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In this dissertation, I examine the relationship between specialisation and stability of plant-pollinator networks, with a focus on two issues: Diversity maintenance in animal-pollinated plant communities and robustness of plant-pollinator systems against disturbances such as those caused by anthropogenic climate change. Chapter 1 of this thesis provides a general introduction to the concepts of ecological stability and specialisation with a focus on plant-pollinator systems, and a brief outline of the following chapters. Chapters 2-5 each consist of a research article addressing a specific question. While chapters 2 and 3 deal with different aspects of diversity maintenance in animal-pollinated plant communities, chapters 4 and 5 are concerned with the consequences of climate change in the form of temporary disturbances caused by extreme climatic events (chapter 4) and shifts in phenology of plants and pollinators (chapter 5). From a methodological perspective, the first three articles (chapter 2-4) can be grouped together as they all employ mathematical models of plant-pollinator systems, whereas chapter 5 describes an empirical study of plant-pollinator interactions along an altitudinal gradient in the Alps. The final chapter (6) provides a review of current knowledge on each of the two main themes of this thesis and places the findings of the four research articles in the context of related studies.
Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -
(2018)
All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: Sönke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the
M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.
Although the concept of botanical carnivory has been known since Darwin's time, the molecular mechanisms that allow animal feeding remain unknown, primarily due to a complete lack of genomic information. Here, we show that the transcriptomic landscape of the Dionaea trap is dramatically shifted toward signal transduction and nutrient transport upon insect feeding, with touch hormone signaling and protein secretion prevailing. At the same time, a massive induction of general defense responses is accompanied by the repression of cell death-related genes/processes. We hypothesize that the carnivory syndrome of Dionaea evolved by exaptation of ancient defense pathways, replacing cell death with nutrient acquisition.
Nuclei assembled in Xenopus egg extract from purified DNA or chromatin resemble their natural counterparts in a number of structural and functional features. However, the most obvious structural element of normal interphase nuclei, the nucleolus, is absent from the in vitro reconstituted nuclei. By EM, cytological silver staining, and immunofluorescence microscopy employing antibodies directed against various nucleolar components we show that nuclei assembled in vitro contain numerous distinct aggregates that resemble prenucleolar bodies (PNBs) by several criteria. Formation of these PNB-like structures requires pore complex-mediated nuclear transport of proteins but is independent of the genetic content of the in vitro nuclei as well as transcriptional and translational events. Our data indicate that nuclei assembled in vitro are capable of initiating early steps of nucleologenesis but that the resulting PNBs are unable to fuse with each other, probably due to the absence of a functional nucleolus organizer. With appropriate modifications, this experimental system should be useful to define and analyze conditions promoting the site-specific assembly of PNBs into a coherent nucleolar body.
Der genetische Code beschreibt die Ver- und Entschlüsselung der Erb-information für das universelle Prinzip der Proteinbiosynthese aus einzelnen Aminosäuren. Durch Erweiterung des genetischen Codes lassen sich unna-türliche Aminosäuren (uAA) mit einzigartigen biophysikalischen Eigenschaf-ten ortsspezifisch in Proteine einführen und ermöglichen die spezifische Ma-nipulation von Proteinen.
Die Click-Reaktion zwischen der unnatürlichen Aminosäure TCO*-Lysin und Tetrazin besitzt eine außergewöhnliche Reaktionskinetik (≥800 M-1s-1) und ermöglicht eine spezifische und bioorthogonale Markierung von Bio-
¬molekülen unter physiologischen Bedingungen.
Im Fokus dieser Arbeit stand zunächst die Markierung von Membran-
¬rezeptoren durch Click-Chemie in lebenden Zellen sowie die Untersuchung der Wechselwirkung 22 bekannter und neuartiger Tetrazin-Farbstoff-
Konjugate. Darüber hinaus wurde die Anwendbarkeit von bioorthogonalen Click-Reaktionen für die hochauflösende Fluoreszenzmikroskopie untersucht. Durch Erweiterung des genetischen Codes in Proteine aus der Klasse der ionotropen Glutamatrezeptoren (iGluR), TNF-Rezeptoren oder Mikrotubu-li-assoziierten Proteinen (MAP) wurde ortspezifisch die unnatürliche Amino-säure TCO*-Lysin eingeführt und dadurch die Fluoreszenzmarkierung durch Tetrazin-Farbstoffe ermöglicht. Die direkte chemische Kopplung von TCO an Liganden wie Phalloidin und Docetaxel, welche spezifisch das Aktin-Zytoskelett bzw. Mikrotubuli-Filamente binden können, ermöglichte zudem die Click-Färbungen von fixierten und lebenden Zellen ohne genetische Ver-änderungen der Zielproteine.
Des Weiteren wurden die spektroskopischen Eigenschaften von 22 Tetrazin-Farbstoffen, verteilt über den gesamten sichtbaren Wellenlängenbereich, untersucht. Ein charakteristisches Kennzeichen der Click-Reaktion mit Tet-razin-Farbstoffen ist dabei ihre Fluorogenität. Das Tetrazin fungiert nicht nur als reaktive Gruppe während der Click-Reaktion mit Alkenen, sondern führt in vielen Tetrazin-Farbstoff-Konjugaten zur Fluoreszenzlöschung. Während bei grün-absorbierenden Farbstoffe vor allem FRET-basierte Löschprozesse dominieren, konnte photoinduzierter Elektronentransfer (PET) vom angeregten Farbstoff zum Tetrazin als Hauptlöschmechanismus bei rot-absorbierenden Oxazin- und Rhodamin-Derivaten identifiziert
werden.
Die effiziente und spezifische Markierung aller untersuchten Tetrazin-
Farbstoffe ermöglichte die Visualisierung von Aktin-Filamenten, Mikrotubuli und Membranrezeptoren sowohl durch konventionelle Fluoreszenzmikrosko-pie als auch durch hochauflösende Verfahren, wie z.B. dSTORM, auf Ein-zelmolekülebene. Die unterschiedliche Zellpermeabilität von
Tetrazin-Farbstoffen kann dabei vorteilhaft für die spezifische intra- und extrazelluläre Markierung von Proteinen in fixierten und lebenden Zellen genutzt werden.
Background: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress. Results: In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurablemetabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration. Conclusions: The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.
In recent years high-throughput experiments provided a vast amount of data from all areas of molecular biology, including genomics, transcriptomics, proteomics and metabolomics. Its analysis using bioinformatics methods has developed accordingly, towards a systematic approach to understand how genes and their resulting proteins give rise to biological form and function. They interact with each other and with other molecules in highly complex structures, which are explored in network biology. The in-depth knowledge of genes and proteins obtained from high-throughput experiments can be complemented by the architecture of molecular networks to gain a deeper understanding of biological processes. This thesis provides methods and statistical analyses for the integration of molecular data into biological networks and the identification of functional modules, as well as its application to distinct biological data. The integrated network approach is implemented as a software package, termed BioNet, for the statistical language R. The package includes the statistics for the integration of transcriptomic and functional data with biological networks, the scoring of nodes and edges of these networks as well as methods for subnetwork search and visualisation. The exact algorithm is extensively tested in a simulation study and outperforms existing heuristic methods for the calculation of this NP-hard problem in accuracy and robustness. The variability of the resulting solutions is assessed on perturbed data, mimicking random or biased factors that obscure the biological signal, generated for the integrated data and the network. An optimal, robust module can be calculated using a consensus approach, based on a resampling method. It summarizes optimally an ensemble of solutions in a robust consensus module with the estimated variability indicated by confidence values for the nodes and edges. The approach is subsequently applied to two gene expression data sets. The first application analyses gene expression data for acute lymphoblastic leukaemia (ALL) and differences between the subgroups with and without an oncogenic BCR/ABL gene fusion. In a second application gene expression and survival data from diffuse large B-cell lymphomas are examined. The identified modules include and extend already existing gene lists and signatures by further significant genes and their interactions. The most important novelty is that these genes are determined and visualised in the context of their interactions as a functional module and not as a list of independent and unrelated transcripts. In a third application the integrative network approach is used to trace changes in tardigrade metabolism to identify pathways responsible for their extreme resistance to environmental changes and endurance in an inactive tun state. For the first time a metabolic network approach is proposed to detect shifts in metabolic pathways, integrating transcriptome and metabolite data. Concluding, the presented integrated network approach is an adequate technique to unite high-throughput experimental data for single molecules and their intermolecular dependencies. It is flexible to apply on diverse data, ranging from gene expression changes over metabolite abundances to protein modifications in a combination with a suitable molecular network. The exact algorithm is accurate and robust in comparison to heuristic approaches and delivers an optimal, robust solution in form of a consensus module with confidence values. By the integration of diverse sources of information and a simultaneous inspection of a molecular event from different points of view, new and exhaustive insights into biological processes can be acquired.
The development of all honey bee castes proceeds through three different life stages all of which encounter microbial infections to a various extent. We have examined the immune strength of honey bees across all developmental stages with emphasis on the temporal expression of cellular and humoral immune responses upon artificial challenge with viable Escherichia coli bacteria. We employed a broad array of methods to investigate defence strategies of infected individuals: (a) fate of bacteria in the haemocoel; (b) nodule formation and (c) induction of antimicrobial peptides (AMPs). Newly emerged adult worker bees and drones were able to activate efficiently all examined immune reactions. The number of viable bacteria circulating in the haemocoel of infected bees declined rapidly by more than two orders of magnitude within the first 4–6 h post-injection (p.i.), coinciding with the occurrence of melanised nodules. Antimicrobial activity, on the other hand, became detectable only after the initial bacterial clearance. These two temporal patterns of defence reactions very likely represent the constitutive cellular and the induced humoral immune response. A unique feature of honey bees is that a fraction of worker bees survives the winter season in a cluster mostly engaged in thermoregulation. We show here that the overall immune strength of winter bees matches that of young summer bees although nodulation reactions are not initiated at all. As expected, high doses of injected viable E.coli bacteria caused no mortality in larvae or adults of each age. However, drone and worker pupae succumbed to challenge with E.coli even at low doses, accompanied by a premature darkening of the pupal body. In contrast to larvae and adults, we observed no fast clearance of viable bacteria and no induction of AMPs but a rapid proliferation of E.coli bacteria in the haemocoel of bee pupae ultimately leading to their death.
Neural processing of a desired moving direction requires the continuous comparison between the current heading and the goal direction. While the neural basis underlying the current heading is well-studied, the coding of the goal direction remains unclear in insects. Here, we used tetrode recordings in tethered flying monarch butterflies to unravel how a goal direction is represented in the insect brain. While recording, the butterflies maintained robust goal directions relative to a virtual sun. By resetting their goal directions, we found neurons whose spatial tuning was tightly linked to the goal directions. Importantly, their tuning was unaffected when the butterflies changed their heading after compass perturbations, showing that these neurons specifically encode the goal direction. Overall, we here discovered invertebrate goal-direction neurons that share functional similarities to goal-direction cells reported in mammals. Our results give insights into the evolutionarily conserved principles of goal-directed spatial orientation in animals.
Echolocation behavior, a navigation strategy based on acoustic signals, allows scientists to explore neural processing of behaviorally relevant stimuli. For the purpose of orientation, bats broadcast echolocation calls and extract spatial information from the echoes. Because bats control call emission and thus the availability of spatial information, the behavioral relevance of these signals is undiscussable. While most neurophysiological studies, conducted in the past, used synthesized acoustic stimuli that mimic portions of the echolocation signals, recent progress has been made to understand how naturalistic echolocation signals are encoded in the bat brain. Here, we review how does stimulus history affect neural processing, how spatial information from multiple objects and how echolocation signals embedded in a naturalistic, noisy environment are processed in the bat brain. We end our review by discussing the huge potential that state-of-the-art recording techniques provide to gain a more complete picture on the neuroethology of echolocation behavior.
Chronobiological studies of individual activity rhythms in social insects can be constrained by the artificial isolation of individuals from their social context. We present a new experimental set-up that simultaneously measures the temperature rhythm in a queen-less but brood raising mini colony and the walking activity rhythms of singly kept honey bees that have indirect social contact with it. Our approach enables monitoring of individual bees in the social context of a mini colony under controlled laboratory conditions. In a pilot experiment, we show that social contact with the mini colony improves the survival of monitored young individuals and affects locomotor activity patterns of young and old bees. When exposed to conflicting Zeitgebers consisting of a light-dark (LD) cycle that is phase-delayed with respect to the mini colony rhythm, rhythms of young and old bees are socially synchronized with the mini colony rhythm, whereas isolated bees synchronize to the LD cycle. We conclude that the social environment is a stronger Zeitgeber than the LD cycle and that our new experimental set-up is well suited for studying the mechanisms of social entrainment in honey bees.
Life on earth adapted to the daily reoccurring changes in environment by evolving an endogenous circadian clock. Although the circadian clock has a crucial impact on survival and behavior of solitary bees, many aspects of solitary bee clock mechanisms remain unknown. Our study is the first to show that the circadian clock governs emergence in Osmia bicornis, a bee species which overwinters as adult inside its cocoon. Therefore, its eclosion from the pupal case is separated by an interjacent diapause from its emergence in spring. We show that this bee species synchronizes its emergence to the morning. The daily rhythms of emergence are triggered by temperature cycles but not by light cycles. In contrast to this, the bee’s daily rhythms in locomotion are synchronized by light cycles. Thus, we show that the circadian clock of O. bicornis is set by either temperature or light, depending on what activity is timed. Light is a valuable cue for setting the circadian clock when bees have left the nest. However, for pre-emerged bees, temperature is the most important cue, which may represent an evolutionary adaptation of the circadian system to the cavity-nesting life style of O. bicornis.
Timing seasonal events, like reproduction or diapause, is crucial for the survival of many species. Global change causes phenologies worldwide to shift, which requires a mechanistic explanation of seasonal time measurement. Day length (photoperiod) is a reliable indicator of winter arrival, but it remains unclear how exactly species measure day length. A reference for time of day could be provided by a circadian clock, by an hourglass clock, or, as some newer models suggest, by a damped circadian clock. However, damping of clock outputs has so far been rarely observed. To study putative clock outputs of Acyrthosiphon pisum aphids, we raised individual nymphs on coloured artificial diet, and measured rhythms in metabolic activity in light-dark illumination cycles of 16:08 hours (LD) and constant conditions (DD). In addition, we kept individuals in a novel monitoring setup and measured locomotor activity. We found that A. pisum is day-active in LD, potentially with a bimodal distribution. In constant darkness rhythmicity of locomotor behaviour persisted in some individuals, but patterns were mostly complex with several predominant periods. Metabolic activity, on the other hand, damped quickly. A damped circadian clock, potentially driven by multiple oscillator populations, is the most likely explanation of our results.
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 fruit fly Drosophila melanogaster is an established model organism in chronobiology, because genetic manipulation and breeding in the laboratory are easy. The circadian clock neuroanatomy in D. melanogaster is one of the best-known clock networks in insects and basic circadian behavior has been characterized in detail in this insect. Another model in chronobiology is the honey bee Apis mellifera, of which diurnal foraging behavior has been described already in the early twentieth century. A. mellifera hallmarks the research on the interplay between the clock and sociality and complex behaviors like sun compass navigation and time-place-learning. Nevertheless, there are aspects of clock structure and function, like for example the role of the clock in photoperiodism and diapause, which can be only insufficiently investigated in these two models. Unlike high-latitude flies such as Chymomyza costata or D. ezoana, cosmopolitan D. melanogaster flies do not display a photoperiodic diapause. Similarly, A. mellifera bees do not go into “real” diapause, but most solitary bee species exhibit an obligatory diapause. Furthermore, sociality evolved in different Hymenoptera independently, wherefore it might be misleading to study the social clock only in one social insect. Consequently, additional research on non-model insects is required to understand the circadian clock in Diptera and Hymenoptera. In this review, we introduce the two chronobiology model insects D. melanogaster and A. mellifera, compare them with other insects and show their advantages and limitations as general models for insect circadian clocks.
Post-embryonic Development of the Circadian Clock Seems to Correlate With Social Life Style in Bees
(2020)
Social life style can influence many aspects of an animal’s daily life, but it has not yet been clarified, whether development of the circadian clock in social and solitary living bees differs. In a comparative study, with the social honey bee, Apis mellifera, and the solitary mason bee, Osmia bicornis, we now found indications for a differentially timed clock development in social and solitary bees. Newly emerged solitary bees showed rhythmic locomotion right away and the number of neurons in the brain that produce the clock component pigment-dispersing factor (PDF) did not change during aging of the adult solitary bee. Honey bees on the other hand, showed no circadian locomotion directly after emergence and the neuronal clock network continued to grow after emergence. Social bees appear to emerge at an early developmental stage at which the circadian clock is still immature, but bees are already able to fulfill in-hive tasks.
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.
Age‐dependent transcriptional and epigenomic responses to light exposure in the honey bee brain
(2016)
Light is a powerful environmental stimulus of special importance in social honey bees that undergo a behavioral transition from in-hive to outdoor foraging duties. Our previous work has shown that light exposure induces structural neuronal plasticity in the mushroom bodies (MBs), a brain center implicated in processing inputs from sensory modalities. Here, we extended these analyses to the molecular level to unravel light-induced transcriptomic and epigenomic changes in the honey bee brain. We have compared gene expression in brain compartments of 1- and 7-day-old light-exposed honey bees with age-matched dark-kept individuals. We have found a number of differentially expressed genes (DEGs), both novel and conserved, including several genes with reported roles in neuronal plasticity. Most of the DEGs show age-related changes in the amplitude of light-induced expression and are likely to be both developmentally and environmentally regulated. Some of the DEGs are either known to be methylated or are implicated in epigenetic processes suggesting that responses to light exposure are at least partly regulated at the epigenome level. Consistent with this idea light alters the DNA methylation pattern of bgm, one of the DEGs affected by light exposure, and the expression of microRNA miR-932. This confirms the usefulness of our approach to identify candidate genes for neuronal plasticity and provides evidence for the role of epigenetic processes in driving the molecular responses to visual stimulation.
The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli.
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2\(^{2+}\)) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca\(^{2+}\) might be desirable. Moreover, there is need for an efficient light-gated potassium (K\(^{+}\)) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca\(^{2+}\) and K\(^{+}\) in cell physiology, light-activated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca\(^{2+}\)-permeant and K\(^{+}\)-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca\(^{2+}\) or for K\(^{+}\), respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca\(^{2+}\)-permeant channel, and to body extension when expressing the light-sensitive K\(^{+}\) channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.