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Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.
The sequencing of the human genome in the early 2000s led to an increased interest in cheap and fast sequencing technologies. This interest culminated in the advent of next generation sequencing (NGS). A number of different NGS platforms have arisen since then all promising to do the same thing, i.e. produce large amounts of genetic information for relatively low costs compared to more traditional methods such as Sanger sequencing. The capabilities of NGS meant that researchers were no longer bound to species for which a lot of previous work had already been done (e.g. model organisms and humans) enabling a shift in research towards more novel and diverse species of interest. This capability has greatly benefitted many fields within the biological sciences, one of which being the field of evolutionary biology. Researchers have begun to move away from the study of laboratory model organisms to wild, natural populations and species which has greatly expanded our knowledge of evolution. NGS boasts a number of benefits over more traditional sequencing approaches. The main benefit comes from the capability to generate information for drastically more loci for a fraction of the cost. This is hugely beneficial to the study of wild animals as, even when large numbers of individuals are unobtainable, the amount of data produced still allows for accurate, reliable population and species level results from a small selection of individuals.
The use of NGS to study species for which little to no previous research has been carried out on and the production of novel evolutionary information and reference datasets for the greater scientific community were the focuses of this thesis. Two studies in this thesis focused on producing novel mitochondrial genomes from shotgun sequencing data through iterative mapping, bypassing the need for a close relative to serve as a reference sequence. These mitochondrial genomes were then used to infer species level relationships through phylogenetic analyses. The first of these studies involved reconstructing a complete mitochondrial genome of the bat eared fox (Otocyon megalotis). Phylogenetic analyses of the mitochondrial genome confidently placed the bat eared fox as sister to the clade consisting of the raccoon dog and true foxes within the canidae family. The next study also involved reconstructing a mitochondrial genome but in this case from the extinct Macrauchenia of South America. As this study utilised ancient DNA, it involved a lot of parameter testing, quality controls and strict thresholds to obtain a near complete mitochondrial genome devoid of contamination known to plague ancient DNA studies. Phylogenetic analyses confidently placed Macrauchenia as sister to all living representatives of Perissodactyla with a divergence time of ~66 million years ago. The third and final study of this thesis involved de novo assemblies of both nuclear and mitochondrial genomes from brown and striped hyena and focussed on demographic, genetic diversity and population genomic analyses within the brown hyena. Previous studies of the brown hyena hinted at very low levels of genomic diversity and, perhaps due to this, were unable to find any notable population structure across its range. By incorporating a large number of genetic loci, in the form of complete nuclear genomes, population structure within the brown hyena was uncovered. On top of this, genomic diversity levels were compared to a number of other species. Results showed the brown hyena to have the lowest genomic diversity out of all species included in the study which was perhaps caused by a continuous and ongoing decline in effective population size that started about one million years ago and dramatically accelerated towards the end of the Pleistocene.
The studies within this thesis show the power NGS sequencing has and its utility within evolutionary biology. The most notable capabilities outlined in this thesis involve the study of species for which no reference data is available and in the production of large amounts of data, providing evolutionary answers at the species and population level that data produced using more traditional techniques simply could not.
Background: Inferring regulatory interactions between genes from transcriptomics time-resolved data, yielding reverse engineered gene regulatory networks, is of paramount importance to systems biology and bioinformatics studies. Accurate methods to address this problem can ultimately provide a deeper insight into the complexity, behavior, and functions of the underlying biological systems. However, the large number of interacting genes coupled with short and often noisy time-resolved read-outs of the system renders the reverse engineering a challenging task. Therefore, the development and assessment of methods which are computationally efficient, robust against noise, applicable to short time series data, and preferably capable of reconstructing the directionality of the regulatory interactions remains a pressing research problem with valuable applications.
Results: Here we perform the largest systematic analysis of a set of similarity measures and scoring schemes within the scope of the relevance network approach which are commonly used for gene regulatory network reconstruction from time series data. In addition, we define and analyze several novel measures and schemes which are particularly suitable for short transcriptomics time series. We also compare the considered 21 measures and 6 scoring schemes according to their ability to correctly reconstruct such networks from short time series data by calculating summary statistics based on the corresponding specificity and sensitivity. Our results demonstrate that rank and symbol based measures have the highest performance in inferring regulatory interactions. In addition, the proposed scoring scheme by asymmetric weighting has shown to be valuable in reducing the number of false positive interactions. On the other hand, Granger causality as well as information-theoretic measures, frequently used in inference of regulatory networks, show low performance on the short time series analyzed in this study.
Conclusions: Our study is intended to serve as a guide for choosing a particular combination of similarity measures and scoring schemes suitable for reconstruction of gene regulatory networks from short time series data. We show that further improvement of algorithms for reverse engineering can be obtained if one considers measures that are rooted in the study of symbolic dynamics or ranks, in contrast to the application of common similarity measures which do not consider the temporal character of the employed data. Moreover, we establish that the asymmetric weighting scoring scheme together with symbol based measures (for low noise level) and rank based measures (for high noise level) are the most suitable choices.
Maize is the cereal crop with the highest production worldwide, and its oil is a key energy resource. Improving the quantity and quality of maize oil requires a better understanding of lipid metabolism. To predict the function of maize genes involved in lipid biosynthesis, we assembled transcriptomic and lipidomic data sets from leaves of B73 and the high-oil line By804 in two distinct time-series experiments. The integrative analysis based on high-dimensional regularized regression yielded lipid-transcript associations indirectly validated by Gene Ontology and promoter motif enrichment analyses. The co-localization of lipid-transcript associations using the genetic mapping of lipid traits in leaves and seedlings of a B73 x By804 recombinant inbred line population uncovered 323 genes involved in the metabolism of phospholipids, galactolipids, sulfolipids and glycerolipids. The resulting association network further supported the involvement of 50 gene candidates in modulating levels of representatives from multiple acyl-lipid classes. Therefore, the proposed approach provides high-confidence candidates for experimental testing in maize and model plant species.
Polyhydroxyalkanoates (PHAs) have attracted attention as degradable (co)polyesters which can be produced by microorganisms with variations in the side chain. This structural variation influences not only the thermomechanical properties of the material but also its degradation behavior. Here, we used Langmuir monolayers at the air-water (A-W) interface as suitable models for evaluating the abiotic degradation of two PHAs with different side-chain lengths and crystallinity. By controlling the polymer state (semi crystalline, amorphous), the packing density, the pH, and the degradation mechanism, we could draw several significant conclusions. (i) The maximum degree of crystallinity for a PHA film to be efficiently degraded up to pH = 12.3 is 40%. (ii) PHA made of repeating units with shorter side-chain length are more easily hydrolyzed under alkaline conditions. The efficiency of alkaline hydrolysis decreased by about 65% when the polymer was 40% crystalline. (iii) In PHA films with a relatively high initial crystallinity, abiotic degradation initiated a chemicrystallization phenomenon, detected as an increase in the storage modulus (E'). This could translate into an increase in brittleness and reduction in the material degradability. Finally, we demonstrate the stability of the measurement system for long-term experiments, which allows degradation conditions for polymers that could closely simulate real-time degradation.
Leaf senescence is an active process required for plant survival, and it is flexibly controlled, allowing plant adaptation to environmental conditions. Although senescence is largely an age-dependent process, it can be triggered by environmental signals and stresses. Leaf senescence coordinates the breakdown and turnover of many cellular components, allowing a massive remobilization and recycling of nutrients from senescing tissues to other organs (e.g., young leaves, roots, and seeds), thus enhancing the fitness of the plant. Such metabolic coordination requires a tight regulation of gene expression. One important mechanism for the regulation of gene expression is at the transcriptional level via transcription factors (TFs). The NAC TF family (NAM, ATAF, CUC) includes various members that show elevated expression during senescence, including ORE1 (ANAC092/AtNAC2) among others. ORE1 was first reported in a screen for mutants with delayed senescence (oresara1, 2, 3, and 11). It was named after the Korean word “oresara,” meaning “long-living,” and abbreviated to ORE1, 2, 3, and 11, respectively. Although the pivotal role of ORE1 in controlling leaf senescence has recently been demonstrated, the underlying molecular mechanisms and the pathways it regulates are still poorly understood. To unravel the signaling cascade through which ORE1 exerts its function, we analyzed particular features of regulatory pathways up-stream and down-stream of ORE1. We identified characteristic spatial and temporal expression patterns of ORE1 that are conserved in Arabidopsis thaliana and Nicotiana tabacum and that link ORE1 expression to senescence as well as to salt stress. We proved that ORE1 positively regulates natural and dark-induced senescence. Molecular characterization of the ORE1 promoter in silico and experimentally suggested a role of the 5’UTR in mediating ORE1 expression. ORE1 is a putative substrate of a calcium-dependent protein kinase named CKOR (unpublished data). Promising data revealed a positive regulation of putative ORE1 targets by CKOR, suggesting the phosphorylation of ORE1 as a requirement for its regulation. Additionally, as part of the ORE1 up-stream regulatory pathway, we identified the NAC TF ATAF1 which was able to transactivate the ORE1 promoter in vivo. Expression studies using chemically inducible ORE1 overexpression lines and transactivation assays employing leaf mesophyll cell protoplasts provided information on target genes whose expression was rapidly induced upon ORE1 induction. First, a set of target genes was established and referred to as early responding in the ORE1 regulatory network. The consensus binding site (BS) of ORE1 was characterized. Analysis of some putative targets revealed the presence of ORE1 BSs in their promoters and the in vitro and in vivo binding of ORE1 to their promoters. Among these putative target genes, BIFUNCTIONAL NUCLEASE I (BFN1) and VND-Interacting2 (VNI2) were further characterized. The expression of BFN1 was found to be dependent on the presence of ORE1. Our results provide convincing data which support a role for BFN1 as a direct target of ORE1. Characterization of VNI2 in age-dependent and stress-induced senescence revealed ORE1 as a key up-stream regulator since it can bind and activate VNI2 expression in vivo and in vitro. Furthermore, VNI2 was able to promote or delay senescence depending on the presence of an activation domain located in its C-terminal region. The plasticity of this gene might include alternative splicing (AS) to regulate its function in different organs and at different developmental stages, particularly during senescence. A model is proposed on the molecular mechanism governing the dual role of VNI2 during senescence.
The Annamites mountain range of Southeast Asia which runs along the border of Viet Nam and Laos is an important biodiversity hotspot with high levels of endemism. However, that biodiversity is threatened by unsustainable hunting, and many protected areas across the region have been emptied of their wildlife. To better protect the unique species in the Annamites, it is crucial to have a better understanding of their ecology and distribution. Additionally, basic genetic information is needed to provide conservation stakeholders with essential information to facilitate conservation breeding and counteract the illegal wildlife trade. To date, this baseline information is lacking for many Annamites species.
This thesis aims to assess the effectiveness of using non-invasive collection methods, i.e. camera-trap surveys and leech-derived wildlife host DNA, in order to improve and enhance our understanding of ecology, distribution, and genetic diversity of the Annamites terrestrial mammals.
In chapter 1, we analysed data from a systematic landscape camera-trap survey using single-species occupancy models to assess the ecology and distribution of two little-known Annamite endemics, the Annamite dark muntjac (Muntiacus rooseveltorum / truongsonensis) and Annamite striped rabbit (Nesolagus timminsi), in multiple protected areas across the Annamites. This chapter provided the first in-depth information on their ecology, as well as distribution patterns at large spatial scales. Most notably, we found that the Annamite dark muntjac was predominantly found at higher elevations, while responses to elevation varied among study areas for the Annamite striped rabbit. We estimated occupancy probabilities for both endemics by using their responses to environmental and anthropogenic influences and used this information to make recommendations for targeted conservation actions. We discuss how the approach we used for these two Annamites endemics can be expanded for other little-known and threatened species in other tropical regions.
As is the case with ecology and distribution, very little is known about the genetic diversity of the Annamite striped rabbit and other mammals of the Annamites. This poor understanding is mainly attributed to the lack of a comprehensive DNA sample collection that covers the species’ entire distribution range, which is believed to be a consequence of the low density of mammals or the remoteness of species’ habitat. In order to overcome the difficulties when trying to collect DNA samples from elusive mammals, we applied invertebrate-derived DNA (iDNA) sampling via hematophagous leeches to indirectly obtain genetic materials of their terrestrial host mammals.
In chapter 2, leech-derived DNA was used to study the genetic diversity of the Annamite striped rabbit population. By analysing the DNA extracted from leech samples collected at multiple study areas of the central Annamites, we found a genetic variation with five haplotypes among nine obtained sequences. Despite this diversity, we found no clear phylogeographic pattern among the lagomorph’s populations in central Annamites. The findings have direct conservation implications for the species, as local stakeholders are currently establishing a conservation rescue and breeding facility for Annamite endemic species. Thus our results suggested that Annamite striped rabbits from multiple protected areas in central Annamites can be used as founders for the breeding program.
In chapter 3, the genetic material of six mammals, which are frequently found in Indochina's illegal wildlife trade, was extracted from leeches collected at six study sites across the Anamites. Species-specific genetic markers were used to obtain DNA fragments that were analysed together with Genbank reference sequences from other parts of the species’ distribution range. Our results showed that invertebrate-derived DNA can be used to fill the sampling gaps and provide genetic reference data that is needed for conservation breeding programmes or to counteract the illegal wildlife trade.
Overal, this dissertation provides the first insights in the ecology, distribution, and genetics of rare and threatened species of the Annamites by utilising camera traps and leech-derived DNA as two non-invasive collection methods. This information is essential for improving conservation efforts of local stakeholders and managers, especially for the Annamite endemics. Results in this dissertation also show the effectiveness of both non-invasive methods for studying terrestrial mammals at a landscape level. By expanding the application of these methods to other protected areas across the Annamites, we will further our understanding of ecology, distribution, and genetics of Annamite endemics. With such landscape-scale surveys, we are able to provide stakeholders with an overview of the current status of wildlife in the Annamites which supports efforts to protect these secretive species from illegal hunting and thus their extinction.
Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the “hmmer” program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes.
Water is essential to life and thus, an essential resource. However, freshwater resources are limited and their maintenance is crucial. Pollution with chemicals and pathogens through urbanization and a growing population impair the quality of freshwater. Furthermore, water can serve as vector for the transmission of pathogens resulting in water-borne illness.
The Interdisciplinary Research Group III – "Water" of the Leibniz alliance project INFECTIONS‘21 investigated water as a hub for pathogens focusing on Clostridioides difficile and avian influenza A viruses that may be shed into the water. Another aim of this study was to characterize the bacterial communities in a wastewater treatment plant (WWTP) of the capital Berlin, Germany to further assess potential health risks associated with wastewater management practices.
Bacterial communities of WWTP inflow and effluent differed significantly. The proportion of fecal/enteric bacteria was relatively low and OTUs related to potential enteric pathogens were largely removed from inflow to effluent. However, a health risk might exist as an increased relative abundance of potential pathogenic Legionella spp. such as L. lytica was observed. Three Clostridioides difficile isolates from wastewater inflow and an urban bathing lake in Berlin (‗Weisser See‘) were obtained and sequenced. The two isolates from the wastewater did not carry toxin genes, whereas the isolate from the lake was positive for the toxin genes. All three isolates were closely related to human strains. This indicates a potential, but rather sporadic health risk. Avian influenza A viruses were detected in 38.8% of sediment samples by PCR, but virus isolation failed. An experiment with inoculated freshwater and sediment samples showed that virus isolation from sediment requires relatively high virus concentrations and worked much better in Madin-Darby Canine Kidney (MDCK) cell cultures than in embryonated chicken eggs, but low titre of influenza contamination in freshwater samples was sufficient to recover virus.
In conclusion, this work revealed potential health risks coming from bacterial groups with pathogenic potential such as Legionella spp. whose relative abundance is higher in the released effluent than in the inflow of the investigated WWTP. It further indicates that water bodies such as wastewater and lake sediments can serve as reservoir and vector, even for non-typical water-borne or water-transmitted pathogens such as C. difficile.
Urbanization promotes specific bacteria in freshwater microbiomes including potential pathogens
(2022)
Freshwater ecosystems are characterized by complex and highly dynamic microbial communities that are strongly structured by their local environment and biota. Accelerating urbanization and growing city populations detrimentally alter freshwater environments. To determine differences in freshwater microbial communities associated with urban-ization, full-length 16S rRNA gene PacBio sequencing was performed in a case study from surface waters and sedi-ments from a wastewater treatment plant, urban and rural lakes in the Berlin-Brandenburg region, Northeast Germany. Water samples exhibited highly habitat specific bacterial communities with multiple genera showing clear urban signatures. We identified potentially harmful bacterial groups associated with environmental parameters specific to urban habitats such as Alistipes, Escherichia/Shigella, Rickettsia and Streptococcus. We demonstrate that urban-ization alters natural microbial communities in lakes and, via simultaneous warming and eutrophication and creates favourable conditions that promote specific bacterial genera including potential pathogens. Our findings are evidence to suggest an increased potential for long-term health risk in urbanized waterbodies, at a time of rapidly expanding global urbanization. The results highlight the urgency for undertaking mitigation measures such as targeted lake restoration projects and sustainable water management efforts.
Using ESTs for phylogenomics
(2008)
Background
While full genome sequences are still only available for a handful of taxa, large collections of partial gene sequences are available for many more. The alignment of partial gene sequences results in a multiple sequence alignment containing large gaps that are arranged in a staggered pattern. The consequences of this pattern of missing data on the accuracy of phylogenetic analysis are not well understood. We conducted a simulation study to determine the accuracy of phylogenetic trees obtained from gappy alignments using three commonly used phylogenetic reconstruction methods (Neighbor Joining, Maximum Parsimony, and Maximum Likelihood) and studied ways to improve the accuracy of trees obtained from such datasets.
Results
We found that the pattern of gappiness in multiple sequence alignments derived from partial gene sequences substantially compromised phylogenetic accuracy even in the absence of alignment error. The decline in accuracy was beyond what would be expected based on the amount of missing data. The decline was particularly dramatic for Neighbor Joining and Maximum Parsimony, where the majority of gappy alignments contained 25% to 40% incorrect quartets. To improve the accuracy of the trees obtained from a gappy multiple sequence alignment, we examined two approaches. In the first approach, alignment masking, potentially problematic columns and input sequences are excluded from from the dataset. Even in the absence of alignment error, masking improved phylogenetic accuracy up to 100-fold. However, masking retained, on average, only 83% of the input sequences. In the second approach, alignment subdivision, the missing data is statistically modelled in order to retain as many sequences as possible in the phylogenetic analysis. Subdivision resulted in more modest improvements to alignment accuracy, but succeeded in including almost all of the input sequences.
Conclusion
These results demonstrate that partial gene sequences and gappy multiple sequence alignments can pose a major problem for phylogenetic analysis. The concern will be greatest for high-throughput phylogenomic analyses, in which Neighbor Joining is often the preferred method due to its computational efficiency. Both approaches can be used to increase the accuracy of phylogenetic inference from a gappy alignment. The choice between the two approaches will depend upon how robust the application is to the loss of sequences from the input set, with alignment masking generally giving a much greater improvement in accuracy but at the cost of discarding a larger number of the input sequences.
Using individual-based modeling to understand grassland diversity and resilience in the Anthropocene
(2020)
The world’s grassland systems are increasingly threatened by anthropogenic change. Susceptible to a variety of different stressors, from land-use intensification to climate change, understanding the mechanisms driving the maintenance of these systems’ biodiversity and stability, and how these mechanisms may shift under human-mediated disturbance, is thus critical for successfully navigating the next century. Within this dissertation, I use an individual-based and spatially-explicit model of grassland community assembly (IBC-grass) to examine several processes, thought key to understanding their biodiversity and stability and how it changes under stress. In the first chapter of my thesis, I examine the conditions under which intraspecific trait variation influences the diversity of simulated grassland communities. In the second and third chapters of my thesis, I shift focus towards understanding how belowground herbivores influence the stability of these grassland systems to either a disturbance that results in increased, stochastic, plant mortality, or eutrophication.
Intraspecific trait variation (ITV), or variation in trait values between individuals of the same species, is fundamental to the structure of ecological communities. However, because it has historically been difficult to incorporate into theoretical and statistical models, it has remained largely overlooked in community-level analyses. This reality is quickly shifting, however, as a consensus of research suggests that it may compose a sizeable proportion of the total variation within an ecological community and that it may play a critical role in determining if species coexist. Despite this increasing awareness that ITV matters, there is little consensus of the magnitude and direction of its influence. Therefore, to better understand how ITV changes the assembly of grassland communities, in the first chapter of my thesis, I incorporate it into an established, individual-based grassland community model, simulating both pairwise invasion experiments as well as the assembly of communities with varying initial diversities. By varying the amount of ITV in these species’ functional traits, I examine the magnitude and direction of ITV’s influence on pairwise invasibility and community coexistence. During pairwise invasion, ITV enables the weakest species to more frequently invade the competitively superior species, however, this influence does not generally scale to the community level. Indeed, unless the community has low alpha- and beta- diversity, there will be little effect of ITV in bolstering diversity. In these situations, since the trait axis is sparsely filled, the competitively inferior may suffer less competition and therefore ITV may buffer the persistence and abundance of these species for some time.
In the second and third chapters of my thesis, I model how one of the most ubiquitous trophic interactions within grasslands, herbivory belowground, influences their diversity and stability. Until recently, the fundamental difficulty in studying a process within the soil has left belowground herbivory “out of sight, out of mind.” This dilemma presents an opportunity for simulation models to explore how this understudied process may alter community dynamics. In the second chapter of my thesis, I implement belowground herbivory – represented by the weekly removal of plant biomass – into IBC-grass. Then, by introducing a pulse disturbance, modelled as the stochastic mortality of some percentage of the plant community, I observe how the presence of belowground herbivores influences the resistance and recovery of Shannon diversity in these communities. I find that high resource, low diversity, communities are significantly more destabilized by the presence of belowground herbivores after disturbance. Depending on the timing of the disturbance and whether the grassland’s seed bank persists for more than one season, the impact of the disturbance – and subsequently the influence of the herbivores – can be greatly reduced. However, because human-mediated eutrophication increases the amount of resources in the soil, thus pressuring grassland systems, our results suggest that the influence of these herbivores may become more important over time.
In the third chapter of my thesis, I delve further into understanding the mechanistic underpinnings of belowground herbivores on the diversity of grasslands by replicating an empirical mesocosm experiment that crosses the presence of herbivores above- and below-ground with eutrophication. I show that while aboveground herbivory, as predicted by theory and frequently observed in experiments, mitigates the impact of eutrophication on species diversity, belowground herbivores counterintuitively reduce biodiversity. Indeed, this influence positively interacts with the eutrophication process, amplifying its negative impact on diversity. I discovered the mechanism underlying this surprising pattern to be that, as the herbivores consume roots, they increase the proportion of root resources to root biomass. Because root competition is often symmetric, herbivory fails to mitigate any asymmetries in the plants’ competitive dynamics. However, since the remaining roots have more abundant access to resources, the plants’ competition shifts aboveground, towards asymmetric competition for light. This leads the community towards a low-diversity state, composed of mostly high-performance, large plant species. We further argue that this pattern will emerge unless the plants’ root competition is asymmetric, in which case, like its counterpart aboveground, belowground herbivory may buffer diversity by reducing this asymmetry between the competitively superior and inferior plants.
I conclude my dissertation by discussing the implications of my research on the state of the art in intraspecific trait variation and belowground herbivory, with emphasis on the necessity of more diverse theory development in the study of these fundamental interactions. My results suggest that the influence of these processes on the biodiversity and stability of grassland systems is underappreciated and multidimensional, and must be thoroughly explored if researchers wish to predict how the world’s grasslands will respond to anthropogenic change. Further, should researchers myopically focus on understanding central ecological interactions through only mathematically tractable analyses, they may miss entire suites of potential coexistence mechanisms that can increase the coviability of species, potentially leading to coexistence over ecologically-significant timespans. Individual-based modelling, therefore, with its focus on individual interactions, will prove a critical tool in the coming decades for understanding how local interactions scale to larger contexts, and how these interactions shape ecological communities and further predicting how these systems will change under human-mediated stress.
The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.
Vacuole integrity maintained by DUF300 proteins is required for brassinosteroid signaling regulation
(2018)
Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.
In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations.
Puumala virus (PUUV) causes many human infections in large parts of Europe and can lead to mild to moderate disease. The bank vole (Myodes glareolus) is the only reservoir of PUUV in Central Europe. A commercial PUUV rapid field test for rodents was validated for bank-vole blood samples collected in two PUUV-endemic regions in Germany (North Rhine-Westphalia and Baden-Wurttemberg). A comparison of the results of the rapid field test and standard ELISAs indicated a test efficacy of 93-95%, largely independent of the origin of the antigens used in the ELISA. In ELISAs, reactivity for the German PUUV strain was higher compared to the Swedish strain but not compared to the Finnish strain, which was used for the rapid field test. In conclusion, the use of the rapid field test can facilitate short-term estimation of PUUV seroprevalence in bank-vole populations in Germany and can aid in assessing human PUUV infection risk.
Plastic pollution is an increasing environmental problem, but a comprehensive understanding of its effect in the environment is still missing. The wide variety of size, shape, and polymer composition of plastics impedes an adequate risk assessment. We investigated the effect of differently sized polystyrene beads (1-, 3-, 6-µm; PS) and polyamide fragments (5–25 µm, PA) and non-plastics items such as silica beads (3-µm, SiO2) on the population growth, reproduction (egg ratio), and survival of two common aquatic micro invertebrates: the rotifer species Brachionus calyciflorus and Brachionus fernandoi. The MPs were combined with food quantity, limiting and saturating food concentration, and with food of different quality. We found variable fitness responses with a significant effect of 3-µm PS on the population growth rate in both rotifer species with respect to food quantity. An interaction between the food quality and the MPs treatments was found in the reproduction of B. calyciflorus. PA and SiO2 beads had no effect on fitness response. This study provides further evidence of the indirect effect of MPs in planktonic rotifers and the importance of testing different environmental conditions that could influence the effect of MPs.
Plastic pollution is an increasing environmental problem, but a comprehensive understanding of its effect in the environment is still missing. The wide variety of size, shape, and polymer composition of plastics impedes an adequate risk assessment. We investigated the effect of differently sized polystyrene beads (1-, 3-, 6-µm; PS) and polyamide fragments (5–25 µm, PA) and non-plastics items such as silica beads (3-µm, SiO2) on the population growth, reproduction (egg ratio), and survival of two common aquatic micro invertebrates: the rotifer species Brachionus calyciflorus and Brachionus fernandoi. The MPs were combined with food quantity, limiting and saturating food concentration, and with food of different quality. We found variable fitness responses with a significant effect of 3-µm PS on the population growth rate in both rotifer species with respect to food quantity. An interaction between the food quality and the MPs treatments was found in the reproduction of B. calyciflorus. PA and SiO2 beads had no effect on fitness response. This study provides further evidence of the indirect effect of MPs in planktonic rotifers and the importance of testing different environmental conditions that could influence the effect of MPs.