Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based on AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone [termed "adipokinetic hormone precursor-related peptide" (APRP)] is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. Here we employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, we provide evidence for a negative autoregulatory loop in Akh gene regulation.KEYWORDS Drosophila; adipokinetic hormone; adipokinetic hormone precursor-related peptide; energy homeostasis; stress resistance E NERGY homeostasis requires continuous compensation for fluctuations in the energy expenditure and availability of food resources. Organisms thus build up reserves under positive energy balance and catabolize them when the balance turns negative to retain stable levels of circulating energy fuel. Insulin signaling induces the uptake of excessive circulating sugars, thus promoting reserve accumulation (reviewed, e.g., in Saltiel and Kahn 2001; Cohen 2006), whereas energy mobilization is under the control of glucagon and glucocorticoid signaling in mammals (reviewed, e.g., in Rui 2014;Charron and Vuguin 2015) and adipokinetic hormone (AKH) signaling in insects (reviewed, e.g., in Van der Horst 2003;Lorenz and Gäde 2009;Bednářová et al. 2013a). Consistent with their fundamental physiological function in energy mobilization, AKHs are found not only in insects, but are common in Protostomia, where they have been identified both in Ecdyszoa (in Arthropoda, Tardigrada, and Priapulida) and Lophotrochozoa (in Mollusca, Rotifera, and Annelida) (Gäde 2009;Hauser and Grimmelikhuijzen 2014). Nevertheless, physiological functions of AKHs have been studied mainly in Arthropoda. Similar to mammals, also insects store lipids in the form of triacylglycerides (TGs) and as carbohydrates in the form of glycogen. The main storage organ for lipid and glycogen in insects is the fat body, which can thus b...

To unravel the evolutionarily conserved genetic network underlying energy homeostasis, we performed a systematic in vivo gene knockdown screen in Drosophila. We used a transgenic RNAi library enriched for fly orthologs of human genes to functionally impair about half of all Drosophila genes specifically in adult fat storage tissue. This approach identified 77 genes, which affect the body fat content of the fly, including 58 previously unknown obesity-associated genes. These genes function in diverse biological processes such as lipid metabolism, vesicle-mediated trafficking, and the universal store-operated calcium entry (SOCE). Impairment of the SOCE core component Stromal interaction molecule (Stim), as well as other components of the pathway, causes adiposity in flies. Acute Stim dysfunction in the fat storage tissue triggers hyperphagia via remote control of the orexigenic short neuropeptide F in the brain, which in turn affects the coordinated lipogenic and lipolytic gene regulation, resulting in adipose tissue hypertrophy.

Factors and mechanisms controlling lipometabolism homeostasis share a remarkable evolutionary conservation between humans and Drosophila flies. Accordingly, the Drosophila model has been successfully used to understand the pathophysiology of human metabolic diseases such as obesity. Body fat stores in species as different as humans and flies consist of neutral lipids, mainly triacylglycerols. Changes in body fat storage are a diagnostic phenotype of lipometabolism imbalances of genetic or environmental origin. Various methods have been developed to quantify Drosophila body fat storage. The most widely used method adopts a commercial coupled colorimetric assay designed for human serum triacylglycerol quantification, which is based on glycerol content determination after enzymatic conversion of glycerides into glycerol. The coupled colorimetric assay is compatible with large-scale genetic screen approaches and has been successfully applied to characterize central regulators of Drosophila lipometabolism. Recently, the applicability of the coupled colorimetric assay for Drosophila storage fat quantification has been questioned in principle. Here we compare the performance of the coupled colorimetric assay on Drosophila samples with thin layer chromatography, the “gold standard” in storage lipid analysis. Our data show that the presented variant of the coupled colorimetric assay reliably discriminates between lean and fat flies and allows robust, quick and cost-effective quantification of Drosophila body fat stores.

Drosophila melanogaster curled, one of the first fly mutants described by T. H. Morgan .90 years ago, is the founding member of a series of curled wing phenotype mutants widely used as markers in fruit fly genetics. The expressivity of the wing phenotype is environmentally modulated, suggesting that the mutation affects the metabolic status of cells rather than a developmental control gene. However, the molecular identity of any of the curled wing marker mutant genes is still unknown. In a screen for starvation-responsive genes, we previously identified the single fly homolog of the vertebrate nocturnin genes, which encode cytoplasmic deadenylases that act in the post-transcriptional control of genes by poly(A) tail removal of target mRNAs prior to their degradation. Here we show that curled encodes Drosophila Nocturnin and that the gene is required at pupal stage for proper wing morphogenesis after eclosion of the fly. Despite the complex ontogenetic expression pattern of the gene, curled is not expressed in the developing wing, and wing-specific curled knockdown mediated by RNAi does not result in the curled wing phenotype, indicating a tissue-nonautonomous, systemic mode of curled gene function. Our study not only presents an entry point into the functional analysis of invertebrate nocturnins but also paves the way for the identification of the still elusive Nocturnin target mRNAs by genetic suppressor screens on the curled wing phenotype.

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carboxylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the α-Esterase cluster. Much in contrast to the vital importance attributed to carboxylesterases in general, the in vivo function of individual α-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila melanogaster fat body. One of the fat body carboxylesterases, α-Esterase-7, was selected for mutational analysis by gene targeting to generate a deletion mutant fly. Phenotypic characterization of α-Esterase-7 null mutants and transgenic flies, which overexpress a chimeric α-Esterase-7:EGFP gene, reveals important functions of α-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented first deletion mutant of any α-Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of α-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.