How larvae feel the world around them.

A complete map of the external sense organs shows how fruit fly larvae detect different aspects of their environment.

Purriato is a conserved small open reading frame gene that interacts with the CASA pathway to regulate muscle homeostasis and epithelial tissue growth in Drosophila.

Recent advances in proteogenomic techniques and bioinformatic pipelines have permitted the detection of thousands of translated small Open Reading Frames (smORFs), which contain less than 100 codons, in eukaryotic genomes. Hundreds of these actively translated smORFs display conserved sequence, structure and evolutionary signatures indicating that the translated peptides could fulfil important biological roles. Despite their abundance, only tens of smORF genes have been fully characterised; these act mainly as regulators of canonical proteins involved in essential cellular processes. Importantly, some of these smORFs display conserved functions with their mutations being associated with pathogenesis. Thus, investigating smORF roles in Drosophila will not only expand our understanding of their functions but it may have an impact in human health. Here we describe the function of a novel and essential Drosophila smORF gene named purriato (prto). prto belongs to an ancient gene family whose members have expanded throughout the Protostomia clade. prto encodes a transmembrane peptide which is localized in endo-lysosomes and perinuclear and plasma membranes. prto is dynamically expressed in mesodermal tissues and imaginal discs. Targeted prto knockdown (KD) in these organs results in changes in nuclear morphology and endo-lysosomal distributions correlating with the loss of sarcomeric homeostasis in muscles and reduction of mitosis in wing discs. Consequently, prto KD mutants display severe reduction of motility, and shorter wings. Finally, our genetic interaction experiments show that prto function is closely associated to the CASA pathway, a conserved mechanism involved in turnover of mis-folded proteins and linked to muscle dystrophies and neurodegenerative diseases. Thus, this study shows the relevance of smORFs in regulating important cellular functions and supports the systematic characterisation of this class of genes to understand their functions and evolution.

Adaptation of Drosophila larva foraging in response to changes in food resources.

All animals face the challenge of finding nutritious resources in a changing environment. To maximize lifetime fitness, the exploratory behavior has to be flexible, but which behavioral elements adapt and what triggers those changes remain elusive. Using experiments and modeling, we characterized extensively how Drosophila larvae foraging adapts to different food quality and distribution and how the foraging genetic background influences this adaptation. Our work shows that different food properties modulated specific motor programs. Food quality controls the traveled distance by modulating crawling speed and frequency of pauses and turns. Food distribution, and in particular the food-no food interface, controls turning behavior, stimulating turns toward the food when reaching the patch border and increasing the proportion of time spent within patches of food. Finally, the polymorphism in the foraging gene (rover-sitter) of the larvae adjusts the magnitude of the behavioral response to different food conditions. This study defines several levels of control of foraging and provides the basis for the systematic identification of the neuronal circuits and mechanisms controlling each behavioral response.

Drosophila para bss Flies as a Screening Model for Traditional Medicine: Anticonvulsant Effects of Annona senegalensis.

Epilepsy is among the most common serious neurological disorders and affects around 50 million people worldwide, 80% of which live in developing countries. Despite the introduction of several new Anti-Epileptic Drugs (AEDs) in the last two decades, one third of treated patients have seizures refractory to pharmacotherapy. This highlights the need to develop new treatments with drugs targeting alternative seizure-induction mechanisms. Traditional medicine (TM) is used for the treatment of epilepsy in many developing countries and could constitute an affordable and accessible alternative to AEDs, but a lack of pre-clinical and clinical testing has so far prevented its wider acceptance worldwide. In this study we used Drosophila melanogaster paralytic bangsensitive (para bss ) mutants as a model for epileptic seizure screening and tested, for the first time, the anti-seizure effect of a non-commercial AED. We evaluated the effect of the African custard-apple, Annona senegalensis, which is commonly used as a TM for the treatment of epilepsy in rural Africa, and compared it with the classical AED phenytoin. Our results showed that a stem bark extract from A. senegalensis was significantly more effective than a leaf extract and similar to phenytoin in the prevention and control of seizure-like behavior. These results support that Drosophila constitutes a robust animal model for the screening of TM with potential value for the treatment of intractable epilepsy.

Optimal searching behaviour generated intrinsically by the central pattern generator for locomotion.

Efficient searching for resources such as food by animals is key to their survival. It has been proposed that diverse animals from insects to sharks and humans adopt searching patterns that resemble a simple Lévy random walk, which is theoretically optimal for 'blind foragers' to locate sparse, patchy resources. To test if such patterns are generated intrinsically, or arise via environmental interactions, we tracked free-moving Drosophila larvae with (and without) blocked synaptic activity in the brain, suboesophageal ganglion (SOG) and sensory neurons. In brain-blocked larvae, we found that extended substrate exploration emerges as multi-scale movement paths similar to truncated Lévy walks. Strikingly, power-law exponents of brain/SOG/sensory-blocked larvae averaged 1.96, close to a theoretical optimum (µ ≅ 2.0) for locating sparse resources. Thus, efficient spatial exploration can emerge from autonomous patterns in neural activity. Our results provide the strongest evidence so far for the intrinsic generation of Lévy-like movement patterns.

MicroRNA-encoded behavior in Drosophila.

The relationship between microRNA (miRNA) regulation and the specification of behavior is only beginning to be explored. We found that mutation of a single miRNA locus (miR-iab4/iab8) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside down (self-righting). One of the miRNA targets involved in this behavior is the Hox gene Ultrabithorax, whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants, whereas thermogenetic manipulation of SRN activity results in changes in self-righting behavior. Our work thus reveals a miRNA-encoded behavior and suggests that other miRNAs might also be involved in behavioral control in Drosophila and other species.

Imaging fictive locomotor patterns in larval Drosophila.

We have established a preparation in larval Drosophila to monitor fictive locomotion simultaneously across abdominal and thoracic segments of the isolated CNS with genetically encoded Ca(2+) indicators. The Ca(2+) signals closely followed spiking activity measured electrophysiologically in nerve roots. Three motor patterns are analyzed. Two comprise waves of Ca(2+) signals that progress along the longitudinal body axis in a posterior-to-anterior or anterior-to-posterior direction. These waves had statistically indistinguishable intersegmental phase delays compared with segmental contractions during forward and backward crawling behavior, despite being ∼10 times slower. During these waves, motor neurons of the dorsal longitudinal and transverse muscles were active in the same order as the muscle groups are recruited during crawling behavior. A third fictive motor pattern exhibits a left-right asymmetry across segments and bears similarities with turning behavior in intact larvae, occurring equally frequently and involving asymmetry in the same segments. Ablation of the segments in which forward and backward waves of Ca(2+) signals were normally initiated did not eliminate production of Ca(2+) waves. When the brain and subesophageal ganglion (SOG) were removed, the remaining ganglia retained the ability to produce both forward and backward waves of motor activity, although the speed and frequency of waves changed. Bilateral asymmetry of activity was reduced when the brain was removed and abolished when the SOG was removed. This work paves the way to studying the neural and genetic underpinnings of segmentally coordinated motor pattern generation in Drosophila with imaging techniques.

Genetic dissection of a regionally differentiated network for exploratory behavior in Drosophila larvae.

An efficient strategy to explore the environment for available resources involves the execution of random walks where straight line locomotion alternates with changes of direction. This strategy is highly conserved in the animal kingdom, from zooplankton to human hunter-gatherers. Drosophila larvae execute a routine of this kind, performing straight line crawling interrupted at intervals by pause turns that halt crawling and redirect the trajectory of movement. The execution of this routine depends solely on the activity of networks located in the thoracic and abdominal segments of the nervous system, while descending input from the brain serves to modify it in a context-dependent fashion. I used a genetic method to investigate the location and function of the circuitry required for the different elements of exploratory crawling. By using the Slit-Robo axon guidance pathway to target neuronal midline crossing defects selectively to particular regions of the thoracic and abdominal networks, it has been possible to define at least three functions required for the performance of the exploratory routine: (1) symmetrical outputs in thoracic and abdominal segments that generate the crawls; (2) asymmetrical output that is uniquely initiated in the thoracic segments and generates the turns; and (3) an intermittent interruption to crawling that determines the time-dependent transition between crawls and turns.

Circadian period integrates network information through activation of the BMP signaling pathway.

Living organisms use biological clocks to maintain their internal temporal order and anticipate daily environmental changes. In Drosophila, circadian regulation of locomotor behavior is controlled by ∼150 neurons; among them, neurons expressing the PIGMENT DISPERSING FACTOR (PDF) set the period of locomotor behavior under free-running conditions. To date, it remains unclear how individual circadian clusters integrate their activity to assemble a distinctive behavioral output. Here we show that the BONE MORPHOGENETIC PROTEIN (BMP) signaling pathway plays a crucial role in setting the circadian period in PDF neurons in the adult brain. Acute deregulation of BMP signaling causes period lengthening through regulation of dClock transcription, providing evidence for a novel function of this pathway in the adult brain. We propose that coherence in the circadian network arises from integration in PDF neurons of both the pace of the cell-autonomous molecular clock and information derived from circadian-relevant neurons through release of BMP ligands.

Neural circuits for peristaltic wave propagation in crawling Drosophila larvae: analysis and modeling.

Drosophila larvae crawl by peristaltic waves of muscle contractions, which propagate along the animal body and involve the simultaneous contraction of the left and right side of each segment. Coordinated propagation of contraction does not require sensory input, suggesting that movement is generated by a central pattern generator (CPG). We characterized crawling behavior of newly hatched Drosophila larvae by quantifying timing and duration of segmental boundary contractions. We developed a CPG network model that recapitulates these patterns based on segmentally repeated units of excitatory and inhibitory (EI) neuronal populations coupled with immediate neighboring segments. A single network with symmetric coupling between neighboring segments succeeded in generating both forward and backward propagation of activity. The CPG network was robust to changes in amplitude and variability of connectivity strength. Introducing sensory feedback via "stretch-sensitive" neurons improved wave propagation properties such as speed of propagation and segmental contraction duration as observed experimentally. Sensory feedback also restored propagating activity patterns when an inappropriately tuned CPG network failed to generate waves. Finally, in a two-sided CPG model we demonstrated that two types of connectivity could synchronize the activity of two independent networks: connections from excitatory neurons on one side to excitatory contralateral neurons (E to E), and connections from inhibitory neurons on one side to excitatory contralateral neurons (I to E). To our knowledge, such I to E connectivity has not yet been found in any experimental system; however, it provides the most robust mechanism to synchronize activity between contralateral CPGs in our model. Our model provides a general framework for studying the conditions under which a single locally coupled network generates bilaterally synchronized and longitudinally propagating waves in either direction.

Autonomous circuitry for substrate exploration in freely moving Drosophila larvae.

BACKGROUND: Many organisms, from bacteria to human hunter-gatherers, use specialized random walk strategies to explore their environment. Such behaviors are an efficient stratagem for sampling the environment and usually consist of an alternation between straight runs and turns that redirect these runs. Drosophila larvae execute an exploratory routine of this kind that consists of sequences of straight crawls, pauses, turns, and redirected crawls. Central pattern generating networks underlying rhythmic movements are distributed along the anteroposterior axis of the nervous system. The way in which the operation of these networks is incorporated into extended behavioral routines such as substrate exploration has not yet been explored. In particular, the part played by the brain in dictating the sequence of movements required is unknown. RESULTS: We report the use of a genetic method to block synaptic activity acutely in the brain and subesophageal ganglia (SOG) of larvae during active exploratory behavior. We show that the brain and SOG are not required for the normal performance of an exploratory routine. Alternation between crawls and turns is an intrinsic property of the abdominal and/or thoracic networks. The brain modifies this autonomous routine during goal-directed movements such as those of chemotaxis. Nonetheless, light avoidance behavior can be mediated in the absence of brain activity solely by the sensorimotor system of the abdomen and thorax. CONCLUSIONS: The sequence of movements for substrate exploration is an autonomous capacity of the thoracic and abdominal nervous system. The brain modulates this exploratory routine in response to environmental cues.

The fundamentals of flying: simple and inexpensive strategies for employing Drosophila genetics in neuroscience teaching laboratories.

Drosophila researchers have developed a powerful suite of genetic techniques for studying the neural basis of animal behavior. Many of these tools can be exported to neuroscience teaching laboratories (Berni et al., 2010; Pulver et al., 2011a,b), but often neuroscience educators lack the basic knowledge and resources to obtain, generate and rear transgenic fruit flies on their own. Fly researchers in turn may take for granted resources that are readily available in research laboratories, but out of reach for educators. Our goal is to provide a primer for neuroscience educators who want to incorporate Drosophila genetics into their teaching, but have limited knowledge of fruit fly genetics, and/or small budgets. First we review the available methods for manipulating gene expression in Drosophila. Then we provide educators with blueprints for obtaining transgenic animals tailored for specific types of teaching modules. We outline simple techniques for rearing transgenic Drosophila, performing genetic crosses, and preparing a teaching laboratory without the use of expensive animal-care facilities. Overall, we try to break down the practical barriers educators may face when integrating modern neurogenetic experiments into teaching laboratories.

Adult-specific electrical silencing of pacemaker neurons uncouples molecular clock from circadian outputs.

BACKGROUND: Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral lateral neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. RESULTS: Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PERIOD (PER) oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. CONCLUSIONS: Although electrical activity of the sLNvs is not a clock component, it coordinates circuit outputs leading to rhythmic behavior.

The enzyme NBAD-synthase plays diverse roles during the life cycle of Drosophila melanogaster.

This report shows the biochemical characterization and life cycle-dependent expression of Drosophila melanogaster N-beta-alanyldopamine synthase (NBAD-synthase or Ebony protein). This enzyme not only catalyzes the synthesis of NBAD, the main sclerotization and pigmentation precursor of insect brown cuticles, but also plays a role in brain neurotransmitter metabolism. In addition to the epidermis expression our immunodetection experiments show the novel localization of NBAD-synthase in different regions of the adult brain, in the foregut of pharate adult and, surprisingly, in the epidermis of the trachea during embryogenesis. These results demonstrate that NBAD-synthase is a versatile enzyme involved in different, previously unknown, time- and tissue-dependent processes.

Using Neurogenetics and the Warmth-Gated Ion Channel TRPA1 to Study the Neural Basis of Behavior in Drosophila.

Here we describe a set of straightforward laboratory exercises that integrate the study of genetics, neuroanatomy, cellular physiology and animal behavior. We use genetic tools in Drosophila for visualizing and remotely activating ensembles of neurons with heat pulses. First, we show how to examine the anatomy of several neuronal populations using genetically encoded green fluorescent protein. Next we demonstrate how to use the warmth gated Drosophila TRPA1 (dTRPA1) cation channel to remotely activate neural circuits in flies. To demonstrate the cellular effects of dTRPA1 activation, we expressed dTRPA1 panneurally and recorded excitatory junctional potentials in muscles in response to warmed (29°C) saline. Finally, we present inexpensive techniques for delivering heat pulses to activate dTRPA1 in the neuronal groups we observed previously while flies are freely behaving. We suggest how to film and quantify resulting behavioral phenotypes with limited resources. Activating all neurons with dTRPA1 caused tetanic paralysis in larvae, while in adults it led to paralysis in males and continuous uncoordinated leg and wing movements in females. Activation of cholinergic neurons produced spasms and writhing in larvae while causing paralysis in adults. When a single class of nociceptive sensory neurons was activated, it caused lateral rolling in larvae, but no discernable effects in adults. Overall, these exercises illustrate principles of modern genetics, neuroanatomy, the ionic basis of neuronal excitability, and quantitative methods in neuroethology. Relatively few research studies have used dTRPA1 to activate neural circuits, so these exercises give students opportunities to test novel hypotheses and make actual contributions to the scientific record.

A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.

Circadian remodeling of neuronal circuits involved in rhythmic behavior.

Clock output pathways are central to convey timing information from the circadian clock to a diversity of physiological systems, ranging from cell-autonomous processes to behavior. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is unclear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the time of day information from core pacemaker cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Here we describe a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit, which display higher complexity during the day and significantly lower complexity at nighttime, both under daily cycles and constant conditions. In support to its circadian nature, cycling is lost in bona fide clockless mutants. We propose this clock-controlled structural plasticity as a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells.

The axon-guidance roundabout gene alters the pace of the Drosophila circadian clock.

Great efforts have been directed to the dissection of the cell-autonomous circadian oscillator in Drosophila. However, less information is available regarding how this oscillator controls rhythmic rest-activity cycles. We have identified a viable allele of roundabout, robo(hy), where the period of locomotor activity is shortened. From its role in axon-pathfinding, we anticipated developmental defects in clock-relevant structures. However, robo(hy) produced minor defects in the architecture of the circuits essential for rhythmic behaviour. ROBO's presence within the circadian circuit strengthened the possibility of a novel role for ROBO at this postdevelopmental stage. Genetic interactions between pdf (01) and robo(hy) suggest that ROBO could alter the communication within different clusters of the circadian network, thus impinging on two basic properties, periodicity and/or rhythmicity. Early translocation of PERIOD to the nucleus in robo(hy) pacemaker cells indicated that shortened activity rhythms were derived from alterations in the molecular oscillator. Herein we present a mutation affecting clock function associated with a molecule involved in circuit assembly and maintenance.

Phloxine B effect on immature stages of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) (Wiedemann).

A laboratory bioassay was developed to determine both the chemical toxicity and the phototoxicity of the xanthene dye, phloxine B (D&C Red No 28), to the immature stages of the Mediterranean fruit fly, Certitis capitata (Wiedemann). An additional goal was to find out which main tissues are affected first. A low, but significant, level of toxicity was observed when the insects were maintained in the dark: at the point of adult ecdysis, the LC50 was 11.03 mM. As expected, after 8-h exposure of late larva III to light, a high level of mortality was produced (LC50 at ecdysis: 0.45 mM) as a dose-dependent function of dye concentration. At sublethal concentrations of the dye, the surviving insects showed a number of physiological abnormalities. Phloxine B appeared to mainly affect the larval longitudinal muscles as well as the abdominal muscles of ecdysing adults, giving rise to abnormal puparia and failed adult ecdysis, respectively. Moreover, a significant phloxine B-dependent delay in the jumping of surviving larvae for dispersal was documented. This could be attributed to a delay in attaining a threshold weight for jumping and/or to abnormalities in neuromuscular coordination, thus reinforcing the idea of pleiotropic effects of the dye.