Genome-wide analysis reveals transcriptional and translational changes during diapause of the Asian corn borer (Ostrinia furnacalis).

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.

Excess Dally-like Induces Malformation of Drosophila Legs.

Glypicans are closely associated with organ development and tumorigenesis in animals. Dally-like (Dlp), a membrane-bound glypican, plays pivotal roles in various biological processes in Drosophila. In this study, we observed that an excess of Dlp led to the malformation of legs, particularly affecting the distal part. Accordingly, the leg disc was shrunken and frequently exhibited aberrant morphology. In addition, elevated Dlp levels induced ectopic cell death with no apparent cell proliferation changes. Furthermore, Dlp overexpression in the posterior compartment significantly altered Wingless (Wg) distribution. We observed a marked expansion of Wg distribution within the posterior compartment, accompanied by a corresponding decrease in the anterior compartment. It appears that excess Dlp guides Wg to diffuse to cells with higher Dlp levels. In addition, the distal-less (dll) gene, which is crucial for leg patterning, was up-regulated significantly. Notably, dachshund (dac) and homothorax (hth) expression, also essential for leg patterning and development, only appeared to be negligibly affected. Based on these findings, we speculate that excess Dlp may contribute to malformations of the distal leg region of Drosophila, possibly through its influence on Wg distribution, dll expression and induced cell death. Our research advances the understanding of Dlp function in Drosophila leg development.

The CoREST complex regulates multiple histone modifications temporal-specifically in clock neurons.

Epigenetic regulation is important for circadian rhythm. In previous studies, multiple histone modifications were found at the Period (Per) locus. However, most of these studies were not conducted in clock neurons. In our screen, we found that a CoREST mutation resulted in defects in circadian rhythm by affecting Per transcription. Based on previous studies, we hypothesized that CoREST regulates circadian rhythm by regulating multiple histone modifiers at the Per locus. Genetic and physical interaction experiments supported these regulatory relationships. Moreover, through tissue-specific chromatin immunoprecipitation assays in clock neurons, we found that the CoREST mutation led to time-dependent changes in corresponding histone modifications at the Per locus. Finally, we proposed a model indicating the role of the CoREST complex in the regulation of circadian rhythm. This study revealed the dynamic changes of histone modifications at the Per locus specifically in clock neurons. Importantly, it provides insights into the role of epigenetic factors in the regulation of dynamic gene expression changes in circadian rhythm.

Lead specifically declines tyrosine hydroxylase activity to induce the onset of Parkinson's disease through disrupting dopamine biosynthesis in fly models.

Parkinson's disease (PD) is one of the fastest-growing neurodegenerative diseases and has been linked to the exposure to numerous environmental neurotoxins. Although lead (Pb) exposure has been related to the development of PD, the molecular target of Pb to cause the onset of PD is insufficiently investigated. Herein, we explored the effects of Pb exposure on behavior, pathophysiology, and gene expression of wild-type (WT) fly (Drosophila melanogaster) by comparison with its PD model. After exposure to Pb, the WT flies showed PD-like locomotor impairments and selective loss of dopaminergic (DAergic) neurons, displaying similar phenotypes to fly PD model (PINK1). Transcriptomic analysis showed the similarity in gene expression profiles between Pb treatment WT flies and PINK1 mutant flies. Moreover, Pb exposure resulted in endogenous dopamine deficits in WT flies. Analyses of gene expression and enzyme activity confirmed that Pb exposure reduced tyrosine hydroxylase (TH) activity and led to failure of dopamine synthesis. Furthermore, molecular dynamics simulation confirmed that Pb was adsorbed by TH and subsequently inhibited the enzymatic activity. Exogenous injection of L-dopa and melatonin could partially rescue the pathological phenotypes of Pb-exposed flies and PD fly model. Antagonist injection of microRNA-133, which negatively regulated the expression of TH gene, ultimately rescued in the manifestation of PD phenotypes in flies. Involvement of TH overexpression mutants of fly strongly promoted the resistance to Pb exposure and rescued both behavior and the number of DAergic neurons. Therefore, our study elucidates the Pb molecular target in dopamine pathway and mechanism underlying the risks of Pb exposure on the occurrence of PD at environmentally-relevant concentrations.

Genome-wide profiles of H3K9me3, H3K27me3 modifications, and DNA methylation during diapause of Asian corn borer (Ostrinia furnacalis).

Diapause represents a crucial adaptive strategy used by insects to cope with changing environmental conditions. In North China, the Asian corn borer (Ostrinia furnacalis) enters a winter larval diapause stage. Although there is growing evidence implicating epigenetic mechanisms in diapause regulation, it remains unclear whether dynamic genome-wide profiles of epigenetic modifications exist during this process. By investigating multiple histone modifications, we have discovered the essential roles of H3K9me3 and H3K27me3 during diapause of the Asian corn borer. Building upon previous findings in vertebrates highlighting the connection between DNA methylation and repressive histone methylations, we have examined changes in the genome-wide profile of H3K9me3, H3K27me3, and DNA methylation at the nondiapause, prediapause, and diapause stages. Data analysis reveals significant alterations in these three modifications during diapause. Moreover, we observe a correlation between the H3K9me3 and H3K27me3 modification sites during diapause, whereas DNA modifications show little association with either H3K9me3 or H3K27me3. Integrative analysis of epigenome and expression data unveils the relationship between these epigenetic modifications and gene expression levels at corresponding diapause stages. Furthermore, by studying the function of histone modifications on genes known to be important in diapause, especially those involved in the juvenile pathway, we discover that the juvenile hormone pathway lies downstream from H3K9me3 and H3K27me3 histone modifications. Finally, the analysis of gene loci with modified modifications unreported in diapause uncovers novel pathways potentially crucial in diapause regulation. This study provides a valuable resource for future investigations aiming to elucidate the underlying mechanisms of diapause.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

Osiris17 is essential for stable integrin localization and function during insect wing epithelia remodeling.

The dynamic adhesion between cells and their extracellular matrix is essential for the development and function of organs. During insect wing development, two epithelial sheets contact each other at their basal sites through the interaction of ßPS integrins with the extracellular matrix. We report that Osiris17 contributes to the maintenance of ßPS integrins localization and function in developing wing of Drosophila and locust. In flies with reduced Osiris17 expression the epithelia sheets fail to maintain the integrity of basal cytoplasmic junctional bridges and basal adhesion. In contrast to the continuous basal integrin localization in control wings, this localization is disrupted during late stages of wing development in Osiris17 depleted flies. In addition, the subcellular localization revealed that Osiris17 co-localizes with the endosomal markers Rab5 and Rab11. This observation suggests an involvement of Osiris17 in endosomal recycling of integrins. Indeed, Osiris17 depletion reduced the numbers of Rab5 and Rab11 positive endosomes. Moreover, overexpression of Osiris17 increased co-localization of Rab5 and ßPS integrins and partially rescued the detachment phenotype in flies with reduced ßPS integrins. Taken together, our data suggest that Osiris17 is an endosome related protein that contributes to epithelial remodeling and morphogenesis by assisting basal integrins localization in insects.

Mechanism of foraging selections regulated by gustatory receptor 43a in Ostrinia furnacalis larvae.

BACKGROUND: Omnivores, including humans, have an inborn tendency to avoid risky or non-nutritious foods. However, relatively little is known about how animals perceive and discriminate nutritious foods from risky substances. In this study, we explored the mechanism of feeding selection in Ostrinia furnacalis larvae, one of the most destructive pests to the maize crop. RESULTS: We identified a gustatory receptor, Gr43a, for feeding regulation in larvae of Ostrinia furnacalis, which highly expresses in the mouthparts of the first- (the period of just hatching out from eggs) and fifth-instar larvae (the period of gluttony). The Gr43a regulates foraging plasticity by discriminating sorbitol, a nonsweet nutritious substance, and sucralose, a sweet non-nutritious substance through the labra of mouthparts, while it differentiates fructose/sucrose and sucralose via the sensilla styloconica of mouthparts. Specially, Gr43a responds to fructose and sucrose via the medial and lateral sensilla styloconica in O. furnacalis, respectively. Furthermore, Gr43a is negatively regulated by the neuropeptide F system, a homologous mammalian neuropeptide Y system. CONCLUSION: This study reveals a smart feeding strategy for animals to meet both nutritional needs and sweet gratification, and offers an insight into complex feeding selections dependent on food resources in the surrounding environment. © 2023 Society of Chemical Industry.

Polycomb regulates circadian rhythms in Drosophila in clock neurons.

Circadian rhythms are essential physiological feature for most living organisms. Previous studies have shown that epigenetic regulation plays a crucial role. There is a knowledge gap in the chromatin state of some key clock neuron clusters. In this study, we show that circadian rhythm is affected by the epigenetic regulator Polycomb (Pc) within the Drosophila clock neurons. To investigate the molecular mechanisms underlying the roles of Pc in these clock neuron clusters, we use targeted DamID (TaDa) to identify genes significantly bound by Pc in the neurons marked by C929-Gal4 (including l-LNvs cluster), R6-Gal4 (including s-LNvs cluster), R18H11-Gal4 (including DN1 cluster), and DVpdf-Gal4, pdf-Gal80 (including LNds cluster). It shows that Pc binds to the genes involved in the circadian rhythm pathways, arguing a direct role for Pc in regulating circadian rhythms through specific clock genes. This study shows the identification of Pc targets in the clock neuron clusters, providing potential resource for understanding the regulatory mechanisms of circadian rhythms by the PcG complex. Thus, this study provided an example for epigenetic regulation of adult behavior.

Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila.

Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. Here, we demonstrate that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals.

NPFR regulates the synthesis and metabolism of lipids and glycogen via AMPK: Novel targets for efficient corn borer management.

RNA biopesticides are regarded as "the third revolution in the history of pesticides" due to their extensive advantages such as precision, high efficiency, green, pollution-free, etc. In the current study, two target genes encoding neuropeptide F receptor (NPFR) and AMP-activated protein kinase (AMPK), which are essential for insect feeding, cellular energy homeostasis and nutrient availability, were selected to design RNA pesticides. We achieved high RNA interference (RNAi) efficiency of npfr via a star polycation nanocarrier-based double-stranded RNA (dsRNA) delivery system. The food consumption of Ostrinia furnacalis is largely suppressed, which leads to a good protective effect on corn leaves. We determined the mechanism of the above genes. NPFR binds to the Gα protein and activates the intracellular second messengers cAMP and Ca2+, which in turn phosphorylate AMPK to regulate the synthesis and metabolism of lipids and glycogen. We then adopted a highly efficient bacteria-based expression system for the production of large amounts of dsRNA segments targeting npfr and ampk simultaneously and subsequently complexed them with nanocarriers to develop a novel dual-target RNA pesticide. Our RNA nanopesticide dramatically inhibits larval feeding, growth and development, and its controlling effect is even better than that of the widely used anti-feedant azadirachtin.

The cholinergic pathway transmits signals of neuropeptide F to regulate feeding of Ostrinia furnacalis larvae.

BACKGROUND: Feeding is the basis of animal survival and reproduction. In insects, the neuropeptide F (NPF), a homologous polypeptide of NPY in vertebrates, plays an important role in regulation of feeding behavior. However, relatively little has been known about the molecular mechanism of feeding. RESULTS: In this study, we show that the cholinergic pathway is very important in signaling transmission of NPF feeding regulation in Ostrinia furnacalis larvae, in which the choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (vAChT) in presynaptic membrane and the nicotinic acetylcholine receptor (nAChR) in postsynaptic membrane are positively regulated by NPF, while the ace1 and ace2 encoding the acetylcholinesterase (AChE) are negatively regulated by NPF, leading to a balance of acetylcholine (ACh)-the excitatory transmitter. More, the cholinergic pathway further transmits signaling to the downstream pathways of the phosphoInositide-3 kinase (PI3K) and the cAMP responsive element binding protein (CREB), respectively. CONCLUSION: The cholinergic transmission, positively regulated by NPF, is involved in feeding of O. furnacalis larvae via downstream PI3K and the CREB pathways, respectively. The deexcitation of cell cholinergic pathway or inhibition of PI3K and CREB lead to decreases of larval feeding amount. © 2023 Society of Chemical Industry.

Design, Synthesis, and Antifungal/Antioomycete Activity of Thiohydantoin Analogues Containing Spirocyclic Butenolide.

Novel fungicidal agents were designed based on the combination of two privileged scaffolds, thiohydantoin and spirocyclic butenolide, which are widely found in natural products. The synthesized compounds were characterized by 1H NMR, 13C NMR, and high-resolution electrospray ionisation mass spectrometry. The in vitro antioomycete activity evaluation showed that most of the compounds exhibited excellent inhibitory activities against different developmental stages in the life cycle of pathogenic oomycete Phytophthora capsici. Compound 5j could inhibit the mycelial growth, sporangium production, zoospore release, and cystospore germination significantly with EC50 values of 0.38, 0.25, 0.11, and 0.026 µg/mL, respectively. The in vivo antifungal/antioomycete bioassay results revealed that the series of compounds generally showed outstanding control efficacies against the pathogenic oomycete Pseudoperonospora cubensis, and compounds 5j, 5l, 7j, 7k, and 7l possessed broad-spectrum antifungal activities against the test phytopathogens. The in vivo protective and curative efficacies against P. capsici of the representative compound 5j were excellent, which were better than those of azoxystrobin. More prominently, 5j significantly promoted the biomass accumulation of the root system and reinforced the cell wall by callose deposition. The pronounced upregulation of immune response-related genes indicated that the active oomycete inhibitor 5j also functioned as a plant elicitor. Transmission electron microscopy observation and the enzyme activity test demonstrated that the mechanism of action of 5j was to bind to the pivotal protein, complex III on the respiratory chain, which resulted in a shortage of energy supply. Molecular docking results exhibited that compound 5j appropriately matched with the Qo pocket and had no interaction with the most commonly mutated site Gly-142, which may be of significant benefit in Qo fungicide resistance management. Compound 5j showed great advantages and potential in oomycete control, resistance management, and induction of disease resistance. A further investigation of 5j with a unique structure might have direct implications for the creation of novel oomycete inhibitors against plant-pathogenic oomycetes.

Synergism of Feeding and Digestion Regulated by the Neuropeptide F System in Ostrinia furnacalis Larvae.

Feeding is crucial for the growth and survival of animals, including humans, but relatively little is known about how it is regulated. Here, we show that larval feeding in Ostrinia furnacalis is regulated by neuropeptide F (NPF, the homologous peptide of mammalian NPY) via the insulin signalling pathway in the midgut. Furthermore, the genes pi3k and mtor in the insulin pathway positively regulate α-amylase and lipase of the midgut by recruiting the transcription factors c-Myc and PPARγ for binding to the promotors of these two enzymes. Importantly, we find that the feeding behaviour and the digestive system of midgut in O. furnacalis larvae are closely related and interactive in that knocking down α-amylase or lipase induces a reduction in larval feeding, while food-deprived larvae lead to fewer expressions of α-amylase and lipase. Importantly, it is the gut NPF that regulates the α-amylase and lipase, while variations of α-amylase and lipase may feed back to the brain NPF. This current study reveals a molecular feedback mechanism between feeding behaviour and the digestive system that is regulated by the conserved NPF via insulin signalling systems in the midgut of O. furnacalis larvae.

Cytosolic and mitochondrial ribosomal proteins mediate the locust phase transition via divergence of translational profiles.

The phase transition from solitary to gregarious locusts is crucial in outbreaks of locust plague, which threaten agricultural yield and food security. Research on the regulatory mechanisms of phase transition in locusts has focused primarily on the transcriptional or posttranslational level. However, the translational regulation of phase transition is unexplored. Here, we show a phase-dependent pattern at the translation level, which exhibits different polysome profiles between gregarious and solitary locusts. The gregarious locusts exhibit significant increases in 60S and polyribosomes, while solitary locusts possess higher peaks of the monoribosome and a specific "halfmer." The polysome profiles, a molecular phenotype, respond to changes in population density. In gregarious locusts, ten genes involved in the cytosolic ribosome pathway exhibited increased translational efficiency (TE). In solitary locusts, five genes from the mitochondrial ribosome pathway displayed increased TE. The high expression of large ribosomal protein 7 at the translational level promotes accumulation of the free 60S ribosomal subunit in gregarious locusts, while solitary locusts employ mitochondrial small ribosomal protein 18c to induce the assembly of mitochondrial ribosomes, causing divergence of the translational profiles and behavioral transition. This study reveals the translational regulatory mechanism of locust phase transition, in which the locusts employ divergent ribosome pathways to cope with changes in population density.

Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae.

BACKGROUND: Feeding by pests is one of the most important reasons for reductions in agricultural crop yield. This study aimed to reveal how juvenile hormone (JH) participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. RESULTS: Larvae of O. furnacalis exhibit a daily circadian feeding rhythm, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a JH active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide against O. furnacalis, controlling feeding and metabolism. CONCLUSION: The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control of O. furnacalis. © 2022 Society of Chemical Industry.

SRP54 mediates circadian rhythm-related, temperature-dependent gene expression in Drosophila.

Recent studies have shown that alternative splicing (AS) plays an important role in regulating circadian rhythm. However, it is not clear whether clock neuron-specific AS is circadian rhythm dependent and what genetic and environmental factors mediate the circadian control of AS. By genome-wide RNA sequencing, we identified SRP54 is one of the Clock (Clk) dependent alternative splicing factors. Genetic interaction between Clock and SRP54 alleles showed that the enhancement of the circadian phenotype increased with temperature, being strongest at 29 °C and weakest at 18 °C. The alternative splicing and differential gene expression profile of Clock and SRP54 overlapped with the circadian-related gene profiles identified in various genome-wide studies, indicating that SRP54 is involved in circadian rhythm. By analyzing of the RNA-seq results at different temperatures, we found the roles of Clock and SRP54 are temperature dependent. We also found multiple novel temperature-dependent transcripts not documented in current databases.

Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster.

Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound ß-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Here, using split-ubiquitin membrane yeast two-hybrid and pull-down assays, we have demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis.

Analysis of sleep in individual Drosophila melanogaster reveals a self-regulatory role for cuticular hydrocarbons pheromones.

It is well established that pheromones are used by insects to transmit information between individuals. However, research has revealed that individual insects can be both the sender and the receiver of some pheromonal signals. It is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on itself. In this study, we monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons (CHs) exerted self-regulatory effects on the amount of sleep experienced by these flies. To identify the physiological significance of this mechanism, we compared the amounts of sleep in individual young flies and individual old flies (flies are known to sleep less as they get older) and compared this data with young and old flies exhibiting mutations that lead to CH reception defects. The differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control flies. Our data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner.

DBT affects sleep in both circadian and non-circadian neurons.

Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, dbt plays a central role in both circadian rhythms and development. We investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discuss the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, we uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, we demonstrate that the PER protein is involved in DBT mediated sleep regulation.