IR76b-expressing neurons in Drosophila melanogaster are necessary for associative reward learning of an amino acid mixture.

Learning where to find nutrients while at the same time avoiding toxic food is essential for survival of any animal. Using Drosophila melanogaster larvae as a study case, we investigate the role of gustatory sensory neurons expressing IR76b for associative learning of amino acids, the building blocks of proteins. We found surprising complexity in the neuronal underpinnings of sensing amino acids, and a functional division of sensory neurons. We found that the IR76b receptor is dispensable for amino acid learning, whereas the neurons expressing IR76b are specifically required for the rewarding but not the punishing effect of amino acids. This unexpected dissociation in neuronal processing of amino acids for different behavioural functions provides a study case for functional divisions of labour in gustatory systems.

The Drosophila Baramicin polypeptide gene protects against fungal infection.

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.

Caffeine improves mitochondrial function in PINK1B9-null mutant Drosophila melanogaster.

Mitochondrial dysfunction plays a central role in Parkinson's disease (PD) and can be triggered by xenobiotics and mutations in mitochondrial quality control genes, such as the PINK1 gene. Caffeine has been proposed as a secondary treatment to relieve PD symptoms mainly by its antagonistic effects on adenosine receptors (ARs). Nonetheless, the potential protective effects of caffeine on mitochondrial dysfunction could be a strategy in PD treatment but need further investigation. In this study, we used high-resolution respirometry (HRR) to test caffeine's effects on mitochondrial dysfunction in PINK1B9-null mutants of Drosophila melanogaster. PINK1 loss-of-function induced mitochondrial dysfunction in PINK1B9-null flies observed by a decrease in O2 flux related to oxidative phosphorylation (OXPHOS) and electron transfer system (ETS), respiratory control ratio (RCR) and ATP synthesis compared to control flies. Caffeine treatment improved OXPHOS and ETS in PINKB9-null mutant flies, increasing the mitochondrial O2 flux compared to untreated PINKB9-null mutant flies. Moreover, caffeine treatment increased O2 flux coupled to ATP synthesis and mitochondrial respiratory control ratio (RCR) in PINK 1B9-null mutant flies. The effects of caffeine on respiratory parameters were abolished by rotenone co-treatment, suggesting that caffeine exerts its beneficial effects mainly by stimulating the mitochondrial complex I (CI). In conclusion, we demonstrate that caffeine may improve mitochondrial function by increasing mitochondrial OXPHOS and ETS respiration in the PD model using PINK1 loss-of-function mutant flies.

Drosophila Free-Flight Odor Tracking is Altered in a Sex-Specific Manner By Preimaginal Sensory Exposure.

In insects such as Drosophila melanogaster, flight guidance is based on converging sensory information provided by several modalities, including chemoperception. Drosophila flies are particularly attracted by complex odors constituting volatile molecules from yeast, pheromones and microbe-metabolized food. Based on a recent study revealing that adult male courtship behavior can be affected by early preimaginal exposure to maternally transmitted egg factors, we wondered whether a similar exposure could affect free-flight odor tracking in flies of both sexes. Our main experiment consisted of testing flies differently conditioned during preimaginal development in a wind tunnel. Each fly was presented with a dual choice of food labeled by groups of each sex of D. melanogaster or D. simulans flies. The combined effect of food with the cis-vaccenyl acetate pheromone (cVA), which is involved in aggregation behavior, was also measured. Moreover, we used the headspace method to determine the "odorant" identity of the different labeled foods tested. We also measured the antennal electrophysiological response to cVA in females and males resulting from the different preimaginal conditioning procedures. Our data indicate that flies differentially modulated their flight response (take off, flight duration, food landing and preference) according to sex, conditioning and food choice. Our headspace analysis revealed that many food-derived volatile molecules diverged between sexes and species. Antennal responses to cVA showed clear sex-specific variation for conditioned flies but not for control flies. In summary, our study indicates that preimaginal conditioning can affect Drosophila free flight behavior in a sex-specific manner.

Neuroprotective Effects of Lycium Barbarum Fruit Extract on Pink1B9Drosophila Melanogaster Genetic Model of Parkinson's Disease.

Lycium barbarum (LB) is a famous traditional Chinese medicinal plant as well as food supplement possessing various pharmacological functions such as anti-aging and antioxidant effects. The Parkinson's disease (PD)-related kinase Pink1 plays vital role in maintaining the neuron cell homeostasis, having been recognized as a potential target for the development of anti-PD drugs. In this work, the neuroprotective effects of methanol extract of LB fruit (LBFE) were investigated using a Drosophila PD model (PINK1B9) and a human neuroblastoma SH-SY5Y cell line. We found that when LBFE was supplied to the PINK1B9 flies at 6, 12, and 18 days of age, it raised the ATP and dopamine levels at all ages, extended life span, improved motor behavior, and rescued olfactory deficits of the PINK1B9 flies. In addition, histopathological examinations indicated that muscle atrophy in thoraces of the mutant flies was significantly repaired. Finally, LBFE was able to rescue the SH-SY5Y cells against MPP+-induced neurotoxicity. This work reports for the first time the anti-PD potential of L. barbarum fruit extract in PINK1 mutant fruit flies, presenting a new viewpoint for studing the mechanism of action of LBFE.

The Drosophila melanogaster antimicrobial peptides Mtk-1 and Mtk-2 are active against the malarial parasite Plasmodium falciparum.

Antimicrobial peptides (AMPs) are important components of the vertebrate and invertebrate innate immune systems. Although AMPs are widely recognized for their broad-spectrum activity against bacteria, fungi, and viruses, their activity against protozoan parasites has not been investigated in detail. In this study, we tested 10 AMPs from three different insect species: the greater wax moth Galleria mellonella (cecropin A-D), the fruit fly Drosophila melanogaster (drosocin, Mtk-1 and Mtk-2), and the blow fly Lucilia sericata (LSerPRP-2, LSerPRP-3 and stomoxyn). We tested each AMP against the protozoan parasite Plasmodium falciparum which is responsible for the most severe form of malaria in humans. We also evaluated the impact of these insect AMPs on mouse and pig erythrocytes. Whereas all AMPs showed low hemolytic effects towards mouse and pig erythrocytes, only D. melanogaster Mtk-1 and Mtk-2 significantly inhibited the growth of P. falciparum at low concentrations. Mtk-1 and Mtk-2 could therefore be considered as leads for the development of antiparasitic drugs targeting the clinically important asexual blood stage of P. falciparum.

Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75.

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric oxide is produced in the Drosophila prothoracic gland, where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates.

The insect-derived antimicrobial peptide metchnikowin targets Fusarium graminearum ß(1,3)glucanosyltransferase Gel1, which is required for the maintenance of cell wall integrity.

Antimicrobial peptides (AMPs) are essential components of the insect innate immune system. Their diversity provides protection against a broad spectrum of microbes and they have several distinct modes of action. Insect-derived AMPs are currently being developed for both medical and agricultural applications, and their expression in transgenic crops confers resistance against numerous plant pathogens. The antifungal peptide metchnikowin (Mtk), which was originally discovered in the fruit fly Drosophila melanogaster, is of particular interest because it has potent activity against economically important phytopathogenic fungi of the phylum Ascomycota, such as Fusarium graminearum, but it does not harm beneficial fungi such as the mycorrhizal basidiomycete Piriformospora indica. To investigate the specificity of Mtk, we used the peptide to screen a F. graminearum yeast two-hybrid library. This revealed that Mtk interacts with the fungal enzyme ß(1,3)-glucanosyltransferase Gel1 (FgBGT), which is one of the enzymes responsible for fungal cell wall synthesis. The interaction was independently confirmed in a second interaction screen using mammalian cells. FgBGT is required for the viability of filamentous fungi by maintaining cell wall integrity. Our study therefore paves the way for further applications of Mtk in formulation of bio fungicides or as a supplement in food preservation.

Switching cell fates in the developing Drosophila eye.

The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. Here, we ectopically supply Lz to precluster cells and concomitantly supply the various RTK/N codes that specify each of three second wave cell fates. We thereby reproduce molecular markers of each of the second wave cell types in precluster cells and draw three inferences. First, we confirm that Lz provides key intrinsic information to second wave cells. We can now combine this with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. Second, the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code. Third, RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate.

Bombyx mori prothoracicostatic peptide receptor is allosterically activated via a Gα(i/o)-protein-biased signalling cascade by Drosophila sex peptide.

In insects, molting and metamorphosis are strictly regulated by ecdysteroids. Ecdysteroid synthesis is positively or negatively controlled by several neuropeptides. The prothoracicostatic peptide (PTSP) BmPTSP (Bombyx mori prothoracicostatic peptide), isolated from the larval brain of B. mori, has been demonstrated to inhibit ecdysteroid synthesis in the prothoracic glands (PGs) [Hua et al. (1999) J. Biol. Chem. 274, 31169-31173]. More recently, the newly recognized B. mori receptor for Drosophila melanogaster sex peptide (DmSP) has been identified as a receptor for BmPTSP. However, details on the signalling pathways and physiological functions of this receptor have remained elusive. In the present paper, we report the functional characterization of the BmPTSP receptor (BmPTSPR)/sex peptide (SP) receptor (SPR) using both mammalian and insect cells. Synthetic DmSP shows the potential to inhibit forskolin (FSK) or adipokinetic hormone (AKH)-induced cAMP-response element (CRE)-driven luciferase (Luc) activity in a manner comparable with synthetic BmPTSP1. However, DmSP displayed a much lower activity in triggering Ca²âº mobilization and internalization than did BmPTSP1. Additionally, 6-carboxy-fluorescein fluorophore (FAM)-labelled DmSP and BmPTSP3 were found to bind specifically to BmPTSPR/SPR. The binding of FAM-DmSP was displaced by unlabelled DmSP, but not by unlabelled BmPTSP1 and, vice versa, the binding of FAM-BmPTSP3 was blocked by unlabelled BmPTSP3, but not by unlabelled DmSP. Moreover, internalization assays demonstrated that BmPTSP1, but not DmSP, evoked recruitment of the Bombyx non-visual arrestin, Kurtz, to the activated BmPTSPR/SPR in the plasma membrane. This was followed by induction of internalization. This suggests that BmPTSP1 is probably an endogenous ligand specific for BmPTSPR/SPR. We therefore designate this receptor BmPTSPR. In contrast, DmSP is an allosteric agonist that is biased towards Gα(i/o)-dependent cAMP production and away from Ca²âº mobilization and arrestin recruitment.

Effect of distal sugar and interglycosidic linkage of disaccharides on the activity of proline rich antimicrobial glycopeptides.

The effect of glycosylation on protein structure and function depends on a variety of intrinsic factors including glycan chain length. We have analyzed the effect of distal sugar and interglycosidic linkage of disaccharides on the properties of proline-rich antimicrobial glycopeptides, formaecin I and drosocin. Their glycosylated analogs-bearing lactose, maltose and cellobiose, as a glycan side chain on their conserved threonine residue, were synthesized where these disaccharides possess identical proximal sugar and vary in the nature of distal sugar and/or interglycosidic linkage. The structural and functional properties of these disaccharide-containing formaecin I and drosocin analogs were compared with their corresponding monoglycosylated forms, ß-D-glucosyl-formaecin I and ß-D-glucosyl-drosocin, respectively. We observed neither major secondary structural alterations studied by circular dichroism nor substantial differences in the toxicity with mammalian cells among all of these analogs. The comparative analyses of antibacterial activities of these analogs of formaecin I and drosocin displayed that ß-D-maltosyl-formaecin I and ß-D-maltosyl-drosocin were more potent than that of respective ß-D-Glc-analog, ß-D-cellobiosyl-analog and ß-D-lactosyl-analog. Despite the differences in their antibacterial activity, all the analogs exhibited comparable binding affinity to DnaK that has been reported as one of the targets for proline-rich class of antibacterial peptides. The comparative-quantitative internalization studies of differentially active analogs revealed the differences in their uptake into bacterial cells. Our results exhibit that the sugar chain length as well as interglycosidic linkage of disaccharide may influence the antibacterial activity of glycosylated analogs of proline-rich antimicrobial peptides and the magnitude of variation in antibacterial activity depends on the peptide sequence.

The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons.

Neuropeptides have widespread effects on behavior, but how these molecules alter the activity of their target cells is poorly understood. We employed a new model system in Drosophila melanogaster to assess the electrophysiological and molecular effects of neuropeptides, recording in situ from larval motor neurons, which transgenically express a receptor of choice. We focused on two neuropeptides, pigment-dispersing factor (PDF) and small neuropeptide F (sNPF), which play important roles in sleep/rhythms and feeding/metabolism. PDF treatment depolarized motor neurons expressing the PDF receptor (PDFR), increasing excitability. sNPF treatment had the opposite effect, hyperpolarizing neurons expressing the sNPF receptor (sNPFR). Live optical imaging using a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor for cyclic AMP (cAMP) showed that PDF induced a large increase in cAMP, whereas sNPF caused a small but significant decrease in cAMP. Coexpression of pertussis toxin or RNAi interference to disrupt the G-protein Gαo blocked the electrophysiological responses to sNPF, showing that sNPFR acts via Gαo signaling. Using a fluorescent sensor for intracellular calcium, we observed that sNPF-induced hyperpolarization blocked spontaneous waves of activity propagating along the ventral nerve cord, demonstrating that the electrical effects of sNPF can cause profound changes in natural network activity in the brain. This new model system provides a platform for mechanistic analysis of how neuropeptides can affect target cells at the electrical and molecular level, allowing for predictions of how they regulate brain circuits that control behaviors such as sleep and feeding.

ß-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development.

ß-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila ß-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of ß-arrestins and present a new mechanism of ERK inhibition by the Drosophila ß-arrestin Krz.

Betulinic Acid Increases the Lifespan of Drosophila melanogaster via Sir2 and FoxO Activation.

Betulinic acid (BetA), a triterpenoid derivative found abundantly in the plant kingdom, has emerged as a promising candidate for promoting longevity. Many research studies have shown its antioxidant, anti-inflammatory, antiviral, and anticancer activities, making it an interesting subject for investigating its potential influence on lifespan. This study aimed to investigate the effects of BetA on longevity and the mechanisms associated with it using the fruit fly Drosophila melanogaster as the organism model. The results showed that 50 µM BetA supplementation extended the mean lifespan of fruit flies by 13% in males and 6% in females without any adverse effects on their physiology, such as fecundity, feeding rate, or locomotion ability reduction. However, 50 µM BetA supplementation failed to increase the lifespan in mutants lacking functional silent information regulator 2 (Sir2) and Forkhead box O (FoxO)-null, implying that the longevity effect of BetA is related to Sir2 and FoxO activation. Our study contributes to the knowledge in the field of anti-aging research and inspires further investigations into natural compounds such as BetA to enhance organismal healthspan.

Inhibition of diuretic stimulation of an insect secretory epithelium by a cGMP-dependent protein kinase.

The rate of urine secretion by insect Malpighian tubules (MTs) is regulated by multiple diuretic and antidiuretic hormones, often working either synergistically or antagonistically. In the Drosophila melanogaster MT, only diuretic factors have been reported. Two such agents are the biogenic amine tyramine (TA) and the peptide drosokinin (DK), both of which act on the stellate cells of the tubule to increase transepithelial chloride conductance. In the current study, TA and DK signaling was quantified by microelectrode recording of the transepithelial potential in isolated Drosophila MTs. Treatment of tubules with cGMP caused a significant reduction in the depolarizing responses to both TA and DK, while cAMP had no effect on these responses. To determine whether a specific cGMP-dependent protein kinase (PKG) was mediating this inhibition, PKG expression was knocked down by RNAi in either the principal cells or the stellate cells. Knockdown of Pkg21D in the stellate cells eliminated the modulation of TA and DK signaling. Knockdown of Pkg21D with a second RNAi construct also reduced the modulation of TA signaling. In contrast, knockdown of the expression of foraging or CG4839, which encodes a known and a putative PKG, respectively, had no effect. These data indicate that cGMP, acting through the Pkg21D gene product in the stellate cells, can inhibit signaling by the diuretic agents TA and DK. This represents a novel function for cGMP and PKG in the Drosophila MT and suggests the existence of an antidiuretic hormone in Drosophila.

Daily rhythms in locomotor circuits in Drosophila involve PDF.

The neuropeptide pigment-dispersing factor (PDF) has been studied extensively in Drosophila, and its role in circadian time-keeping has been firmly established. The role of PDF outside of the clock circuit, however, is poorly understood. A recent study suggested that PDF may act on the ellipsoid body (EB) to link the clock and sleep/activity circuits. We performed whole brain optical imaging with the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps expressed under control of the pdfR promoter to address how the clock and sleep deprivation affect the physiology of these cells. Basal cAMP levels in EB were regulated both by PDF and synaptic inputs that are controlled by the circadian clock. Acute application of PDF to the brain caused a significant, and PDF-receptor-dependent, increase in cAMP in EB cells. Application of TTX to block circuit-mediated effects of PDF increased the morning response but not the response at night, implying the existence of a temporally regulated, PDF-stimulated input that blocks cAMP generation. ACh produced both direct (TTX-insensitive) and indirect (TTX-sensitive) increases in cAMP during the day but was totally TTX-insensitive at night, indicating that ACh-stimulated inputs to the EB are suppressed at night. Sleep deprivation did not affect the cAMP responses of these cells to either PDF or ACh. These results suggest a novel role for PDF as a modulator of activity outside of the clock circuit. By elucidating the mechanisms by which the neuropeptide PDF act on its target cells, our work contributes to our understating of how the central clock coordinates activity and sleep.

Deciphering the role of Hippo pathway in lung cancer.

Hippo pathway has been initially recognized as a regulatory mechanism for modulation of organ size in fruitfly. Subsequently, its involvement in the regulation of homeostasis and tumorigenesis has been identified. This pathway contains some tumor suppressor genes such as hippo (hpo) and warts (wts), as well as a number of oncogenic ones such as yorkie (yki). Recent studies have shown participation of Hippo pathway in the lung carcinogenesis. This pathway can affect lung cancer via different mechanisms. The interaction between some miRNAs and Hippo pathway is a possible mechanism for carcinogenic processes. Moreover, some other types of non-coding RNAs including PVT1, SFTA1P, NSCLCAT1 and circ_0067741 are implicated in this process. Besides, anti-cancer effects of gallic acid, icotinib hydrochloride, curcumin, ginsenoside Rg3, cryptotanshinone, nitidine chloride, cucurbitacin E, erlotinib, verteporfin, sophoridine, cisplatin and verteporfin in lung cancer are mediated through modulation of Hippo pathway. Here, we summarize the results of recent studies that investigated the role of Hippo signaling in the progression of lung cancer, the impact of non-coding RNAs on this pathway and the effects of anti-cancer agents on Hippo signaling in the context of lung cancer.

Protective effect of agar oligosaccharide on male Drosophila melanogaster suffering from oxidative stress via intestinal microflora activating the Keap1-Nrf2 signaling pathway.

Agar oligosaccharide (AOS) is a new kind of marine functional oligosaccharide with generous biological activities. To investigate the antioxidative effects of AOS in vivo, 3 % aqueous hydrogen peroxide (H2O2) was used to induce oxidative stress in male Drosophila melanogaster (D. melanogaster) fed 5 % sucrose (SUC). AOS (0.125 %) in the medium extended the lifespan of D. melanogaster suffering from oxidative stress by improving antioxidant capacity and intestinal function. Electron microscopic observation of epithelial cells showed that AOS alleviated the damage caused by H2O2 challenge in the intestine of D. melanogaster, including a reduction of gut leakage and maintenance of intestinal length and cell ultrastructure. The Keap1-Nrf2 (analogues of CncC gene in D. melanogaster) signaling pathway was significantly activated based on gene expression levels and a reduction in ROS content in the intestine of D. melanogaster suffering from oxidative stress. The improvement of antioxidant capacity may be related to the regulation of intestinal microflora with AOS supplementation for D. melanogaster. Nrf2-RNAi, sterile and gnotobiotic D. melanogaster were used to validate the hypothesis that AOS activated the Keap1-Nrf2 signaling pathway to achieve antioxidant effects by regulating intestinal microflora. The above results contribute to our understanding of the antioxidative mechanism of AOS and promote its application in the food industry.

Activated FOXO-mediated insulin resistance is blocked by reduction of TOR activity.

Reducing insulin/IGF signaling allows for organismal survival during periods of inhospitable conditions by regulating the diapause state, whereby the organism stockpiles lipids, reduces fertility, increases stress resistance, and has an increased lifespan. The Target of Rapamycin (TOR) responds to changes in growth factors, amino acids, oxygen tension, and energy status; however, it is unclear how TOR contributes to physiological homeostasis and disease conditions. Here, we show that reducing the function of Drosophila TOR results in decreased lipid stores and glucose levels. Importantly, this reduction of dTOR activity blocks the insulin resistance and metabolic syndrome phenotypes associated with increased activity of the insulin responsive transcription factor, dFOXO. Reduction in dTOR function also protects against age-dependent decline in heart function and increases longevity. Thus, the regulation of dTOR activity may be an ancient "systems biological" means of regulating metabolism and senescence, that has important evolutionary, physiological, and clinical implications.

The Drosophila copper transporter Ctr1C functions in male fertility.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.