | Assessing mitochondrial dysfunction in cells[END_REF]. In antibiotic-treated flies, mitochondrial functions were clearly impaired, as shown by the significant decrease of oxidation rate of pyruvate+proline. It is however not possible to decipher if the reduced oxidation of these two compounds resulted from a dysfunction of state 2 or state 3, or both.

An alteration of the state 3, for instance, by alteration of the activity of the Krebs cycle enzyme ɑ-ketoglutarate dehydrogenase, could have lowered the demand for substrate for oxidative phosphorylation.

Physiological underpinnings of the effects of antibiotics

We observed two distinct survival patterns, for flies injected with cefradine, chloramphenicol, or tetracycline on the one hand, and flies injected with ampicillin or cycloheximide on the other hand. In parallel, we demonstrated that all five antibiotics caused mitochondria dysfunctions. To date, metabolic changes resulting from antibiotic treatments have been mostly investigated in microorganisms [START_REF] Olszewska | Mitochondria as a pharmacological target: magnum overview[END_REF]. For the metabolomics approach, we restricted our analyses to flies treated with two doses of chloramphenicol, cycloheximide and tetracycline, in addition to control and saline injected flies. The metabolic profiles of the two untreated groups (controls, saline-injected flies) overlaped and clustered in the same branch of the HCA. Metabolism of the organisms is often very sensitive to environmental stressors and/or injuries, by mediating adequate responses to maintain physiological homeostasis [START_REF] Colinet | Metabolic effects of CO 2 anaesthesia in Drosophila melanogaster[END_REF][START_REF] Hidalgo | Metabolic fingerprinting to salinity in the invasive ground Merizodus soledadinus at the Kerguelen Islands: does physiological plasticity and ecological range match?[END_REF][START_REF] Colinet | Similar post-stress metabolic trajectories in young and old flies[END_REF]. Importantly, the quasi superposition of the metabolic phenotypes of the two control groups ensures that the observed repatterning of the metabolism of antibiotic-treated flies depicts the sole effects of the drugs. Interestingly, the metabolic changes were not affected by drug dosage, and were specific to the antibiotics that were injected.

Consistent with survival patterns, metabolic phenotypes of flies treated with chloramphenicol and tetracycline were similar, but distinct from the one measured from flies treated with cycloheximide. Injection of cycloheximide, an antifungal antibiotics that inhibits protein synthesis and that is used in agriculture [START_REF] Mcmanus | Antibiotic use in plant agriculture[END_REF][START_REF] Wang | Conversion of forest to agricultural land affects the relative contribution of bacteria and fungi to nitrification in humid subtropical soils[END_REF], had the most striking effect on the metabolic phenotype of the flies and provoked a decrease of the levels of most metabolites. In C. erythrocephala, Ashour et al. (1980a) found that cycloheximide can inhibit amino acid incorporation in the young flies, a finding consistent with the inhibition of RNA translocation induced by cycloheximide [START_REF] Schneider-Poetsch | Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin[END_REF]. The depletion of the quantities of most metabolites in flies treated with cycloheximide suggests that intermediary metabolism was globally depressed. The increased levels of fumaric acid measured from flies treated with cycloheximide should also be highlighted. We speculate that the conversion of succinic acid to fumaric acid remained functional in these flies, generating FADH 2 , but the transfer of the electrons, and their entry into the electrontransfer chain at the complex II, may have been blocked. This could have resulted in a greater production of fumaric acid and FADH 2 in regards to their demand, leading to the accumulation of fumaric acid.

Increased levels of putrescine were observed in chloramphenicol-and cycloheximidetreated flies. Putrescine often accumulates during anoxia in plant cells, and indirectly contributes to H 2 O 2 production under oxygen shortage [START_REF] Blokhina | Antioxidants, oxidative damage and oxygen deprivation stress: a review[END_REF].

Commonalities in the metabolic profiles were observed between chloramphenicol and tetracycline, with amounts of glucose, fructose-6-P, free phosphate, glucose-6-P and trehalose being increased in flies that received one of these two drugs. Studies conducted on microorganisms reported that bactericidal drugs alter the oxidative phosphorylation and the activity of the Krebs cycle, and resulted in increased amounts of metabolites from energetic metabolism [START_REF] Belenky | Bactericidal antibiotics induce toxic metabolic perturbations that lead to cellular damage[END_REF]. Yet, in our study, the amounts of TCA cycle intermediates were overall decreased, and more particularly at the intermediate antibiotic dose, which is consistent with our measurements of lower oxidative rates in these conditions. The reduction of the activity of the TCA cycle can be compensated by an increased activity of the glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT), which can provide alpha ketoglutarate [START_REF] Huang | Aspartate Aminotransferase (AST/GOT) and Alanine Aminotransferase (ALT/GPT) detection techniques[END_REF]. Our results are in accordance with this assumption, as the activity of the transaminases was significantly increased at 200 µg chloramphenicol/fly, likely accompanying the increased amounts of succinic, fumaric and malic acids, whereas levels of citric acid were reduced. In parallel, levels of alanine and aspartic acid remained high, suggesting the higher demand for these compounds that may be converted to glutamic acid instantaneously transformed to alpha ketoglutarate.

Transamination permits the biosynthesis of amino acids and the shift from sugar to protein metabolism according to the ecological situations encountered by organisms [START_REF] Bursell | The role of proline in energy metabolism[END_REF][START_REF] Cooper | Metabolism of Glutathione S-Conjugates: Multiple Pathways[END_REF]. The ratio GOT/GPT is often used as a biomarker of the health status of liver in human [START_REF] Kim | Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease[END_REF], and has been used to assess the level of xenobiotic-induced stress in insects [START_REF] Içen | Biochemical stress indicators of greater wax moth exposure to organophosphorus insecticides[END_REF][START_REF] Zibaee | A juvenile hormone analog, pyriproxifen, affects some biochemical components in the hemolymph and fat bodies of Eurygaster integriceps Puton (Hemiptera: Scutelleridae)[END_REF]. In our study, the large increase of GPT activity in flies treated with chloramphenicol altered GOT/GPT ratio may have revealed cell lesion to the flight muscles tissues of the treated flies. Lactic acid amounts were measured in high amounts from flight muscles of untreated flies. Lactic acid is a well-known product of anaerobic glycolysis but has also been experimentally demonstrated as a prominent oxidized substrate by muscles of rainbow trouts during glycogenesis [START_REF] Kam | Fuel use during glycogenesis in rainbow trout (Oncorhynchus mykiss Walbaum) white muscle studied in vitro[END_REF]. Levels of lactic acid were highly significantly reduced in flies treated with chloramphenicol and cycloheximide, and we speculated that this reduction affects the energy (ATP) required for the glycogenesis in muscles [START_REF] Kam | Fuel use during glycogenesis in rainbow trout (Oncorhynchus mykiss Walbaum) white muscle studied in vitro[END_REF][START_REF] Kane | Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work[END_REF]. Further studies should compare the amount of stored glycogen in treated and non-treated fly muscles to comfort this assumption. The activity of lactate dehydrogenase and lactate transporters [START_REF] Kane | Lactate oxidation at the mitochondria: a lactate-malate-aspartate shuttle at work[END_REF] that may have been greatly altered by chloramphenicol and cycloheximide treatments could also be quantified.

Conclusion

Low doses of antibiotics had limited effects on the survival of newly emerged flies of Ch.

albiceps. Yet, the extracellular footprints and physiological intracellular fingerprints we used demonstrated that these drugs have multiple direct and cascading effects on the structure of the flight muscles and physiology of the flies. The time-dependent sequence of antibioticassociated effects was drug-specific, with LD 50 that greatly varied for ampicillin and cycloheximide from day 1 to 4 post injection. Flies treated with chloramphenicol and tetracycline had altered oxidative phosphorylation and activity of the Krebs cycle, likely compensated by an increased activity of transaminase enzymes. The drop of oxidative phosphorylation correlated with damages of mitochondria. Cycloheximide had the most pronounced physiological effects on the phenotype of the flies, and quickly provoked their death, even at low doses. Overall, our findings suggest that antibiotic pollution could greatly affect insect population dynamics, which could be particularly problematic for beneficial insects and insect community structure. The effects on the trophic network should be explored in future studies.

FUNDING

Figure legends FIGURE 1

 legends1 Figure legends

FIGURE 2

 2 FIGURE 2 Concentrations of mitochondrial proteins (mean ± S.E.; N = 4 replicates per

FIGURE 3 FIGURE 4

 34 FIGURE 3 Oxidation rate of pyruvate+proline (mean ± S.E.) from flight muscle

FIGURE 5

 5 FIGURE 5 Scores and contribution plots of principal component analysis (PCA) of flight

AUTHOR CONTRIBUTIONS

Conceptualization

CONFLICT OF INTEREST

The authors declare no conflict of interest.

This study was supported by The Research Support Fund Program of the Faculty of Science, Cairo University, Egypt and by the grant 'ENVIE' from the Institut Universitaire de France (Paris, France). Part of the biochemical analyses were performed at the analytical platform 'PLAY' (University of Rennes 1, UMR CNRS EcoBio, Rennes, France).