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.

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.

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.

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octß2R cascade.

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octß2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octß2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octß2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octß2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octß2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octß2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.

AKH-FOXO pathway regulates starvation-induced sleep loss through remodeling of the small ventral lateral neuron dorsal projections.

Starvation caused by adverse feeding stresses or food shortages has been reported to result in sleep loss in animals. However, how the starvation signal interacts with the central nervous system is still unknown. Here, the adipokinetic hormone (AKH)-Fork head Box-O (FOXO) pathway is shown to respond to energy change and adjust the sleep of Drosophila through remodeling of the s-LNv (small ventral lateral neurons) dorsal projections. Our results show that starvation prevents flies from going to sleep after the first light-dark transition. The LNvs are required for starvation-induced sleep loss through extension of the pigment dispersing factor (PDF)-containing s-LNv dorsal projections. Further studies reveal that loss of AKH or AKHR (akh receptor) function blocks starvation-induced extension of s-LNv dorsal projections and rescues sleep suppression during food deprivation. FOXO, which has been reported to regulate synapse plasticity of neurons, acts as starvation response factor downstream of AKH, and down regulation of FOXO level considerably alleviates the influence of starvation on s-LNv dorsal projections and sleep. Taking together, our results outline the transduction pathways between starvation signal and sleep, and reveal a novel functional site for sleep regulation.

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.

Extra sex combs buffers sleep-related stresses through regulating Heat shock proteins.

The impact of global warming on the life of the earth is increasingly concerned. Previous studies indicated that temperature changes have a serious impact on insect sleep. Sleep is critical for animals as it has many important physiological functions. It is of great significance to study the regulation mechanism of temperature-induced sleep changes for understanding the impact of global warming on insects. More importantly, understanding how these pressures regulate sleep can provide insights into improving sleep. In this study, we found that extra sex combs (ESC) are a regulatory factor in this process. Our data showed that ESC was an upstream negative regulatory factor of Heat shock proteins (Hsps), and it could regulate sleep in mushroom and ellipsoid of Drosophila. ESC mutation exaggerates the sleep change caused by temperature, while buffering the shortening of life caused by sleep deprivation. These phenotypes can be rescued by Hsps mutants. Therefore, we concluded that the ESC buffers sleep-related stresses through regulating Hsps.

Regulatory mechanism of daily sleep by miR-276a.

MiRNAs have attracted more attention in recent years as regulators of sleep and circadian rhythms after their roles in circadian rhythm and sleep were discovered. In this study, we explored the roles of the miR-276a on daily sleep in Drosophila melanogaster, and found a regulatory cycle for the miR-276a pathway, in which miR-276a, regulated by the core CLOCK/CYCLE (CLK/CYC) transcription factor upstream, regulates sleep via suppressing targets TIM and NPFR1. (a) Loss of miR-276a function makes the flies sleep more during both daytime and nighttime, while flies with gain of miR-276a function sleep less; (b) MiR-276a is widely expressed in the mushroom body (MB), the pars intercerebralis (PI) and some clock neurons lateral dorsal neurons (LNds), in which tim neurons is important for sleep regulation; (c) MiR-276a promoter is identified to locate in the 8th fragment (aFrag8) of the pre-miR-276a, and this fragment is directly activated and regulated by CLK/CYC; (4) MiR-276a is rhythmically oscillating in heads of the wild-type w1118 , but this oscillation disappears in the loss of function mutant clkjrk ; (5) The neuropeptide F receptor 1 (npfr1) was found to be a downstream target of miR-276a. These results clarify that the miR-276a is a very important factor for sleep regulation.

Regulation of circadian rhythm and sleep by miR-375-timeless interaction in Drosophila.

MicroRNAs are important coordinators of circadian regulation that mediate the fine-tuning of gene expression. Although many studies have shown the effects of individual miRNAs on the circadian clock, the global functional miRNA-mRNA interaction network involved in the circadian system remains poorly understood. Here, we used CLEAR (Covalent Ligation of Endogenous Argonaute-bound RNAs)-CLIP (Cross-Linking and Immuno-Precipitation) to explore the regulatory functions of miRNAs in the circadian system by comparing the miRNA-mRNA interactions between Drosophila wild-type strain W1118 and a mutant of the key circadian transcriptional regulator Clock (Clkjrk ). This experimental approach unambiguously identified tens of thousands of miRNA-mRNA interactions in both the head and body. The miRNA-mRNA interactome showed dramatic changes in the Clkjrk flies. Particularly, among ~300 miRNA-mRNA circadian relevant interactions, multiple interactions involving core clock genes pdp1, tim, and vri displayed distinct changes as a result of the Clk mutation. Based on the CLEAR-CLIP analysis, we found a novel regulation of the circadian rhythm and sleep by the miR-375-timeless interaction. The results indicated that Clk disruption abolished normal rhythmic expression of miR-375 and the functional regulation occurred in the l-LNv neurons, where miR-375 modulated the circadian rhythm and sleep via targeting timeless. This work provides the first global view of miRNA regulation in the circadian rhythm.

Sexual dimorphism of sleep regulated by juvenile hormone signaling in Drosophila.

Sexually dimorphic phenotypes are a universal phenomenon in animals. In the model animal fruit fly Drosophila, males and females exhibit long- and short-sleep phenotypes, respectively. However, the mechanism is still a mystery. In this study, we showed that juvenile hormone (JH) is involved in regulation of sexually dimorphic sleep in Drosophila, in which gain of JH function enlarges differences of the dimorphic sleep phenotype with higher sleep in males and lower sleep in females, while loss of JH function blurs these differences and results in feminization of male sleep and masculinization of female sleep. Further studies indicate that germ cell-expressed (GCE), one of the JH receptors, mediates the response in the JH pathway because the sexually dimorphic sleep phenotypes cannot be rescued by JH hormone in a gce deletion mutant. The JH-GCE regulated sleep dimorphism is generated through the sex differentiation-related genes -fruitless (fru) and doublesex (dsx) in males and sex-lethal (sxl), transformer (tra) and doublesex (dsx) in females. These are the "switch" genes that separately control the sleep pattern in males and females. Moreover, analysis of sleep deprivation and circadian behaviors showed that the sexually dimorphic sleep induced by JH signals is a change of sleep drive and independent of the circadian clock. Furthermore, we found that JH seems to also play an unanticipated role in antagonism of an aging-induced sleep decrease in male flies. Taken together, these results indicate that the JH signal pathway is critical for maintenance of sexually dimorphic sleep by regulating sex-relevant genes.

Integrative Role of 14-3-3ε in Sleep Regulation.

Sleep is a crucial factor for health and survival in all animals. In this study, we found by proteomic analysis that some cancer related proteins were impacted by the circadian clock. The 14-3-3ε protein, expression of which is activated by the circadian transcription factor Clock, regulates adult sleep of Drosophila independent of circadian rhythm. Detailed analysis of the sleep regulatory mechanism shows that 14-3-3ε directly targets the Ultrabithorax (Ubx) gene to activate transcription of the pigment dispersing factor (PDF). The dopamine receptor (Dop1R1) and the octopamine receptor (Oamb), are also involved in the 14-3-3ε pathway, which in 14-3-3ε mutant flies causes increases in the dopR1 and OAMB, while downregulation of the DopR1 and Oamb can restore the sleep phenotype caused by the 14-3-3ε mutation. In conclusion, 14-3-3ε is necessary for sleep regulation in Drosophila.

Feeding regulation by neuropeptide Y on Asian corn borer Ostrinia furnacalis.

The Asian Corn Borer Ostrinia furnacalis is a major agricultural pest. In this study, a full-length neuropeptide Y (npy) gene in O. furnacalis was sequenced and cloned from cDNA library, which contains an ORF of 273 bp by encoding 90 amino acid residues. The mature OfurNPY is composed of 29 amino acids with amidation in C-terminal. The spatiotemporal expression analysis showed that npy highest expression level was in the midgut of the fifth instar larvae (the gluttony period). When the expression of npy was knocked down by feeding or injecting dsNPY, larval food consumption, body size, and body weight were significantly inhibited compared to controls. These results indicate that NPY is an important regulator in the control of feeding of O. furnacalis.

Circadian Rhythm Neuropeptides in Drosophila: Signals for Normal Circadian Function and Circadian Neurodegenerative Disease.

Circadian rhythm is a ubiquitous phenomenon in many organisms ranging from prokaryotes to eukaryotes. During more than four decades, the intrinsic and exogenous regulations of circadian rhythm have been studied. This review summarizes the core endogenous oscillation in Drosophila and then focuses on the neuropeptides, neurotransmitters and hormones that mediate its outputs and integration in Drosophila and the links between several of these (pigment dispersing factor (PDF) and insulin-like peptides) and neurodegenerative disease. These signaling molecules convey important network connectivity and signaling information for normal circadian function, but PDF and insulin-like peptides can also convey signals that lead to apoptosis, enhanced neurodegeneration and cognitive decline in flies carrying circadian mutations or in a senescent state.

Development of a novel-type transgenic cotton plant for control of cotton bollworm.

The transgenic Bt cotton plant has been widely planted throughout the world for the control of cotton budworm Helicoverpa armigera (Hubner). However, a shift towards insect tolerance of Bt cotton is now apparent. In this study, the gene encoding neuropeptide F (NPF) was cloned from cotton budworm H. armigera, an important agricultural pest. The npf gene produces two splicing mRNA variants-npf1 and npf2 (with a 120-bp segment inserted into the npf1 sequence). These are predicted to form the mature NPF1 and NPF2 peptides, and they were found to regulate feeding behaviour. Knock down of larval npf with dsNPF in vitro resulted in decreases of food consumption and body weight, and dsNPF also caused a decrease of glycogen and an increase of trehalose. Moreover, we produced transgenic tobacco plants transiently expressing dsNPF and transgenic cotton plants with stably expressed dsNPF. Results showed that H. armigera larvae fed on these transgenic plants or leaves had lower food consumption, body size and body weight compared to controls. These results indicate that NPF is important in the control of feeding of H. armigera and valuable for production of potential transgenic cotton.

SUPPRESSING A PEROXIDASE GENE REDUCES SURVIVAL IN THE WHEAT APHID Sitobion avenae.

Peroxidases (POXs) make up a large superfamily of enzymes that act in a wide range of biological mechanisms, including maintaining appropriate redox balances within cells, among other actions. In this study, we cloned a sequence that encodes a POX protein, SaPOX, from wheat aphids, Sitobion avenae. Amino acid sequence alignment showed the SaPOX sequence was conserved with POXs from other insect species. SaPOX mRNA accumulations were present in all nymphal and adult stages, at higher levels during the first and second instar, and lower during later stages in the life cycle. Ingestion of dsRNA specific to POX led to reduced SaPOX mRNA accumulation. Sitobion avenae nymphs continuously exposed to dietary dsPOX via an artificial diet led to reduced survival rate and ecdysis index. We infer that POX is important to maintain the growth and development of S. avenae.

Influence of catalase gene silencing on the survivability of Sitobion avenae.

Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide produced in cell metabolism, result in the disruption of cellular function and structure. Catalase (CAT), an enzyme which exists in almost all organisms including plants, invertebrates and vertebrates, acts in scavenging ROS. In this study, a sequence fragment encoding a CAT-like protein from wheat aphids ( Sitobion avenae) was cloned. Amino acid sequence alignment showed this CAT shared relatively high conservation with CAT sequences from other insects. We detected cat mRNA levels at nymphs of different stages and adults and results showed that cat expression in adults was significantly higher compared to juvenile stages. At the third instar stage, ingestion of dsCAT significantly knocked down CAT expression. Continuous feeding of dsCAT mixed in an artificial diet led to reduced survival rate and ecdysis index. This study indicates that cat, a potential target gene for management of insect pests, is important for maintaining the survival of S. avenae.

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.

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.

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.

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.