Pigment-dispersing Factor Research Articles

Circadian plasticity evolves through regulatory changes in a neuropeptide gene.

Many organisms, including cosmopolitan drosophilids, show circadian plasticity, varying their activity with changing dawn-dusk intervals1. How this behaviour evolves is unclear. Here we compare Drosophila melanogaster with Drosophila sechellia, an equatorial, ecological specialist that experiences minimal photoperiod variation, to investigate the mechanistic basis of circadian plasticity evolution2. D. sechellia has lost the ability to delay its evening activity peak time under long photoperiods. Screening of circadian mutants in D. melanogaster/D. sechellia hybrids identifies a contribution of the neuropeptide pigment-dispersing factor (Pdf) to this loss. Pdf exhibits species-specific temporal expression, due in part to cis-regulatory divergence. RNA interference and rescue experiments in D. melanogaster using species-specific Pdf regulatory sequences demonstrate that modulation of this neuropeptide's expression affects the degree of behavioural plasticity. The Pdf regulatory region exhibits signals of selection in D. sechellia and across populations of D. melanogaster from different latitudes. We provide evidence that plasticity confers a selective advantage for D. melanogaster at elevated latitude, whereas D. sechellia probably suffers fitness costs through reduced copulation success outside its range. Our findings highlight this neuropeptide gene as a hotspot locus for circadian plasticity evolution that might have contributed to both D. melanogaster's global distribution and D. sechellia's specialization.

Efficient CRISPR/Cas9-mediated genome editing in the European corn borer, Ostrinia nubilalis.

The European corn borer (Ostrinia nubilalis) is an agricultural pest and burgeoning model for research on speciation, seasonal adaptation and insect resistance management. Although previous work in O. nubilalis has identified genes associated with differences in life cycle, reproduction, and resistance to Bt toxins, the general lack of a robust gene-editing protocol for O. nubilalis has been a barrier to functional validation of candidate genes. Here, we demonstrate an efficient and practical methodology for heritable gene mutagenesis in O. nubilalis using the CRISPR/Cas9 genome editing system. Precise loss-of-function (LOF) mutations were generated at two circadian clock genes, period (per) and pigment-dispersing factor receptor (pdfr), and a developmental gene, prothoracicotropic hormone (ptth). Precluding the need for a visible genetic marker, gene-editing efficiency remained high across different single guide RNAs (sgRNA) and germline transmission of mutations to F1 offspring approached 100%. When single or dual sgRNAs were injected at a high concentration, gene-specific phenotypic differences in behaviour and development were identified in F0 mutants. Specifically, F0 gene mutants demonstrated that PER, but not PDFR, is essential for normal timing of eclosion. PTTH F0 mutants were significantly heavier and exhibited a higher incidence of diapause. This work will accelerate future studies of gene function in O. nubilalis and facilitate the development of similar screens in other Lepidopteran and non-model insects.

Exploring the Asymmetric Body's Influence on Interval Timing Behaviors of Drosophila melanogaster.

The roles of brain asymmetry in Drosophila are diverse, encompassing the regulation of behavior, the creation of memory, neurodevelopment, and evolution. A comprehensive examination of the Drosophila brain has the potential to enhance our understanding of the functional significance of brain asymmetry in cognitive and behavioral processes, as well as its role in evolutionary perspectives. This study explores the influence of brain asymmetry on interval timing behaviors in Drosophila, with a specific focus on the asymmetric body (AB) structure. Despite being bilaterally symmetric, the AB exhibits functional asymmetry and is located within the central complex of the fly brain. Interval timing behaviors, such as rival-induced prolonged mating duration: longer mating duration behavior (LMD) and sexual experience-mediated shorter mating duration behavior (SMD), are essential for Drosophila. We utilize genetic manipulations to selectively activate or inhibit AB neurons and evaluates their impact on LMD and SMD behaviors. The results indicate that specific populations of AB neurons play unique roles in orchestrating these interval timing behaviors. Notably, inhibiting GAL4R38D01-labeled AB neurons disrupts both LMD and SMD, while GAL4R42C09 neuron inhibition affects only LMD. Moreover, hyperexcitation of GAL4R72A10-labeled AB neurons perturbs SMD. Our study identifies NetrinB (NetB) and Abdominal-B (Abd-B) are important genes for AB neurons in LMD and highlights the role of 5-HT1B neurons in generating LMD through peptidergic Pigment-dispersing factor (PDF) signaling. In summary, this study underscores the importance of AB neuron asymmetry in mediating interval timing behaviors and provides insights into the underlying mechanisms of memory formation and function in Drosophila.

Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

The neuropeptide pigment-dispersing factor signals independently of Bruchpilot-labelled active zones in daily remodelled terminals of Drosophila clock neurons.

The small ventrolateral neurons (sLNvs) are key components of the central clock in the Drosophila brain. They signal via the neuropeptide pigment-dispersing factor (PDF) to align the molecular clockwork of different central clock neurons and to modulate downstream circuits. The dorsal terminals of the sLNvs undergo daily morphological changes that affect presynaptic sites organised by the active zone protein Bruchpilot (BRP), a homolog of mammalian ELKS proteins. However, the role of these presynaptic sites for PDF release is ill-defined. Here, we combined expansion microscopy with labelling of active zones by endogenously tagged BRP to examine the spatial correlation between PDF-containing dense-core vesicles and BRP-labelled active zones. We found that the number of BRP-labelled puncta in the sLNv terminals was similar while their density differed between Zeitgeber time (ZT) 2 and 14. The relative distance between BRP- and PDF-labelled puncta was increased in the morning, around the reported time of PDF release. Spontaneous dense-core vesicle release profiles of sLNvs in a publicly available ssTEM dataset (FAFB) consistently lacked spatial correlation to BRP-organised active zones. RNAi-mediated downregulation of brp and other active zone proteins expressed by the sLNvs did not affect PDF-dependent locomotor rhythmicity. In contrast, down-regulation of genes encoding proteins of the canonical vesicle release machinery, the dense-core vesicle-related protein CADPS, as well as PDF impaired locomotor rhythmicity. Taken together, our study suggests that PDF release from the sLNvs is independent of BRP-organised active zones, while BRP may be redistributed to active zones in a time-dependent manner.

MiR-277 regulates the phase of circadian activity-rest rhythm in Drosophila melanogaster.

Circadian clocks temporally organize behaviour and physiology of organisms with a rhythmicity of about 24h. In Drosophila, the circadian clock is composed of mainly four clock genes: period (per), timeless (tim), Clock (Clk) and cycle (cyc) which constitutes the transcription-translation feedback loop. The circadian clock is further regulated via post-transcriptional and post-translational mechanisms among which microRNAs (miRNAs) are well known post-transcriptional regulatory molecules. Here, we identified and characterized the role of miRNA-277 (miR-277) expressed in the clock neurons in regulating the circadian rhythm. Downregulation of miR-277 in the pacemaker neurons expressing circadian neuropeptide, pigment dispersing factor (PDF) advanced the phase of the morning activity peak under 12h light: 12h dark cycles (LD) at lower light intensities and these flies exhibited less robust rhythms compared to the controls under constant darkness. In addition, downregulation of miR-277 in the PDF expressing neurons abolished the Clk gene transcript oscillation under LD. Our study points to the potential role of miR-277 in fine tuning the Clk expression and in maintaining the phase of the circadian rhythm in Drosophila.

Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body

A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, we examined whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons.

Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause.

Insects from high latitudes spend the winter in a state of overwintering diapause, which is characterized by arrested reproduction, reduced food intake and metabolism, and increased life span. The main trigger to enter diapause is the decreasing day length in summer-autumn. It is thus assumed that the circadian clock acts as an internal sensor for measuring photoperiod and orchestrates appropriate seasonal changes in physiology and metabolism through various neurohormones. However, little is known about the neuronal organization of the circadian clock network and the neurosecretory system that controls diapause in high-latitude insects. We addressed this here by mapping the expression of clock proteins and neuropeptides/neurohormones in the high-latitude fly Drosophila littoralis. We found that the principal organization of both systems is similar to that in Drosophila melanogaster, but with some striking differences in neuropeptide expression levels and patterns. The small ventrolateral clock neurons that express pigment-dispersing factor (PDF) and short neuropeptide F (sNPF) and are most important for robust circadian rhythmicity in D. melanogaster virtually lack PDF and sNPF expression in D. littoralis. In contrast, dorsolateral clock neurons that express ion transport peptide in D. melanogaster additionally express allatostatin-C and appear suited to transfer day-length information to the neurosecretory system of D. littoralis. The lateral neurosecretory cells of D. littoralis contain more neuropeptides than D. melanogaster. Among them, the cells that coexpress corazonin, PDF, and diuretic hormone 44 appear most suited to control diapause. Our work sets the stage to investigate the roles of these diverse neuropeptides in regulating insect diapause.

In vivo characterization of the maturation steps of a pigment dispersing factor neuropeptide precursor in the Drosophila circadian pacemaker neurons.

Pigment dispersing factor (PDF) is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of 2 motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, we generated various mutations that eliminate putative processing sites and then analyzed the effect of each mutation on the production of cPAP and mPDF by 4 different antibodies in both ectopic and endogenous conditions. We also assessed the knockdown effects of processing enzymes on the proPDF maturation. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, we confirm the roles of key enzymes and their target residues: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. Our results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, the PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

Pigment-dispersing factor and CCHamide1 in the Drosophila circadian clock network

ABSTRACT Animals possess a circadian central clock in the brain, where circadian behavioural rhythms are generated. In the fruit fly (Drosophila melanogaster), the central clock comprises a network of approximately 150 clock neurons, which is important for the maintenance of a coherent and robust rhythm. Several neuropeptides involved in the network have been identified, including Pigment-dispersing factor (PDF) and CCHamide1 (CCHa1) neuropeptides. PDF signals bidirectionally to CCHa1-positive clock neurons; thus, the clock neuron groups expressing PDF and CCHa1 interact reciprocally. However, the role of these interactions in molecular and behavioural rhythms remains elusive. In this study, we generated Pdf 01 and CCHa1SK8 double mutants and examined their locomotor activity-related rhythms. The single mutants of Pdf 01 or CCHa1SK8 displayed free-running rhythms under constant dark conditions, whereas approximately 98% of the double mutants were arrhythmic. In light-dark conditions, the evening activity of the double mutants was phase-advanced compared with that of the single mutants. In contrast, both the single and double mutants had diminished morning activity. These results suggest that the effects of the double mutation varied in behavioural parameters. The double and triple mutants of per 01, Pdf 01 , and CCHa1SK8 further revealed that PDF signalling plays a role in the suppression of activity during the daytime under a clock-less background. Our results provide insights into the interactions between PDF and CCHa1 signalling and their roles in activity rhythms.

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.

Mating disrupts morning anticipation in Drosophila melanogaster females.

After mating, the physiology of Drosophila females undergo several important changes, some of which are reflected in their rest-activity cycles. To explore the hypothesis that mating modifies the temporal organization of locomotor activity patterns, we recorded fly activity by a video tracking method. Monitoring rest-activity patterns under light/dark (LD) cycles indicated that mated females lose their ability to anticipate the night-day transition, in stark contrast to males and virgins. This postmating response is mediated by the activation of the sex peptide receptor (SPR) mainly on pickpocket (ppk) expressing neurons, since reducing expression of this receptor in these neurons restores the ability to anticipate the LD transition in mated females. Furthermore, we provide evidence of connectivity between ppk+ neurons and the pigment-dispersing factor (PDF)-positive ventral lateral neurons (sLNv), which play a central role in the temporal organization of daily activity. Since PDF has been associated to the generation of the morning activity peak, we hypothesized that the mating signal could modulate PDF levels. Indeed, we confirm that mated females have reduced PDF levels at the dorsal protocerebrum; moreover, SPR downregulation in ppk+ neurons mimics PDF levels observed in males. In sum, our results are consistent with a model whereby mating-triggered signals reach clock neurons in the fly central nervous system to modulate the temporal organization of circadian behavior according to the needs of the new status.

Morphology and synaptic connections of pigment-dispersing factor-immunoreactive neurons projecting to the lateral protocerebrum in the large black chafer, Holotrichia parallela.

Pigment-dispersing factor (PDF) is a well-known output neuropeptide modulator of circadian pacemakers in insects. Here, we investigated PDF-immunoreactive (ir) neurons in the brain of the large black chafer Holotrichia parallela to search for circadian neural components, which are potentially involved in its circabidian rhythm. PDF-ir cells were exclusively detected near the accessory medulla (AME) as a cluster of ∼ 100 cells with almost homogeneous size. No other cells exhibited immunoreactivity. The PDF-ir cells send beaded fibers into the proximal half of the AME and ventral elongation in an anterior region between the medulla (ME) and lobula (LO). Neither the lamina, ME, LO, nor lobula plate receives PDF-ir fibers. Primary axons derived from the PDF-ir cells extend toward the contralateral hemisphere through the dorsolateral protocerebrum anterior to the calyx to connect the bilateral AME. The axons form varicose outgrowths exclusively in the lateral protocerebrum. Double labeling with antisynapsin revealed partial overlaps between PDF-ir varicosities and synapsin-ir puncta. Thus, it was assumed that the PDF-ir fibers form output synapses there. To verify this, we investigated the ultrastructure of the PDF-ir varicosities in the lateral protocerebrum by preembedding immunoelectron microscopy. The PDF-ir profiles contain small clear synaptic vesicles as well as both PDF-positive and PDF-negative dense-core vesicles, and the profiles form output synapses upon unknown profiles and receive synapses from other PDF-ir profiles. PDF neurons near the AME are considered to be prominent circadian pacemakers in the cockroach and flies. Their possible function in the circabidian rhythm was discussed based on these anatomical insights.

Circadian Neuropeptide-Expressing Clock Neurons as Regulators of Long-Term Memory: Molecular and Cellular Perspectives.

The neuropeptide pigment-dispersing factor (Pdf) is critically involved in the regulation of circadian rhythms in various insects. The function of Pdf in circadian rhythms has been best studied in the fruitfly, i.e., Drosophila melanogaster. Drosophila Pdf is produced in a small subset of circadian clock neurons in the adult brain and functions as a circadian output signal. Recently, however, Pdf has been shown to play important roles not only in regulating circadian rhythms but also in innate and learned behaviors in Drosophila. In this mini-review, we will focus on the current findings that Pdf signaling and Pdf-producing neurons are essential for consolidating and maintaining long-term memory induced by the courtship conditioning in Drosophila and discuss the mechanisms of courtship memory processing through Pdf-producing neurons.

Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus.

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect’s circadian and photoperiodic systems.

Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor.

Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. We show here that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment.

Pigment-dispersing factor is involved in photoperiodic control of reproduction in the brown-winged green bug, Plautia stali

Animals in temperate regions breed in the appropriate season by sensing seasonal changes through photoperiodism. Many studies suggest the involvement of a circadian clock system in the photoperiodic regulation of reproduction. Pigment-dispersing factor (PDF) is a known brain neuropeptide involved in the circadian control in various insects. Here, we investigated the localization and projection of PDF neurons in the brain and their involvement in the photoperiodic control of reproduction in the females of the brown-winged green bug, Plautia stali. Immunohistochemical analyses revealed a dense cluster of PDF-immunoreactive cells localized in the proximal medulla of the optic lobe, which corresponded to the cluster known as PDFMe cells. PDF-immunoreactive cells projected their fibers to the lamina through the medulla surface. PDF-immunoreactive fibers were also found in the protocerebrum and seemed to connect both PDF cell bodies in the optic lobes. RNA interference-mediated knockdown of pdf inhibited oviposition arrest induced by the transfer from long- to short-day conditions. Additionally, the knockdown of pdf delayed oviposition onset after the change from short- to long-day conditions. In conclusion, the study results indicate that PDF is locally expressed in a cell cluster at the proximal medulla and involved in the photoperiodic control of reproduction in P. stali females.

The pigment-dispersing factor neuronal network systematically grows in developing honey bees.

The neuropeptide pigment-dispersing factor (PDF) plays a prominent role in the circadian clock of many insects including honey bees. In the honey bee brain, PDF is expressed in about 15 clock neurons per hemisphere that lie between the central brain and the optic lobes. As in other insects, the bee PDF neurons form wide arborizations in the brain, but certain differences are evident. For example, they arborize only sparsely in the accessory medulla (AME), which serves as important communication center of the circadian clock in cockroaches and flies. Furthermore, all bee PDF neurons cluster together, which makes it impossible to distinguish individual projections. Here, we investigated the developing bee PDF network and found that the first three PDF neurons arise in the third larval instar and form a dense network of varicose fibers at the base of the developing medulla that strongly resembles the AME of hemimetabolous insects. In addition, they send faint fibers toward the lateral superior protocerebrum. In last larval instar, PDF cells with larger somata appear and send fibers toward the distal medulla and the medial protocerebrum. In the dorsal part of the medulla serpentine layer, a small PDF knot evolves from which PDF fibers extend ventrally. This knot disappears during metamorphosis and the varicose arborizations in the putative AME become fainter. Instead, a new strongly stained PDF fiber hub appears in front of the lobula. Simultaneously, the number of PDF neurons increases and the PDF neuronal network in the brain gets continuously more complex.

The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.