Wild-type Drosophila Research Articles

A119: Intermittent Hypoxic Training Promotes Whole-Body HIF-1Α Expression to Improve Exercise Capacity

Research Context: It has been shown that a hypoxic environment induces up-regulation of HIF-1α and changes in its downstream gene sequences, and that exercising under hypoxic conditions improves athletes' sprint performance. However, there is more controversy regarding the effects of exercise under hypoxic conditions on exercise capacity, and the molecular mechanisms have been less well studied. Therefore, the aim of this study was to investigate the link between HIF-1α expression and exercise capacity under hypoxic conditions of exercise, and to provide a realistic theoretical basis for intermittent hypoxic training to improve exercise capacity. Research Methodology: A total of 480 wild-type female Drosophila flies that were fledged within 12 hours were collected and randomly divided into control (NC), hypoxic quiet (HC), normoxic exercise (NE), and intermittent hypoxic training (HE) groups, with 120 flies in each group. At 7 days of age, intermittent hypoxic, normoxic and hypoxic combined exercise interventions were performed for 5 consecutive days, lasting 1 hour per day, respectively. Gases used in both the HC and HE groups were a mixture of 6% O2 and 94% N2. RT-qPCR was used to detect the expression of HIF-1α throughout the body, and the average speed, acceleration, and distance moved per unit time of Drosophila were quantified using LoliTrack Animal Behavioral Analysis Software, as well as the jumping ability and climbing ability of Drosophila. Findings: Intermittent hypoxic exposure, normoxic exercise, and intermittent hypoxic training all significantly upregulated HIF-1α expression compared with the NC group (P < 0.001). In terms of jumping ability, both intermittent hypoxic training and normoxic exercise improved the climbing ability of Drosophila compared to the NC group (P < 0.001), whereas intermittent hypoxic exposure alone did not play a role. In terms of climbing ability, Drosophila's jumping ability was significantly improved after intermittent hypoxic training compared to the NC group (P < 0.001); however, no significant changes could be observed in experiments with normoxic exercise as well as intermittent hypoxic exposure. In terms of behavioral analysis, the mean speed of Drosophila movement was increased in the HE group compared to the NC group (P < 0.001), acceleration increased (P < 0.001), distance traveled increased (P < 0.001), and normoxic exercise increased the distance traveled by Drosophila (P < 0.05). Conclusion: Intermittent hypoxic training promotes HIF-1Α expression and improves locomotor performance in Drosophila.

Brown rice attenuates iron-induced Parkinson's disease phenotypes in male wild-type drosophila: insights into antioxidant and iron metabolism modulation.

Parkinson's disease (PD) is a progressive movement disorder associated with brain iron (Fe) accumulation and free radicals. Brown rice (BR) is antioxidant-rich and has been shown to ameliorate oxidative stress-induced damage. The aim of this study was to investigate the effects of BR compared to white rice (WR) on Fe-induced PD in a fruit fly model. Three-day-old male adult flies were divided into two groups: one on a normal diet and the other on Fe-diet (1 mmol/L) for 10 days to induce PD. After 10 days, the Fe-fed flies were redistributed into four groups: one on normal diet (Fe group), while the others were treated with BR (Fe+BR group), WR (Fe+WR group), or L-3,4-dihydroxyphenylalanine (L-dopa) (Fe+L-dopa group) for 5 days. Similarly, the flies initially on a normal diet were separated into four groups: one on normal diet (Control group), while the others were treated with BR (BR group), WR (WR group), or L-dopa (L-dopa group) for 5 days. Finally, Fe levels, dopamine, malonaldehyde (MDA), and antioxidant enzymes were measured, and the mRNA levels of antioxidant and Fe metabolism genes were assessed. BR significantly improved motor and cognitive functions, decreased fly head MDA and Fe levels, and increased antioxidant enzyme levels in comparison to the Fe and WR groups. Similarly, BR upregulated the mRNA levels of antioxidant genes: catalase, GPx, Nrf2, and DJ-1. The results suggest that BR could potentially reduce morbidities associated with PD possibly due to its bioactive compounds compared to WR.

Unique tau- and synuclein-dependent metabolic reprogramming in neurons distinct from normal aging.

Neuronal cells are highly specialized cells and have a specific metabolic profile to support their function. It has been demonstrated that the metabolic profiles of different cells/tissues undergo significant reprogramming with advancing age, which has often been considered a contributing factor towards aging-related diseases including Alzheimer's (AD) and Parkinson's (PD) diseases. However, it is unclear if the metabolic changes associated with normal aging predispose neurons to disease conditions or a distinct set of metabolic alterations happen in neurons in AD or PD which might contribute to disease pathologies. To decipher the changes in neuronal metabolism with age, in AD, or in PD, we performed high-throughput steady-state metabolite profiling on heads in wildtype Drosophila and in Drosophila models relevant to AD and PD. Intriguingly, we found that the spectrum of affected metabolic pathways is dramatically different between normal aging, Tau, or Synuclein overexpressing neurons. Genetic targeting of the purine and glutamate metabolism pathways, which were dysregulated in both old age and disease conditions partially rescued the neurodegenerative phenotype associated with the overexpression of wildtype and mutant tau. Our findings support a "two-hit model" to explain the pathological manifestations associated with AD where both aging- and Tau/Synuclein- driven metabolic reprogramming events cooperate with each other, and targeting both could be a potent therapeutic strategy.

Time-restricted feeding mediated modulation of microbiota leads to changes in muscle physiology in Drosophila obesity models.

Recent research has highlighted the essential role of the microbiome in maintaining skeletal muscle physiology. The microbiota influences muscle health by regulating lipid metabolism, protein synthesis, and insulin sensitivity. However, metabolic disturbances such as obesity can lead to dysbiosis, impairing muscle function. Time-restricted feeding (TRF) has been shown to mitigate obesity-related muscle dysfunction, but its effects on restoring healthy microbiomes remain poorly understood. This study utilizes 16S microbiome analysis and bacterial supplementation to investigate the bacterial communities influenced by TRF that may benefit skeletal muscle physiology. In wild-type and obese Drosophila models (axenic models devoid of natural microbial communities), the absence of microbiota influence muscle performance and metabolism differently. Specifically, axenic wild-type Drosophila exhibited reduced muscle performance, higher glucose levels, insulin resistance, ectopic lipid accumulation, and decreased ATP levels. Interestingly, in obese Drosophila (induced by a high-fat diet or predisposed obesity mutant Sk2), the absence of microbiota improved muscle performance, lowered glucose levels, reduced insulin resistance, and increased ATP levels. TRF was found to modulate microbiota composition, notably increasing Acetobacter pasteurianus (AP) and decreasing Staphylococcus aureus (SA) in both obesity models. Supplementation with AP improved muscle performance and reduced glucose and insulin resistance, while SA supplementation had the opposite effect. This study provides novel insights into the complex interactions between TRF, microbiota, and skeletal muscle physiology in different Drosophila models.

Aging impaired locomotor and biochemical activities in Drosophila melanogaster Oregon R (fruit fly) model

Despite advancements in healthcare and increased lifespan, aging populations face numerous challenges, including declining cognitive function, increased susceptibility to chronic diseases, and reduced quality of life. This study investigated Aging impaired Locomotors and Biochemical Activities in Drosophila melanogaster Oregon R (Fruit Fly) Model with the aim to elucidate the mechanism involved. Adult wild-type Drosophila melanogaster Oregon R was used for this study. Survival assay, antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and malondialdehyde (MDA)) and total protein (TP) concentration were investigated. Data obtained were analyzed using R studio and GraphPad Prism. The result indicated low survival in male flies compared to female flies and the highest survival rate was observed with both flies reared together in the same vial. There was impaired locomotor activity in the flies with age. There was a significant decrease in the level of SOD, CAT, GSH and TP with age with a corresponding significant increase in the level of MDA. This finding demonstrated that locomotor activity decreased with aging with decrease performance index and also established the involvement of oxidation through the activities of antioxidant enzymes in aging; decreased (p < 0.05) concentration of antioxidant enzymes and increased (p < 0.05) lipid peroxidation. Also, it demonstrated that female species had longer lifespan compared to males while co-habiting of male and female species extended lifespan.

High sugar diet promotes tumor progression paradoxically through aberrant upregulation of pepck1

High dietary sugar (HDS), a contemporary dietary concern due to excessive intake of added sugars and carbohydrates, escalates the risk of metabolic disorders and concomitant cancers. However, the molecular mechanisms underlying HDS-induced cancer progression are not completely understood. We found that phosphoenolpyruvate carboxykinase 1 (PEPCK1), a pivotal enzyme in gluconeogenesis, is paradoxically upregulated in tumors by HDS, but not by normal dietary sugar (NDS), during tumor progression. Targeted knockdown of pepck1, but not pepck2, specifically in tumor tissue in Drosophila in vivo, not only attenuates HDS-induced tumor growth but also significantly improves the survival of Ras/Src tumor-bearing animals fed HDS. Interestingly, HP1a-mediated heterochromatin interacts directly with the pepck1 gene and downregulates pepck1 gene expression in wild-type Drosophila. Mechanistically, we demonstrated that, under HDS conditions, pepck1 knockdown reduces both wingless and TOR signaling, decreases evasion of apoptosis, reduces genome instability, and suppresses glucose uptake and trehalose levels in tumor cells in vivo. Moreover, rational pharmacological inhibition of PEPCK1, using hydrazinium sulfate, greatly improves the survival of tumor-bearing animals with pepck1 knockdown under HDS. This study is the first to show that elevated levels of dietary sugar induce aberrant upregulation of PEPCK1, which promotes tumor progression through altered cell signaling, evasion of apoptosis, genome instability, and reprogramming of carbohydrate metabolism. These findings contribute to our understanding of the complex relationship between diet and cancer at the molecular, cellular, and organismal levels and reveal PEPCK1 as a potential target for the prevention and treatment of cancers associated with metabolic disorders.

Anti-aging and immunomodulatory role of caffeine in Drosophila larvae.

Drug repurposing is a promising approach to identify new pharmacological indications for drugs that have already been established. However, there is still a limitation in the availability of a high-throughput in vivo preclinical system that is suitable for screening and investigating new pharmacological indications. The aim of this study was to introduce the application of Drosophila larvae as an in vivo platform to screen drug candidates with anti-aging and immunomodulatory activities. To determine whether Drosophila larvae can be utilized for assessing anti-aging and immunomodulatory activities, phenotypical and molecular assays were conducted using wildtype and mutant lines of Drosophila. The utilization of mutant lines (PGRP-LBΔ and Psh[1];;ModSP[KO]) mimics the autoinflammatory and immunodeficient conditions in humans, thereby enabling a thorough investigation of the effects of various compounds. The phenotypical assay was carried out using survival and locomotor observation in Drosophila larvae and adult flies. Meanwhile, the molecular assay was conducted using the RT-qPCR method. In vivo survival analysis revealed that caffeine was relatively safe for Drosophila larvae and exhibited the ability to extend Drosophila lifespan compared to the untreated controls, suggesting its anti-aging properties. Further analysis using the RT-qPCR method demonstrated that caffeine treatment induced transcriptional changes in the Drosophila larvae, particularly in the downstream of NF-κB and JAK-STAT pathways, two distinct immune-related pathways homologue to humans. In addition, caffeine enhanced the survival of Drosophila autoinflammatory model, further implying its immunosuppressive activity. Nevertheless, this compound had minimal to no effect on the survival of Staphylococcus aureus-infected wildtype and immunodeficient Drosophila, refuting its antibacterial and immunostimulant activities. Overall, our results suggest that the anti-aging and immunosuppressive activities of caffeine observed in Drosophila larvae align with those reported in mammalian model systems, emphasizing the suitability of Drosophila larvae as a model organism in drug repurposing endeavors, particularly for the screening of newly discovered chemical entities to assess their immunomodulatory activities before proceedings to investigations in mammalian animal models.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila

Background Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. Methods We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. Results We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Conclusions Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Withania somnifera showed neuroprotective effect and increased longevity in Drosophila Alzheimer’s disease model

Alzheimer’s disease is a unique neurodegenerative condition in humans that is characterized by amyloid beta accumulation in the brain parenchyma. Withania somnifera, commonly known as Ashwagandha, is a plant that has been used for centuries in Indian Ayurvedic medicine to treat various health conditions. The active compound in Ashwagandha has shown to be beneficial in treating several neurodegenerative diseases, including Alzheimer's disease (AD). This study used a Drosophila melanogaster AD model to investigate the effect of Ashwagandha in reducing amyloid beta toxicity and promoting longevity. The findings showed that 20 mg/mL of Ashwagandha root powder effectively rescued the "rough eye phenotype" in AD Drosophila and increased longevity in both AD model and wild-type Drosophila. Overall, the results suggest that Ashwagandha may have potent therapeutic potential for treating AD and maintaining cellular well-being.

Fertility differences between two wild-type Drosophila melanogaster lines correlate with differences in the expression of the Jheh1 gene, which codes for an enzyme degrading juvenile hormone.

Juvenile hormone plays a "status quo" role in Drosophila melanogaster larvae, preventing the untimely metamorphosis, and performs a gonadotropic function in imagoes, ensuring the ovaries' preparedness for vitellogenesis. The decreased level of juvenile hormone results in reproductive disorders in D. melanogaster females including a delay in the oviposition onset and a fertility decrease. Another factor that can affect the insect reproduction is an infection with the maternally inherited symbiotic α-proteobacterium Wolbachia. The present study is devoted to the analysis of the expression of two juvenile hormone metabolism genes encoding enzymes of its synthesis and degradation, juvenile hormone acid O-methyltransferase ( jhamt) and juvenile hormone epoxide hydrase (Jheh1), respectively, in four wild-type D. melanogaster lines, two of them being infected with Wolbachia. Lines w153 and Bi90 were both derived from an individual wild-caught females infected with Wolbachia, while lines w153T and Bi90T were derived from them by tetracycline treatment and are free of infection. Line Bi90 is known to be infected with the Wolbachia strain wMel, and line w153, with the Wolbachia strain wMelPlus belonging to the wMelCS genotype. It was found that infection with either Wolbachia strain does not affect the expression of the studied genes. At the same time, it was shown that the w153 and w153T lines differ from the Bi90 and Bi90T lines by an increased level of the Jheh1 gene expression and do not differ in the jhamt gene expression level. Analysis of the fertility of these four lines showed that it does not depend on Wolbachia infection either, but differs between lines with different nuclear genotypes: in w153 and w153T, it is significantly lower than in lines Bi90 and Bi90T. The data obtained allow us to reasonably propose that the inter-line D. melanogaster polymorphism in the metabolism of the juvenile hormone is determined by its degradation (not by its synthesis) and correlates with the fertility level.

ZnO nanoparticles: A key ingredient of sunscreen shows absence of adverse effects on Drosophila melanization pathway

The rapid development of zinc oxide nanoparticle (ZnO NP) based sunscreens, shampoos and other skin care products has raised a concern about their safety at the cellular level. The melanization pathway, being an important pathway of skin cells, is prone to being affected by ZnO NPs. Therefore it is necessary to understand the ZnO NP mediated effect on the melanin synthetic pathway. Our previous study on bulk zinc oxide has shown that large sized zinc oxide particles did not have any adverse effect on melanization in Drosophila, hence it would be interesting to study the behavior of ZnO when present in the nano-form by virtue of the changed size and thereby changed properties. The present study was undertaken to investigate ZnO NP mediated effects on key metabolites of the Drosophila melanin pathway. Spectrophotometric analysis of melanin was performed. followed by a study of the enzyme activity of phenol oxidase and HPLC quantification of dopa and tyrosine. FTIR spectroscopy was carried out to examine the interaction of the ZnO NPs and precursor amines of the melanin synthetic pathway. Pigmentation mutants of Drosophila were also investigated for validation of the results under a different genetic background. The ZnO NPs did not show any decrement in the levels of melanin after the treatment. Moreover there was no adverse effect on the phenol oxidase activity, Tyrosine and L-Dopa. The FTIR studies showed minimal interaction between the ZnO NPs and the metabolites of the melanin pathway. The results of the present study have clearly indicated the absence of any adverse effects on key metabolites of the melanin pathway after ZnO NP treatment in both wild-type and mutant Drosophila. This is the first report of a detailed analysis of ZnO NP mediated effects on key constituents of the Drosophila melanization pathway, providing information about its safety for the usage in cosmetics.

NUS1 Variants Cause Lennox-Gastaut Syndrome Related to Unfolded Protein Reaction Activation.

NUS1 encodes the Nogo-B receptor, a critical regulator for unfolded protein reaction (UPR) signaling. Although several loss-of-function variants of NUS1 have been identified in patients with developmental and epileptic encephalopathy (DEE), the role of the NUS1 variant in Lennox-Gastaut syndrome (LGS), a severe child-onset DEE, remains unknown. In this study, we identified two de novo variants of NUS1, a missense variant (c.868C > T/p.R290C) and a splice site variant (c.792-2A > G), in two unrelated LGS patients using trio-based whole-exome sequencing performed in a cohort of 165 LGS patients. Both variants were absent in the gnomAD population and showed a significantly higher observed number of variants than expected genome-wide. The R290C variant was predicted to damage NUS1 and decrease its protein stability. The c.792-2A > G variant caused premature termination of the protein. Knockdown of NUS1 activated the UPR pathway, resulting in apoptosis of HEK293T cells. Supplementing cells with expression of wild-type NUS1, but not the mutant (R290C), rescued UPR activation and apoptosis in NUS1 knockdown cells. Compared to wild-type Drosophila, seizure-like behaviors and excitability in projection neurons were significantly increased in Tango14 (homolog of human NUS1) knockdown and Tango14R290C/+ knock-in Drosophila. Additionally, abnormal development and a small body size were observed in both mutants. Activated UPR signaling was also detected in both mutants. Thus, NUS1 is a causative gene for LGS with dominant inheritance. The pathogenicity of these variants is related to the UPR signaling activation, which may be a common pathogenic mechanism of DEE.

Synthesis, Characterization, In‐Vitro and In‐Vivo Anti‐Cancer Evaluation of Novel Benzopyrylium Salts on Colorectal Cancer Model of Drosophila Melanogaster

AbstractA total of 10 benzopyrylium salts, 7 being novel, were synthesized by Claisen‐Schmidt condensation reaction and characterized by 1H and 13C NMR, High‐Resolution Mass Spectrometry, FT‐IR, and UV‐Vis spectroscopy. The purity of these synthesized compounds was determined by HPLC. The in‐vitro anti‐cancer evaluation of these synthesized benzopyrylium salts was done at National Cancer Institute (NCI), USA, against 60 human cancer cell lines. The compounds 4 and 8 showed promising anti‐cancer activities against various human cancer cell lines. The docking studies of the compounds were carried out against the oncogenic target JNK and their docking scores were compared to that of the known JNK inhibitor SP600125 (Pyrazolanthrone). Compound 4 exhibited an outstanding docking score of −9.0 kcal/mol, comparable to the docking score of SP600125 (−9.3 kcal/mol). The ex‐vivo studies in the hind‐gut of Drosophila were carried out and the inhibitory activity of compound 4 supported the findings of the docking studies. Furthermore, the potent compounds 4 and 8 were taken up for their in‐vivo anti‐cancer evaluation in the colorectal cancer model (CRC) of Drosophila melanogaster. The toxicity studies of these potent compounds were carried out both in wild type and mutant type Drosophila and the lethal doses were determined to be 200 μM.

Wings of Change: aPKC/FoxP-dependent plasticity in steering motor neurons underlies operant self-learning in Drosophila.

Motor learning is central to human existence, such as learning to speak or walk, sports moves, or rehabilitation after injury. Evidence suggests that all forms of motor learning share an evolutionarily conserved molecular plasticity pathway. Here, we present novel insights into the neural processes underlying operant self-learning, a form of motor learning in the fruit fly Drosophila. We operantly trained wild type and transgenic Drosophila fruit flies, tethered at the torque meter, in a motor learning task that required them to initiate and maintain turning maneuvers around their vertical body axis (yaw torque). We combined this behavioral experiment with transgenic peptide expression, CRISPR/Cas9-mediated, spatio-temporally controlled gene knock-out and confocal microscopy. We find that expression of atypical protein kinase C (aPKC) in direct wing steering motoneurons co-expressing the transcription factor FoxP is necessary for this type of motor learning and that aPKC likely acts via non-canonical pathways. We also found that it takes more than a week for CRISPR/Cas9-mediated knockout of FoxP in adult animals to impair motor learning, suggesting that adult FoxP expression is required for operant self-learning. Our experiments suggest that, for operant self-learning, a type of motor learning in Drosophila, co-expression of atypical protein kinase C (aPKC) and the transcription factor FoxP is necessary in direct wing steering motoneurons. Some of these neurons control the wing beat amplitude when generating optomotor responses, and we have discovered modulation of optomotor behavior after operant self-learning. We also discovered that aPKC likely acts via non-canonical pathways and that FoxP expression is also required in adult flies.

Atractylodes macrocephala Koidz. and Cuscuta chinensis Lam. extract relieves insulin resistance via PI3K/Akt signalling in diabetic Drosophila

Background and aimType-2 diabetes mellitus (T2DM) is mainly characterized by insulin resistance (IR) induced by hyperglycaemia and insufficient insulin secretion. We employed a diabetic fly model to examine the effect and molecular mechanism of Atractylodes macrocephala Koidz. and Cuscuta chinensis Lam. (AMK–CCL) extract as traditional Chinese medicine in treating IR and T2DM. Experimental procedureThe contents of the active ingredients (rhamnose, xylose, mannose, and hyperoside) in AMK–CCL extract were determined by high-performance liquid chromatography. Wild-type (Cg-GAL4/+) or diabetic (Cg > InRK1409A) Drosophila flies were divided into the control group or metformin group and AMK–CCL (0.0125, 0.025, 0.05, 0.1 g/ml) groups. Food intake, haemolymph glucose and trehalose, protein, weight, triglycerides (TAG), and glycogen were measured to assess glycolipid metabolism. Phosphatidylinositol-3-kinase (PI3K)/Akt signalling was detected using fluorescent reporters [tGPH, Drosophila forkhead box O (dFoxO)–green fluorescent protein (GFP), Glut1–GFP, 2-NBDG] in vivo. Glut1/3 mRNA levels and Akt phosphorylation levels were detected by quantitative polymerase chain reaction and western blotting, respectively, in vitro. ResultsAMK–CCL extract contained 0.038 % rhamnose, 0.017 % xylose, 0.69 % mannose, and 0.039 % hyperoside. AMK–CCL at 0.0125 g/mL significantly suppressed the increase in circulating glucose, and the decrease in body weight, TAG, and glycogen contents of diabetic flies. AMK–CCL improved PI3K activity, Akt phosphorylation, Glut1/3 expression, and glucose uptake in diabetic flies, and also rescued diabetes-induced dFoxO nuclear localisation. ConclusionsThese findings indicate that AMK–CCL extract ameliorates IR-induced diabetes via the PI3K/Akt signalling pathway, providing an experimental basis for clinical treatment.

Opposing chemosensory functions of closely related gustatory receptors

In the fruit fly Drosophila melanogaster, gustatory sensory neurons express taste receptors that are tuned to distinct groups of chemicals, thereby activating neural ensembles that elicit either feeding or avoidance behavior. Members of a family of ligand -gated receptor channels, the Gustatory receptors (Grs), play a central role in these behaviors. In general, closely related, evolutionarily conserved Gr proteins are co-expressed in the same type of taste neurons, tuned to chemically related compounds, and therefore triggering the same behavioral response. Here, we report that members of the Gr28 subfamily are expressed in largely non-overlapping sets of taste neurons in Drosophila larvae, detect chemicals of different valence, and trigger opposing feeding behaviors. We determined the intrinsic properties of Gr28 neurons by expressing the mammalian Vanilloid Receptor 1 (VR1), which is activated by capsaicin, a chemical to which wild-type Drosophila larvae do not respond. When VR1 is expressed in Gr28a neurons, larvae become attracted to capsaicin, consistent with reports showing that Gr28a itself encodes a receptor for nutritious RNA. In contrast, expression of VR1 in two pairs of Gr28b.c neurons triggers avoidance to capsaicin. Moreover, neuronal inactivation experiments show that the Gr28b.c neurons are necessary for avoidance of several bitter compounds. Lastly, behavioral experiments of Gr28 deficient larvae and live Ca2+ imaging studies of Gr28b.c neurons revealed that denatonium benzoate, a synthetic bitter compound that shares structural similarities with natural bitter chemicals, is a ligand for a receptor complex containing a Gr28b.c or Gr28b.a subunit. Thus, the Gr28 proteins, which have been evolutionarily conserved over 260 million years in insects, represent the first taste receptor subfamily in which specific members mediate behavior with opposite valence.

Opposing chemosensory functions of closely related gustatory receptors.

In the fruit fly Drosophila melanogaster, gustatory sensory neurons express taste receptors that are tuned to distinct groups of chemicals, thereby activating neural ensembles that elicit either feeding or avoidance behavior. Members of a family of ligand -gated receptor channels, the Gustatory receptors (Grs), play a central role in these behaviors. In general, closely related, evolutionarily conserved Gr proteins are co-expressed in the same type of taste neurons, tuned to chemically related compounds, and therefore triggering the same behavioral response. Here, we report that members of the Gr28 subfamily are expressed in largely non-overlapping sets of taste neurons in Drosophila larvae, detect chemicals of different valence, and trigger opposing feeding behaviors. We determined the intrinsic properties of Gr28 neurons by expressing the mammalian Vanilloid Receptor 1 (VR1), which is activated by capsaicin, a chemical to which wild-type Drosophila larvae do not respond. When VR1 is expressed in Gr28a neurons, larvae become attracted to capsaicin, consistent with reports showing that Gr28a itself encodes a receptor for nutritious RNA. In contrast, expression of VR1 in two pairs of Gr28b.c neurons triggers avoidance to capsaicin. Moreover, neuronal inactivation experiments show that the Gr28b.c neurons are necessary for avoidance of several bitter compounds. Lastly, behavioral experiments of Gr28 deficient larvae and live Ca2+ imaging studies of Gr28b.c neurons revealed that denatonium benzoate, a synthetic bitter compound that shares structural similarities with natural bitter chemicals, is a ligand for a receptor complex containing a Gr28b.c or Gr28b.a subunit. Thus, the Gr28 proteins, which have been evolutionarily conserved over 260 million years in insects, represent the first taste receptor subfamily in which specific members mediate behavior with opposite valence.

A glutamate-gated chloride channel as the mite-specific target-site of dicofol and other diphenylcarbinol acaricides.

Dicofol has been widely used to control phytophagous mites. Although dicofol is chemically related to DDT, its mode of action has remained elusive. Here, we mapped dicofol resistance in the spider mite Tetranychus urticae to two genomic regions. Each region harbored a glutamate-gated chloride channel (GluCl) gene that contained a mutation-G314D or G326E-known to confer resistance against the unrelated acaricide abamectin. Using electrophysiology assays we showed that dicofol and other diphenylcarbinol acaricides-bromopropylate and chlorobenzilate-induce persistent currents in Xenopus oocytes expressing wild-type T. urticae GluCl3 receptors and potentiate glutamate responses. In contrast, the G326E substitution abolished the agonistic activity of all three compounds. Assays with the wild-type Drosophila GluClα revealed that this receptor was unresponsive to dicofol. Homology modeling combined with ligand-docking confirmed the specificity of electrophysiology assays. Altogether, this work elucidates the mode of action of diphenylcarbinols as mite-specific agonists of GluCl.

Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle

Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the questions of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wildtype Drosophila and cardiolipin deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C615N2-lysine or 13C6-glucose) and determined the relative abundance of heavy isotopomers in protein and lipid species by mass spectrometry. To minimize confounding effects of tissue regeneration, we restricted our analysis to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48±16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27±6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.

The Larval Plate Assay

The larval plate test has been used by researchers as a choice test to study the response of terrestrial larvae to various external cues. We describe here a laboratory exercise using third instar larvae of wild-type Drosophila to study the response toward multiple cues including olfactory as well as physical environmental cues such as light. The experiment can be easily carried out in an undergraduate lab and does not require any sophisticated instruments or equipment. The results of a series of experiments indicate the extent of response that each of the cue generates relative to the other. The highlight of the article however is the pre-lab discussion that can be done with students by putting forth various possible designs followed by discussion regarding the benefits and/or errors that could creep into the results obtained by using a particular design. Such a discussion would prove to be helpful in enhancing student understanding of experimental design, especially in designing experiments in animal behavior. The article also includes an extended theoretical problem set that can be used by facilitators in an undergraduate classroom activity.