Alternative splicing of clock transcript mediates the response of circadian clocks to temperature changes.

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although clock (clk) gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whether clk exhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed that clk transcripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternative clk transcript, hereinafter termed clk-cold, which encodes a CLK isoform with an in-frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, and in vitro experiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock.

Characterization of light-dependent rhythm of courtship vibrational signals in Nilaparvata lugens: essential involvement of cryptochrome genes.

BACKGROUND: Vibrational signal plays a crucial role in courtship communication in many insects. However, it remains unclear whether insect vibrational signals exhibit daily rhythmicity in response to changes in environmental cues. RESULTS: In this study, we observed daily rhythms of both female vibrational signals (FVS) and male vibrational signals (MVS) in the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most notorious rice pests across Asia. Notably, oscillations of FVS and MVS in paired BPHs were synchronized as part of male-female duetting interactions, displaying significant day-night rhythmicity. Furthermore, we observed light dependency of FVS emissions under different photoperiodic regimes (18 L:6 D and 6 L:18 D) and illumination intensity levels (>300 lx, 50 lx, and 25 lx). Subsequently, the potential role of circadian clock genes cryptochromes (Nlcry1 and Nlcry2) in regulating FVS daily oscillations was examined using gene knockdown via RNA interference. We observed sharp declines and disrupted rhythms in FVS frequencies when either of the Nlcrys was downregulated, with Nlcry2 knockdown showing a more prominent effect. Moreover, we recorded a novel FVS variant (with a dominant frequency of 361.76 ± 4.31 Hz) emitted by dsNlcry1-treated BPH females, which significantly diminished the impact of courtship stimuli on receptive males. CONCLUSION: We observed light-dependent daily rhythms of substrate-borne vibrational signals (SBVS) in BPH and demonstrated essential yet distinct roles of the two Nlcrys. These findings enhanced our understanding of insect SBVS and illustrated the potential of novel precision physical control strategies for disrupting mating behaviors in this rice pest. © 2023 Society of Chemical Industry.

Regulation of protein O-GlcNAcylation by circadian, metabolic, and cellular signals.

O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a dynamic post-translational modification that regulates thousands of proteins and almost all cellular processes. Aberrant O-GlcNAcylation has been associated with numerous diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and type 2 diabetes. O-GlcNAcylation is highly nutrient-sensitive since it is dependent on UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). We previously observed daily rhythmicity of protein O-GlcNAcylation in a Drosophila model that is sensitive to the timing of food consumption. We showed that the circadian clock is pivotal in regulating daily O-GlcNAcylation rhythms given its control of the feeding-fasting cycle and hence nutrient availability. Interestingly, we reported that the circadian clock also modulates daily O-GlcNAcylation rhythm by regulating molecular mechanisms beyond the regulation of food consumption time. A large body of work now indicates that O-GlcNAcylation is likely a generalized cellular status effector as it responds to various cellular signals and conditions, such as ER stress, apoptosis, and infection. In this review, we summarize the metabolic regulation of protein O-GlcNAcylation through nutrient availability, HBP enzymes, and O-GlcNAc processing enzymes. We discuss the emerging roles of circadian clocks in regulating daily O-GlcNAcylation rhythm. Finally, we provide an overview of other cellular signals or conditions that impact O-GlcNAcylation. Many of these cellular pathways are themselves regulated by the clock and/or metabolism. Our review highlights the importance of maintaining optimal O-GlcNAc rhythm by restricting eating activity to the active period under physiological conditions and provides insights into potential therapeutic targets of O-GlcNAc homeostasis under pathological conditions.

Integration of photoperiodic and temperature cues by the circadian clock to regulate insect seasonal adaptations.

Organisms adapt to unfavorable seasonal conditions to survive. These seasonal adaptations rely on the correct interpretation of environmental cues such as photoperiod, and temperature. Genetic studies in several organisms, including the genetic powerhouse Drosophila melanogaster, indicate that circadian clock components, such as period and timeless, are involved in photoperiodic-dependent seasonal adaptations, but our understanding of this process is far from complete. In particular, the role of temperature as a key factor to complement photoperiodic response is not well understood. The development of new sequencing technologies has proven extremely useful in understanding the plastic changes that the clock and other cellular components undergo in different environmental conditions, including changes in gene expression and alternative splicing. This article discusses the integration of photoperiod and temperature for seasonal biology as well as downstream molecular and cellular pathways involved in the regulation of physiological adaptations that occur with changing seasons. We focus our discussion on the current understanding of the involvement of the molecular clock and the circadian clock neuronal circuits in these adaptations in D. melanogaster.

CRUMB: a shiny-based app to analyze rhythmic feeding in Drosophila using the FLIC system.

Rhythmic feeding activity has become an important research area for circadian biologists as it is now clear that metabolic input is critical for regulating circadian rhythms, and chrononutrition has been shown to promote health span. In contrast to locomotor activity rhythm, studies conducting high throughput analysis of Drosophila rhythmic food intake have been limited and few monitoring system options are available. One monitoring system, the Fly Liquid-Food Interaction Counter (FLIC) has become popular, but there is a lack of efficient analysis toolkits to facilitate scalability and ensure reproducibility by using unified parameters for data analysis. Here, we developed Circadian Rhythm Using Mealtime Behavior (CRUMB), a user-friendly Shiny app to analyze data collected using the FLIC system. CRUMB leverages the 'plotly' and 'DT' packages to enable interactive raw data review as well as the generation of easily manipulable graphs and data tables. We used the main features of the FLIC master code provided with the system to retrieve feeding events and provide a simplified pipeline to conduct circadian analysis. We also replaced the use of base functions in time-consuming processes such as 'rle' and 'read.csv' with faster versions available from other packages to optimize computing time. We expect CRUMB to facilitate analysis of feeding-fasting rhythm as a robust output of the circadian clock.

PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period.

PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

Probe-based quantitative PCR and RPA-Cas12a molecular diagnostics for detection of the tomato pest Phthorimaea absoluta (Lepidoptera: Gelechiidae).

The tomato pest Phthorimaea absoluta Meyrick is highly invasive but has not yet invaded North America. However, several morphologically similar species are already present, making detection of P. absoluta presence and invasion challenging. We designed a quantitative PCR molecular diagnostic to differentiate P. absoluta, P. operculella (Zeller), or Keiferia lycopersicella (Walsingham) (Lepidoptera: Gelechiidae) DNA. Additionally, we developed an RPA-Cas12a molecular diagnostic that allows for the isothermal detection of P. absoluta DNA, eliminating the need for a thermocycler. The results of the RPA-Cas12a diagnostic can be visualized simply using a UV light source and cell phone camera. We expect these diagnostics to improve quarantine and prevention measures against this serious agricultural threat.

Genome Assembly and Population Sequencing Reveal Three Populations and Signatures of Insecticide Resistance of Tuta absoluta in Latin America.

Tuta absoluta is one of the largest threats to tomato agriculture worldwide. Native to South America, it has rapidly spread throughout Europe, Africa, and Asia over the past two decades. To understand how T. absoluta has been so successful and to improve containment strategies, high-quality genomic resources and an understanding of population history are critical. Here, we describe a highly contiguous annotated genome assembly, as well as a genome-wide population analysis of samples collected across Latin America. The new genome assembly has an L50 of 17 with only 132 contigs. Based on hundreds of thousands of single nucleotide polymorphisms, we detect three major population clusters in Latin America with some evidence of admixture along the Andes Mountain range. Based on coalescent simulations, we find these clusters diverged from each other tens of thousands of generations ago prior to domestication of tomatoes. We further identify several genomic loci with patterns consistent with positive selection and that are related to insecticide resistance, immunity, and metabolism. This data will further future research toward genetic control strategies and inform future containment policies.

Conformational changes in the negative arm of the circadian clock correlate with dynamic interactomes involved in post-transcriptional regulation.

Biology is tuned to the Earth's diurnal cycle by the circadian clock, a transcriptional/translational negative feedback loop that regulates physiology via transcriptional activation and other post-transcriptional mechanisms. We hypothesize that circadian post-transcriptional regulation might stem from conformational shifts in the intrinsically disordered proteins that comprise the negative arm of the feedback loop to coordinate variation in negative-arm-centered macromolecular complexes. This work demonstrates temporal conformational fluidity in the negative arm that correlates with 24-h variation in physiologically diverse macromolecular complex components in eukaryotic clock proteins. Short linear motifs on the negative-arm proteins that correspond with the interactors localized to disordered regions and known temporal phosphorylation sites suggesting changes in these macromolecular complexes could be due to conformational changes imparted by the temporal phospho-state. Interactors that oscillate in the macromolecular complexes over circadian time correlate with post-transcriptionally regulated proteins, highlighting how time-of-day variation in the negative-arm protein complexes may tune cellular physiology.

CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters.

Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.

Seasonal cues act through the circadian clock and pigment-dispersing factor to control EYES ABSENT and downstream physiological changes.

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain to alter seasonal biology are poorly understood. We previously reported that EYES ABSENT (EYA) shows higher levels in cold temperature or short photoperiod and promotes winter physiology in Drosophila. Nevertheless, how EYA senses seasonal cues is unclear. Pigment-dispersing factor (PDF) is a neuropeptide important for regulating circadian output rhythms. Interestingly, PDF has also been shown to regulate seasonality, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of EYA in mediating the function of PDF on seasonal biology. We observed that PDF abundance is lower on cold and short days as compared with warm and long days, contrary to what was previously observed for EYA. We observed that manipulating PDF signaling in eya+ fly brain neurons, where EYA and PDF receptor are co-expressed, modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and EYA-independent mechanisms. At the molecular level, altering PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA promoting its degradation, thus explaining the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations.

Glutamate and clock help bean bugs track seasonal reproductive changes.

Animals adapt their seasonal physiology by measuring photoperiodic changes over the calendar year. A new study in PLOS Biology uncovers changes in glutamate dynamics in the bean bug that are dependent on photoperiod and a clock gene.

Nutrient-sensitive protein O-GlcNAcylation shapes daily biological rhythms.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a nutrient-sensitive protein modification that alters the structure and function of a wide range of proteins involved in diverse cellular processes. Similar to phosphorylation, another protein modification that targets serine and threonine residues, O-GlcNAcylation occupancy on cellular proteins exhibits daily rhythmicity and has been shown to play critical roles in regulating daily rhythms in biology by modifying circadian clock proteins and downstream effectors. We recently reported that daily rhythm in global O-GlcNAcylation observed in Drosophila tissues is regulated via the integration of circadian and metabolic signals. Significantly, mistimed feeding, which disrupts coordination of these signals, is sufficient to dampen daily O-GlcNAcylation rhythm and is predicted to negatively impact animal biological rhythms and health span. In this review, we provide an overview of published and potential mechanisms by which metabolic and circadian signals regulate hexosamine biosynthetic pathway metabolites and enzymes, as well as O-GlcNAc processing enzymes to shape daily O-GlcNAcylation rhythms. We also discuss the significance of functional interactions between O-GlcNAcylation and other post-translational modifications in regulating biological rhythms. Finally, we highlight organ/tissue-specific cellular processes and molecular pathways that could be modulated by rhythmic O-GlcNAcylation to regulate time-of-day-specific biology.

Characterization of Field-Derived Drosophila suzukii (Diptera: Drosophilidae) Resistance to Pyrethroids in California Berry Production.

The spotted-wing drosophila, Drosophila suzukii (Matsumura), is a global economic pest of berry crops and stone fruit. Since management of this pest primarily relies on calendar insecticide applications, and field-derived resistance to spinosad has already been documented in California caneberry production, there is significant concern for development of resistance to other insecticides. In this study, susceptibility of D. suzukii populations collected from caneberry and strawberry fields to two pyrethroids, zeta-cypermethrin, and bifenthrin, was assessed in 2019 and 2020. Resistance to both pyrethroids were observed in flies from all sampling sites. For flies collected from caneberries in 2019, the LC50 values ranged from 4.5 to 5.2 mg liter-1 with RR50s ranging from 7.5- to 8.7-fold. Our 2020 assays showed that susceptibility of flies to the discriminating dose of zeta-cypermethrin decreased significantly as the season progressed. For flies collected from strawberries in 2020, the LC50s ranged from 19.0 to 36.1 mg liter-1 and from 30.3 to 90.7 mg liter-1 for zeta-cypermethrin and bifenthrin, respectively. The RR50 values varied from 19.0- to 36.1-fold for zeta-cypermethrin and from 15.9- to 47.7-fold for bifenthrin. This study is the first report of field-derived pyrethroid resistance in D. suzukii from two major California berry production areas. Adoption of informed insecticide resistance management practices would be essential to prolong the efficacy of products available to control D. suzukii. Future molecular work is needed to unravel the underlying genetic mechanisms conferring the observed pyrethroid resistance and to develop robust diagnostics that can inform integrated pest management of this pest.

PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock.

Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

Spatio-temporal Variation of Spinosad Susceptibility in Drosophila suzukii (Diptera: Drosophilidae), a Three-year Study in California's Monterey Bay Region.

Spinosyn insecticides are widely used in conventional berry production, and spinosad is regarded as the most effective insecticide for managing Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), spotted-wing drosophila, in organic berry crops. Following the 2017 identification of spinosad resistance in caneberry fields in the Watsonville area, Santa Cruz Co., California, we conducted a study to examine the seasonal and annual susceptibility of D. suzukii over a three-year period. Adult flies were collected from two conventional and two organic caneberry fields in the Monterey Bay region, California, at 'early', 'middle', and 'late' time points during the 2018-2020 growing seasons, and their susceptibility to spinosad was assessed. Results demonstrated that spinosad susceptibility in the D. suzukii field populations generally decreased during the fruit production season (from June through November), and over consecutive seasons. LC50 values of adults from the conventional sites were determined to be as high as 228.7 mg l-1 in 2018, 665.6 mg l-1 in 2019, and 2700.8 mg l-1 in 2020. For the organically managed fields, LC50s of adults were as great as 300.0 mg l-1 in 2018, 1291.5 mg l-1 in 2019, and 2547.1 mg l-1 in 2020. Resistance ratios based on the LC50 values were as high as 10.7-, 13.2-, and 16.9-fold in 2018, 2019, and 2020, respectively. These results should serve as a caution for growers in other production areas, facilitate informed choice of insecticides used in D. suzukii management, and emphasize the need to develop effective insecticide resistance management strategies for this insect.

Timeless in animal circadian clocks and beyond.

TIMELESS (TIM) was first identified as a molecular cog in the Drosophila circadian clock. Almost three decades of investigations have resulted in an insightful model describing the critical role of Drosophila TIM (dTIM) in circadian timekeeping in insects, including its function in mediating light entrainment and temperature compensation of the molecular clock. Furthermore, exciting discoveries on its sequence polymorphism and thermosensitive alternative RNA splicing have also established its role in regulating seasonal biology. Although mammalian TIM (mTIM), its mammalian paralog, was first identified as a potential circadian clock component in 1990s due to sequence similarity to dTIM, its role in clock regulation has been more controversial. Mammalian TIM has now been characterized as a DNA replication fork component and has been shown to promote fork progression and participate in cell cycle checkpoint signaling in response to DNA damage. Despite defective circadian rhythms displayed by mtim mutants, it remains controversial whether the regulation of circadian clocks by mTIM is direct, especially given the interconnection between the cell cycle and circadian clocks. In this review, we provide a historical perspective on the identification of animal tim genes, summarize the roles of TIM proteins in biological timing and genomic stability, and draw parallels between dTIM and mTIM despite apparent functional divergence.

Population genomics of Drosophila suzukii reveal longitudinal population structure and signals of migrations in and out of the continental United States.

Drosophila suzukii, or spotted-wing drosophila, is now an established pest in many parts of the world, causing significant damage to numerous fruit crop industries. Native to East Asia, D. suzukii infestations started in the United States a decade ago, occupying a wide range of climates. To better understand invasion ecology of this pest, knowledge of past migration events, population structure, and genetic diversity is needed. In this study, we sequenced whole genomes of 237 individual flies collected across the continental United States, as well as several sites in Europe, Brazil, and Asia, to identify and analyze hundreds of thousands of genetic markers. We observed strong population structure between Western and Eastern US populations, but no evidence of any population structure between different latitudes within the continental United States, suggesting that there are no broad-scale adaptations occurring in response to differences in winter climates. We detect admixture from Hawaii to the Western United States and from the Eastern United States to Europe, in agreement with previously identified introduction routes inferred from microsatellite analysis. We also detect potential signals of admixture from the Western United States back to Asia, which could have important implications for shipping and quarantine policies for exported agriculture. We anticipate this large genomic dataset will spur future research into the genomic adaptations underlying D. suzukii pest activity and development of novel control methods for this agricultural pest.