Energy Homeostasis Control in Drosophila Adipokinetic Hormone Mutants

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.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.     

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.     

Low doses of paraquat and polyphenols prolong life span and locomotor activity in knock-down parkin Drosophila melanogaster exposed to oxidative stress stimuli: Implication in autosomal recessive juvenile Parkinsonism

Previous studies have shown that polyphenols might be potent neuroprotective agents in Drosophila melanogaster wild type Canton-S acutely or chronically treated with paraquat (PQ), a selective toxin for elimination of dopaminergic (DAergic) neurons by oxidative stress (OS), as model of Parkinson's disease (PD). This study reports for the first time that knock-down (K-D) parkin Drosophila melanogaster (TH-GAL4; UAS-RNAi-parkin) chronically exposed to PQ (0.1–0.25 mM), FeSO₄ (Fe, 0.1 mM), deferoxamine (DFO, 0.01 mM) alone or (0.1 mM) PQ in combination with polyphenols propyl gallate (PG, 0.1 mM) and epigallocathecin gallate (EGCG, 0.1, 0.5 mM) showed significantly higher life span and locomotor activity than untreated K-D flies or treated with (1, 5, 20 mM) PQ alone. Whilst gallic acid (GA, 0.1, 0.5 mM) alone or in the presence of PQ provoked no effect on K-D flies, epicathecin (EC, 0.5 mM) only showed a positive effect on prolonging K-D flies’ life span. It is shown that PG (and EGCG) protected protocerebral posterolateral 1 (PPL1) DAergic neurons against PQ. Interestingly, the protective effect of low PQ concentrations, DFO and iron might be explained by a phenomenon known as “hormesis.” However, pre-fed K-D flies with (0.1 mM) PQ for 7 days and then exposed to (0.25 mM) for additional 8 days affect neither survival nor climbing of K-D Drosophila compared to flies treated with (0.25 mM) PQ alone. Remarkably, K-D flies treated with 0.1 mM PQ (7 days) and then with (0.25 mM) PQ plus PG (8 days) behaved almost as flies treated with (0.25 mM) PQ. Taken these data suggest that antioxidant supplements that synergistically act with low pro-oxidant stimuli to prolong and increase locomotor activity become inefficient once a threshold of OS has been reached in K-D flies. Our present findings support the notion that genetically altered Drosophila melanogaster as suitable model to study genetic and environmental factors as causal and/or modulators in the development of autosomal recessive juvenile Parkinsonism (AR-JD)/PD. Most importantly, we have shown for the first time that low amounts of stressors induce a health-promoting extending effect in K-D parkin flies. Altogether our present results open new avenues for the screening, testing and development of novel antioxidant drugs against OS stimuli in neurodegenerative disorders.     

Dmp53, basket and drICE gene knockdown and polyphenol gallic acid increase life span and locomotor activity in a Drosophila Parkinson’s disease model

Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson’s disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ² test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively “switching off” death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition.     

Efficacy of methuselah gene mutation toward tolerance of dichlorvos exposure in Drosophila melanogaster

Adverse reports on the exposure of organisms to dichlorvos (DDVP; an organophosphate insecticide) necessitate studies of organismal resistance/tolerance by way of pharmacological or genetic means. In the context of genetic modulation, a mutation in methuselah (mth; encodes a class II G-protein-coupled receptor (GPCR)) is reported to extend (~35%) the life span of Drosophila melanogaster and enhance their resistance to oxidative stress induced by paraquat exposure (short term, high level). A lack of studies on organismal tolerance of DDVP by genetic modulation prompted us to examine the protective efficacy of mth mutation in exposed Drosophila. Flies were exposed to 1.5 and 15.0 ng/ml DDVP for 12–48 h to examine oxidative stress endpoints and chemical resistance. After prolonged exposure of flies to DDVP, antioxidant enzyme activities, oxidative stress, glutathione content, and locomotor performance were assayed at various days (0, 10, 20, 30, 40, 50) of age. Flies with the mth mutation (mth¹) showed improved chemical resistance and rescued redox impairment after acute DDVP exposure. Exposed mth¹ flies exhibited improved life span along with enhanced antioxidant enzyme activities and rescued oxidative perturbations and locomotor insufficiency up to middle age (~20 days) over similarly exposed w¹¹¹⁸ flies. However, at late (≥30 days) age, these benefits were undermined. Further, similarly exposed mth-knockdown flies showed effects similar to those observed in mth¹ flies. This study provides evidence of tolerance in organisms carrying a mth mutation against prolonged DDVP exposure and further warrants examination of similar class II GPCR signaling facets toward better organismal health.     

Neuroprotective Effect of Decalepis hamiltonii in Paraquat-Induced Neurotoxicity in Drosophila melanogaster: Biochemical and Behavioral Evidences

In this paper, we have demonstrated for the first time, the antioxidant and neuroprotective effects of Decalepis hamiltonii (Dh) root extract against paraquat (PQ)-induced oxidative stress and neurotoxicity in Drosophila melanogaster. Exposure of adult D. melanogaster (Oregon K) to PQ induced oxidative stress as evidenced by glutathione depletion, lipid peroxidation and enhanced activities of antioxidant enzymes such as catalase, superoxide dismutase as well as elevated levels of acetylcholine esterase. Pretreatment of flies by feeding with Dh extract (0.1, 0.5 %) for 14 days boosted the activities of antioxidant enzymes and prevented the PQ-induced oxidative stress. Dietary feeding of Dh extract prior to PQ exposure showed a lower incidence of mortality and enhanced motor activities of flies in a negative geotaxis assay; both suggesting the neuroprotective potential of Dh. Based on the results, we contemplate that the roots of Dh might prevent and ameliorate the human diseases caused by oxidative stress. The neuroprotective action of Dh can be attributed to the antioxidant constituents while the precise mechanism of its action needs further investigations.     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.     

Adipokinetic hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus hemolymph in response to oxidative stress

The in vivo effects of oxidative stress on adipokinetic hormone (AKH) titer in short-winged (brachypterous) males of the firebug Pyrrhocoris apterus were tested using paraquat (PQ), a bipyridilium herbicide. PQ undergoes a cyclic redox reaction with oxygen during microsomal and electron transfer reactions forming free radicals in the insect body. Oxidative insult (40 pmol PQ) resulted in enhanced protein carbonylation (a biomarker for oxidative stress) and a depletion of glutathione (GSH) pool in the hemolymph. Interestingly, AKH titer was significantly enhanced in hemolymph at 4 h post inoculation of PQ, while its content in CNS (brain with corpora cardiaca) showed non-specific changes in comparable period. Co-injection of AKH with PQ (40 pmol each) reversed these effects by decreasing protein carbonyl formation, increasing reduced GSH levels, and enhancing the total antioxidant capacity of cell free plasma. Our results indicate that there is a positive feedback regulation between an oxidative stressor action and the level of AKH in insect body, and that AKHs might be involved in the activation of antioxidant protection mechanism.     

ACUTE EXPOSURE OF Drosophila melanogaster TO PARAQUAT CAUSES OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION

Paraquat (PQ; 1, 1′‐dimethyl‐4‐4′‐bipyridinium), an herbicide and model neurotoxicant, is identified to be one of the prime risk factors in Parkinson's disease (PD). In the Drosophila system, PQ is commonly used to measure acquired resistance against oxidative stress (PQ resistance test). Despite this, under acute PQ exposure, data on the oxidative stress response and associated impact on mitochondria among flies is limited. Accordingly, in this study, we measured markers of oxidative stress and mitochondrial dysfunctions among adult male flies (8–10 days old) exposed to varying concentrations of PQ (10, 20, and 40 mM in 5% sucrose solution) employing a conventional filter disc method for 24 h. PQ exposure resulted in significant elevation in the levels of oxidative stress biomarkers (malondialdehyde: 43% increase: hydroperoxide: 32–39% increase), with concomitant enhancement in reduced glutathione and total thiol levels in cytosol. Higher activity of antioxidant enzymes were also evident along with increased free iron levels. Furthermore, PQ exposure caused a concentration‐dependent increase in mitochondrial superoxide generation and activity of manganese‐superoxide dismutase (Mn‐SOD). The activity levels of complex I‐III, complex II‐III, and Mg⁺² adinosine triphosphatase (ATPase) were also decreased significantly. A robust diminution in the activity of succinate dehydrogenase and moderate decline in the citrate synthase activity suggested a specific effect on citric acid cycle enzymes. Collectively, these data suggest that acute PQ exposure causes significant oxidative stress and mitochondrial dysfunction among flies in vivo. It is suggested that in various experimental settings, while conducting the “PQ resistance stress test” incorporation of selected biochemical end points is likely to enhance the quality of the data.     

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of Drosophila melanogaster

The composition of the intestinal microbiota of Drosophila has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in Drosophila melanogaster. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that Chd1 deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although Acetobacteraceae dominated the microbiota of both Chd1 wild-type and mutant guts, Chd1 mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between Chd1 mutant and control flies. Finally, diet supplementation experiments with Lactobacillus plantarum revealed that, in contrast to wild-type flies, Chd1 mutant flies were unable to maintain higher L. plantarum titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of Drosophila melanogaster.     

Water loss through the integument in the desiccation-sensitive mutant, Parched, of Drosophila melanogaster

Two desiccation-sensitive mutants of Drosophila melanogaster were isolated. Genetic analysis showed that the phenotype is controlled by a recessive gene parched located in 1A1-8 of the X-chromosome. In a desiccated environment without any water supply, the survival time of the mutant flies was considerably shorter than that of the wild-type flies. The rate of water loss in the mutant flies was significantly higher than that of the wild-type flies, whether dead or alive. The survival time of the mosaic flies, which have the mutant and wild-type cuticle, was prolonged in proportion to the amount of wild-type cuticle which they possessed. These results suggest that the mutant has a defect in some waterproofing mechanism of the integument. The mutant flies drank much more water than the wild-type flies, to compensate for the rapid water loss. The hydrocarbons, which are the predominant constituent of cuticular lipids, were analyzed by gas-liquid chromatography, but there were no significant quantitative nor qualitative differences between the wild-type and the mutant flies.     

Role of Bacopa monnieri in the temporal regulation of oxidative stress in clock mutant (cryb) of Drosophila melanogaster

Accruing evidences imply that circadian organization of biochemical, endocrinological, cellular and physiological processes contribute to wellness of organisms and in the development of pathologies such as malignancy, sleep and endocrine disorders. Oxidative stress is known to mediate a number of diseases and it is notable to comprehend the orchestration of circadian clock of a model organism of circadian biology, Drosophila melanogaster, under oxidative stress. We investigated the nexus between circadian clock and oxidative stress susceptibility by exposing D. melanogaster to hydrogen peroxide (H₂O₂) or rotenone; the reversibility of rhythms following exposure to Bacopa monnieri extract (ayurvedic medicine rich in antioxidants) was also investigated. Abolishment of 24 h rhythms in physiological response (negative geotaxis), oxidative stress markers (protein carbonyl and thiobarbituric acid reactive substances) and antioxidants (superoxide dismutase, catalase, glutathione-S-transferase and reduced glutathione) were observed under oxidative stress. Furthermore, abolishment of per mRNA rhythm in H₂O₂ treated wild type flies and augmented susceptibility to oxidative stress in clock mutant (cry^b) flies connotes the role of circadian clock in reactive oxygen species (ROS) homeostasis. Significant reversibility of rhythms was noted following B. monnieri treatment in wild type flies than cry^b flies. Our experimental approach revealed a relationship involving oxidative stress and circadian clock in fruit fly and the utility of Drosophila model in screening putative antioxidative phytomedicines prior to their use in mammalian systems.     

Strain‐specific effects of parental age on offspring in Drosophila melanogaster

Maternal age is generally known to be negatively correlated with the lifespan of offspring in several animal models including yeast, rotifers, flies, and possibly in humans. However, several reports have shown positive effects of parental age on offspring lifespan. Thus, there was a need to investigate further the inconsistent results on the effect of parental age on lifespan. In this study, the effects of parental age on offspring fitness and lifespan were examined by using Drosophila melanogaster. The lifespan of offspring from old parents was significantly increased compared with that of the young counterparts in the Canton‐S (CS) strain but not in other D. melanogaster strains, such as Oregon‐R (OR) and w¹¹¹⁸. To find out why the lifespan is increased in the offspring from old parents in CS flies, fitness components that could modulate lifespan were examined in CS flies. Egg weight and body weight were reduced by parental aging and the offspring of old fathers or old mothers developed faster than that of the young. In addition, the offspring of old parents had increased resistance to oxidative and heat shock stresses. However, reproductive capacity, mating preference, and food intake were unaffected by parental aging. These results indicate that parental aging in CS strain D. melanogaster has beneficial effects on the lifespan and fitness of offspring. The presence of strain‐specific manner effects suggests that genetic background might be a significant factor in the parental age effect.