Virtually all organisms exhibit circadian rhythms, this includes mosquitoes. Many aspects of their biology are under the control of their circadian clocks like their vision, olfaction, host-seeking, mating, oviposition, metabolism, locomotion, and more. However, how the circadian clock regulates mosquito-host interactions and adapts to specific environmental conditions remains largely unknown, despite its importance to vector disease control. Here, we relied on a multidisciplinary, integrative, and comparative approach to shed light on the mechanisms underlying mosquitoes adaptations to various temporal niches. We use CRISPR/Cas9 to knockout timeless in Aedes aegypti and show this mutation causes an increase in their free running periods under continuous darkness conditions. External factors can also influence a vector's activity pattern like climate, light pollution, as well as host preference and availability. To investigate the influence these factors have on activity patterns, we compare the activity patterns of multiple lab rear strains of Aedes albopictus as well as two field collected populations of Aedes japonicus. Our results suggest host availability and light pollution could cause the differences in activity profiles that we observed. With vision playing an important role in both circadian rhythms, host seeking, and threat detection, we compared a nocturnal mosquito (Aedes japonicus) and a diurnal mosquito (Aedes aegypti). We introduced a looming visual stimulus in an LED arena and found Aedes aegypti to be more responsive to the looming stimulus than Aedes japonicus. Finally, the underlying mechanisms that mediate this threat detection and escape behavior were examined, revealing a possible candidate for a giant fiber neuron in Aedes aegypti. Overall these results provide novel insights into mosquito biology that have the potential to be applied towards vector control methods. / Doctor of Philosophy / Most living things, including mosquitoes, follow a daily pattern called a circadian rhythm. This rhythm affects many parts of their lives including their vision, smell, and activity. Understanding how their circadian clock functions can help us control populations of mosquitoes and prevent the transmission of diseases they may carry. By studying different species and populations of mosquitoes in the lab and from the wild, we have found that factors like light pollution and host availability could affect when mosquitoes are active. In addition to these results we have also demonstrated that mosquitoes that are active during the day and mosquitoes that are active during the night will respond differently to a looming visual stimulus. We examined the brain of the mosquitoes that were most responsive to the looming stimulus and found a system of large neurons that could potentially be responsible for eliciting their escape behaviors. Overall these results provide new and important information about mosquito biology and can be applied to mosquito control.

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Summary Bees, like many other organisms, evolved an endogenous circadian clock, which enables them to foresee daily environmental changes and exactly time foraging flights to periods of floral resource availability. The social lifestyle of a honey bee colony has been shown to influence circadian behavior in nurse bees, which do not exhibit rhythmic behavior when they are nursing. On the other hand, forager bees display strong circadian rhythms. Solitary bees, like the mason bee, do not nurse their offspring and do not live in hive communities, but face the same daily environmental changes as honey bees. Besides their lifestyle mason and honey bees differ in their development and life history, because mason bees overwinter after eclosion as adults in their cocoons until they emerge in spring. Honey bees do not undergo diapause and have a relatively short development of a few weeks until they emerge. In my thesis, I present a comparison of the circadian clock of social honey bees (Apis mellifera) and solitary mason bees (Osmia bicornis and Osmia cornuta) on the neuroanatomical level and behavioral output level. I firstly characterized in detail the localization of the circadian clock in the bee brain via the expression pattern of two clock components, namely the clock protein PERIOD (PER) and the neuropeptide Pigment Dispersing Factor (PDF), in the brain of honey bee and mason bee. PER is localized in lateral neuron clusters (which we called lateral neurons 1 and 2: LN1 and LN2) and dorsal neuron clusters (we called dorsal lateral neurons and dorsal neurons: DLN, DN), many glia cells and photoreceptor cells. This expression pattern is similar to the one in other insect species and indicates a common ground plan of clock cells among insects. In the LN2 neuron cluster with cell bodies located in the lateral brain, PER is co-expressed with PDF. These cells build a complex arborization network throughout the brain and provide the perfect structure to convey time information to brain centers, where complex behavior, e.g. sun-compass orientation and time memory, is controlled. The PDF arborizations centralize in a dense network (we named it anterio-lobular PDF hub: ALO) which is located in front of the lobula. In other insects, this fiber center is associated with the medulla (accessory medulla: AME). Few PDF cells build the ALO already in very early larval development and the cell number and complexity of the network grows throughout honey bee development. Thereby, dorsal regions are innervated first by PDF fibers and, in late larval development, the fibers grow laterally to the optic lobe and central brain. The overall expression pattern of PER and PDF are similar in adult social and solitary bees, but I found a few differences in the PDF network density in the posterior protocerebrum and the lamina, which may be associated with evolution of sociality in bees. Secondly, I monitored activity rhythms, for which I developed and established a device to monitor locomotor activity rhythms of individual honey bees with contact to a mini colony in the laboratory. This revealed new aspects of social synchronization and survival of young bees with indirect social contact to the mini colony (no trophalaxis was possible). For mason bees, I established a method to monitor emergence and locomotor activity rhythms and I could show that circadian emergence rhythms are entrainable by daily temperature cycles. Furthermore, I present the first locomotor activity rhythms of solitary bees, which show strong circadian rhythms in their behavior right after emergence. Honey bees needed several days to develop circadian locomotor rhythms in my experiments. I hypothesized that honey bees do not emerge with a fully matured circadian system in the hive, while solitary bees, without the protection of a colony, would need a fully matured circadian clock right away after emergence. Several indices in published work and preliminary studies support my hypothesis and future studies on PDF expression in different developmental stages in solitary bees may provide hard evidence. / Zusammenfassung Bienen, sowie viele andere Organismen, evolvierten eine innere circadiane Uhr, die es ihnen ermöglicht, tägliche Umweltveränderungen voraus zu sehen und ihre Foragierflüge zu Tageszeiten durchzuführen, wenn sie möglichst viele Blüten besuchen können. Es zeigte sich, dass der soziale Lebensstil der Honigbiene Einfluss auf das rhythmische Verhalten der Ammenbienen hat, die während der Brutpflege keinen täglichen Rhythmus im Verhalten aufweisen. Sammlerbienen auf der anderen Seite zeigen ein stark rhythmisches Verhalten. Solitäre Bienen, wie die Mauerbiene, betreiben keine Brutpflege und leben nicht in einer Staatengemeinschaft, aber sind den gleichen Umweltveränderungen ausgesetzt. Nicht nur Lebensstil, sondern auch Entwicklung und Lebenszyklus unterscheiden sich zwischen Honig- und Mauerbienen. Mauerbienen überwintern als adulte Insekten in einem Kokon bis sie im Frühjahr schlüpfen. Honigbienen durchleben keine Diapause und schlüpfen nach wenigen Wochen der Entwicklung im Bienenstock. In meiner Dissertation vergleiche ich die circadiane Uhr von sozialen Honigbienen (Apis mellifera) und solitären Mauerbienen (Osmia bicornis und Osmia cornuta) auf Ebene der Neuroanatomie und das durch die innere Uhr verursachte rhythmische Verhalten. Erstens charakterisierte ich detailliert die Lage der circadianen Uhr im Gehirn von Honig- und Mauerbiene anhand des Expressionsmusters von zwei Uhrkomponenten. Diese sind das Uhrprotein PERIOD (PER) und das Neuropeptid Pigment Dispersing Factor (PDF). PER wird exprimiert in lateralen Neuronen-Gruppen (die wir laterale Neurone 1 und 2 nannten: LN1 und LN2) und dorsalen Neuronen-Gruppen (benannt dorsal laterale Neurone und dorsale Neurone: DLN und DN), sowie in vielen Gliazellen und Fotorezeptorzellen. Dieses Expressionsmuster liegt ähnlich in anderen Insektengruppen vor und deutet auf einen Grundbauplan der Inneren Uhr im Gehirn von Insekten hin. In der LN2 Neuronen-Gruppe, deren Zellkörper im lateralen Gehirn liegen, sind PER und PDF in den gleichen Zellen co-lokalisiert. Diese Zellen bilden ein komplexes Netzwerk aus Verzweigungen durch das gesamte Gehirn und liefern damit die perfekte Infrastruktur, um Zeitinformation an Gehirnregionen weiterzuleiten, die komplexe Verhaltensweisen, wie Sonnenkompass-Orientierung und Zeitgedächtnis, steuern. Alle PDF Neuriten laufen in einer anterior zur Lobula liegenden Region zusammen (sie wurde ALO, anterio-lobular PDF Knotenpunkt, genannt). Dieser Knotenpunkt ist in anderen Insekten mit der Medulla assoziiert und wird akzessorische Medulla (AME) genannt. Wenige PDF Zellen bilden bereits im frühen Larvalstadium diesen ALO und die Zellzahl sowie die Komplexität des Netzwerks wächst die gesamte Entwicklung der Honigbiene hindurch. Dabei werden zuerst die dorsalen Gehirnregionen von PDF Neuronen innerviert und in der späteren Larvalentwicklung wachsen die Neurite lateral in Richtung der optischen Loben und des Zentralgehirns. Das generelle Expressionsmuster von PER und PDF in adulten sozialen und solitären Bienen ähnelt sich stark, aber ich identifizierte kleine Unterschiede in der PDF Netzwerkdichte im posterioren Protocerebrum und in der Lamina. Diese könnten mit der Evolution von sozialen Bienen assoziiert sein. Zweitens entwickelte und etablierte ich eine Methode, Lokomotionsrhythmen von individuellen Bienen im Labor aufzunehmen, die in Kontakt mit einem Miniaturvolk standen. Diese Methode enthüllte neue Aspekte der sozialen Synchronisation unter Honigbienen und des Überlebens von jungen Bienen, die indirekten sozialen Kontakt zu dem Miniaturvolk hatten (Trophalaxis war nicht möglich). Für Mauerbienen etablierte ich eine Methode Schlupf- und lokomotorische Aktivitätsrhythmik aufzuzeichnen und konnte damit zeigen, dass tägliche Rhythmen im Schlupf durch Synchronisation der circadianen Uhr in Mauerbienen durch Tagestemperatur-Zyklen erzielt werden kann. Des Weiteren präsentiere ich die ersten lokomotorischen Aktivitätsrhythmen von solitären Bienen, die sofort nach ihrem Schlupf einen starken circadianen Rhythmus im Verhalten aufwiesen. Honigbienen brauchten in meinen Experimenten mehrere Tage, um circadiane Rhythmen in Lokomotion zu entwickeln. Ich erstellte die Hypothese, dass Honigbienen zum Zeitpunkt des Schlupfes im Bienenvolk ein noch nicht vollständig ausgereiftes circadianes System besitzen, während solitäre Bienen, die ohne den Schutz eines Volkes sind, direkt nach dem Schlupf eine vollständig ausgereifte Uhr brauchen. Mehrere Hinweise in Publikationen und Vorversuchen unterstützen meine Hypothese. Zukünftige Studien der Entwicklung des PDF Neuronen-Netzwerkes in solitären Bienen unterschiedlicher Entwicklungsstufen könnten dies nachweisen.

We examined the circadian rhythms of locomotor activity in three spider species in the Family Theridiidae under light–dark cycles and constant darkness. Contrary to previous findings in other organisms, we found exceptionally high variability in endogenous circadian period both within and among species. Many individuals exhibited circadian periods much lower (19–22 h) or much higher (26–30 h) than the archetypal circadian period. These results suggest relaxed selection on circadian period as well as an ability to succeed in nature despite a lack of circadian resonance with the 24-h daily cycle. Although displaying similar entrainment waveforms under light–dark cycles, there were remarkable differences among the three species with respect to levels of apparent masking and dispersion of activity under constant dark conditions. These behavioral differences suggest an aspect of chronotype adapted to the particular ecologies of the different species.

Chrononutrition, or the circadian timing of food intake, has garnered attention as a topic of study due to its associations with health (e.g., weight gain); however, a valid assessment of chrononutrition in daily life has not yet been developed. The present study therefore aimed to develop and validate both a diary and questionnaire version of the Chrononutrition Profile which assess 6 components of chrononutrition that have been associated with poor health (breakfast skipping, night eating, eating window, evening latency, largest meal, and evening eating). The measure demonstrated preliminary evidence of test-retest reliability and convergent validity, though concurrent validity was not interpretable. Based on analyses, the final diary and questionnaire versions of the CP assess 5 components of chrononutrition: breakfast skipping, night eating, eating window, evening latency, and evening eating. This measure offers health care professionals, researchers, and stakeholders a cost-effective method of evaluating chrononutrition and identifying targets for health improvement.

<p>  </p> <p>Cyanobacteria are among the simplest organisms to display circadian rhythms that synchronize endogenous physiological activities with a ~12-hour-light:12-hour-dark (12L:12D) cycle of the external environment. Detected by the input pathway composed of CikA and LdpA proteins, light is transduced to the central circadian oscillator encoded by the gene cluster <em>kaiABC. </em>While KaiC phosphorylation is primarily regulated by KaiA and KaiB proteins, two key components of the output pathway, RpaA and SasA proteins, mediate between KaiC phosphorylation, genome-wide expression, and control of cell division. In this study, <em>Synechococcus elongatus </em>UTEX 2973 showed similar growth patterns when subjected to white light only and white light supplemented with ultraviolet A (UV-A) radiation under 12L:12D intervals, although UV-A radiation hindered growth during light periods. Under continuous illumination, growth rates of <em>S. elongatus </em>UTEX 2973 were reduced by UV-A radiation but reflected intrinsic circadian rhythmicity. To elucidate the critical role of the circadian clock, a mutant void of <em>kaiABC</em> was generated via the CRISPR/Cpf1 system. A dysfunctional clock severely disrupted inherent growth rhythmicity, which was exacerbated by UV-A radiation. To investigate the effects of UV-A radiation on transcription patterns in <em>S. elongatus </em>UTEX 2973, expression levels of circadian genes, specifically <em>kaiABC</em>, <em>cikA</em>, <em>lpdA</em>, <em>rpaA</em>, and <em>sasA</em>, were assessed by qPCR analysis. For the UV-A-treated wild-type strain, <em>kaiA</em> and <em>kaiB</em> expression was generally downregulated, <em>kaiC</em> expression was upregulated during the second dark period, and <em>rpaA</em> expression was either upregulated or downregulated depending on the period. For the UV-A-treated Δ<em>kaiABC </em>strain, <em>lpaA</em> expression was upregulated in darkness, whereas <em>rpaA</em> and <em>sasA</em> expression was downregulated during light periods. When Δ<em>kaiABC </em>and wild-type strains were examined in the presence and absence of UV-A radiation, expression of <em>lpaA</em>, <em>rpaA</em>, and <em>sasA</em> was universally downregulated, yet <em>cikA</em> expression was upregulated in the dark. This study was the first to evaluate the impact of UV-A radiation on cyanobacterial circadian rhythms, in which UV-A radiation negatively affected cyanobacterial growth and strongly altered gene expression patterns over time. Without the circadian clock, rhythmicity of growth and transcription was demolished, such that the consequences were aggravated for the output pathway that relayed signals downstream from the central oscillator. </p>

Light not only allows us to see but is also fundamental to our health and well-being. Several parameters of light exposure, such as wavelengths, intensity, and timing of exposure, all play an important role on its effects on psychophysiological functions. The way in which studies have previously quantified light (based on intensity), has been found to be inadequate since it does not consider the spectrum of light which influences non-visual effects. Among these non-visual effects, light has been found to have antidepressant effects, however, these effects remain inconsistent for non-seasonal depression and their underlying mechanisms remain elusive. The three research studies in this thesis investigated the effects of light on mood. The first study focused on the direct pathway between light and mood, aiming to predict mood outcomes based on the amount of intrinsically photosensitive retinal ganglion cells (ipRGC) stimulation from polychromatic light in healthy individuals. The second and third studies focused on the indirect pathways, exploring predictors and underlying mechanisms of mood improvements by means of sleep and circadian re-alignment in the context of non-seasonal depression. These studies explored the antidepressant mechanisms of monochromatic light therapy and predictive models of mood improvement. The results from study I (systematic review) suggest that ipRGCs may not be as involved in the mood improvement associated with polychromatic light. Drawing strong conclusions from these results are, however, cautioned. Mood metrics used across the studies were inconsistent and the light sources were not designed to maximally stimulate ipRGCs. The results from study II (open-label trial) support the notion that light therapy does have antidepressant effects in people with non-seasonal depression. The underlying mechanisms for these antidepressant effects may involve improvements in sleep initiation and daytime functioning. Individuals with difficulties with fallings asleep and waking-up may be those that respond most prominently to light therapy. The results from study III (randomized controlled trial) indicate that depression symptoms improved slightly more in the active light therapy condition as opposed to a placebo condition. Although this effect was modest across the overall group, there were considerable inter-individual variations in treatment response. The degree of improvement in mood was associated with improvement in pre-sleep thoughts and the circadian rhythmicity of skin temperature. Short REM latency and worst global subjective sleep were predictive of greater response to light therapy. Overall, further research is required to disentangle the involvement of the different photoreceptors in the mood response to polychromatic light in healthy individuals. Monochromatic light therapy for non-seasonal depression yields overall modest antidepressant effects. Clinical applications of light therapy may benefit from further research investigating differential effects in sub-groups of depression and underlying mechanisms in larger studies.

Parkinson’s disease (PD) is a neurodegenerative disorder associated with motor disturbance and non-motor symptoms (NMS). Although NMS such as sleep and circadian disruption, cognitive impairment, and mood disturbance are prevalent and debilitating, treatments are limited, owing to lack of knowledge about their etiologies and interactions. This project comprised two studies of NMS in mild to moderate idiopathic PD without dementia. The hypothesis of Study 1 was that disruption of rest-activity circadian rhythm contributes to cognitive impairment in PD independently of sleep disruption. Thirty-five participants underwent 7-10 days of actigraphy monitoring, which provided estimates of sleep and circadian functioning, and then neuropsychological testing. Hierarchical regression showed that circadian interdaily stability predicted executive function, visuospatial function, and psychomotor speed, though not memory, independently of sleep. Sleep efficiency independently predicted executive but not other cognitive function. Study 2 examined daily temporal associations between sleep, subjective cognition, and mood (anxiety, positive affect) in PD. Twenty participants used a smartphone app to engage in 14 days of ecological momentary assessment (EMA) of daytime symptoms and subjective sleep quantity and quality. They wore actigraphs for objective sleep assessment. The hypothesis was that sleep quality would predict cognition and mood. Multilevel modeling indicated that subjective sleep quality was bidirectionally associated with mood on a day-to-day basis (i.e., last night’s perceived sleep affected today’s mood, which in turn will affect tonight’s sleep). There was also a trend for subjective sleep quality to predict the next day’s subjective cognitive function. Objective sleep was not associated with any daytime symptoms. Study 1 provided the first evidence that circadian rhythm contributes to cognition in PD independently of sleep. Study 2 indicated that, on a daily timescale, subjective sleep is relevant to mood and possibly cognition in PD, whereas objective sleep is either insufficiently sensitive or not relevant. Together these findings support the use of EMA to investigate small-timescale interactions among NMS, and present circadian rhythms and sleep perception as intervention targets for improving cognition and mood, and ultimately for enhancing quality of life in PD. / 2020-02-15T00:00:00Z