The Drosophila NPY-like system protects against chronic stress-induced learning deficit by preventing the disruption of autophagic flux.

Chronic stress may induce learning and memory deficits that are associated with a depression-like state in Drosophila melanogaster. The molecular and neural mechanisms underlying the etiology of chronic stress-induced learning deficit (CSLD) remain elusive. Here, we show that the autophagy-lysosomal pathway, a conserved cellular signaling mechanism, is associated with chronic stress in Drosophila, as indicated by time-series transcriptome profiling. Our findings demonstrate that chronic stress induces the disruption of autophagic flux, and chronic disruption of autophagic flux could lead to a learning deficit. Remarkably, preventing the disruption of autophagic flux by up-regulating the basal autophagy level is sufficient to protect against CSLD. Consistent with the essential role of the dopaminergic system in modulating susceptibility to CSLD, dopamine neuronal activity is also indispensable for chronic stress to induce the disruption of autophagic flux. By screening knockout mutants, we found that neuropeptide F, the Drosophila homolog of neuropeptide Y, is necessary for normal autophagic flux and promotes resilience to CSLD. Moreover, neuropeptide F signaling during chronic stress treatment promotes resilience to CSLD by preventing the disruption of autophagic flux. Importantly, neuropeptide F receptor activity in dopamine neurons also promotes resilience to CSLD. Together, our data elucidate a mechanism by which stress-induced excessive dopaminergic activity precipitates the disruption of autophagic flux, and chronic disruption of autophagic flux leads to CSLD, while inhibitory neuropeptide F signaling to dopamine neurons promotes resilience to CSLD by preventing the disruption of autophagic flux.

Differential expression of neuropeptide F in the digestive organs of female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle.

Neuropeptide F is a key hormone that controls feeding in invertebrates, including decapod crustaceans. We investigated the differential expression of Macrobrachium rosenbergii neuropeptide F (MrNPF) in the digestive organs of female prawns, M. rosenbergii, during the ovarian cycle. By using RT-qPCR, the expression of MrNPF mRNA in the esophagus (ESO), cardia (CD), and pylorus (PY) of the foregut (FG) gradually increased from stage II and peaked at stage III. In the midgut (MG), hindgut (HG), and hepatopancreas (HP), MrNPF mRNA increased from stage I, reaching a maximal level at stage II, and declined by about half at stages III and IV (P < 0.05). In the ESO, CD, and PY, strong MrNPF-immunoreactivities were seen in the epithelium, muscle, and lamina propria. Intense MrNPF-ir was found in the MG cells and the muscular layer. In the HG, MrNPF-ir was detected in the epithelium of the villi and gland regions, while MrNPF-ir was also more intense in the F-, R-, and B-cells in the HP. However, we found little colocalization between the MrNPF and PGP9.5/ChAT in digestive tissues, implying that most of the positive cells might not be neurons but could be digestive tract-associated endocrine cells that produce and secrete MrNPF to control digestive organ functions in feeding and utilizing feed. Taken together, our first findings indicated that MrNPF was differentially expressed in digestive organs in correlation with the ovarian cycle, suggesting an important link between MrNPF, the physiology of various digestive organs in feeding, and possibly ovarian maturation in female M. rosenbergii.

Differential expression of sNPF in male and female eyestalk leading to sex dimorphism of AMP expression in Procambarus clarkii intestine.

Antimicrobial peptide (AMP) is an important component of crustaceans' innate immune system. In this study, a short neuropeptide F (sNPF) gene (Pc-sNPF) and a Forkhead box O (FOXO) gene (PcFOXO) from Procambarus clarkii were identified. Analysis findings showed that the expression level of AMP genes differed between male and female P. clarkii. Furthermore, Pc-sNPF and PcFOXO were related to the sex dimorphism of AMP. Knockdown of Pc-sNPF in the eyestalk significantly upregulated the expression of PcFOXO and two anti-lipopolysaccharide factors (PcALF4 and PcALFL) in the intestine of P. clarkii. The expression of PcFOXO in the intestine of female P. clarkii was higher than in that of males. Results from RNA interference revealed that PcFOXO positively regulated the expression of PcALF4 and PcALFL in the intestine of male and female P. clarkii. In summary, our study showed that differences in Pc-sNPF expression in eyestalk of male and female P. clarkii leading to sex dimorphism of AMP expression in the intestine are mediated by the sNPF-FOXO-AMP signal pathway called the eyestalk-intestine axis.

Oscillations in the central brain of Drosophila are phase locked to attended visual features.

Object-based attention describes the brain's capacity to prioritize one set of stimuli while ignoring others. Human research suggests that the binding of diverse stimuli into one attended percept requires phase-locked oscillatory activity in the brain. Even insects display oscillatory brain activity during visual attention tasks, but it is unclear if neural oscillations in insects are selectively correlated to different features of attended objects. We addressed this question by recording local field potentials in the Drosophila central complex, a brain structure involved in visual navigation and decision making. We found that attention selectively increased the neural gain of visual features associated with attended objects and that attention could be redirected to unattended objects by activation of a reward circuit. Attention was associated with increased beta (20- to 30-Hz) oscillations that selectively locked onto temporal features of the attended visual objects. Our results suggest a conserved function for the beta frequency range in regulating selective attention to salient visual features.

Mechanisms of feeding cessation in Helicoverpa armigera larvae exposed to Bacillus thuringiensis Cry1Ac toxin.

Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been applied in sprayable formulations and expressed in transgenic crops for the control of pests in the field. When exposed to Bt proteins insect larvae display feeding cessation, yet the mechanism for this phenomenon remains unknown. In this study, we investigated the feeding behavior and underlying mechanisms of cotton bollworm (Helicoverpa armigera) larvae after exposure to the Cry1Ac protein from Bt. Three H. armigera strains were studied: the susceptible SCD strain, the C2/3-KO strain with HaABCC2 and HaABCC3 knocked out and high-level resistance to Cry1Ac (>15,000-fold), and the SCD-KI strain with a T92C point mutation in tetraspanin (HaTSPAN1) and medium-level resistance to Cry1Ac (125-fold). When determining the percentage of insects that continued feeding after various exposure times to Cry1Ac, we observed quick cessation of feeding in larvae from the susceptible SCD strain, whereas larvae from the C2/3-KO strain did not display feeding cessation. In contrast, larvae from the SCD-KI strain rapidly recovered from the initial feeding cessation. Histopathological analyses and qRT-PCR in midguts of SCD larvae after Cry1Ac exposure detected serious epithelial damage and significantly reduced expression of the neuropeptide F gene (NPF) and its potential receptor gene NPFR, which are reported to promote insect feeding. Neither epithelial damage nor altered NPF and NPFR expression appeared in midguts of C2/3-KO larvae after Cry1Ac treatment. The same treatment in SCD-KI larvae resulted in milder epithelial damage and subsequent repair, and a decrease followed by an initial increase in NPF and NPFR expression. These results demonstrate that the feeding cessation response to Cry1Ac in cotton bollworm larvae is closely associated with midgut epithelial damage and downregulation of NPF and NPFR expression. This information provides clues to the mechanism of feeding cessation in response to Bt intoxication and contributes to the mode of action of the Cry1Ac toxin in target pests.

A short neuropeptide F analog (sNPF), III-2 may particularly regulate juvenile hormone III to influence Spodoptera frugiperda metamorphosis and development.

Allatostatin (AS) or Allatotropin (AT) is a class of insect short neuropeptide F (sNPF) that affects insect growth and development by inhibiting or promote the synthesis of juvenile hormone (JH) in different insects. III-2 is a novel sNPF analog derived from a group of nitroaromatic groups connected by different amino acids. In this study, we found that III-2 showed high insecticidal activity against S. frugiperda larvae with a LC50 of 18.7 mg L-1. As demonstrated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), III-2 particularly facilitated JH III and hindered 20E synthesis in S. frugiperda. The results of RNA-Seq and quantitative real-time polymerase chain reaction (qPCR) showed that III-2 treatment promoted the expression of key genes such as SfCYP15C1 in JH synthesis pathway and inhibited the expression of SfCYP314A1 and other genes in the 20E synthetic pathway. Significant differences were also observed in the expression of the genes related to cuticle formation. We report for the first time that sNPF compounds specifically interfere with the synthesis and secretion of a certain JH in insects, thus affecting the ecdysis and growth of insects, and leading to death. This study may provide a new plant conservation concept for us to seek the targeted control of certain insects based on specific interference with different JH.

The short neuropeptide F (sNPF) promotes the formation of appetitive visual memories in honey bees.

Motivation can critically influence learning and memory. Multiple neural mechanisms regulate motivational states, among which signalling via specific neuropeptides, such as NPY in vertebrates and NPF and its short variant sNPF in invertebrates, plays an essential role. The honey bee (Apis mellifera) is a privileged model for the study of appetitive learning and memory. Bees learn and memorize sensory cues associated with nectar reward while foraging, and their learning is affected by their feeding state. However, the neural underpinnings of their motivational states remain poorly known. Here we focused on the short neuropeptide F (sNPF) and studied if it modulates the acquisition and formation of colour memories. Artificially increasing sNPF levels in partially fed foragers with a reduced motivation to learn colours resulted in significant colour learning and memory above the levels exhibited by starved foragers. Our results thus identify sNPF as a critical component of motivational processes involved in foraging and in the cognitive processes associated with this activity in honey bees.

Appetitive Memory with Survival Benefit Is Robust Across Aging in Drosophila.

The formation of memory declines with advancing age. However, susceptibility to memory impairments depends on several factors, including the robustness of memory, the responsible neural circuits, and the internal state of aged individuals. How age-dependent changes in internal states and neural circuits affect memory formation remains unclear. Here, we show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which suppresses their high mortality rates during starvation. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. We found that aging enhanced the preference for nutritious sugar over non-nutritious sugar correlated with an age-dependent increase in the expression of Drosophila neuropeptide F, an ortholog of mammalian neuropeptide Y. Furthermore, a subset of dopaminergic neurons that signal the sweet taste of sugar decreases its function with aging, while a subset of dopaminergic neurons that signal the nutritional value of sugar maintains its function with age. Our results suggest that aging impairs the ability to form memories without survival benefits; however, the ability to form memories with survival benefits is maintained through age-dependent changes in the neural circuits and neuropeptides.SIGNIFICANCE STATEMENT The susceptibility to age-dependent memory impairments depends on the strength of the memory, changes in the responsible neurons, and internal states of aged individuals. How age-dependent changes in such internal states affect neural activity and memory formation remains unclear. We show in Drosophila melanogaster that aged flies of both sexes form robust appetitive memory conditioned with nutritious sugar, which has survival benefits for aged flies. In contrast, aging impairs the formation of appetitive memory conditioned with non-nutritious sugar that lacks survival benefits for the flies. Aging changes the neural circuits including dopamine neurons and neuropeptide F-expressing neurons, leading to the age-dependent impairment in memory with insufficient survival benefits and the preservation of the ability to form memory with survival benefits.

Insulin signalling elicits hunger-induced feeding in Drosophila.

Insulin, a highly conserved peptide hormone, links nutrient availability to metabolism and growth in animals. In fed states insulin levels remain high and in animals that are food deprived insulin signalling drops. Here, we report that in Drosophila, feeding elicited by short periods of starvation is dependent on insulin signalling. The activity of insulin signalling pathway in the abdominal fatbody aids in feeding during short periods of starvation. A feedback regulatory signalling that involves cells that express the Drosophila hunger hormone short-neuropeptide-F (sNPF) and insulin-producing cells sustain the orexigenic function of insulin. Furthermore, the orexigenic phase of insulin activity aids in the efficient management of nutrient stores and survival of flies during starvation.

Neuropeptide F from endocrine cells in Plutella xylostella midgut modulates feeding and synergizes Cry1Ac action.

With the wide cultivation of transgenic plants throughout the world and the rising risk of resistance to Bacillus thuringiensis crystal (Cry) toxins, it is essential to design an adaptive resistance management strategy for continued use. Neuropeptide F (NPF) of insects has proven to be valuable for the production of novel-type transgenic plants via its important role in the control of feeding behavior. In this study, the gene encoding NPF was cloned from the diamondback moth, Plutella xylostella, an important agricultural pest. Real-time quantitative reverse transcription-polymerase chain reaction and in situ hybridization showed a relatively high expression of P. xylostella-npf (P. x-npf) in endocrine cells of the midgut of fourth instar larvae, and it was found to participate in P. xylostella feeding behavior and Cry1Ac-induced feeding inhibition. Prokaryotic expression and purification provided structure unfolded P. x-npf from inclusion bodies for diet surface overlay bioassays and the results demonstrated a significant synergistic effect of P. x-npf on Cry1Ac toxicity by increasing intake of noxious food which contains Cry toxins, especially quick death at an early stage of feeding. Our findings provided a potential new way to efficiently control pests by increasing intake of lower dose Cry toxins and a novel hint for the complex Cry toxin mechanism.

Molecular cloning, characterization and functional analysis of two neuropeptide F genes from the oriental river prawn (Macrobrachium nipponense).

In invertebrates, neuropeptide F (NPF) has many regulatory functions, similar to NPY, its homologous peptide. In this study, two neuropeptide F genes were identified in Macrobrachium nipponense: Mn-NPF1 and Mn-NPF2. Mn-NPF2 shared the same amino acid sequence with Mn-NPF1, except for a 37 amino acid insert in the middle of the NPF region. The quantitative-PCR (qPCR) results indicated that Mn-NPF1 expression was positively correlated with ovarian maturation, whereas Mn-NPF2 had opposing expression patterns. Both Mn-NPFs were poorly expressed at early embryonic stages, but enhanced expression levels were observed up to day 10 after hatching, when the gonads began to differentiate. Ovary in situ hybridization (ISH) analyses showed that both Mn-NPFs were present at all stages, but were differentially localized to distinct compartments. Temperature gradient studies showed that both Mn-NPFs were implicated in the seasonal regulation of reproduction. A double-stranded (ds) RNA-Mn-NPF2 injection led to a significant 38.5% increase in the vitellogenin (VG) transcript (P < 0.05). These results demonstrated that Mn-NPF2 plays an important role in inhibiting ovarian maturation.

Leucokinin and Associated Neuropeptides Regulate Multiple Aspects of Physiology and Behavior in Drosophila.

Leucokinins (LKs) constitute a family of neuropeptides identified in numerous insects and many other invertebrates. LKs act on G-protein-coupled receptors that display only distant relations to other known receptors. In adult Drosophila, 26 neurons/neurosecretory cells of three main types express LK. The four brain interneurons are of two types, and these are implicated in several important functions in the fly's behavior and physiology, including feeding, sleep-metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. The 22 neurosecretory cells (abdominal LK neurons, ABLKs) of the abdominal neuromeres co-express LK and a diuretic hormone (DH44), and together, these regulate water and ion homeostasis and associated stress as well as food intake. In Drosophila larvae, LK neurons modulate locomotion, escape responses and aspects of ecdysis behavior. A set of lateral neurosecretory cells, ALKs (anterior LK neurons), in the brain express LK in larvae, but inconsistently so in adults. These ALKs co-express three other neuropeptides and regulate water and ion homeostasis, feeding, and drinking, but the specific role of LK is not yet known. This review summarizes Drosophila data on embryonic lineages of LK neurons, functional roles of individual LK neuron types, interactions with other peptidergic systems, and orchestrating functions of LK.

Short neuropeptide F enhances the immune response in the hepatopancreas of mud crab (Scylla paramamosain).

Short neuropeptide F (sNPF), a highly conserved neuropeptide, displays pleiotropic functions on multiple aspects of physiological processes, such as feeding, metabolic stress, locomotion, circadian clock and reproduction. However, to date there has no any report on the possible immunoregulation of sNPF in crustaceans. In the present study, we found that the Sp-sNPF was mainly expressed in the nervous tissue in the mud crab Scylla paramamosain, while the sNPF receptor gene (Sp-sNPF-R) was expressed in a wide variety of tissues, including the hepatopancreas. In situ hybridization further showed that the Sp-sNPF-R positive signal mainly localized in the F-cells of the hepatopancreas. Moreover, the Sp-sNPF-R transcription could be significantly up-regulated after the challenge of bacteria-analog LPS or virus-analog Poly (I:C). Both in vitro and in vivo experiments showed that the synthetic sNPF peptide significantly increased the gene expressions of sNPF-R, nuclear factor-κB (NF-κB) signaling genes and antimicrobial peptides (AMPs) in the hepatopancreas. Simultaneously, the administration of sNPF peptide in vitro also increased the concentration of nitric oxide (NO) and the bacteriostasis of the culture medium of hepatopancreas. These results indicated that sNPF up-regulated hepatopancreas immune responses, which may bring new insight into the neuroendocrine-immune regulatory system in crustacean species, and could potentially provide a new strategy for disease prevention and control for mud crab aquaculture.

A cyclic nucleotide-gated channel in the brain regulates olfactory modulation through neuropeptide F in fruit fly Drosophila melanogaster.

Olfactory sensing and its modulation are important for the insects in recognizing diverse odors from the environment and in making correct decisions to survive. Identifying new genes involved in olfactory modulation and unveiling their mechanisms may lead us to understand decision making processes in the central nervous system. Here, we report a novel olfactory function of the cyclic nucleotide-gated (CNG) channel CG42260 in modulating ab3A olfactory sensory neurons, which specifically respond to food-derived odors in fruit fly Drosophila melanogaster. We found that two independent CG42260 mutants show reduced responses in the ab3A neurons. Unlike mammalian CNGs, CG42260 is not expressed in the odorant sensory neurons but broadly in the central nervous system including neuropeptide-producing cells. By using molecular genetic tools, we identified CG42260 expression in one pair of neuropeptide F (NPF) positive L1-l cells known to modulate food odor responsiveness. Knockdown of CG42260 in the NPF neurons reduced production of NPF in Ll-1 cells, which in turn, led to reduction of neuronal responses of the ab3A neurons. Our findings show the novel biological function of CG42260 in modulating olfactory responses to food odor through NPF.

Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay.

In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.

Abdominal contact of fluvalinate induces olfactory deficit in Apis mellifera.

τ-Fluvalinate (fluvalinate) is a highly selective pyrethroid insecticide compound used for controlling ectoparasitic mites that cause major damages in honey bee colonies. Although honey bees have resistance and low toxicity to this xenobiotic chemical, little is known about the effects of this chemical on sensory modulation and behaviors in honey bees. Here we addressed the effect on olfactory cognition at the behavioral, molecular, and neurophysiological levels. First, we found that topical application of fluvalinate to honeybee abdomen elicited somewhat severe toxicity to honey bees. Furthermore, honeybees treated with sublethal doses of fluvalinate showed a significant decrease in olfactory responses. At the molecular level, there was no change in gene expression levels of odorant receptor co-receptor (Orco), which is important for electrical conductivity induced by odorant binding in insects. Rather, small neuropeptide F (sNPF) signaling pathway was involved in olfactory fluctuation after treatment of fluvalinate. This indicates that olfactory deficits by abdominal contact of fluvalinate may stem from various internal molecular pathways in honey bees.

Distribution of neuropeptide F in the ventral nerve cord and its possible role on testicular development and germ cell proliferation in the giant freshwater prawn, Macrobrachium rosenbergii.

Neuropeptide F in invertebrates is a homolog of neuropeptide Y in mammals and it is a member of FMRFamide-related peptides. In arthropods, such as insects, there are two types of neuropeptide F comprising long neuropeptide F (NPF) and short neuropeptide F (sNPF). Both NPFs are known to play a crucial role in the regulations of foraging, feeding-related behaviors, circadian rhythm, stress responses, aggression and reproduction in invertebrates. We have earlier found that in the giant freshwater prawn, Macrobrachium rosenbergii, there are three isoforms of NPF and four isoforms of sNPF and that NPFs are expressed in the eyestalks and brain. In the present study, we investigate further the tissue distribution of NPF-I in the ventral nerve cord (VNC) and its role in the development of testes in small male (SM) Macrobrachium rosenbergii. By immunolocalization, using the rabbit polyclonal antibody against NPF-I as a probe, we could detect NPF-I immunoreactivity in the neuropils and neuronal clusters of the subesophageal ganglia (SEG), thoracic ganglia (TG) and abdominal ganglia (AG) of the SM prawns. In functional assays, the administrations of synthetic NPF-I (KPDPTQLAAMADALKYLQELDKYYSQVSRPRFamide) and sNPF (APALRLRFamide) peptides significantly increased the growth rates of SM prawns and significantly increased the gonadosomatic index (GSI) and proliferations of early germ cells in the seminiferous tubules of their testes. It is, therefore, suggestive that NPFs may play critical roles in energy homeostasis towards promoting growth as well as testicular development in prawns that could be applied in the aquaculture of this species.

Antagonistic Effect of Short Neuropeptide F on Allatotropin-Inhibited Feeding Motivation of the Silkworm Larva, Bombyx mori.

Here, we demonstrated an antagonistic effect of short neuropeptide F (sNPF) in modulating feeding motivation in the silkworm Bombyx mori; sNPF reduced the feeding-delaying effects caused by administration of an inhibitory peptide, allatotropin (AT). In situ hybridization and MALDI-TOF MS analysis revealed the presence of three subtypes of sNPFs (sNPF-1, -2, and -3) in the midgut enteroendocrine cells. Ca2+-imaging analyses revealed that three subtypes of sNPF receptors (sNPFRs) (BNGR-A7, -A10, and -A11) showed different affinities with the three subtypes of sNPFs. In addition, sNPF activated its signaling via ERK phosphorylation in the midgut, while mixture of sNPF and AT reduced the phosphorylation level, agreeing with the results of behavioral assay. Together, our current findings suggest that intestinal sNPF positively modulates the feeding motivation by reducing the inhibitory effects by AT within the midgut.

Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm (Bombyx mori) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca2+ mobilization via a Gi/o-dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo-sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities.

Innate visual preferences and behavioral flexibility in Drosophila.

Visual decision making in animals is influenced by innate preferences as well as experience. Interaction between hard-wired responses and changing motivational states determines whether a visual stimulus is attractive, aversive or neutral. It is, however, difficult to separate the relative contribution of nature versus nurture in experimental paradigms, especially for more complex visual parameters such as the shape of objects. We used a closed-loop virtual reality paradigm for walking Drosophila to uncover innate visual preferences for the shape and size of objects, in a recursive choice scenario allowing the flies to reveal their visual preferences over time. We found that Drosophila melanogaster display a robust attraction/repulsion profile for a range of object sizes in this paradigm, and that this visual preference profile remains evident under a variety of conditions and persists into old age. We also demonstrate a level of flexibility in this behavior: innate repulsion to certain objects could be transiently overridden if these were novel, although this effect was only evident in younger flies. Finally, we show that a neuromodulatory circuit in the fly brain, Drosophila neuropeptide F (dNPF), can be recruited to guide visual decision making. Optogenetic activation of dNPF-expressing neurons converted a visually repulsive object into a more attractive object. This suggests that dNPF activity in the Drosophila brain guides ongoing visual choices, to override innate preferences and thereby provide a necessary level of behavioral flexibility in visual decision making.