Timed receptor tyrosine kinase signaling couples the central and a peripheral circadian clock in Drosophila.

Circadian clocks impose daily periodicities to behavior, physiology, and metabolism. This control is mediated by a central clock and by peripheral clocks, which are synchronized to provide the organism with a unified time through mechanisms that are not fully understood. Here, we characterized in Drosophila the cellular and molecular mechanisms involved in coupling the central clock and the peripheral clock located in the prothoracic gland (PG), which together control the circadian rhythm of emergence of adult flies. The time signal from central clock neurons is transmitted via small neuropeptide F (sNPF) to neurons that produce the neuropeptide Prothoracicotropic Hormone (PTTH), which is then translated into daily oscillations of Ca2+ concentration and PTTH levels. PTTH signaling is required at the end of metamorphosis and transmits time information to the PG through changes in the expression of the PTTH receptor tyrosine kinase (RTK), TORSO, and of ERK phosphorylation, a key component of PTTH transduction. In addition to PTTH, we demonstrate that signaling mediated by other RTKs contributes to the rhythmicity of emergence. Interestingly, the ligand to one of these receptors (Pvf2) plays an autocrine role in the PG, which may explain why both central brain and PG clocks are required for the circadian gating of emergence. Our findings show that the coupling between the central and the PG clock is unexpectedly complex and involves several RTKs that act in concert and could serve as a paradigm to understand how circadian clocks are coordinated.

The circadian clock gates Drosophila adult emergence by controlling the timecourse of metamorphosis.

The daily rhythm of adult emergence of holometabolous insects is one of the first circadian rhythms to be studied. In these insects, the circadian clock imposes a daily pattern of emergence by allowing or stimulating eclosion during certain windows of time and inhibiting emergence during others, a process that has been described as "gating." Although the circadian rhythm of insect emergence provided many of the key concepts of chronobiology, little progress has been made in understanding the bases of the gating process itself, although the term "gating" suggests that it is separate from the developmental process of metamorphosis. Here, we follow the progression through the final stages of Drosophila adult development with single-animal resolution and show that the circadian clock imposes a daily rhythmicity to the pattern of emergence by controlling when the insect initiates the final steps of metamorphosis itself. Circadian rhythmicity of emergence depends on the coupling between the central clock located in the brain and a peripheral clock located in the prothoracic gland (PG), an endocrine gland whose only known function is the production of the molting hormone, ecdysone. Here, we show that the clock exerts its action by regulating not the levels of ecdysone but that of its actions mediated by the ecdysone receptor. Our findings may also provide insights for understanding the mechanisms by which the daily rhythms of glucocorticoids are produced in mammals, which result from the coupling between the central clock in the suprachiasmatic nucleus and a peripheral clock located in the suprarenal gland.

Mutations in trpγ, the homologue of TRPC6 autism candidate gene, causes autism-like behavioral deficits in Drosophila.

Autism Spectrum Disorder (ASD) is characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. The TRPC6 (transient receptor potential channel 6) represents an ASD candidate gene under an oligogenic/multifactorial model based on the initial description and cellular characterization of an individual with ASD bearing a de novo heterozygous mutation disrupting TRPC6, together with the enrichment of disruptive TRPC6 variants in ASD cases as compared to controls. Here, we perform a clinical re-evaluation of the initial non-verbal patient, and also present eight newly reported individuals ascertained for ASD and bearing predicted loss-of-function mutations in TRPC6. In order to understand the consequences of mutations in TRPC6 on nervous system function, we used the fruit fly, Drosophila melanogaster, to show that null mutations in transient receptor gamma (trpγ; the fly gene most similar to TRPC6), cause a number of behavioral defects that mirror features seen in ASD patients, including deficits in social interactions (based on courtship behavior), impaired sleep homeostasis (without affecting the circadian control of sleep), hyperactivity in both young and old flies, and defects in learning and memory. Some defects, most notably in sleep, differed in severity between males and females and became normal with age. Interestingly, hyperforin, a TRPC6 agonist and the primary active component of the St. John's wort antidepressant, attenuated many of the deficits expressed by trpγ mutant flies. In summary, our results provide further evidence that the TRPC6 gene is a risk factor for ASD. In addition, they show that the behavioral defects caused by mutations in TRPC6 can be modeled in Drosophila, thereby establishing a paradigm to examine the impact of mutations in other candidate genes.

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior.

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.

The Drosophila foraging gene plays a vital role at the start of metamorphosis for subsequent adult emergence.

The foraging (for) gene has been extensively studied in many species for its functions in development, physiology, and behavior. It is common for genes that influence behavior and development to be essential genes, and for has been found to be an essential gene in both fruit flies and mammals, with for mutants dying before reaching the adult stage. However, the biological process underlying the lethality associated with this gene is not known. Here, we show that in Drosophila melanogaster, some but not all gene products of for are essential for survival. Specifically, we show that promoter 3 of for, but not promoters 1, 2, and 4 are required for survival past pupal stage. We use full and partial genetic deletions of for, and temperature-restricted knock-down of the gene to further investigate the stage of lethality. While deletion analysis shows that flies lacking for die at the end of pupal development, as pharate adults, temperature-restricted knock-down shows that for is only required at the start of pupal development, for normal adult emergence (AE) and viability. We further show that the inability of these mutants to emerge from their pupal cases is linked to deficiencies in emergence behaviors, caused by a possible energy deficiency, and finally, that the lethality of for mutants seems to be linked to protein isoform P3, transcribed from for promoter 3.

The impact of the gut microbiome on memory and sleep in Drosophila.

The gut microbiome has been proposed to influence diverse behavioral traits of animals, although the experimental evidence is limited and often contradictory. Here, we made use of the tractability of Drosophila melanogaster for both behavioral analyses and microbiome studies to test how elimination of microorganisms affects a number of behavioral traits. Relative to conventional flies (i.e. with unaltered microbiome), microbiologically sterile (axenic) flies displayed a moderate reduction in memory performance in olfactory appetitive conditioning and courtship assays. The microbiological status of the flies had a small or no effect on anxiety-like behavior (centrophobism) or circadian rhythmicity of locomotor activity, but axenic flies tended to sleep for longer and displayed reduced sleep rebound after sleep deprivation. These last two effects were robust for most tests conducted on both wild-type Canton S and w1118 strains, as well for tests using an isogenized panel of flies with mutations in the period gene, which causes altered circadian rhythmicity. Interestingly, the effect of absence of microbiota on a few behavioral features, most notably instantaneous locomotor activity speed, varied among wild-type strains. Taken together, our findings demonstrate that the microbiome can have subtle but significant effects on specific aspects of Drosophila behavior, some of which are dependent on genetic background.

Calcium and cAMP directly modulate the speed of the Drosophila circadian clock.

Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. Here we investigated this question focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. We show that genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Our results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence.

The tanning hormone, bursicon, does not act directly on the epidermis to tan the Drosophila exoskeleton.

BACKGROUND: In insects, continuous growth requires the periodic replacement of the exoskeleton. Once the remains of the exoskeleton from the previous stage have been shed during ecdysis, the new one is rapidly sclerotized (hardened) and melanized (pigmented), a process collectively known as tanning. The rapid tanning that occurs after ecdysis is critical for insect survival, as it reduces desiccation, and gives the exoskeleton the rigidity needed to support the internal organs and to provide a solid anchor for the muscles. This rapid postecdysial tanning is triggered by the "tanning hormone", bursicon. Since bursicon is released into the hemolymph, it has naturally been assumed that it would act on the epidermal cells to cause the tanning of the overlying exoskeleton. RESULTS: Here we investigated the site of bursicon action in Drosophila by examining the consequences on tanning of disabling the bursicon receptor (encoded by the rickets gene) in different tissues. To our surprise, we found that rapid tanning does not require rickets function in the epidermis but requires it instead in peptidergic neurons of the ventral nervous system (VNS). Although we were unable to identify the signal that is transmitted from the VNS to the epidermis, we show that neurons that express the Drosophila insulin-like peptide ILP7, but not the ILP7 peptide itself, are involved. In addition, we found that some of the bursicon targets involved in melanization are different from those that cause sclerotization. CONCLUSIONS: Our findings show that bursicon does not act directly on the epidermis to cause the tanning of the overlying exoskeleton but instead requires an intermediary messenger produced by peptidergic neurons within the central nervous system. Thus, this work has uncovered an unexpected layer of control in a process that is critical for insect survival, which will significantly alter the direction of future research aimed at understanding how rapid postecdysial tanning occurs.

Neuroendocrinology of reproduction: Is gonadotropin-releasing hormone (GnRH) dispensable?

Gonadotropin releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. First identified in mammals, the GnRH signaling pathway is found in all classes of vertebrates; homologues of GnRH have also been identified in invertebrates. In addition to its role as a hypothalamic releasing hormone, GnRH has multiple functions including modulating neural activity within specific regions of the brain. These various functions are mediated by multiple isoforms, which are expressed at diverse locations within the central nervous system. Here we discuss the GnRH signaling pathways in light of new reports that reveal that some vertebrate genomes lack GnRH1. Not only do other isoforms of GnRH not compensate for this gene loss, but elements upstream of GnRH1, including kisspeptins, appear to also be dispensable. We discuss routes that may compensate for the loss of the GnRH1 pathway.

Genetic analysis of Eclosion hormone action during Drosophila larval ecdysis.

Insect growth is punctuated by molts, during which the animal produces a new exoskeleton. The molt culminates in ecdysis, an ordered sequence of behaviors that causes the old cuticle to be shed. This sequence is activated by Ecdysis triggering hormone (ETH), which acts on the CNS to activate neurons that produce neuropeptides implicated in ecdysis, including Eclosion hormone (EH), Crustacean cardioactive peptide (CCAP) and Bursicon. Despite more than 40 years of research on ecdysis, our understanding of the precise roles of these neurohormones remains rudimentary. Of particular interest is EH; although it is known to upregulate ETH release, other roles for EH have remained elusive. We isolated an Eh null mutant in Drosophila and used it to investigate the role of EH in larval ecdysis. We found that null mutant animals invariably died at around the time of ecdysis, revealing an essential role in its control. Further analyses showed that these animals failed to express the preparatory behavior of pre-ecdysis while directly expressing the motor program of ecdysis. Although ETH release could not be detected, the lack of pre-ecdysis could not be rescued by injections of ETH, suggesting that EH is required within the CNS for ETH to trigger the normal ecdysial sequence. Using a genetically encoded calcium probe, we showed that EH configured the response of the CNS to ETH. These findings show that EH plays an essential role in the Drosophila CNS in the control of ecdysis, in addition to its known role in the periphery of triggering ETH release.

The genome of Tetranychus urticae reveals herbivorous pest adaptations.

The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant-herbivore interactions, and provides unique opportunities for developing novel plant protection strategies.

Genetic variants associated with neurodegenerative Alzheimer disease in natural models.

The use of transgenic models for the study of neurodegenerative diseases has made valuable contributions to the field. However, some important limitations, including protein overexpression and general systemic compensation for the missing genes, has caused researchers to seek natural models that show the main biomarkers of neurodegenerative diseases during aging. Here we review some of these models-most of them rodents, focusing especially on the genetic variations in biomarkers for Alzheimer diseases, in order to explain their relationships with variants associated with the occurrence of the disease in humans.

Model Organisms in G Protein-Coupled Receptor Research.

The study of G protein-coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor's native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism.

Neural and hormonal control of postecdysial behaviors in insects.

The shedding of the old exoskeleton that occurs in insects at the end of a molt (a process called ecdysis) is typically followed by the expansion and tanning of a new one. At the adult molt, these postecdysial processes include expansion and hardening of the wings. Here we describe recent advances in understanding the neural and hormonal control of wing expansion and hardening, focusing on work using Drosophila melanogaster in which genetic manipulations have permitted detailed investigation of postecdysial processes and their modulation by sensory input. To place this work in context, we briefly review recent progress in understanding the neuroendocrine regulation of ecdysis, which appears to be largely conserved across insect species. Investigations into the neuroendocrine networks that regulate ecdysial and postecdysial behaviors provide insights into how stereotyped, yet environmentally responsive, sequences are generated and how they develop and evolve.

Genetic analysis of ecdysis behavior in Drosophila reveals partially overlapping functions of two unrelated neuropeptides.

Ecdysis behavior allows insects to shed their old exoskeleton at the end of every molt. It is controlled by a suite of interacting hormones and neuropeptides, and has served as a useful behavior for understanding how bioactive peptides regulate CNS function. Previous findings suggest that crustacean cardioactive peptide (CCAP) activates the ecdysis motor program; the hormone bursicon is believed to then act downstream of CCAP to inflate, pigment, and harden the exoskeleton of the next stage. However, the exact roles of these signaling molecules in regulating ecdysis remain unclear. Here we use a genetic approach to investigate the functions of CCAP and bursicon in Drosophila ecdysis. We show that null mutants in CCAP express no apparent defects in ecdysis and postecdysis, producing normal adults. By contrast, a substantial fraction of flies genetically null for one of the two subunits of bursicon [encoded by the partner of bursicon gene (pburs)] show severe defects in ecdysis, with escaper adults exhibiting the expected failures in wing expansion and exoskeleton pigmentation and hardening. Furthermore, flies lacking both CCAP and bursicon show much more severe defects at ecdysis than do animals null for either neuropeptide alone. Our results show that the functions thought to be subserved by CCAP are partially effected by bursicon, and that bursicon plays an important and heretofore undescribed role in ecdysis behavior itself. These findings have important implications for understanding the regulation of this vital insect behavior and the mechanisms by which hormones and neuropeptides control the physiology and behavior of animals.

A coupled Computational Fluid Dynamics and Wells-Riley model to predict COVID-19 infection probability for passengers on long-distance trains.

Coupled Wells-Riley (WR) and Computational Fluid Dynamics (CFD) modelling (WR-CFD) facilitates a detailed analysis of COVID-19 infection probability (IP). This approach overcomes issues associated with the WR 'well-mixed' assumption. The WR-CFD model, which makes uses of a scalar approach to simulate quanta dispersal, is applied to Chinese long-distance trains (G-train). Predicted IPs, at multiple locations, are validated using statistically derived (SD) IPs from reported infections on G-trains. This is the first known attempt to validate a coupled WR-CFD approach using reported COVID-19 infections derived from the rail environment. There is reasonable agreement between trends in predicted and SD IPs, with the maximum SD IP being 10.3% while maximum predicted IP was 14.8%. Additionally, predicted locations of highest and lowest IP, agree with those identified in the statistical analysis. Furthermore, the study demonstrates that the distribution of infectious aerosols is non-uniform and dependent on the nature of the ventilation. This suggests that modelling techniques neglecting these differences are inappropriate for assessing mitigation measures such as physical distancing. A range of mitigation strategies were analysed; the most effective being the majority (90%) of passengers correctly wearing high efficiency masks (e.g. N95). Compared to the base case (40% of passengers wearing low efficiency masks) there was a 95% reduction in average IP. Surprisingly, HEPA filtration was only effective for passengers distant from an index patient, having almost no effect for those in close proximity. Finally, as the approach is based on CFD it can be applied to a range of other indoor environments.

Source terms for benchmarking models of SARS-CoV-2 transmission via aerosols and droplets.

There is ongoing and rapid advancement in approaches to modelling the fate of exhaled particles in different environments relevant to disease transmission. It is important that models are verified by comparison with each other using a common set of input parameters to ensure that model differences can be interpreted in terms of model physics rather than unspecified differences in model input parameters. In this paper, we define parameters necessary for such benchmarking of models of airborne particles exhaled by humans and transported in the environment during breathing and speaking.

Reciprocal Relationship Between Calcium Signaling and Circadian Clocks: Implications for Calcium Homeostasis, Clock Function, and Therapeutics.

In animals, circadian clocks impose a daily rhythmicity to many behaviors and physiological processes. At the molecular level, circadian rhythms are driven by intracellular transcriptional/translational feedback loops (TTFL). Interestingly, emerging evidence indicates that they can also be modulated by multiple signaling pathways. Among these, Ca2+ signaling plays a key role in regulating the molecular rhythms of clock genes and of the resulting circadian behavior. In addition, the application of in vivo imaging approaches has revealed that Ca2+ is fundamental to the synchronization of the neuronal networks that make up circadian pacemakers. Conversely, the activity of circadian clocks may influence Ca2+ signaling. For instance, several genes that encode Ca2+ channels and Ca2+-binding proteins display a rhythmic expression, and a disruption of this cycling affects circadian function, underscoring their reciprocal relationship. Here, we review recent advances in our understanding of how Ca2+ signaling both modulates and is modulated by circadian clocks, focusing on the regulatory mechanisms described in Drosophila and mice. In particular, we examine findings related to the oscillations in intracellular Ca2+ levels in circadian pacemakers and how they are regulated by canonical clock genes, neuropeptides, and light stimuli. In addition, we discuss how Ca2+ rhythms and their associated signaling pathways modulate clock gene expression at the transcriptional and post-translational levels. We also review evidence based on transcriptomic analyzes that suggests that mammalian Ca2+ channels and transporters (e.g., ryanodine receptor, ip3r, serca, L- and T-type Ca2+ channels) as well as Ca2+-binding proteins (e.g., camk, cask, and calcineurin) show rhythmic expression in the central brain clock and in peripheral tissues such as the heart and skeletal muscles. Finally, we discuss how the discovery that Ca2+ signaling is regulated by the circadian clock could influence the efficacy of pharmacotherapy and the outcomes of clinical interventions.

Inflight transmission of COVID-19 based on experimental aerosol dispersion data.

BACKGROUND: An issue of concern to the travelling public is the possibility of in-flight transmission of coronavirus disease 2019 (COVID-19) during long- and short-haul flights. The aviation industry maintains that the probability of contracting the illness is small based on reported cases, modelling and data from aerosol dispersion experiments conducted on-board aircraft. METHODS: Using experimentally derived aerosol dispersion data for a B777-200 aircraft and a modified version of the Wells-Riley equation we estimate inflight infection probability for a range of scenarios involving quanta generation rate and face mask efficiency. Quanta generation rates were selected based on COVID-19 events reported in the literature while mask efficiency was determined from the aerosol dispersion experiments. RESULTS: The MID-AFT cabin exhibits the highest infection probability. The calculated maximum individual infection probability (without masks) for a 2-hour flight in this section varies from 4.5% for the 'Mild Scenario' to 60.2% for the 'Severe Scenario' although the corresponding average infection probability varies from 0.1% to 2.5%. For a 12-hour flight, the corresponding maximum individual infection probability varies from 24.1% to 99.6% and the average infection probability varies from 0.8% to 10.8%. If all passengers wear face masks throughout the 12-hour flight, the average infection probability can be reduced by ~73%/32% for high/low efficiency masks. If face masks are worn by all passengers except during a one-hour meal service, the average infection probability is increased by 59%/8% compared to the situation where the mask is not removed. CONCLUSIONS: This analysis has demonstrated that while there is a significant reduction in aerosol concentration due to the nature of the cabin ventilation and filtration system, this does not necessarily mean that there is a low probability or risk of in-flight infection. However, mask wearing, particularly high-efficiency ones, significantly reduces this risk.

Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster.

In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. In the present work, we made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. We found that RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, our results suggest an important role in reproductive success.