Tracking Sugar-Elicited Local Searching Behavior in Drosophila.

Foraging behavior is essential for the survival of organisms as it enables them to locate and acquire essential food resources. In Drosophila, hunger triggers a distinct search behavior following the consumption of small quantities of a sugar solution. This report presents a simple experimental setup to study sugar-elicited search behavior with the aim of uncovering the underlying mechanisms. Minute quantities of concentrated sugar solution elicit sustained searching behavior in flies. The involvement of path integration in this behavior has been established, as flies utilize their trajectory to return to the sugar location. The most recent findings provide evidence of temporal modulation in the initiation and intensity of the search behavior after sugar intake. We have also used this setup for artificial activation of specific taste-receptor neurons in the pharynx, which elicits the search behavior. The Drosophila neurogenetic toolkit offers a diverse array of tools and techniques that can be combined with the sugar-elicited search behavior paradigm to study the neural and genetic mechanisms underlying foraging. Understanding the neural basis of hunger-driven searching behavior in flies contributes to the field of neurobiology as a whole, offering insights into the regulatory mechanisms that govern feeding behaviors not only in other organisms but also in humans.

A gain-of-function mutation in head involution defective , Wrinkled, causes precocious cell death of wing epidermal cells in Drosophila.

In Drosophila , wing epidermal cells undergo programmed cell death as the last step of metamorphosis. The aim of this study was to evaluate the role of hid , particularly the Wrinkled mutation ( hid W ), an allele of hid , in the cell death. The wing epithelial cell death is suppressed by loss-of-function mutation of hid , indicating that the death is governed by a cascade involving hid . Examination of the cell death in hid W showed that precocious death started at G stage, 3 h before eclosion. Thus, mutated-HID in the hid W mutant was activated at G stage, supporting the gain-of-function effect of hid W mutation.

A conserved odorant binding protein is required for essential amino acid detection in Drosophila.

Animals need to detect in the food essential amino acids that they cannot synthesize. We found that the odorant binding protein OBP19b, which is highly expressed in Drosophila melanogaster taste sensilla, is necessary for the detection of several amino acids including the essential l-phenylalanine. The recombinant OBP19b protein was produced and characterized for its binding properties: it stereoselectively binds to several amino acids. Using a feeding-choice assay, we found that OBP19b is necessary for detecting l-phenylalanine and l-glutamine, but not l-alanine or D-phenylalanine. We mapped the cells expressing OBP19b and compared the electrophysiological responses of a single taste sensillum to several amino acids: OBP19b mutant flies showed a reduced response compared to control flies when tested to preferred amino acids, but not to the other ones. OBP19b is well conserved in phylogenetically distant species suggesting that this protein is necessary for detection of specific amino acids in insects.

Softness sensing and learning in Drosophila larvae.

Mechanosensation provides animals with important sensory information in addition to olfaction and gustation during feeding behavior. Here, we used Drosophila melanogaster larvae to investigate the role of softness sensing in behavior and learning. In the natural environment, larvae need to dig into soft foods for feeding. Finding foods that are soft enough to dig into is likely to be essential for their survival. We report that larvae can discriminate between different agar concentrations and prefer softer agar. Interestingly, we show that larvae on a harder surface search for a softer surface using memory associated with an odor, and that they evaluate foods by balancing softness and sweetness. These findings suggest that larvae integrate mechanosensory information with chemosensory input while foraging. Moreover, we found that the larval preference for softness is affected by genetic background.

Sugar Intake Elicits Intelligent Searching Behavior in Flies and Honey Bees.

We present a comparison of the sugar-elicited search behavior in Drosophila melanogaster and Apis mellifera. In both species, intake of sugar-water elicits a complex of searching responses. The most obvious response was an increase in turning frequency. However, we also found that flies and honey bees returned to the location of the sugar drop. They even returned to the food location when we prevented them from using visual and chemosensory cues. Analyses of the recorded trajectories indicated that flies and bees use two mechanisms, a locomotor pattern involving an increased turning frequency and path integration to increase the probability to stay close or even return to the sugar drop location. However, evidence for the use of path integration in honey bees was less clear. In general, walking trajectories of honey bees showed a higher degree of curvature and were more spacious; two characters which likely masked evidence for the use of path integration in our experiments. Visual cues, i.e., a black dot, presented underneath the sugar drop made flies and honey bees stay closer to the starting point of the search. In honey bees, vertical black columns close to the sugar drop increased the probability to visit similar cues in the vicinity. An additional one trial learning experiment suggested that the intake of sugar-water likely has the potential to initiate an associative learning process. Together, our experiments indicate that the sugar-elicited local search is more complex than previously assumed. Most importantly, this local search behavior appeared to exhibit major behavioral capabilities of large-scale navigation. Thus, we propose that sugar-elicited search behavior has the potential to become a fruitful behavioral paradigm to identify neural and molecular mechanisms involved in general mechanisms of navigation.

Deciphering the Genes for Taste Receptors for Fructose in Drosophila.

Taste sensitivity to sugars plays an essential role in the initiation of feeding behavior. In Drosophila melanogaster, recent studies have identified several gustatory receptor (Gr) genes required for sensing sweet compounds. However, it is as yet undetermined how these GRs function as taste receptors tuned to a wide range of sugars. Among sugars, fructose has been suggested to be detected by a distinct receptor from other sugars. While GR43A has been reported to sense fructose in the brain, it is not expressed in labellar gustatory receptor neurons that show taste response to fructose. In contrast, the Gr64a-Gr64f gene cluster was recently shown to be associated with fructose sensitivity. Here we sought to decipher the genes required for fructose response among Gr64a-Gr64f genes. Unexpectedly, the qPCR analyses for these genes show that labellar expression levels of Gr64d and Gr64e are higher in fructose low-sensitivity flies than in high-sensitivity flies. Moreover, gustatory nerve responses to fructose in labellar sensilla are higher in Gr64d and Gr64f mutant lines than in mutant flies of the other Gr64a-Gr64f genes. These data suggest the possibility that deletion of GR64D or GR64F may indirectly induce enhanced fructose sensitivity in the labellum. Finally, we conclude that response to fructose cannot be explained by a single one of the Gr64a-Gr64f genes.

Pharyngeal stimulation with sugar triggers local searching behavior in Drosophila.

Foraging behavior is essential for all organisms to find food containing nutritional chemicals. A hungry Drosophila melanogaster fly performs local searching behavior after drinking a small amount of sugar solution. Using video tracking, we examined how the searching behavior is regulated in D. melanogaster We found that a small amount of highly concentrated sugar solution induced a long-lasting searching behavior. After the intake of sugar solution, a fly moved around in circles and repeatedly returned to the position where the sugar droplet had been placed. The non-nutritious sugar d-arabinose, but not the non-sweet nutritious sugar d-sorbitol, was effective in inducing the behavior, indicating that sweet sensation is essential. Furthermore, pox-neuro mutant flies, which have no external taste bristles, showed local searching behavior, suggesting the involvement of the pharyngeal taste organ. Experimental activation of pharyngeal sugar-sensitive gustatory receptor neurons by capsaicin using the GAL4/UAS system induced local searching behavior. In contrast, inhibition of pharyngeal sugar-responsive gustatory receptor neurons abolished the searching behavior. Together, our results indicate that, in Drosophila, the pharyngeal taste-receptor neurons trigger searching behavior immediately after ingestion.

The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Ol1mpiad, we probed stage 1 Drosophila larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages.

Pavlovian Conditioning of Larval Drosophila: An Illustrated, Multilingual, Hands-On Manual for Odor-Taste Associative Learning in Maggots.

Larval Drosophila offer a study case for behavioral neurogenetics that is simple enough to be experimentally tractable, yet complex enough to be worth the effort. We provide a detailed, hands-on manual for Pavlovian odor-reward learning in these animals. Given the versatility of Drosophila for genetic analyses, combined with the evolutionarily shared genetic heritage with humans, the paradigm has utility not only in behavioral neurogenetics and experimental psychology, but for translational biomedicine as well. Together with the upcoming total synaptic connectome of the Drosophila nervous system and the possibilities of single-cell-specific transgene expression, it offers enticing opportunities for research. Indeed, the paradigm has already been adopted by a number of labs and is robust enough to be used for teaching in classroom settings. This has given rise to a demand for a detailed, hands-on manual directed at newcomers and/or at laboratory novices, and this is what we here provide. The paradigm and the present manual have a unique set of features: The paradigm is cheap, easy, and robust;The manual is detailed enough for newcomers or laboratory novices;It briefly covers the essential scientific context;It includes sheets for scoring, data analysis, and display;It is multilingual: in addition to an English version we provide German, French, Japanese, Spanish and Italian language versions as well.The present manual can thus foster science education at an earlier age and enable research by a broader community than has been the case to date.

cryptochrome genes form an oscillatory loop independent of the per/tim loop in the circadian clockwork of the cricket Gryllus bimaculatus.

BACKGROUND: Animals exhibit circadian rhythms with a period of approximately 24 h in various physiological functions, including locomotor activity. This rhythm is controlled by an endogenous oscillatory mechanism, or circadian clock, which consists of cyclically expressed clock genes and their product proteins. cryptochrome (cry) genes are thought to be involved in the clock mechanism, and their functions have been examined extensively in holometabolous insects, but in hemimetabolous insects their role is less well understood. RESULTS: In the present study, the role of cry genes was investigated using RNAi technology in a hemimetabolous insect, the cricket Gryllus bimaculatus. Using a molecular cloning approach, we obtained cDNAs for two cry genes: Drosophila-type cry1 (Gb'cry1) and mammalian-type cry2 (Gb'cry2). Gb'cry2 has six splicing variants, most of which showed rhythmic mRNA expression. Gb'cry1RNAi treatment had only a limited effect at the behavioral and molecular levels, while Gb'cry2RNAi had a significant effect on behavioral rhythms and molecular oscillatory machinery, alone or in combination with Gb'cry1RNAi. In Gb'cry1/Gb'cry2 double-RNAi crickets, most clock genes showed arrhythmic expression, except for timeless, which retained clear rhythmic expression. Molecular analysis revealed that some combination of Gb'cry1 and Gb'cry2 variants suppressed CLK/CYC transcriptional activity in cultured cells. CONCLUSION: Based on these results, we propose a new model of the cricket's circadian clock, including a molecular oscillatory loop for Gb'cry2, which can operate independent of the Gb'per/Gb'tim loop.

Preference for and learning of amino acids in larval Drosophila.

Relative to other nutrients, less is known about how animals sense amino acids and how behaviour is organized accordingly. This is a significant gap in our knowledge because amino acids are required for protein synthesis - and hence for life as we know it. Choosing Drosophila larvae as a case study, we provide the first systematic analysis of both the preference behaviour for, and the learning of, all 20 canonical amino acids in Drosophila We report that preference for individual amino acids differs according to the kind of amino acid, both in first-instar and in third-instar larvae. Our data suggest that this preference profile changes across larval instars, and that starvation during the third instar also alters this profile. Only aspartic acid turns out to be robustly attractive across all our experiments. The essentiality of amino acids does not appear to be a determinant of preference. Interestingly, although amino acids thus differ in their innate attractiveness, we find that all amino acids are equally rewarding. Similar discrepancies between innate attractiveness and reinforcing effect have previously been reported for other tastants, including sugars, bitter substances and salt. The present analyses will facilitate the ongoing search for the receptors, sensory neurons, and internal, homeostatic amino acid sensors in Drosophila.

Mated Drosophila melanogaster females consume more amino acids during the dark phase.

To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly Drosophila melanogaster, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional period gene (per0), a well-characterized clock gene in Drosophila, found no difference between the light and dark phases in amino acid consumption by per0 flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a per0-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase via the circadian clock.

Genetic Variation in Taste Sensitivity to Sugars in Drosophila melanogaster.

Taste sensitivity plays a major role in controlling feeding behavior, and alterations in feeding habit induced by changes in taste sensitivity can drive speciation. We investigated variability in taste preferences in wild-derived inbred lines from the Drosophila melanogaster Genetic Reference Panel. Preferences for different sugars, which are essential nutrients for fruit flies, were assessed using two-choice preference tests that paired glucose with fructose, sucrose, or trehalose. The two-choice tests revealed that individual lines have differential and widely variable sugar preferences, and that sugar taste sensitivity is polygenic in the inbred population tested. We focused on 2 strains that exhibited opposing preferences for glucose and fructose, and performed proboscis extension reflex tests and electrophysiological recordings on taste sensilla upon exposure to fructose and glucose. The results indicated that taste sensitivity to fructose is dimorphic between the 2 lines. Genetic analysis showed that high sensitivity to fructose is autosomal dominant over low sensitivity, and that multiple loci on chromosomes 2 and 3 influence sensitivity. Further genetic complementation tests for fructose sensitivity on putative gustatory receptor (Gr) genes for sugars suggested that the Gr64a-Gr64f locus, not the fructose receptor gene Gr43a, might contribute to the dimorphic sensitivity to fructose between the 2 lines.

Octopamine and Tyramine Contribute Separately to the Counter-Regulatory Response to Sugar Deficit in Drosophila.

All animals constantly negotiate external with internal demands before and during action selection. Energy homeostasis is a major internal factor biasing action selection. For instance, in addition to physiologically regulating carbohydrate mobilization, starvation-induced sugar shortage also biases action selection toward food-seeking and food consumption behaviors (the counter-regulatory response). Biogenic amines are often involved when such widespread behavioral biases need to be orchestrated. In mammals, norepinephrine (noradrenalin) is involved in the counterregulatory response to starvation-induced drops in glucose levels. The invertebrate homolog of noradrenalin, octopamine (OA) and its precursor tyramine (TA) are neuromodulators operating in many different neuronal and physiological processes. Tyrosine-ß-hydroxylase (tßh) mutants are unable to convert TA into OA. We hypothesized that tßh mutant flies may be aberrant in some or all of the counter-regulatory responses to starvation and that techniques restoring gene function or amine signaling may elucidate potential mechanisms and sites of action. Corroborating our hypothesis, starved mutants show a reduced sugar response and their hemolymph sugar concentration is elevated compared to control flies. When starved, they survive longer. Temporally controlled rescue experiments revealed an action of the OA/TA-system during the sugar response, while spatially controlled rescue experiments suggest actions also outside of the nervous system. Additionally, the analysis of two OA- and four TA-receptor mutants suggests an involvement of both receptor types in the animals' physiological and neuronal response to starvation. These results complement the investigations in Apis mellifera described in our companion paper (Buckemüller et al., 2017).

Function of desiccate in gustatory sensilla of drosophila melanogaster.

Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

Learning the specific quality of taste reinforcement in larval Drosophila.

The only property of reinforcement insects are commonly thought to learn about is its value. We show that larval Drosophila not only remember the value of reinforcement (How much?), but also its quality (What?). This is demonstrated both within the appetitive domain by using sugar vs amino acid as different reward qualities, and within the aversive domain by using bitter vs high-concentration salt as different qualities of punishment. From the available literature, such nuanced memories for the quality of reinforcement are unexpected and pose a challenge to present models of how insect memory is organized. Given that animals as simple as larval Drosophila, endowed with but 10,000 neurons, operate with both reinforcement value and quality, we suggest that both are fundamental aspects of mnemonic processing-in any brain.

Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila.

A diverse range of organisms shows physiological and behavioural rhythms with various periods. Extensive studies have been performed to elucidate the molecular mechanisms of circadian rhythms with an approximately 24 h period in both Drosophila and mammals, while less attention has been paid to ultradian rhythms with shorter periods. We used a video-tracking method to monitor the movement of single flies, and clear ultradian rhythms were detected in the locomotor behaviour of wild type and clock mutant flies kept under constant dark conditions. In particular, the Pigment-dispersing factor mutant (Pdf 01) demonstrated a precise and robust ultradian rhythmicity, which was not temperature compensated. Our results suggest that Drosophila has an endogenous ultradian oscillator that is masked by circadian rhythmic behaviours.

Genetic variation in food choice behaviour of amino acid-deprived Drosophila.

To understand homeostatic regulation in insects, we need to understand the mechanisms by which they respond to external stimuli to maintain the internal milieu. Our previous study showed that Drosophila melanogaster exhibit specific amino acid preferences. Here, we used the D.melanogaster Genetic Reference Panel (DGRP), which is comprised of multiple inbred lines derived from a natural population, to examine how amino acid preference changes depending on the internal nutritional state in different lines. We performed a two-choice preference test and observed genetic variations in the response to amino acid deprivation. For example, a high-responding line showed an enhanced preference for amino acids even after only 1day of deprivation and responded to a fairly low concentration of amino acids. Conversely, a low-responding line showed no increased preference for amino acids after deprivation. We compared the gene expression profiles between selected high- and the low-responding lines and performed SNP analyses. We found several groups of genes putatively involved in altering amino acid preference. These results will contribute to future studies designed to explore how the genetic architecture of an organism evolves to adapt to different nutritional environments.

C-terminal binding protein (CtBP) activates the expression of E-box clock genes with CLOCK/CYCLE in Drosophila.

In Drosophila, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as "E-box clock genes"). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. In vivo overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, period (per), vrille (vri), and PAR domain protein 1ε (Pdp1ε). Co-expression of dCtBP with CLK in vitro also increased the promoter activity of per, vri, Pdp1ε and cwo depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes in vitro was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD+ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.

Suppression of conditioned odor approach by feeding is independent of taste and nutritional value in Drosophila.

Motivation controls behavior [1]. A variety of food-related behaviors undergo motivational modulation by hunger, satiety, and other states [2-4]. Here we searched for critical satiation factors modulating approach to an odor associated with sugar reward in Drosophila melanogaster. We selectively manipulated different parameters associated with feeding, such as internal glucose levels, and determined which are required for suppressing conditioned odor approach. Surprisingly, glucose levels in the hemolymph, nutritional value, sweetness of the food, and ingested volume (above a minimal threshold) did not influence behavior suppression. Instead, we found that the total osmolarity of ingested food is a critical satiation factor. In parallel, we found that conditioned approach is transiently suppressed by artificial stimulation of adipokinetic hormone (AKH) expressing corpora cardiaca cells, which causes elevation of hemolymph carbohydrate and lipid concentrations [5, 6]. This result implies that a rise in hemolymph osmolarity, without the experience of feeding, is sufficient to satiate conditioned odor approach. AKH stimulation did not affect innate sugar preference, suggesting that multiple satiation signals control different sets of appetitive behaviors.