Bitter compounds elicit an aversive response. In Drosophila, bitter-sensitive taste neurons coexpress many members of the Gr family of taste receptors. However, the molecular logic of bitter signaling is unknown. We used an in vivo expression approach to analyze the logic of bitter taste signaling. Ectopic or overexpression of bitter Grs increased endogenous responses or conferred novel responses. Surprisingly, expression of Grs also suppressed many endogenous bitter responses. Conversely, deletion of an endogenous Gr led to novel responses. Expression of individual Grs conferred strikingly different effects in different neurons. The results support a model in which bitter Grs interact, exhibiting competition, inhibition, or activation. The results have broad implications for the problem of how taste systems evolve to detect new environmental dangers.DOI: http://dx.doi.org/10.7554/eLife.11181.001

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadianregulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. Here, O’Connor et al. find that Drosophila Fmr1 mutants, a model for Fragile X syndrome, exhibit defects in phagocytosis by innate immune cells in both the body and the brain.

Ammonia is both a building block and a breakdown product of amino acids and is found widely in the environment. The odor of ammonia is attractive to many insects, including insect vectors of disease. The olfactory response of Drosophila to ammonia has been studied in some detail, but the taste response has received remarkably little attention. Here, we show that ammonia is a taste cue for Drosophila. Nearly all sensilla of the major taste organ of the Drosophila head house a neuron that responds to neutral solutions of ammonia. Ammonia is toxic at high levels to many organisms, and we find that it has a negative valence in two paradigms of taste behavior, one operating over hours and the other over seconds. Physiological and behavioral responses to ammonia depend at least in part on Gr66a+ bitter-sensing taste neurons, which activate a circuit that deters feeding. The Amt transporter, a critical component of olfactory responses to ammonia, is widely expressed in taste neurons but is not required for taste responses. This work establishes ammonia as an ecologically important taste cue in Drosophila, and shows that it can activate circuits that promote opposite behavioral outcomes via different sensory systems.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called ‘master pacemakers’ because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these clock neurons secrete the neuropeptide Pdf and have been called "master pacemakers" because they are essential for circadian rhythms. A subset of Pdf + neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf + neurons, including a subset called the evening oscillator. It is assumed that the molecular clock in Pdf + neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient for circadian locomotor activity. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds. Video LinkThe video component of this article can be found at

As educators strive to incorporate more active learning and inquiry-driven exercises into STEM curricula, Course-based Undergraduate Research Experiences (CUREs) are becoming more common in undergraduate laboratory courses. Here we detail a CURE developed in an upper-level undergraduate genetics course at Yeshiva University, centered on the Drosophila melanogaster ortholog of the human neurodegeneration locus PLA2G6/PARK14. Drosophila PLA2G6 mutants exhibit symptoms of neurodegeneration, such as attenuated lifespan and decreased climbing ability with age, which can be replicated by neuron-specific knockdown of PLA2G6. To ask whether the neurodegeneration phenotype could be caused by loss of PLA2G6 in specific neuronal subtypes, students used GAL4-UAS to perform RNAi knockdown of PLA2G6 in subsets of neurons in the Drosophila central nervous system and measured age-dependent climbing ability. We organized our learning objectives for the CURE into three broad goals of having students think, communicate, and perform like scientists. To assess how well students achieved these goals, we developed a detailed rubric to analyze written lab reports, administered pre-and post-course surveys, and solicited written feedback. We observed striking gains related to all three learning goals, and students reported a high degree of satisfaction. We also observed significantly improved understanding of the scientific method by students in the CURE as compared to the prior year's non-CURE genetics lab students. Thus, this CURE can serve as a template to successfully engage students in novel research, improve understanding of the scientific process, and expose students to the use of Drosophila as a model for human neurodegenerative disease.