Institut für Biologie

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Taeniidae in Namibian wildlife with emphasis on lion, cheetah, and African wild dog
(2024) Aschenborn, Ortwin; Mackenstedt, Ute
An opportunic survey for Echinococcus spp. in wild mammals was conducted in seven distinct stuy areas throughout Namibia, representing all major ecosystems, between 2012 and 2021. In total, 184 individually attributable faeces and 40 intestines were collected from eight species of carnivores, and 300 carcasses or organs of thirteen species of ungulates were examined for Echinococcus cysts. Nested PCR and sequencing of the mitochondrial nad1 gene led to the identification of five species of the Echinococcus granulosus sensu lato complex. Echinococcus canadensis G6/7 was found throughout Namibia at low frequency in lions, cheetahs, African wild dogs, black-backed jackals and oryx antelopes. Echinococcus equinus was present only in northern Namibia, locally at high frequency in lions, black-backed jackals and plains zebras. Echinococcus felidis was found only in one small area in the north-east of Namibia, but with high frequency in lions and warthogs. Echinococcus granulosus sensu stricto was identified only in two African wild dogs in the north-east of Namibia, and Echinococcus ortleppi occurred in central and southern Namibia in black-backed jackals and oryx antelopes. The development of fertile cysts indicated active intermediate host roles of oryx antelopes for E. canadensis and E. ortleppi, of warthogs for E. felidis, and of plains zebras for E. equinus. Our data support earlier hypotheses of exclusive or predominant wildlife life-cycles for E. felidis involving lions and warthogs, and – in Namibia – for E. equinus involving lions and/or black-backed jackals and plains zebras. Our data further support an interlink of wild and domestic transmission for E. ortleppi. A possible involvement of livestock and domestic dogs in transmission of E. canadensis G6/7 and E. granulosus s.s., the two parasite species with highest zoonotic potential, is uncertain for Namibia and needs further investigation. The present study was conducted in the isolated desert town of Oranjemund in the far south of Namibia. It is an extremely arid region where no livestock husbandry is practiced and only animals adapted to the desert can be found. However, in and around the city, artifi cial irrigation maintains lush green patches of grass that attract wild animals, in particular oryx antelopes (Oryx gazella). In 2015 four oryx antelopes were euthanised due to poor conditions and a post-mortem examination was conducted. Two were found positive for cystic echinococcosis and 16 cysts were collected for molecular analyses. In addition, faecal samples from black-backed jackals (n=5) and domestic dogs (n=9), which were regularly observed to feed on oryx carcasses, were collected and taeniid eggs isolated. Parasite species identifi cation of the cysts and eggs was done by amplifying and se- quencing the mitochondrial nad1 gene. Both oryx antelopes were found infected with E. ortleppi and one co-infected with E. canadensis G6/7. Both Echinococcus species were able to develop fertile cysts in oryx, making oryx antelopes competent hosts for these parasites. Therefore, the analysis of faecal samples was of high interest and although the numbers were quite small, taeniid eggs were found in three out of fi ve faecal samples of jackals and in all nine dog samples. However, species determination was only successful with two jackal and one dog sample. All three were positive for E. canadensis G6/7. The absence of E. ortleppi may be due to the low number of faecal samples examined. In our small study, we discovered a rather unique lifecycle of Echinococcus spp. between jackals and domestic dogs as defi nitive hosts and oryx antelopes as intermediate hosts. Here, the presence of E. canadensis G6/7 is of particular concern, as it is the second most important causative agent of CE in humans.
The emergence and dynamics of tick-borne Encephalitis Virus in a new endemic region in Southern Germany
(2022) Lang, Daniel; Chitimia-Dobler, Lidia; Bestehorn-Willmann, Malena; Lindau, Alexander; Drehmann, Marco; Stroppel, Gabriele; Hengge, Helga; Mackenstedt, Ute; Kaier, Klaus; Dobler, Gerhard; Borde, Johannes
Tick-borne encephalitis (TBE) is the most important viral tick-borne infection in Europe and Asia. It is emerging in new areas. The mechanisms of emergence are fairly unknown or speculative. In the Ravensburg district in southern Germany, TBE emerged, mainly over the last five years. Here, we analyzed the underlying epidemiology in humans. The resulting identified natural foci of the causal TBE virus (TBEV) were genetically characterized. We sampled 13 potential infection sites at these foci and detected TBEV in ticks (Ixodes ricinus) at eight sites. Phylogenetic analysis spurred the introduction of at least four distinct TBEV lineages of the European subtype into the Ravensburg district over the last few years. In two instances, a continuous spread of these virus strains over up to 10 km was observed.
Central carbon metabolism, sodium-motive electron ransfer, and ammonium formation by the vaginal pathogen Prevotella bivia
(2021) Schleicher, Lena; Herdan, Sebastian; Fritz, Günter; Trautmann, Andrej; Seifert, Jana; Steuber, Julia
Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.
Rickettsia spp. in ticks of South Luangwa valley, Eastern Province, Zambia
(2023) Phiri, Bruno S. J.; Kattner, Simone; Chitimia-Dobler, Lidia; Woelfel, Silke; Albanus, Celina; Dobler, Gerhard; Küpper, Thomas
Ticks are important vectors for Rickettsia spp. belonging to the Spotted Fever Group responsible for causing Rickettsiosis worldwide. Rickettsioses pose an underestimated health risk to tourists and local inhabitants. There is evidence of the presence of Rickettsia spp. in Zambia, however there is limited data. A total of 1465 ticks were collected in 20 different locations from dogs and cattle including one cat. Ticks were identified by morphological features or by sequencing of the 16S mitochondrial rRNA gene. Individual ticks were further tested for rickettsiae using a pan-Rickettsia real-time-PCR. Rickettsia species in PCR-positive ticks were identified by sequencing the 23S-5S intergenic spacer region or partial ompA gene, respectively. Seven tick species belonging to three different tick genera were found, namely: Amblyomma variegatum, Rhipicephalus appendiculatus, Rhipicephalus (Boophilus) microplus, Rhipicephalus simus, Rhipicephalus sanguineus, Rhipicephalus zambesiensis and Haemaphysalis elliptica. Out of the 1465 ticks collected, 67 (4.6%) tested positive in the pan-Rickettsia PCR. This study provides detailed data about the presence of Rickettsia species in South Luangwa Valley, Eastern Province, Zambia for the first time. High prevalence of Rickettsia africae in Amblyomma variegatum was found, which indicates the potential risk of infection in the investigated area. Furthermore, to our best knowledge, this is the first time Rickettsia massiliae, a human pathogen causing spotted fever, has been detected in Zambia.
Serological protection rates against TBEV infection in blood donors from a highly endemic region in Southern Germany
(2023) Dobler, Gerhard; Euringer, Kathrin; Kaier, Klaus; Borde, Johannes P.
Background: Tick-borne encephalitis (TBE) is the most significant tick-borne disease in Europe and Asia, with more than 10,000 cases per year worldwide. A surge of reported TBE cases can be observed despite the availability of highly efficient vaccines. There is little known about the serological immune protection rate of the population in Germany. The seroprotection rate is defined as the presence of neutralizing antibodies. In contrast, the vaccination rate, as defined by public health agencies, may differ from the true protection rate in a population. Materials and Methods: 2220 blood samples from inhabitants of the county Ortenaukreis in the Federal State of Baden-Württemberg in Germany were included in the study. These were tested for anti-TBEV IgG antibodies by an anti-TBEV-IgG-ELISA. Subsequently, all TBEV-IgG positive samples were confirmed for neutralizing antibodies in the micro serum neutralization assay. Results: From the overall 2220 samples, 2104 were included in the comparison because of the selection of specific age groups (ages 20–69). In our sample size, we found an average serological protection rate (presence of neutralizing antibodies) of 57% (518/908) for the female blood donors and of 52% (632/1196) for the male blood donors. Discussion: In this study, we present new findings on a highly endemic region in southern Germany. Additionally, we present current data regarding the serological TBEV protection rates in the Ortenaukreis in southern Germany and compare these with a dataset published by the RKI, which is based on vaccination reports of the primary care providers and health care insurers, and with a self-reporting study conducted by a vaccine manufacturer. Our results significantly exceed the official numbers of average active vaccination status by 23.2% for females and by 21% for males. This might indicate an even longer persistence of TBE-vaccination-induced antibody titers than previously assumed.
Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility
(2023) Waegneer, Evelien; Rombauts, Stephane; Baert, Joost; Dauchot, Nicolas; De Keyser, Annick; Eeckhaut, Tom; Haegeman, Annelies; Liu, Chang; Maudoux, Olivier; Notté, Christine; Staelens, Ariane; van der Veken, Jeroen; van Laere, Katrijn; Ruttink, Tom
Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.
DNA barcoding resolves quantitative multi‐trophic interaction networks and reveals pest species in trap nests
(2023) Fornoff, Felix; Halla, Wenzel; Geiger, Sarah; Klein, Alexandra‐Maria; Sann, Manuela
Insects, as one of the most species‐rich taxa with enormous taxonomic, behavioural and functional diversity, are in decline. Bees and wasps are especially crucial for ecosystems as pollinators or to control populations of other insects. To understand population drivers, comprehensive knowledge about top‐down and bottom‐up interactions, including all interaction partners, is needed. Nests of trap‐nesting bees and wasps include multi‐trophic interactions between bees, wasps, their food resources and natural enemies, simultaneously, however, up to today, all trophic interactions are not yet included in trap nest research because of challenges to identify the food used by nesting bees and wasps. Here, we reconstructed quantitative three‐ and four‐trophic interaction networks of species in three apoid wasp families using DNA barcoding. The obtained tripartite and quadripartite networks encompassed natural enemy‐wasp‐spider and natural enemy‐wasp‐herbivore‐plant interactions. Moreover, we identified so far undescribed Hymenoptera‐prey interactions, including prey species known as agricultural and forest pests. More extensive research on bee and wasp multitrophic interaction networks will provide valuable insights to better understand responses to environmental and biodiversity change, to investigate ecological theory and to reveal so far unknown feeding links.
The Bacillus phage SPβ : a model system to study the lysis-lysogeny regulatory network and antiphage defense systems
(2024) Kohm, Katharina; Commichau, Fabian
Although bacteriophages are considered the most abundant biological entities on our planet, they are less well-studied compared to their host. Being intracellular parasites, phages rely on the metabolic processes of their bacterial hosts for their replication. Phages that use the host exclusively to produce virions are called virulent phages and the reproduction cycle is called the lytic cycle. The lytic cycle is accompanied by lysis and, thus, the killing of the host cell. Temperate phages can choose between the virulent or lysogenic lifecycle. Lysogeny or the lysogenic cycle is a type of viral reproduction in which no virus particles are produced, instead, the genetic material of the phage is replicated and then passed on to the daughter cells. The viral genome can be present as part of the bacterial chromosome or as a circular or linear plasmid molecule and is referred to as a prophage. Since temperate phages can influence the mutual interactions with other bacteria, growth, metabolic pathways or pathogenicity of their host, it is important to understand how temperate phages control their lysogenic life cycle and which genes are involved. Repression usually occurs through the interaction between a repressor and specific binding sites, which are mostly located in the promoter regions of the lytic genes. SPβ is a temperate phage of the model bacterium Bacillus subtilis. In contrast to its host, many aspects of the life cycle of SPβ have been little studied and many genes have not been assigned a function. Not only are SPβ-like phages widespread within the genus Bacillus and of greater importance to their hosts than previously thought, but they also exhibit a novel lysogeny management system. With regard to the control and regulation of the lysis-lysogeny network, it is already partially known which gene products are involved in the decision, establishment and resolvement of lysogeny. The maintenance and resolvement of lysogeny of SPβ was investigated in more detail in this thesis. To gain more insight into the regulation and control of lysogeny, the SPβ c2 mutant was characterized in this work. This mutant is unable to maintain its lysogenic state when exposed to heat, suggesting the alteration of a key regulatory element. This work demonstrated that the SPβ c2 phenotype is due to a single nucleotide exchange in the mrpR (yopR) gene that renders the encoded MrpRG136E protein temperature-sensitive. Furthermore, it was shown that this protein acts as a repressor of lytic gene expression. This occurs through the binding of the repressor to several conserved elements in the genome of the SPβ prophage. Further biochemical analysis revealed that the G136E exchange makes MrpR less stable and reduces its affinity for DNA binding. Structural characterization of MrpR revealed that the protein is a DNA-binding protein with a similar protein fold to tyrosine recombinases. However, the repressor function is independent of functional recombinase activity. In addition, a mutagenesis approach was used to identify the region within the protein that is essential for the function of the repressor. This work also identified further players in the lysogeny management system, with the YosL protein being crucial for the induction of the lytic cycle. However, YosL cannot activate the lytic cycle of SPβ alone. In addition, the core genome of SPβ-like phages was defined and new integration loci were identified in this work. Apart from a better understanding of lysis-lysogeny regulation and phagehost relationships, the characterization of the SPβ c2 mutant also led to the identification of a previously unknown phage defense system. The defense system is encoded on a plasmid and leads to a decrease in phage titer and a change in plaque morphology. It could be shown that the spbB locus, which ensures the segregation stability of the plasmid and codes for two open reading frames, is also responsible for the resistance to SPβ c2 and related phages. Further studies have shown that the spbB gene and the downstream region, which presumably encodes an RNA element and a terminator, play a crucial role in mediating resistance. The second open reading frame of the spbB locus is irrelevant for the mediation of phage resistance. Overall, this work contributes to a better understanding of the phage-host relationship.
A shift towards succinate‐producing Prevotella in the ruminal microbiome challenged with monensin
(2022) Trautmann, Andrej; Schleicher, Lena; Koch, Ariane; Günther, Johannes; Steuber, Julia; Seifert, Jana
The time‐resolved impact of monensin on the active rumen microbiome was studied in a rumen‐simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched‐chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane‐bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.
Comparison of five serological methods for the detection of West Nile Virus antibodies
(2024) Girl, Philipp; Euringer, Kathrin; Coroian, Mircea; Mihalca, Andrei Daniel; Borde, Johannes P.; Dobler, Gerhard
The West Nile Virus (WNV), a member of the family Flaviviridae, is an emerging mosquito-borne flavivirus causing potentially severe infections in humans and animals involving the central nervous system (CNS). Due to its emerging tendency, WNV now occurs in many areas where other flaviviruses are co-occurring. Cross-reactive antibodies with flavivirus infections or vaccination (e.g., tick-borne encephalitis virus (TBEV), Usutu virus (USUV), yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV)) therefore remain a major challenge in diagnosing flavivirus infections. Virus neutralization tests are considered as reference tests for the detection of specific flavivirus antibodies, but are elaborate, time-consuming and need biosafety level 3 facilities. A simple and straightforward assay for the differentiation and detection of specific WNV IgG antibodies for the routine laboratory is urgently needed. In this study, we compared two commercially available enzyme-linked immunosorbent assays (anti-IgG WNV ELISA and anti-NS1-IgG WNV), a commercially available indirect immunofluorescence assay, and a newly developed in-house ELISA for the detection of WNV-NS1-IgG antibodies. All four tests were compared to an in-house NT to determine both the sensitivity and specificity of the four test systems. None of the assays could match the specificity of the NT, although the two NS1-IgG based ELISAs were very close to the specificity of the NT at 97.3% and 94.6%. The in-house WNV-NS1-IgG ELISA had the best performance regarding sensitivity and specificity. The specificities of the ELISA assays and the indirect immunofluorescence assays could not meet the necessary specificity and/or sensitivity.
Numerous Serine/Threonine kinases affect blood cell homeostasis in Drosophila melanogaster
(2024) Deichsel, Sebastian; Gahr, Bernd M.; Mastel, Helena; Preiss, Anette; Nagel, Anja C.
Blood cells in Drosophila serve primarily innate immune responses. Various stressors influence blood cell homeostasis regarding both numbers and the proportion of blood cell types. The principle molecular mechanisms governing hematopoiesis are conserved amongst species and involve major signaling pathways like Notch, Toll, JNK, JAK/Stat or RTK. Albeit signaling pathways generally rely on the activity of protein kinases, their specific contribution to hematopoiesis remains understudied. Here, we assess the role of Serine/Threonine kinases with the potential to phosphorylate the transcription factor Su(H) in crystal cell homeostasis. Su(H) is central to Notch signal transduction, and its inhibition by phosphorylation impedes crystal cell formation. Overall, nearly twenty percent of all Drosophila Serine/Threonine kinases were studied in two assays, global and hemocyte-specific overexpression and downregulation, respectively. Unexpectedly, the majority of kinases influenced crystal cell numbers, albeit only a few were related to hematopoiesis so far. Four kinases appeared essential for crystal cell formation, whereas most kinases restrained crystal cell development. This group comprises all kinase classes, indicative of the complex regulatory network underlying blood cell homeostasis. The rather indiscriminative response we observed opens the possibility that blood cells measure their overall phospho-status as a proxy for stress-signals, and activate an adaptive immune response accordingly.
Novel genome-engineered H alleles differentially affect lateral inhibition and cell dichotomy processes during bristle organ development
(2024) Mönch, Tanja C.; Smylla, Thomas K.; Brändle, Franziska; Preiss, Anette; Nagel, Anja C.
Hairless (H) encodes the major antagonist in the Notch signaling pathway, which governs cellular differentiation of various tissues in Drosophila. By binding to the Notch signal transducer Suppressor of Hairless (Su(H)), H assembles repressor complexes onto Notch target genes. Using genome engineering, three new H alleles, HFA, HLLAA and HWA were generated and a phenotypic series was established by several parameters, reflecting the residual H-Su(H) binding capacity. Occasionally, homozygous HWA flies develop to adulthood. They were compared with the likewise semi-viable HNN allele affecting H-Su(H) nuclear entry. The H homozygotes were short-lived, sterile and flightless, yet showed largely normal expression of several mitochondrial genes. Typical for H mutants, both HWA and HNN homozygous alleles displayed strong defects in wing venation and mechano-sensory bristle development. Strikingly, however, HWA displayed only a loss of bristles, whereas bristle organs of HNN flies showed a complete shaft-to-socket transformation. Apparently, the impact of HWA is restricted to lateral inhibition, whereas that of HNN also affects the respective cell type specification. Notably, reduction in Su(H) gene dosage only suppressed the HNN bristle phenotype, but amplified that of HWA. We interpret these differences as to the role of H regarding Su(H) stability and availability.
Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites
(2024) Musa, Sandrine; Hemberle, Theo; Bensch, Staffan; Palinauskas, Vaidas; Baltrūnaitė, Laima; Woog, Friederike; Mackenstedt, Ute
Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.
Identification of new microfoci and genetic characterization of tick-borne encephalitis virus isolates from Eastern Germany and Western Poland
(2024) Król, Nina; Chitimia-Dobler, Lidia; Dobler, Gerhard; Kiewra, Dorota; Czułowska, Aleksandra; Obiegala, Anna; Zajkowska, Joanna; Juretzek, Thomas; Pfeffer, Martin
(1) Background: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia, although effective vaccines are available. Caused by the tick-borne encephalitis virus (TBEV, syn. Orthoflavivirus encephalitidis), in Europe, it is transmitted by ticks like Ixodes ricinus and Dermacentor reticulatus. TBEV circulates in natural foci, making it endemic to specific regions, such as southern Germany and northeastern Poland. Our study aimed to identify new TBEV natural foci and genetically characterize strains in ticks in previously nonendemic areas in Eastern Germany and Western Poland. (2) Methods: Ticks were collected from vegetation in areas reported by TBE patients. After identification, ticks were tested for TBEV in pools of a maximum of 10 specimens using real-time RT-PCR. From the positive TBEV samples, E genes were sequenced. (3) Results: Among 8400 ticks from 19 sites, I. ricinus (n = 4784; 56.9%) was predominant, followed by D. reticulatus (n = 3506; 41.7%), Haemaphysalis concinna (n = 108; 1.3%), and I. frontalis (n = 2; <0.1%). TBEV was detected in 19 pools originating in six sites. The phylogenetic analyses revealed that TBEV strains from Germany and Poland clustered with other German strains, as well as those from Finland and Estonia. (4) Conclusions: Although there are still only a few cases are reported from these areas, people spending much time outdoors should consider TBE vaccination.
Plastid phylogenomics reveals evolutionary relationships in the mycoheterotrophic orchid genus Dipodium and provides insights into plastid gene degeneration
(2024) Goedderz, Stephanie; Clements, Mark A.; Bent, Stephen J.; Nicholls, James A.; Patel, Vidushi S.; Crayn, Darren M.; Schlüter, Philipp M.; Nargar, Katharina; Goedderz, Stephanie; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia; Clements, Mark A.; Centre for Australian National Biodiversity Research (joint venture between Parks Australia and CSIRO), Canberra, ACT, Australia; Bent, Stephen J.; Data61, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Brisbane, QLD, Australia; Nicholls, James A.; Australian National Insect Collection, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Canberra, ACT, Australia; Patel, Vidushi S.; National Research Collections Australia, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Canberra, ACT, Australia; Crayn, Darren M.; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia; Schlüter, Philipp M.; Department of Plant Evolutionary Biology, Institute of Biology, University of Hohenheim, Stuttgart, Germany; Nargar, Katharina; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia
The orchid genus Dipodium R.Br. (Epidendroideae) comprises leafy autotrophic and leafless mycoheterotrophic species, with the latter confined to sect. Dipodium . This study examined plastome degeneration in Dipodium in a phylogenomic and temporal context. Whole plastomes were reconstructed and annotated for 24 Dipodium samples representing 14 species and two putatively new species, encompassing over 80% of species diversity in sect. Dipodium . Phylogenomic analysis based on 68 plastid loci including a broad outgroup sampling across Orchidaceae found that sect. Leopardanthus is the sister lineage to sect. Dipodium. Dipodium ensifolium , the only leafy autotrophic species in sect. Dipodium , was found to be a sister to all leafless, mycoheterotrophic species, supporting a single evolutionary origin of mycoheterotrophy in the genus. Divergence-time estimations found that Dipodium arose ca. 33.3 Ma near the lower boundary of the Oligocene and that crown diversification commenced in the late Miocene, ca. 11.3 Ma. Mycoheterotrophy in the genus was estimated to have evolved in the late Miocene, ca. 7.3 Ma, in sect. Dipodium . The comparative assessment of plastome structure and gene degradation in Dipodium revealed that plastid ndh genes were pseudogenised or physically lost in all Dipodium species, including in leafy autotrophic species of both Dipodium sections. Levels of plastid ndh gene degradation were found to vary among species as well as within species, providing evidence of relaxed selection for retention of the NADH dehydrogenase complex within the genus. Dipodium exhibits an early stage of plastid genome degradation, as all species were found to have retained a full set of functional photosynthesis-related genes and housekeeping genes. This study provides important insights into plastid genome degradation along the transition from autotrophy to mycoheterotrophy in a phylogenomic and temporal context.
Genomic adaptation of Burkholderia anthina to glyphosate uncovers a novel herbicide resistance mechanism
(2023) Schwedt, Inge; Collignon, Madeline; Mittelstädt, Carolin; Giudici, Florian; Rapp, Johanna; Meißner, Janek; Link, Hannes; Hertel, Robert; Commichau, Fabian M.
Glyphosate (GS) specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate to EPSP in the shikimate pathway of bacteria and other organisms. The inhibition of the EPSP synthase depletes the cell of the EPSP-derived aromatic amino acids as well as of folate and quinones. A variety of mechanisms (e.g., EPSP synthase modification) has been described that confer GS resistance to bacteria. Here, we show that the Burkholderia anthina strain DSM 16086 quickly evolves GS resistance by the acquisition of mutations in the ppsR gene. ppsR codes for the pyruvate/ortho-Pi dikinase PpsR that physically interacts and regulates the activity of the PEP synthetase PpsA. The mutational inactivation of ppsR causes an increase in the cellular PEP concentration, thereby abolishing the inhibition of the EPSP synthase by GS that competes with PEP for binding to the enzyme. Since the overexpression of the Escherichia coli ppsA gene in Bacillus subtilis and E. coli did not increase GS resistance in these organisms, the mutational inactivation of the ppsR gene resulting in PpsA overactivity is a GS resistance mechanism that is probably unique to B. anthina.
The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates
(2023) Schwedt, Inge; Schöne, Kerstin; Eckert, Maike; Pizzinato, Manon; Winkler, Laura; Knotkova, Barbora; Richts, Björn; Hau, Jann-Louis; Steuber, Julia; Mireles, Raul; Noda‐Garcia, Lianet; Fritz, Günter; Mittelstädt, Carolin; Hertel, Robert; Commichau, Fabian M.
Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.
Uptake of pathogenic Escherichia coli into crop plants
(2023) Detert, Katharina Margarete; Schmidt, Herbert
Contaminations of fresh produce by enterohemorrhagic Escherichia coli (EHEC) have been an increasing source of human disease outbreaks in the past few decades. In particular, the large disease outbreak in 2011 in Germany has left many questions about the survival of EHEC in soil and the colonization of plants unanswered. Agricultural soil as a reservoir for EHEC strains represents an important contamination source for crop plants. The use of cattle manure for soil fertilization or contaminated irrigation water can result in the introduction of pathogens in fields. To prevent the transfer of pathogens into the food chain, the German fertilizer ordinance disallows the use of organic fertilizer 12 weeks before harvesting the crop plants. However, it is expected that EHEC survives for longer and therefore continue to pose a risk for crop plant contamination. In this study, the overall survival ability of the pathogenic E. coli O104:H4 strain C227/11Φcu, which represents a stx2a-negative derivative of the 2011 outbreak strain C227/11, in agricultural soil was investigated. Thereby, different environmental conditions, soil types and genetic factors of the bacterial strain were identified as influencing factors. Furthermore, the ability of E. coli O104:H4 strain C227/11Φcu to colonize lamb’s lettuce via the root system was investigated to demonstrate that contamination of soil can result in crop plant contamination. In the first publication, the survival of E. coli O104:H4 strain C227/11Φcu in soil microenvironments containing either diluvial sand or alluvial loam was investigated. Two different temperatures (4°C and 22°C) were used and the samples were incubated for more than 12 weeks. The study aimed to analyze whether cattle manure addition prolongs EHEC survival in the soil microenvironments. In the last step, the survival studies were performed using ΔrpoS and ΔfliC deletion mutant strains of C227/11Φcu. The results demonstrated that E. coli O104:H4 strain C227/11Φcu survived for at least 12 weeks in the soil microenvironment model. The survival rate was influenced by the soil type and the temperature. In more detail, the incubation at lower temperature prolonged the survival rate and pathogens were detected up to 20 weeks after inoculation. The application of contaminated cattle manure increased the survival ability at 22°C. Sigma factor RpoS was recognized as an important factor for soil survival. The rpoS deletion mutants showed significant reduction of the survival period while FliC did not influence the overall survival ability in these experiments. To investigate the influence of further genetic factors of the bacterial strain in more detail and thereby characterize all transcription activities, transcriptome analysis was performed in the second publication. Since the strain showed the best survival in alluvial loam at 4°C, this condition was used for the next experiments. To decrease the amount of competing soil microbiota, the soil samples were autoclaved prior inoculation. After inoculation and after 1 and 4 weeks, samples were taken for RNA isolation. The differential expression analysis was performed using the sample from time point 0 as control. The analysis revealed that stress response genes and genes of the primary metabolism were upregulated after 1 and 4 weeks. In addition, genes and gene sets for the uptake of various carbohydrates or amino acids were upregulated, indicating adaption to an environment with low nutrient availability. Moreover, the results of the second publication demonstrated that persistence of C227/11Φcu in soil is related with a complex interface of metabolic networks. The third paper focused on the colonization of lamb’s lettuce by E. coli O104:H4 strain C227/11Φcu via the root system. Surface-sterilized seeds were cultivated on Murashige-Skoog agar or in autoclaved agricultural soil and migration into the edible portions of the plants was analyzed after 2, 4 and 8 weeks. The results of this publication demonstrated that migration into the edible parts occurred when the surrounding agar or soil was contaminated. This highlighted the threat of plant contamination with pathogenic E. coli on the field as a result of soil contamination.
Integrative taxonomy, systematics and biogeography of geometrid moths in a Middle Eastern biodiversity hotspot
(2023) Wanke, Dominic; Krogmann, Lars
Iran is an important biodiversity hotspot in the world. Recent studies have shown that two of the 36 global biodiversity hotspots are located in Iran: The Irano-Anatolian and the Caucasian hotspots. These two hotspots include parts of the two mountain ranges in Iran, the Alborz Mountains and the Zagros Mountains, which are crucial for the biodiversity, hosting a large number of endemic species. However, climate change and anthropogenic activities threaten its diversity. This study uses geometrid moths as a model group to better understand general patterns of biodiversity and zoogeography in Iran. Geometridae are suitable for such studies and scientifically interesting for several reasons: The family is species-rich with nearly 24,000 known species worldwide (539 known species in Iran), the species have short life-cycles and thus react quickly to environmental changes, and they occupy specialized ecological niches. Knowledge of Palearctic geometrid moths is rather advanced compared to other regions. The Western Palearctic, in particular, has been the target of considerable research. However, this is not the case for regions in the Middle East and Central Asia, where much is still unknown and further research is crucial. To fill this gap for geometrid moths in this region, data on their species richness and distribution patterns were collected to reveal regions with special faunal elements. Therefore, this dissertation consists of three parts, each of which contributes an essential element to achieve these goals. The first part deals with the taxonomic problems of partially species-rich and morphologically very difficult genera within the three subfamilies Sterrhinae (Problepsis, Scopula, Cinglis, Pseudocinglis, Scopuloides, Glossotrophia, Zygophyxia); Geometrinae (Xenochlorodes); and Ennominae (Nychiodes, Synopsia, Synopsidia, Eumera). Type specimens and original descriptions were used for critical revisions to understand the diagnostic characters of the species. Additionally, large series of specimens from many different museums and private collections were examined to highlight morphological variations. Using an integrative taxonomic approach that includes morphological and molecular data, a total of one new genus and four new species were described and 37 taxonomic changes (e.g., new synonyms, new combinations) were made. The second part addresses genera with uncertain tribal affiliation or questionable taxonomic status, which were also partially targets of the taxonomic revisions. A multi-gene phylogenetic analysis was performed using one mitochondrial gene and up to nine nuclear genes, sequences generated as part of this work and sequences from published phylogenetic studies were taken to run the analyses. As a result, the genus Eumera was determined to belong to the tribe Prosopolophini, the genera Cinglis and Scopuloides were removed from synonymy with Scopula, two genera were synonymized (Glossotrophia, Pseudocinglis), and two species were transferred to a different genus (Problepsis wiltshirei, Aphilopota tyttha). In the third part, distribution data of Iranian Geometridae was used to identify biodiversity hotspots and regions of high endemism. In addition, a network-based method was used to divide the country into unique bioregions and highlight areas with specific faunal elements. As a result, an exceptional species richness was found along the two main mountain ranges, Zagros in the west and south and Alborz in the north. Considering only the endemic species, the southern mountain areas were identified as the most species-rich regions. The bioregionalization analysis also identified six main bioregions. Most of these bioregions reflect specific faunal structures and are in accordance with previous studies. This highlights the complex species composition in Iran and demonstrates the exceptional biodiversity of the country. In addition, our results indicated two transition zones between zoogeographical realms. Of the six zoogeographical realms defined by Wallace, three occur in Iran meeting in the south of the country: The Palearctic and Saharo-Arabian along the foothills of the Zagros Mountains and the Palearctic and Oriental in southeastern Iran. At these transition zones, Iran has very specific faunal elements of the Geometridae, which makes these zones important for conservation. Overall, this work contributes to a better understanding of the biodiversity of geometrids in Iran and neighboring countries. It serves as a resource for the identification of species, their distribution and habitats, which are of great interest for conservation efforts in Iran and neighboring countries.
Reproductive success and escape behaviour in urban greylag geese (Anser anser)
(2023) Mai, Sabrina; Steidle, Johannes
Urbanisation and its effect on animals and plants is an important factor to analyse in behavioural studies. Warmer temperatures and an increased availability of food provide benefits to animals occurring in urban habitats. In contrast, these animals also have to tolerate the disturbance caused by artificial lights or noise. This work studies an urban local population of greylag geese Anser anser. Greylag geese live in wetland landscapes, but also in urban parks with wide lawns and water bodies. Since the nineties, the city of Stuttgart in southwest Germany is home to a breeding population of greylag geese. As a result of a long-term ringing project, a large percentage of the population is ringed. This data shows that the population is non-migratory, as the geese generally remain in the area year-round. The individual identification offers the opportunity of tracing data, such as reproduction or behaviour, for the same animal over a longer period. This data can then be individually connected to genetic information, which is a large benefit in behavioural studies. The first chapter of this work focuses on reproductive success as one of the two key parameters which influence population change. As reproductive success itself is influenced by a variety of factors, this study analysed the effect of factors such as population size, brood size or dispersal by using two different measures of reproductive success: fledging success (the relation between hatched and fledged young of a brood ) and hatchling survival (the likelihood of a hatchling to survive to fledging). Fledging success of pairs initially increased with the number of times pairs bred together but decreased again in later broods. While the experience of a pair is therefore beneficial for their reproductive success, the subsequent decrease may be caused by the increasing age of the parents. The brood size also influenced reproductive success, as fledging success was higher in larger broods and hatchling survival was also positively influenced by brood size. In some years, several goose families dispersed from the breeding ground to a different brood rearing area. Hatchling survival was higher in these families, though this effect decreased again when the broods were large. The total population size had a negative effect on hatchling survival, indicating density dependence. To determine if there is a genetic basis for escape behaviour in greylag geese, the second chapter of this work compares Stuttgart’s urban local population of greylag geese with a rural local population in the countryside near Ludwigsburg. Animals occurring in urban habitats need a higher tolerance towards disturbance. This tolerance may be due to the animal’s personality. Animal personality is defined as consistent behaviour across time or context and is expressed for example through risk-prone behaviour. Urban animals are likely more risk-prone than their rural counterparts and thus have a bolder personality. There is increasing evidence that personality and thus behaviour have a genetic basis. This study analyses the dopamine D4 receptor (DRD4) as a candidate gene associated with boldness / shyness and fear. The gene showed a high variation with eleven alleles and 35 genotypes being detected in the analysed greylag geese. Allele and genotype frequencies were skewed, with one allele and two genotypes occurring more often than the others. Based on the detected frequencies, these two genotypes were defined as common and the remaining 33 as rare genotypes. Common genotypes were found more frequently in geese from urban areas, while rare genotypes were found more frequently in geese from rural areas. To determine if the detected genotypes could be associated with behaviour, the flight initiation distance (FID) of individual geese was measured. As the geese are ringed, FID measurements could be directly assigned to a specific goose and thus to its genotype. A high FID indicates geese which are more cautious, while a low FID indicates boldness. Indeed, greylag geese of the rural location show increased FID, while those in urban areas in Stuttgart have lower FIDs and thus a bolder personality. Despite these results, there was no significant correlation between DRD4 genotype frequency and FID. This may be explained by methodological effects. Alternatively, DRD4 has also been associated with other behaviours such as novelty seeking and exploratory behaviour. These behaviours may also differ between urban and rural animals and may thus be the reason for the detected frequencies. It is therefore likely that local habitat selection may be influenced by the DRD4 genotype. Overall, this work demonstrates that different factors, including genetics, should be taken into account when managing urban populations of wild animals, as their behaviour can differ significantly from their rural counterparts, even within a single species.

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