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Wild bee species are important pollinators in agricultural landscapes. However, population decline was reported over the last decades and is still ongoing. While agricultural intensification is a major driver of the rapid loss of pollinating species, transition zones between arable fields and forest or grassland patches, i.e., agricultural buffer zones, are frequently mentioned as suitable mitigation measures to support wild bee populations and other pollinator species. Despite the reported general positive effect, it remains unclear which amount of buffer zones is needed to ensure a sustainable and permanent impact for enhancing bee diversity and abundance. To address this question at a pollinator community level, we implemented a process-based, spatially explicit simulation model of functional bee diversity dynamics in an agricultural landscape. More specifically, we introduced a variable amount of agricultural buffer zones (ABZs) at the transition of arable to grassland, or arable to forest patches to analyze the impact on bee functional diversity and functional richness. We focused our study on solitary bees in a typical agricultural area in the Northeast of Germany. Our results showed positive effects with at least 25% of virtually implemented agricultural buffer zones. However, higher amounts of ABZs of at least 75% should be considered to ensure a sufficient increase in Shannon diversity and decrease in quasi-extinction risks. These high amounts of ABZs represent effective conservation measures to safeguard the stability of pollination services provided by solitary bee species. As the model structure can be easily adapted to other mobile species in agricultural landscapes, our community approach offers the chance to compare the effectiveness of conservation measures also for other pollinator communities in future.
Artificial light at night (ALAN) is altering the behaviour of nocturnal animals in a manifold of ways. Nocturnal invertebrates are particularly affected, due to their fatal attraction to ALAN. This selective pressure has the potential to reduce the strength of the flight-to-light response in insects, as shown recently in a moth species. Here we investigated light attraction of ground beetles (Coleoptera: Carabidae).We compared among animals (three genera) from a highly light polluted (HLP) grassland in the centre of Berlin and animals collected at a low-polluted area in a Dark Sky Reserve (DSR), captured using odour bait. In an arena setting tested at night time, HLP beetles (n = 75 across all genera) showed a reduced attraction towards ALAN. Tested during daytime, HLP beetles were less active in an open field test (measured as latency to start moving), compared to DSR (n = 143). However, we did not observe a reduced attraction towards ALAN within the species most common at both sides, Calathus fuscipes (HLP = 37, DSR = 118 individuals) indicating that not all species may be equally affected by ALAN. Reduced attraction to ALAN in urban beetles may either be a result of phenotypic selection in each generation removing HLP individuals that are attracted to light, or an indication for ongoing evolutionary differentiation among city and rural populations in their light response. Reduced attraction to light sources may directly enhance survival and reproductive success of urban individuals. However, decrease in mobility may negatively influence dispersal, reproduction and foraging success, highlighting the selective pressure that light pollution may have on fitness, by shaping and modifying the behaviour of insects.
The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
Extreme habitats often harbor specific communities that differ substantially from non-extreme habitats. In many cases, these communities are characterized by archaea, bacteria and protists, whereas the number of species of metazoa and higher plants is relatively low. In extremely acidic habitats, mostly prokaryotes and protists thrive, and only very few metazoa thrive, for example, rotifers. Since many studies have investigated the physiology and ecology of individual species, there is still a gap in research on direct, trophic interactions among extremophiles. To fill this gap, we experimentally studied the trophic interactions between a predatory protist (Actinophrys sol, Heliozoa) and its prey, the rotifers Elosa woralli and Cephalodella sp., the ciliate Urosomoida sp. and the mixotrophic protist Chlamydomonas acidophila (a green phytoflagellate, Chlorophyta). We found substantial predation pressure on all animal prey. High densities of Chlamydomonas acidophila reduced the predation impact on the rotifers by interfering with the feeding behaviour of A. sol. These trophic relations represent a natural case of intraguild predation, with Chlamydomonas acidophila being the common prey and the rotifers/ciliate and A. sol being the intraguild prey and predator, respectively. We further studied this intraguild predation along a resource gradient using Cephalodella sp. as the intraguild prey. The interactions among the three species led to an increase in relative rotifer abundance with increasing resource (Chlamydomonas) densities. By applying a series of laboratory experiments, we revealed the complexity of trophic interactions within a natural extremophilic community.
Dictyostelium cells undergo a semi-closed mitosis, during which the nuclear envelope (NE) persists; however, free diffusion between the cytoplasm and the nucleus takes place. To permit the formation of the mitotic spindle, the nuclear envelope must be permeabilized in order to allow diffusion of tubulin dimers and spindle assembly factors into the nucleus. In Aspergillus, free diffusion of proteins between the cytoplasm and the nucleus is achieved by a partial disassembly of the nuclear pore complexes (NPCs) prior to spindle assembly. In order to determine whether this is also the case in Dictyostelium, we analysed components of the NPC by immunofluorescence microscopy and live cell imaging and studied their behaviour during interphase and mitosis. We observed that the NPCs are absent from the contact area of the nucleoli and that some nucleoporins also localize to the centrosome and the spindle poles. In addition, we could show that, during mitosis, the central FG protein NUP62, two inner ring components and Gle1 depart from the NPCs, while all other tested NUPs remained at the NE. This leads to the conclusion that indeed a partial disassembly of the NPCs takes place, which contributes to permeabilisation of the NE during semi-closed mitosis.
Malagasy shrew tenrecs (Microgale) have increasingly been used to study speciation genetics over the last years. A previous study recently uncovered gene flow between the Shrew-toothed shrew tenrec (M. soricoides) and sympatric southern population of the Pale shrew tenrec (M. fotsifotsy). This gene flow has been suggested to be accompanied by complete mitochondrial replacement in M. fotsifotsy. To explore the temporal framework of this replacement, we assembled mitogenomes from publicly available sequencing data of ultra-conserved elements. We were able to assemble complete and partial mitogenomes for 19 specimens from five species of shrew tenrecs, which represents a multifold increase in mitogenomic resources available for all tenrecs. Phylogenetic inferences and sequence simulations support the close relationship between the mitochondrial lineages of M. soricoides and the southern population of M. fotsifotsy. Based on the nuclear divergence of northern and southern populations of M. fotsifotsy and the mitochondrial divergence between the latter and M. soricoides, there was a mean time window for replacement of similar to 350,000 years. This timeframe implies that the effective size of the ancestral M. fotsifotsy southern population was less 70,000.
Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.
A temperature-inducible epigenome editing system to knock down histone methylation can be used to study the role of histone H3K4 methylation during heat stress memory in Arabidopsis. <br /> Histone modifications play a crucial role in the integration of environmental signals to mediate gene expression outcomes. However, genetic and pharmacological interference often causes pleiotropic effects, creating the urgent need for methods that allow locus-specific manipulation of histone modifications, preferably in an inducible manner. Here, we report an inducible system for epigenome editing in Arabidopsis (Arabidopsis thaliana) using a heat-inducible dCas9 to target a JUMONJI (JMJ) histone H3 lysine 4 (H3K4) demethylase domain to a locus of interest. As a model locus, we target the ASCORBATE PEROXIDASE2 (APX2) gene that shows transcriptional memory after heat stress (HS), correlating with H3K4 hyper-methylation. We show that dCas9-JMJ is targeted in a HS-dependent manner to APX2 and that the HS-induced overaccumulation of H3K4 trimethylation (H3K4me3) decreases when dCas9-JMJ binds to the locus. This results in reduced HS-mediated transcriptional memory at the APX2 locus. Targeting an enzymatically inactive JMJ protein in an analogous manner affected transcriptional memory less than the active JMJ protein; however, we still observed a decrease in H3K4 methylation levels. Thus, the inducible targeting of dCas9-JMJ to APX2 was effective in reducing H3K4 methylation levels. As the effect was not fully dependent on enzyme activity of the eraser domain, the dCas9-JMJ fusion protein may act in part independently of its demethylase activity. This underlines the need for caution in the design and interpretation of epigenome editing studies. We expect our versatile inducible epigenome editing system to be especially useful for studying temporal dynamics of chromatin modifications.
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.
Uncovering the interplay between nutrient availability and cellulose biosynthesis inhibitor activity
(2022)
All plant cells are surrounded by a dynamic, carbohydrate-rich extracellular matrix known as the cell wall. Nutrient availability affects cell wall composition via uncharacterized regulatory mechanisms, and cellulose deficient mutants develop a hypersensitive root response to growth on high concentrations of nitrate. Since cell walls account for the bulk of plant biomass, it is important to understand how nutrients regulate cell walls. This could provide important knowledge for directing fertilizer treatments and engineering plants with higher nutrient use efficiency. The direct effect of nitrate on cell wall synthesis was investigated through growth assays on varying concentrations of nitrate, measuring cellulose content of roots and shoots, and assessing cellulose synthase activity (CESA) using live cell imaging with spinning disk confocal microscopy. A forward genetic screen was developed to isolate mutants impaired in nutrient-mediated cell wall regulation, revealing that cellulose biosynthesis inhibitor (CBI) activity is modulated by nutrient availability. Various non-CESA mutants were isolated that displayed CBI resistance, with the majority of mutations causing perturbation of mitochondria-localized proteins. To investigate mitochondrial involvement, the CBI mechanism of action was investigated using a reverse genetic screen, a targeted pharmacological screen, and -omics approaches. The results generated suggest that CBI-induced cellulose inhibition is due to off-target effects. This provides the groundwork to investigate uncharacterized processes of CESA regulation and adds valuable knowledge to the understanding of CBI activity, which could be harnessed to develop new and improved herbicides.
Biological invasions may result from multiple introductions, which might compensate for reduced gene pools caused by bottleneck events, but could also dilute adaptive processes. A previous common-garden experiment showed heritable latitudinal clines in fitness-related traits in the invasive goldenrod Solidago canadensis in Central Europe. These latitudinal clines remained stable even in plants chemically treated with zebularine to reduce epigenetic variation. However, despite the heritability of traits investigated, genetic isolation-by-distance was non-significant. Utilizing the same specimens, we applied a molecular analysis of (epi)genetic differentiation with standard and methylation-sensitive (MSAP) AFLPs. We tested whether this variation was spatially structured among populations and whether zebularine had altered epigenetic variation. Additionally, we used genome scans to mine for putative outlier loci susceptible to selection processes in the invaded range. Despite the absence of isolation-by-distance, we found spatial genetic neighborhoods among populations and two AFLP clusters differentiating northern and southern Solidago populations. Genetic and epigenetic diversity were significantly correlated, but not linked to phenotypic variation. Hence, no spatial epigenetic patterns were detected along the latitudinal gradient sampled. Applying genome-scan approaches (BAYESCAN, BAYESCENV, RDA, and LFMM), we found 51 genetic and epigenetic loci putatively responding to selection. One of these genetic loci was significantly more frequent in populations at the northern range. Also, one epigenetic locus was more frequent in populations in the southern range, but this pattern was lost under zebularine treatment. Our results point to some genetic, but not epigenetic adaptation processes along a large-scale latitudinal gradient of S. canadensis in its invasive range.
Phenotypic plasticity can increase individual fitness when environmental conditions change over time. Inducible defences are a striking example, allowing species to react to fluctuating predation pressure by only expressing their costly defended phenotype under high predation risk. Previous theoretical investigations have focused on how this affects predator–prey dynamics, but the impact on competitive outcomes and broader community dynamics has received less attention. Here we use a small food web model, consisting of two competing plastic autotrophic species exploited by a shared consumer, to study how the speed of inducible defences across three trade-off constellations affects autotroph coexistence, biomasses across trophic levels, and temporal variability. Contrary to the intuitive idea that faster adaptation increases autotroph fitness, we found that higher switching rates reduced individual fitness as it consistently provoked more maladaptive switching towards undefended phenotypes under high predation pressure. This had an unexpected positive impact on the consumer, increasing consumer biomass and lowering total autotroph biomass. Additionally, maladaptive switching strongly reduced autotroph coexistence through an emerging source-sink dynamic between defended and undefended phenotypes. The striking impact of maladaptive switching on species and food web dynamics indicates that this mechanism may be of more critical importance than previously recognized.
For starch metabolism to take place correctly, various enzymes and proteins acting on the starch granule surface are crucial. Recently, two non-catalytic starch-binding proteins, pivotal for normal starch turnover in Arabidopsis leaves, namely, EARLY STARVATION 1 (ESV1) and its homolog LIKE EARLY STARVATION 1 (LESV), have been identified. Both share nearly 38% sequence homology. As ESV1 has been found to influence glucan phosphorylation via two starch-related dikinases, alpha-glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD), through modulating the surface glucan structures of the starch granules and thus affecting starch degradation, we assess the impact of its homolog LESV on starch metabolism. Thus, the 65-kDa recombinant protein LESV and the 50-kDa ESV1 were analyzed regarding their influence on the action of GWD and PWD on the surface of the starch granules. We included starches from various sources and additionally assessed the effect of these non-enzymatic proteins on other starch-related enzymes, such as starch synthases (SSI and SSIII), starch phosphorylases (PHS1), isoamylase and beta-amylase. The data obtained indicate that starch phosphorylation, hydrolyses and synthesis were affected by LESV and ESV1. Furthermore, incubation with LESV and ESV1 together exerted an additive effect on starch phosphorylation. In addition, a stable alteration of the glucan structures at the starch granule surface following treatment with LESV and ESV1 was observed. Here, we discuss all the observed changes that point to modifications in the glucan structures at the surface of the native starch granules and present a model to explain the existing processes.
Patterns of space use are often subject to large temporal and individual-level variation, due to seasonality in behaviour and environmental conditions as well as age- or sex-specific needs. Especially in temperate regions, seasonality likely influences space use even in non-migratory birds. In waterfowl of the family Anatidae, however, few studies have analyzed space use of the same individuals across the full annual cycle. We used a resident population of Mandarin Ducks (Aix galericulata) in northeast Germany to study their year-round space use in relation to season, sex, and age. We marked 172 birds with colour rings and surveyed relevant water bodies for re-encounters for several years. As space-use patterns we derived home ranges from minimum convex polygons and the number of water bodies used by individual birds. Our analysis revealed that individuals shifted their space use between seasons, in particular extending their home ranges during the non-breeding season. Between years, in contrast, birds tended to show season-specific site fidelity. Sex differences were apparent during both breeding and non-breeding season, males consistently having larger home ranges and using slightly more water bodies. No difference was found between first-year and adult birds. Our study demonstrates that mark-resighting can provide valuable information about space use in species with suitable behaviour and readily accessible habitat. In such cases, it may be a valid alternative to more expensive GPS-tracking or short-term manual radio telemetry, particularly within citizen-science projects.