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Chapter I – Introduction
Global trade of beans of the cacao tree (Theobroma cacao), of which chocolate is produced, contributes to the livelihoods of millions of smallholder farmers. The understorey tree is native to South America but is nowadays cultivated in many tropical regions. In Peru, a South American country with a particularly high cacao diversity, it is common to find the tree cultivated alongside non-crop trees that provide shade, in so-called agroforestry systems. Because of the small scale and low management intensity of such systems, agroforestry is one of the most wildlife-friendly land-use types, harbouring the potential for species conservation. Studying wildlife-friendly land-use is of special importance for species conservation in biodiversity-rich tropical regions such as Peru, where agricultural expansion and intensification are threatening biodiversity. Moreover, there is a growing body of evidence that shows co-occurrence of high biodiversity levels and high yield in wildlife-friendly cacao farming. Yet studies are restricted to non-native cacao countries, and since patterns might be different among continents, it is important to improve knowledge on wildlife-friendly agroforestry in native countries.
Because studies of wildlife-friendly cultivation processes are still largely lacking for South America, we set out to study multiple aspects of cacao productivity in agroforests in Peru, part of cacao´s region of origin. The natural pollination process of cacao, which is critically understudied, was investigated by trapping flower visitors and studying pollen deposition from macrophotographs (Chapter II). Next, we excluded birds, bats, ants and flying insects and squirrels from cacao trees in a full-factorial field experiment and quantified these animals´ contribution to cacao fruit set, fruit loss and yield (Chapter III). Lastly, we aimed to assess whether fruit quantity and quality of native cacao increases through manually supplementing pollen (Chapter II and IV), and whether microclimatic conditions and the genetic background of the studied varieties limit fruit set (Chapter IV).
Chapter II – Cacao flower visitation: Low pollen deposition, low fruit set and dominance of herbivores
Given the importance of cacao pollination for the global chocolate production, it is remarkable that fruit set limitations are still understudied. Knowledge on flower visitation and the effect of landscape context and local management are lacking, especially in the crop’s region of origin. Moreover, the role of pollen deposition in limiting fruit set as well as the benefits of hand pollination in native cacao are unknown. In this chapter, we aimed to close the current knowledge gaps on cacao pollination biology and sampled flower visitors in 20 Peruvian agroforests with native cacao, along gradients of shade cover and forest distance. We also assessed pollen quantities and compared fruit set between manually and naturally pollinated flowers. We found that herbivores were the most abundant flower visitors in both northern and southern Peru, but we could not conclude which insects are effective cacao pollinators. Fruit set was remarkably low (2%) but improved to 7% due to pollen supplementation. Other factors such as a lack of effective pollinators, genetic pollen incompatibility or resource unavailability could be causing fruit set limitations. We conclude that revealing those causes and the effective pollinators of cacao will be key to improve pollination services in cacao.
Chapter III – Quantifying services and disservices provided by insects and vertebrates in cacao agroforestry landscapes
Pollination and pest control, two ecosystem services that support cacao yield, are provided by insects and vertebrates. However, animals also generate disservices, and their combined contribution is still unclear. Therefore, we excluded flying insects, ants, birds and bats, and as a side effect also squirrels from cacao trees and we assessed fruit set, fruit loss and final yield. Local management and landscape context can influence animal occurrence in cacao agroforestry landscapes; therefore, shade cover and forest distance were included in the analyses. Flying insects benefitted cacao fruit set, with largest gains in agroforests with intermediate shade cover. Birds and bats were also associated with improved fruit set rates and with a 114% increase in yield, potentially due to pest control services provided by these animals. The role of ants was complicated: these insects had a positive effect on yield, but only close to forest. We also evidenced disservices generated by ants and squirrels, causing 7% and 10% of harvest loss, respectively. Even though the benefits provided by animals outweighed the disservices, trade-offs between services and disservices still should be integrated in cacao agroforestry management.
Chapter IV – Cross-pollination improves fruit set and yield quality of Peruvian native cacao
Because yields of the cacao tree are restricted by pollination, hand pollination has been proposed to improve yield quantity and potentially, also quality. However, low self- and cross-compatibility of native cacao, and abiotic conditions could cancel out hand pollination benefits. Yet, the impact of genetic constraints and abiotic conditions on fruit set have not been assessed in native cacao so far. To increase our understanding of the factors that limit fruit set in native cacao, we compared manual self- and cross-pollination with five native genotypes selected for their sensorial quality and simultaneously tested for effects of soil water content, temperature, and relative air humidity. We also compared quality traits between manually and naturally pollinated fruits. Success rates of self-pollination were low (0.5%), but increased three- to eightfold due to cross-pollination, depending on the genotype of the pollen donor. Fruit set was also affected by the interaction between relative air humidity and temperature, and we found heavier and more premium seeds in fruits resulting from manual than natural pollination. Together, these findings show that reproductive traits of native cacao are constrained by genetic compatibility and abiotic conditions. We argue that because of the high costs of hand pollination, natural cross-pollination with native pollen donors should be promoted so that quality improvements can result in optimal economic gains for smallholder farmers.
Chapter V – Discussion
In this thesis, we demonstrated that the presence of flying insects, ants and vertebrates, local and landscape management practices, and pollen supplementation interactively affected cacao yield, at different stages of the development from flower to fruit. First, we showed that fruit set improved by intermediate shade levels and flower visitation by flying insects. Because the effective cacao pollinators remain unknown, we recommend shade cover management to safeguard fruit set rates. The importance of integrating trade-offs in wildlife-friendly management was highlighted by lower harvest losses due to ants and squirrels than the yield benefits provided by birds and bats. The maintenance of forest in the landscape might further promote occurrence of beneficial animals, because in proximity to forest, ants were positively associated with cacao yields. Therefore, an integrated wildlife-friendly farming approach in which shade cover is managed and forest is maintained or restored to optimize ecosystem service provision, while minimizing fruit loss, might benefit yields of native cacao. Finally, manual cross-pollination with native genotypes could be recommended, due to improved yield quantity and quality. However, large costs associated with hand pollination might cancel out these benefits. Instead, we argue that in an integrated management, natural cross-pollination should be promoted by employing compatible genotypes in order to improve yield quantity and quality of native cacao.
For all animals the cold represents a dreadful danger. In the event of severe heat loss, animals
fall into a chill coma. If this state persists, it is inevitably followed by death. In poikilotherms
(e.g. insects), the optimal temperature range is narrow compared to homeotherms
(e.g. mammals), resulting in a critical core temperature being reached more quickly. As a
consequence, poikilotherms either had to develop survival strategies, migrate or die. Unlike
the majority of insects, the Western honeybee (Apis mellifera) is able to organize itself into
a superorganism. In this process, worker bees warm and cool the colony by coordinated
use of their flight muscles. This enables precise control of the core temperature in the hive,
analogous to the core body temperature in homeothermic animals. However, to survive the
harsh temperatures in the northern hemisphere, the thermogenic mechanism of honeybees
must be in constant readiness. This mechanism is called shivering thermogenesis, in which
honeybees generate heat using their flight muscles.
My thesis presents the molecular and neurochemical background underlying shivering thermogenesis
in worker honeybees. In this context, I investigated biogenic amine signaling.
I found that the depletion of vesicular monoamines impairs thermogenesis, resulting in
a decrease in thoracic temperature. Subsequent investigations involving various biogenic
amines showed that octopamine can reverse this effect. This clearly indicates the involvement
of the octopaminergic system. Proceeding from these results, the next step was to elucidate
the honeybee thoracic octopaminergic system. This required a multidisciplinary approach to
ultimately provide profound insights into the function and action of octopamine at the flight
muscles. This led to the identification of octopaminergic flight muscle controlling neurons,
which presumably transport octopamine to the flight muscle release sites. These neurons
most likely innervate octopamine β receptors and their activation may stimulate intracellular
glycolytic pathways, which ensure sufficient energy supply to the muscles.
Next, I examined the response of the thoracic octopaminergic system to cold stress conditions.
I found that the thoracic octopaminergic system tends towards an equilibrium,
even though the initial stress response leads to fluctuations of octopamine signaling. My
results indicate the importance of the neuro-muscular octopaminergic system and thus the need for its robustness. Moreover, cold sensitivity was observed for the expression of one
transcript of the octopamine receptor gene AmOARβ2. Furthermore, I found that honeybees
without colony context show a physiological disruption within the octopaminergic system.
This disruption has profound effects on the honeybees protection against the cold.
I could show how important the neuro-muscular octopaminergic system is for thermogenesis
in honeybees. In this context, the previously unknown neurochemical modulation of the
honeybee thorax has now been revealed. I also provide a broad basis to conduct further
experiments regarding honeybee thermogenesis and muscle physiology.
T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antikörper und CAR T Zellen haben in den vergangen Jahren die Therapiemöglichkeiten für Tumorpatienten erweitert. Diese Therapeutika verknüpfen T-Zellen mit malignen Zellen über je ein spezifisches Oberflächenmolekül und initiieren, über eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. Häufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivität limitieren, sondern zu geringen bis letalen Begleiterscheinungen führen können. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese Lücke soll durch ein neues Antikörperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antikörpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplementären Molekülen mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antikörpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer Nähe an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antikörperkonstrukte über das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle.
Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolekülen, wie BCMA, CD38, CD138, CD229 und SLAMF7, und für das MM unbekannte Oberflächenmolekülen, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfläche der MM Zelle führte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend über eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilität von Antigen-einfach-positiven gesunden Körper- oder Effektor-Zellen zu beeinflussen. Zusätzlich führte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antikörperformat für eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen.
Cellular growth and proliferation are among the most important processes for cells and
organisms. One of the major determinants of these processes is the amount of proteins
and consequently also the amount of ribosomes. Their synthesis involves several hundred
proteins and four different ribosomal RNA species, is highly coordinated and very
energy-demanding. However, the molecular mechanims of transcriptional regulation of
the protein-coding genes involved, is only poorly understood in mammals.
In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming
to identify previously unknown transcriptional regulators of ribosome biogenesis
factors (RiBis), which are important for the assembly and maturation of ribosomes,
and ribosomal proteins (RPs), which are ribosomal components themself. With that
approach and follow-up (validation) experiments, ALDOA and RBM8A among others,
could be identified as regulators of ribosome biogenesis.
Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter
driven reporters on protein and transcript level, as well as to a downregulation
of ribosome biogenesis gene transcripts and of mRNAs of other genes important for
proliferation.
Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent
downregulation of one of the fluorescent reporters, but this regulation was independent
of the promoter driving the expression of the reporter. However, acute protein
depletion experiments in combination with nascent RNA sequencing (4sU-Seq)
revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon
acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of
RP genes, but also to other chromatin regions. Total POL II or elongating and initiating
POL II levels were not altered upon acute RBM8A depletion.
These data provide a starting point for further research on the mechanisms of transcriptional
regulation of RP and RiBi genes in mammals.
Honeybees are among the few animals that rely on eusociality to survive. While the
task of queen and drones is only reproduction, all other tasks are accomplished by sterile
female worker bees. Different tasks are mostly divided by worker bees of different ages
(temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and
nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding
the hive entrance. The oldest honeybees eventually leave the hive to forage for resources
until they die. However, uncontrollable circumstances might force the colony to adapt or
perish. For example, the introduced Varroa destructor mite or the deformed wing virus
might erase a lot of in-hive bees. On the other hand, environmental events might kill a
lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of
task allocation must be a priority for a honeybee colony.
In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of
the honeybee division of labor, especially in conjunction with honeybee malnourishment.
The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to
be important for the transition from in-hive tasks to foraging. I have studied in detail
expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus
lay on gene expression in the honeybee brain and fat body. I found an increase in the
AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to
foraging and a decrease in expression of the other genes in both tissues. Interestingly,
I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the
honeybee fat body during orientation flights. Furthermore, I closely observed juvenile
hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers
increased with the transition from in-hive tasks to foraging and triglyceride levels decreased.
Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level.
For example, foragers are more responsive towards light and sucrose. I proposed that
modulation via biogenic amines, especially via octopamine and tyramine, can increase
or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified
1
using electroretinography. In addition, I studied the behavioral response of the bees to
light using a phototaxis assay. Injecting octopamine increased the receptor response and
tyramine decreased it. Also, both groups of honeybees showed an increased phototactic
response when injected with octopamine and a decreased response when injected with
tyramine, independent of locomotion.
Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to
cope with the missing resources. Furthermore, larval undernourishment has also been
implied to speed up the transition from in-hive bees to foragers, due to increasing levels
of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared
honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to
bees reared under the standard in-vitro rearing diet. However, first I had to investigate
whether the in-vitro rearing method affects adult honeybees.
I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging
earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was
unaffected.
Afterwards, I investigated the effects of different larval diets on adult honeybee workers.
I found no effects of malnourishment on behavioral or physiological factors besides a
difference in weight. Honeybee weight increased with increasing amounts of larval food,
but the effect seemed to vanish after a week.
These results show the complexity and adaptability of the honeybee division of labor.
They show the importance of the biogenic amines octopamine and tyramine and of the
corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on
nursing behavior, but that it speeds up the transition from nursing to foraging, showing
strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems
that larval malnourishment can be easily compensated during the early lifetime of adult
honeybees.
The western honeybee (Apis mellifera) is widely known as the honey producer and pollinator managed by beekeepers but neglected as a wild bee species. Central European honeybee populations have been anthropogenically disturbed since about 1850 through introgression and moderate artificial selection but have never been truly domesticated due to a lack of mating control. While their decline in the wild was historically attributed to the scarcity of nesting cavities, a contemporary view considers the invasion of the parasitic mite Varroa destructor in the 1970s as the major driver. However, there are no longitudinal population data available that could substantiate either claim. Based on the insight that introduced European honeybees form viable wild populations in eastern North America and reports on the occurrence of wild-living colonies from various European countries, we systematically studied the ecology of wild-living honeybees in Germany. First, we investigated whether wild-living honeybees colonising German forests form a self-sustaining population. Second, we asked how the parasite burden of wild-living colonies relates to that of managed colonies. And third, we explored whether the winter mortality of wild-living colonies is associated with parasite burden, nest depredation, or the lack of resources on the landscape scale.
Between 2017 and 2021, we monitored listed trees with black woodpecker cavities for honeybees in the managed forests of three study regions (Swabian Alb, counties Coburg and Lichtenfels, county Weilheim-Schongau). Continuity of occupation was determined using microsatellite genetic markers. Wild-living colonies predictably colonised forests in summer, when about 10% of all cavities were occupied. The annual colony survival rate and colony lifespan (based on N=112 colonies) were 10.6% and 0.6 years, with 90% of colonies surviving summer (July–September), 16% surviving winter (September–April), and 72% surviving spring (April–July). The average maximum and minimum colony densities were 0.23 (July) and 0.02 (April) colonies per km^2. During the (re-)colonisation of forests in spring, swarms preferred cavities that had already been occupied by other honeybee colonies. We estimate the net reproductive rate of the population to be R0= 0.318, meaning that it is currently not self-sustaining but maintained by the annual immigration of swarms from managed hives. The wild-living colonies are feral in a behavioural sense.
We compared the occurrence of 18 microparasites among feral colonies (N=64) and managed colonies (N=74) using qPCR. Samples were collected in four regions (the three regions mentioned above and the city of Munich) in July 2020; they consisted of 20 workers per colony captured at flight entrances. We distinguished five colony types representing differences in colony age and management histories. Besides strong regional variation, feral colonies consistently hosted fewer microparasite taxa (median: 5, range 1–8) than managed colonies (median: 6, range 4–9) and had different parasite communities. Microparasites that were notably less prevalent among feral colonies were Trypanosomatidae, Chronic bee paralysis virus, and Deformed wing viruses A and B. In the comparison of five colony types, parasite burden was lowest in newly founded feral colonies, intermediate in overwintered feral colonies and managed nucleus colonies, and highest in overwintered managed colonies and hived swarms. This suggests that the natural mode of colony reproduction by swarming, which creates pauses in brood production, and well-dispersed nests, which reduce horizontal transmission, explain the reduced parasite burden in feral compared to managed colonies.
To explore the roles of three potential drivers of feral colony winter mortality, we combined colony observations gathered during the monitoring study with data on colony-level parasite burden, observations and experiments on nest depredation, and landscape analyses. There was no evidence for an effect of summertime parasite burden on subsequent winter mortality: colonies that died (N=57) did not have a higher parasite burden than colonies that survived (N=10). Camera traps (N=15) installed on cavity trees revealed that honeybee nests are visited by a range of vertebrate species throughout the winter at rates of up to 10 visits per week. Four woodpecker species, great tits, and pine martens acted as true nest depredators. The winter survival rate of colonies whose nest entrances were protected by screens of wire mesh (N=32) was 50% higher than that of colonies with unmanipulated entrances (N=40). Analyses of land cover maps revealed that the landscapes surrounding surviving colonies (N=19) contained on average 6.4 percentage points more resource-rich cropland than landscapes surrounding dying colonies (N=94).
We estimate that tens of thousands of swarms escape from apiaries each year to occupy black woodpecker cavities and other hollow spaces in Germany and that feral colonies make up about 5% of the regional honeybee populations. They are unlikely to contribute disproportionately to the spread of bee diseases. Instead, by spatially complementing managed colonies, they contribute to the pollination of wild plants in forests. Honeybees occupying tree cavities likely have various effects on forest communities by acting as nest site competitors or prey, and by accumulating biomass in tree holes. Nest depredation (a consequence of a lack of well-protected nest sites) and food resource limitation seem to be more important than parasites in hampering feral colony survival. The outstanding question is how environmental and intrinsic factors interact in preventing population establishment. Nest boxes with movable frames could be used to better study the environmental drivers of feral colonies’ mortality. Pairs of wild (self-sustaining) and managed populations known to exist outside Europe could provide answers to whether modern apiculture creates honeybee populations maladapted to life in the wild. In Europe, large continuous forests might represent evolutionary refuges for wild honeybees.
Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
The behavior of honeybees and bumblebees relies on a constant sensory integration of abiotic or biotic stimuli. As eusocial insects, a sophisticated intraspecific communication as well as the processing of multisensory cues during foraging is of utter importance. To tackle the arising challenges, both honeybees and bumblebees have evolved a sophisticated olfactory and visual processing system.
In both organisms, olfactory reception starts at the antennae, where olfactory sensilla cover the antennal surface in a sex-specific manner. These sensilla house olfactory receptor neurons (ORN) that express olfactory receptors. ORNs send their axons via four tracts to the antennal lobe (AL), the prime olfactory processing center in the bee brain. Here, ORNs specifically innervate spheroidal structures, so-called glomeruli, in which they form synapses with local interneurons and projection neurons (PN). PNs subsequently project the olfactory information via two distinct tracts, the medial and the lateral antennal-lobe tract, to the mushroom body (MB), the main center of sensory integration and memory formation. In the honeybee calyx, the sensory input region of the MB, PNs synapse on Kenyon cells (KC), the principal neuron type of the MB. Olfactory PNs mainly innervate the lip and basal ring layer of the calyx. In addition, the basal ring receives input from visual PNs, making it the first site of integration of visual and olfactory information. Visual PNs, carrying sensory information from the optic lobes, send their terminals not only to the to the basal ring compartment but also to the collar of the calyx. Receiving olfactory or visual input, KCs send their axons along the MB peduncle and terminate in the main output regions of the MB, the medial and the vertical lobe (VL) in a layer-specific manner. In the MB lobes, KCs synapse onto mushroom body output neurons (MBON). In so far barely understood processes, multimodal information is integrated by the MBONs and then relayed further into the protocerebral lobes, the contralateral brain hemisphere, or the central brain among others.
This dissertation comprises a dichotomous structure that (i) aims to gain more insight into the olfactory processing in bumblebees and (ii) sets out to broaden our understanding of visual processing in honeybee MBONs.
The first manuscript examines the olfactory processing of Bombus terrestris and specifically investigates sex-specific differences. We used behavioral (absolute conditioning) and electrophysiological approaches to elaborate the processing of ecologically relevant odors (components of plant odors and pheromones) at three distinct levels, in the periphery, in the AL and during olfactory conditioning. We found both sexes to form robust memories after absolute conditioning and to generalize towards the carbon chain length of the presented odors. On the contrary, electroantennographic (EAG) activity showed distinct stimulus and sex-specific activity, e.g. reduced activity towards citronellol in drones. Interestingly, extracellular multi-unit recordings in the AL confirmed stimulus and sex-specific differences in olfactory processing, but did not reflect the differences previously found in the EAG. Here, farnesol and 2,3-dihydrofarnesol, components of sex-specific pheromones, show a distinct representation, especially in workers, corroborating the results of a previous study. This explicitly different representation suggests that the peripheral stimulus representation is an imperfect indication for neuronal representation in high-order neuropils and ecological importance of a specific odor.
The second manuscript investigates MBONs in honeybees to gain more insights into visual processing in the VL. Honeybee MBONs can be categorized into visually responsive, olfactory responsive and multimodal. To clarify which visual features are represented at this high-order integration center, we used extracellular multi-unit recordings in combination with visual and olfactory stimulation. We show for the first time that information about brightness and wavelength is preserved in the VL. Furthermore, we defined three specific classes of visual MBONs that distinctly encode the intensity, identity or simply the onset of a stimulus. The identity-subgroup exhibits a specific tuning towards UV light. These results support the view of the MB as the center of multimodal integration that categorizes sensory input and subsequently channels this information into specific MBON populations.
Finally, I discuss differences between the peripheral representations of stimuli and their distinct processing in high-order neuropils. The unique activity of farnesol in manuscript 1 or the representation of UV light in manuscript 2 suggest that the peripheral representation of a stimulus is insufficient as a sole indicator for its neural activity in subsequent neuropils or its putative behavioral importance. In addition, I discuss the influence of hard-wired concepts or plasticity induced changes in the sensory pathways on the processing of such key stimuli in the peripheral reception as well as in high-order centers like the AL or the MB. The MB as the center of multisensory integration has been broadly examined for its olfactory processing capabilities and receives increasing interest about its visual coding properties. To further unravel its role of sensory integration and to include neglected modalities, future studies need to combine additional approaches and gain more insights on the multimodal aspects in both the input and output region.
Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host.
Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression.
A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts.
The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes.
In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection.
After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host.
In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.
The cancer stem cell hypothesis is a cancer development model which elicited great interest in the last decades stating that cancer heterogeneity arises from a stem cell through asymmetrical division. The Cancer Stem Cell subset is described as the only population to be tumorigenic and having the potential to renew. Conventional therapy often fails to eradicate CSC resulting in tumor relapse. Consequently, it is of great inter-est to eliminate this subset of cells to provide the best patient outcome. In the last years several approaches to target CSC were developed, one of them being immunotherapeu-tic targeting with antibodies. Since markers associated with CSC are also expressed on normal stem cells or healthy adjacent tissue in colorectal cancer, dual targeting strate-gies are preferred over targeting only a single antigen. Subsequently, the idea of dual targeting two CSC markers in parallel by a newly developed split T cell-engaging anti-body format termed as Hemibodies emerged. In a preliminary single cell RNA sequenc-ing analysis of colorectal cancer cells CD133, CD24, CD166 and CEA were identified as suitable targets for the combinatorial targeting strategy. Therefore, this study focused on trispecific and trivalent Hemibodies comprising a split binding moiety against CD3 and a binding moiety against either CD133, CD24, CD166 or CEA to overcome the occurrence of resistance and to efficiently eradicate all tumor cells including the CSC compartment. The study showed that the Hemibody combinations CD133xCD24, CD133xCD166 and CD133xCEA are able to eliminate double positive CHO cells with high efficacy while having a high specificity indicated by no killing of single antigen positive cells. A thera-peutic window ranging between one to two log levels could be achieved for all combina-tions mentioned above. The combinations CD133xCD24 and CD133xCD166 further-more proved its efficacy and specificity on established colorectal cancer cell lines. Be-sides the evaluation of specificity and efficacy the already introduced 1st generation of Hemibodies could be improved into a 2nd generation Hemibody format with increased half-life, stability and production yield. In future experiments the applicability of above-mentioned Hemibodies will be proven on patient-derived micro tumors to also include variables like tumor microenvironment and infiltration.
Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc
(2023)
Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis.
Die Fanconi-Anämie (FA) ist eine seltene, heterogene Erbkrankheit. Sie weist ein sehr variables klinisches Erscheinungsbild auf, das sich aus angeborenen Fehlbildungen, hämatologischen Funktionsstörungen, einem erhöhten Risiko für Tumorentwicklung und endokrinen Pathologien zusammensetzt. Die Erkrankung zählt zu den genomischen Instabilitätssyndromen, welche durch eine fehlerhafte DNA-Schadensreparatur gekennzeichnet sind. Bei der FA zeigt sich dies vor allem in einer charakteristischen Hypersensitivität gegenüber DNA-quervernetzenden Substanzen (z. B. Mitomycin C, Cisplatin). Der zelluläre FA-Phänotyp zeichnet sich durch eine erhöhte Chromosomenbrüchigkeit und einen Zellzyklusarrest in der G2-Phase aus. Diese Charakteristika sind bereits spontan vorhanden und werden durch Induktion mit DNA-quervernetzenden Substanzen verstärkt. Der Gendefekt ist dabei in einem der 22 bekannten FA-Gene (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) oder in noch unbekannten FA-Genen zu finden. Die FA-Gendefekte werden mit Ausnahme von FANCR (dominant-negative de novo Mutationen) und FANCB (X-chromosomal) autosomal rezessiv vererbt. Die FA-Genprodukte bilden zusammen mit weiteren Proteinen den FA/BRCA-Signalweg. Das Schlüsselereignis dieses Signalwegs stellt die Monoubiquitinierung von FANCD2 und FANCI (ID2-Komplex) dar. Ausgehend davon lässt sich zwischen upstream- und downstream-gelegenen FA-Proteinen unterscheiden. Letztere sind direkt an der DNA-Schadensreparatur beteiligt. Zu den upstream-gelegenen Proteinen zählt der FA-Kernkomplex, der sich aus bekannten FA-Proteinen und aus FA-assoziierten-Proteinen (FAAPs) zusammensetzt und für die Monoubiquitinierung des ID2-Komplexes verantwortlich ist. Für FAAPs wurden bisher keine pathogenen humanen Mutationen beschrieben. Zu diesen Proteinen gehört auch FAAP100, das mit FANCB und FANCL innerhalb des FA-Kernkomplexes den Subkomplex LBP100 bildet.
Durch die vorliegende Arbeit wurde eine nähere Charakterisierung dieses Proteins erreicht. In einer Amnion-Zelllinie konnte eine homozygote Missense-Mutation identifiziert werden. Der Fetus zeigte einen typischen FA-Phänotyp und auch seine Zellen wiesen charakteristische FA-Merkmale auf. Der zelluläre Phänotyp ließ sich durch FAAP100WT komplementieren, sodass die Pathogenität der Mutation bewiesen war. Unterstützend dazu wurden mithilfe des CRISPR/Cas9-Systems weitere FAAP100-defiziente Zelllinien generiert. Diese zeigten ebenfalls einen typischen FA-Phänotyp, welcher sich durch FAAP100WT komplementieren ließ. Die in vitro-Modelle dienten als Grundlage dafür, die Funktion des FA-Kernkomplexes im Allgemeinen und die des Subkomplexes LBP100 im Besonderen besser zu verstehen. Dabei kann nur durch intaktes FAAP100 das LBP100-Modul gebildet und dieses an die DNA-Schadensstelle transportiert werden. Dort leistet FAAP100 einen essentiellen Beitrag für den FANCD2-Monoubiquitinierungsprozess und somit für die Aktivierung der FA-abhängigen DNA-Schadensreparatur. Um die Funktion von FAAP100 auch in vivo zu untersuchen, wurde ein Faap100-/--Mausmodell generiert, das einen mit anderen FA-Mausmodellen vergleichbaren, relativ schweren FA-Phänotyp aufwies. Aufgrund der Ergebnisse lässt sich FAAP100 als neues FA-Gen klassifizieren. Zudem wurde die Rolle des Subkomplexes LBP100 innerhalb des FA-Kernkomplexes weiter aufgeklärt. Beides trägt zu einem besseren Verständnis des FA/BRCA-Signalweges bei. Ein weiterer Teil der vorliegenden Arbeit beschäftigt sich mit der Charakterisierung von FAAP100138, einer bisher nicht validierten Isoform von FAAP100. Durch dieses Protein konnte der zelluläre FA-Phänotyp von FAAP100-defizienten Zelllinien nicht komplementiert werden, jedoch wurden Hinweise auf einen dominant-negativen Effekt von FAAP100138 auf den FA/BRCA-Signalweg gefunden. Dies könnte zu der Erklärung beitragen, warum und wie der Signalweg, beispielsweise in bestimmtem Gewebearten, herunterreguliert wird. Zudem wäre eine Verwendung in der Krebstherapie denkbar.
Forests are multi-functional system, which have to fulfil different objectives at the same time. The main functions include the production of wood, storage of carbon, the promotion of biological diversity and the provision of recreational space. Yet, global forests are affected by large and intense natural disturbances, like bark beetle infestations. While natural disturbances threaten wood production and are perceived as ‘catastrophe’ diminishing recreational value, biodiversity can benefit from the disturbance-induced changes in forest structures. This trade-off poses a dilemma to managers of bark beetle affected stands, particularly in protected areas designated to both nature conservation and recreation. Forest landscapes need a sustainable management concept aligning these different objectives. In order to support this goal with scientific knowledge, the aim of this work is to analyse ecological and social effects along a gradient of different disturbance severities. In this context, I studied the effects of a disturbance severity gradient on the diversity of different taxonomic groups including vascular plants, mosses, lichens, fungi, arthropods and birds in five national parks in Central Europe. To analyse the recreational value of the landscape I conducted visitor surveys in the same study areas in which the biodiversity surveys were performed. To analyse possible psychological or demographic effects on preferences for certain disturbance intensities, an additional online survey was carried out.
The synaptic cleft is of central importance for synaptic transmission, neuronal plasticity and memory and thus well studied in neurobiology. To target proteins of interest with high specificity and strong signal to noise conventional immunohistochemistry relies on the use of fluorescently labeled antibodies. However, investigations on synaptic receptors remain challenging due to the defined size of the synaptic cleft of ~20 nm between opposing pre- and postsynaptic membranes. At this limited space, antibodies bear unwanted side effects such as crosslinking, accessibility issues and a considerable linkage error between fluorophore and target of ~10 nm. With recent single molecule localization microscopy (SMLM) methods enabling localization precisions of a few nanometers, the demand for labeling approaches with minimal linkage error and reliable recognition of the target molecules rises.
Within the scope of this work, different labeling techniques for super-resolution fluorescence microscopy were utilized allowing site-specific labeling of a single amino acid in synaptic proteins like kainate receptors (KARs), transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory proteins (TARPs), γ-aminobutyric acid type A receptors (GABA-ARs) and neuroligin 2 (NL2). The method exploits the incorporation of unnatural amino acids (uAAs) in the protein of interest using genetic code expansion (GCE) via amber suppression technology and subsequent labeling with tetrazine functionalized fluorophores. Implementing this technique, hard-to-target proteins such as KARs, TARPs and GABA-ARs could be labeled successfully, which could only be imaged insufficiently with conventional labeling approaches. Furthermore, functional studies involving electrophysiological characterization, as well as FRAP and FRET experiments validated that incorporation of uAAs maintains the native character of the targeted proteins. Next, the method was transferred into primary hippocampal neurons and in combination with super-resolution microscopy it was possible to resolve the nanoscale organization of γ2 and γ8 TARPs. Cluster analysis of dSTORM localization data verified synaptic accumulation of γ2, while γ8 was homogenously distributed along the neuron. Additionally, GCE and bioorthogonal labeling allowed visualization of clickable GABA-A receptors located at postsynaptic compartments in dissociated hippocampal neurons. Moreover, saturation experiments and FRET imaging of clickable multimeric receptors revealed successful binding of multiple tetrazine functionalized fluorophores to uAA-modified dimeric GABA-AR α2 subunits in close proximity (~5 nm). Further utilization of tetrazine-dyes via super-resolution microscopy methods such as dSTORM and click-ExM will provide insights to subunit arrangement in receptors in the future.
This work investigated the nanoscale organization of synaptic proteins with minimal linkage error enabling new insights into receptor assembly, trafficking and recycling, as well as protein-protein interactions at synapses. Ultimately, bioorthogonal labeling can help to understand pathologies such as the limbic encephalitis associated with GABA-AR autoantibodies and is already in application for cancer therapies.
Summary
Chapters I & II: General Introduction & General Methods
Agriculture is confronted with a rampant loss of biodiversity potentially eroding ecosystem service potentials and adding up to other stressors like climate change or the consequences of land-use change and intensive management. To counter this ‘biodiversity crisis’, agri-environment schemes (AES) have been introduced as part of ecological intensification efforts. These AES combine special management regimes with the establishment of tailored habitats to create refuges for biodiversity in agricultural landscapes and thus ensure biodiversity mediated ecosystem services such as pest control. However, little is known about how well different AES habitats fulfil this purpose and whether they benefit ecosystem services in adjacent crop fields. Here I investigated how effective different AES habitats are for restoring biodiversity in different agricultural landscapes (Chapter V) and whether they benefit natural pest control in adjacent oilseed rape (Chapter VI) and winter cereal fields (Chapter VII). I recorded biodiversity and pest control potentials using a variety of different methods (Chapters II, V, VI & VII). Moreover, I validated the methodology I used to assess predator assemblages and predation rates (Chapters III & IV).
Chapter III: How to record ground dwelling predators?
Testing methodology is critical as it ensures scientific standards and trustworthy results. Pitfall traps are widely used to record ground dwelling predators, but little is known about how different trap types affect catches. I compared different types of pitfall traps that had been used in previous studies in respect to resulting carabid beetle assemblages. While barrier traps collected more species and deliver more complete species inventories, conventional simple pitfall traps provide reliable results with comparatively little handling effort. Placing several simple pitfall traps in the field can compensate the difference while still saving handling effort.
Chapter IV: How to record predation rates?
A plethora of methods has been proposed and used for recording predation rates, but these have rarely been validated before use. I assessed whether a novel approach to record predation, the use of sentinel prey cards with glued on aphids, delivers realistic results. I compared different sampling efforts and showed that obtained predation rates were similar and could be linked to predator (carabid beetle) densities and body-sizes (a proxy often used for food intake rates). Thus, the method delivers reliable and meaningful predation rates.
Chapter V: Do AES habitats benefit multi-taxa biodiversity?
The main goal of AES is the conservation of biodiversity in agricultural landscapes. I investigated how effectively AES habitats with different temporal continuity fulfil this goal in differently structured landscapes. The different AES habitats investigated had variable effects on local biodiversity. Temporal continuity of AES habitats was the most important predictor with older, more temporally continuous habitats harbouring higher overall biodiversity and different species assemblages in most taxonomic groups than younger AES habitats. Results however varied among taxonomic groups and natural enemies were equally supported by younger habitats. Semi-natural habitats in the surrounding landscape and AES habitat size were of minor importance for local biodiversity and had limited effects. This stresses that newly established AES habitats alone cannot restore farmland biodiversity. Both AES habitats as well as more continuous semi-natural habitats synergistically increase overall biodiversity in agricultural landscapes.
Chapter VI: The effects of AES habitats on predators in adjacent oilseed rape fields
Apart from biodiversity conservation, ensuring ecosystem service delivery in agricultural landscapes is a crucial goal of AES. I therefore investigated the effects of adjacent AES habitats on ground dwelling predator assemblages in oilseed rape fields. I found clear distance decay effects from the field edges into the field centres on both richness and densities of ground dwelling predators. Direct effects of adjacent AES habitats on assemblages in oilseed rape fields however were limited and only visible in functional traits of carabid beetle assemblages. Adjacent AES habitats doubled the proportion of predatory carabid beetles indicating a beneficial role for pest control. My results show that pest control potentials are largest close to the field edges and beneficial effects are comparably short ranged.
Chapter VII: The effects of AES habitats on pest control in adjacent cereal fields
Whether distance functions and potential effects of AES habitats are universal across crops is unknown. Therefore, I assessed distance functions of predators, pests, predation rates and yields after crop rotation in winter cereals using the same study design as in the previous year. Resulting distance functions were not uniform and differed from those found in oilseed rape in the previous year, indicating that the interactions between certain adjacent habitats vary with habitat and crop types. Distance functions of cereal-leaf beetles (important cereal pests) and parasitoid wasps were moreover modulated by semi-natural habitat proportion in the surrounding landscapes. Field edges buffered assemblage changes in carabid beetle assemblages over crop rotation confirming their important function as refuges for natural enemies. My results emphasize the beneficial role of field edges for pest control potentials. These findings back the calls for smaller field sizes and more diverse, more heterogeneously structured agricultural landscapes.
Chapter VIII: General Discussion
Countering biodiversity loss and ensuring ecosystem service provision in agricultural landscapes is intricate and requires strategic planning and restructuring of these landscapes. I showed that agricultural landscapes could benefit maximally from (i) a mixture of AES habitats and semi-natural habitats to support high levels of overall biodiversity and from (ii) smaller continuously managed agricultural areas (i.e. smaller field sizes or the insertion of AES elements within large fields) to maximize natural pest control potentials in crop fields. I propose a mosaic of younger AES habitats and semi-natural habitats to support ecosystem service providers and increase edge density for ecosystem service spillover into adjacent crops. The optimal extent and density of this network as well as the location in which AES and semi-natural habitats interact most beneficially with adjacent crops need further investigation. My results provide a further step towards more sustainable agricultural landscapes that simultaneously allow biodiversity to persist and maintain agricultural production under the framework of ecological intensification.
New insights into the histone variant H2A.Z incorporation pathway in \(Trypanosoma\) \(brucei\)
(2022)
The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general.
One of the pronounced global challenges facing ecologists is how to feed the current growing human population while sustaining biodiversity and ecosystem services. To shed light on this, I investigated the impact of human land use on bee diversity and plant-pollinator interactions in Tanzania Savannah ecosystems. The thesis comprises the following chapters:
Chapter I: General Introduction
This chapter provides the background information including the study objectives and hypotheses. It highlights the ecological importance of bees and the main threats facing bee pollinators with a focus on two land-use practices namely livestock grazing and agriculture. It also highlights the diversity and global distribution of bees. It further introduces the tropical savannah ecosystem, its climate, and vegetation characteristics and explains spectacular megafauna species of the system that form centers of wildlife tourism and inadequacy knowledge on pollinators diversity of the system. Finally, this chapter describes the study methodology including, the description of the study area, study design, and data collection.
Chapter II: Positive effects of low livestock grazing intensity on East African bee assemblages mediated by increases in floral resources
The impact of livestock grazing intensity on bee assemblage has been subjected to research over decades. Moreover, most of these studies have been conducted in temperate Europe and America leaving the huge tropical savannah of East Africa less studied. Using sweep netting and pan traps, a total of 183 species (from 2,691 individuals) representing 55 genera and five families were collected from 24 study sites representing three levels of livestock grazing intensity in savannah ecosystem of northern Tanzania. Results have shown that moderate livestock grazing slightly increased bee species richness. However, high livestock grazing intensity led to a strong decline. Besides, results revealed a unimodal distribution pattern of bee species richness and mean annual temperature. It was also found that the effect of livestock grazing and environmental temperature on bee species richness was mediated by a positive effect of moderate grazing on floral resource richness. The study, therefore, reveals that bee communities of the African savannah zone may benefit from low levels of livestock grazing as this favors the growth of flowering plant species. A high level of livestock grazing intensity will cause significant species losses, an effect that may increase with climatic warming.
Chapter III: Agricultural intensification with seasonal fallow land promotes high bee diversity in Afrotropical drylands
This study investigated the impact of local agriculture intensification on bee diversity in the Afro tropical drylands of northern Tanzania. Using sweep netting and pan traps, a total of 219 species (from 3,428 individuals) representing 58 genera and six families were collected from 24 study sites (distributed from 702 to 1708 m. asl) representing three levels of agriculture intensity spanning an extensive gradient of mean annual temperature. Results showed that bee species richness increased with agricultural intensity and with increasing temperature. However, the effects of agriculture intensity and temperature on bee species richness were mediated by the positive effects of agriculture and temperature on floral resource richness used by bee pollinators. Moreover, results showed that variation of bee body sizes increases with agricultural intensification, “that effect”, however, diminished in environments with higher temperatures. This study reveals that bee assemblages in Afrotropical drylands benefit from agriculture intensification in the way it is currently practiced. Further intensification, including year-round irrigated crop monocultures and extensive use of agrochemicals, is likely to exert a negative impact on bee diversity and pollination services, as reported in temperate regions. Moreover, several bee species were restricted to natural savannah habitats. Therefore, to conserve bee communities in Afro tropical drylands and guarantee pollination services, a mixture of savannah and agriculture, with long periods of fallow land should be maintained.
Chapter IV: Impact of land use intensification and local features on plants and pollinators in Sub-Saharan smallholder farms
For the first time in the region, this study explores the impact of land-use intensification on plants and pollinators in Sub-Saharan smallholder farms. The study complemented field surveys of bees with a modern DNA metabarcoding approach to characterize the foraged plants and thus built networks describing plant-pollinator interactions at the individual insect level. This information was coupled with quantitative traits of landscape composition and floral availability surrounding each farm. The study found that pollinator richness decreased with increasing impervious and agricultural cover in the landscape, whereas the flower density at each farm correlated with pollinator richness. The intensification of agricultural land use and urbanization correlated with a higher foraging niche overlap among pollinators due to the convergence of individuals' flower-visiting strategies. Furthermore, within farms, the higher availability of floral resources drove lower niche overlap among individuals, greater abundance of flower visitors shaped higher generalization at the networks level (H2I), possibly due to increased competition. These mechanistic understandings leading to individuals’ foraging niche overlap and generalism at the network level, could imply stability of interactions and the pollination ecosystem service. The integrative survey proved that plant-pollinator systems are largely affected by land use intensification and by local factors in smallholder farms of Sub-Saharan Africa. Thus, policies promoting nature-based solutions, among which the introduction of more pollinator-friendly practices by smallholder farmers, could be effective in mitigating the intensification of both urban and rural landscapes in this region, as well as in similar Sub-Saharan contexts.
Chapter V: A synopsis of the Bee occurrence data of northern Tanzania
This study represents a synopsis of the bee occurrence data of northern Tanzania obtained from a survey in the Kilimanjaro, Arusha, and Manyara regions. Bees were sampled using two standardized methods, sweep netting and colored pan traps. The study summed up 953 species occurrences of 45 species belonging to 20 genera and four families (Halictidae, Apidae, Megachilidae, and andrenidae) A. This study serves as the baseline information in understanding the diversity and distribution of bees in the northern parts of the country. Understanding the richness and distribution of bees is a critical step in devising robust conservation and monitoring strategies for their populations since limited taxonomic information of the existing and unidentified bee species makes their conservation haphazard.
Chapter VI: General discussion
In general, findings obtained in these studies suggest that livestock grazing and agriculture intensification affects bee assemblages and floral resources used by bee pollinators. Results have shown that moderate livestock grazing intensity may be important in preserving bee diversity. However, high level of livestock grazing intensity may result in a strong decline in bee species richness and abundance. Moreover, findings indicate that agriculture intensification with seasonal fallow lands supports high floral resource richness promoting high bee diversity in Afrotropical drylands. Nonetheless, natural savannahs were found to contain unique bee species. Therefore, agriculture intensification with seasonal fallow should go in hand with conserving remnant savannah in the landscapes to increase bee diversity and ensure pollination services. Likewise, findings suggest that increasing urbanization and agriculture cover at the landscape level reduce plant and pollinator biodiversity with negative impacts on their complex interactions with plants. Conversely, local scale availability of floral resources has shown the positive effects in buffering pollinators decline and mitigating all detrimental effects induced by land-use intensification. Moreover, findings suggest that the impact of human land use (livestock grazing and agriculture) do not act in isolation but synergistically interacts with climatic factors such as mean annual temperature, MAT. The impact of MAT on bee species richness in grazing gradient showed to be more detrimental than in agriculture habitats. This could probably be explained by the remaining vegetation cover following anthropogenic disturbance. Meaning that the remaining vegetation cover in the agricultural gradient probably absorbs the solar radiations hence reducing detrimental effect of mean annual temperature on bee species richness. This one is not the case in grazing gradient since the impact of livestock grazing is severe, leaving the bare land with no vegetation cover. Finally, our findings conclude that understanding the interplay of multiple anthropogenic activities and their interaction with MAT as a consequence of ongoing climate change is necessary for mitigating their potential consequences on bee assemblages and the provision of ecosystem services. Morever, future increases in livestock grazing and agriculture intensification (including year-round crop irrigated monocultures and excessive use of agrochemicals) may lead to undesirable consequences such as species loss and impair provision of pollination services.
Die Fluoreszenzmikroskopie ist eine vielseitig einsetzbare Untersuchungsmethode für biologische Proben, bei der Biomoleküle selektiv mit Fluoreszenzfarbstoffen markiert werden, um sie dann mit sehr gutem Kontrast abzubilden. Dies ist auch mit mehreren verschiedenartigen Zielmolekülen gleichzeitig möglich, wobei üblicherweise verschiedene Farbstoffe eingesetzt werden, die über ihre Spektren unterschieden werden können.
Um die Anzahl gleichzeitig verwendbarer Färbungen zu maximieren, wird in dieser Arbeit zusätzlich zur spektralen Information auch das zeitliche Abklingverhalten der Fluoreszenzfarbstoffe mittels spektral aufgelöster Fluoreszenzlebensdauer-Mikroskopie (spectrally resolved fluorescence lifetime imaging microscopy, sFLIM) vermessen. Dazu wird die Probe in einem Konfokalmikroskop von drei abwechselnd gepulsten Lasern mit Wellenlängen von 485 nm, 532nm und 640nm angeregt. Die Detektion des Fluoreszenzlichtes erfolgt mit einer hohen spektralen Auflösung von 32 Kanälen und gleichzeitig mit sehr hoher zeitlicher Auflösung von einigen Picosekunden. Damit wird zu jedem detektierten Fluoreszenzphoton der Anregungslaser, der spektrale Kanal und die Ankunftszeit registriert. Diese detaillierte multidimensionale Information wird von einem Pattern-Matching-Algorithmus ausgewertet, der das Fluoreszenzsignal mit zuvor erstellten Referenzpattern der einzelnen Farbstoffe vergleicht. Der Algorithmus bestimmt so für jedes Pixel die Beiträge der einzelnen Farbstoffe.
Mit dieser Technik konnten pro Anregungslaser fünf verschiedene Färbungen gleichzeitig dargestellt werden, also theoretisch insgesamt 15 Färbungen. In der Praxis konnten mit allen drei Lasern zusammen insgesamt neun Färbungen abgebildet werden, wobei die Anzahl der Farben vor allem durch die anspruchsvolle Probenvorbereitung limitiert war. In anderen Versuchen konnte die sehr hohe Sensitivität des sFLIM-Systems genutzt werden, um verschiedene Zielmoleküle voneinander zu unterscheiden, obwohl sie alle mit demselben Farbstoff markiert waren. Dies war möglich, weil sich die Fluoreszenzeigenschaften eines Farbstoffmoleküls geringfügig in Abhängigkeit von seiner Umgebung ändern. Weiterhin konnte die sFLIM-Technik mit der hochauflösenden STED-Mikroskopie (STED: stimulated emission depletion) kombiniert werden, um so hochaufgelöste zweifarbige Bilder zu erzeugen, wobei nur ein einziger gemeinsamer STED-Laser benötigt wurde.
Die gleichzeitige Erfassung von mehreren photophysikalischen Messgrößen sowie deren Auswertung durch den Pattern-Matching-Algorithmus ermöglichten somit die Entwicklung von neuen Methoden der Fluoreszenzmikroskopie für Mehrfachfärbungen.
Biodiversity is in rapid decline worldwide. These declines are more pronounced in areas that are currently biodiversity rich, but economically poor – essentially describing many tropical regions in the Global South where landscapes are dominated by smallholder agriculture. Agriculture is an important driver of biodiversity decline, through habitat destruction and unsustainable practices. Ironically, agriculture itself is dependent on a range of ecosystem services, such as pollination and pest control, provided by biodiversity. Biodiversity on fields and the delivery of ecosystem services to crops is often closely tied to the composition of the surrounding landscape – complex landscapes with a higher proportion of (semi-)natural habitats tend to support a high abundances and biodiversity of pollinators and natural enemies that are beneficial to crop production. However, past landscape scale studies have focused primarily on industrialized agricultural landscapes in the Global North, and context dependent differences between regions and agricultural systems are understudied. Smallholder agriculture supports 2 billion people worldwide and contributes to over half the world’s food supply. Yet smallholders, particularly in sub-Saharan Africa, are underrepresented in research investigating the consequences of landscape change and agricultural practices. Where research in smallholder agriculture is conducted, the focus is often on commodity crops, such as cacao, and less on crops that are directly consumed by smallholder households, though the loss of services to these crops could potentially impact the most vulnerable farmers the hardest. Agroecology – a holistic and nature-based approach to agriculture, provides an alternative to unsustainable input-intensive agriculture. Agroecology has been found to benefit smallholders through improved agronomical and food-security outcomes. Co-benefits of agroecological practices with biodiversity and ecosystem services are assumed, but not often empirically tested. In addition, the local and landscape effects on biodiversity and ecosystem services are more commonly studied in isolation, but their potentially interactive effects are so far little explored. Our study region in northern Malawi exemplifies many challenges experienced by smallholder farmers throughout sub-Saharan Africa and more generally in the Global South. Malawi is located in a global biodiversity hotspot, but biodiversity is threatened by rapid habitat loss and a push for input-intensive agriculture by government and other stakeholders. In contrast, agroecology has been effectively promoted and implemented in the study region. We investigated how land-use differences and the agroecological practices affects biodiversity and ecosystem services of multiple taxa in a maize-bean intercropping system (Chapter 2), and pollination of pumpkin (Chapter 3) and pigeon pea (Chapter 4). Additionally, the effects of local and landscape scale shrub- to farmland habitat conversion was investigated on butterfly communities, as well as the potential for agroecology to mitigate these effects (Chapter 5).