Mare-MAGE curated reference database of fish mitochondrial genes.

Biodiversity assessment approaches based on molecular biology techniques such as metabarcoding, RAD-seq, or SnaPshot sequencing are increasingly applied in assessing marine and aquatic ecosystems. Here we present a new reference database for fish meta-barcoding based on mitochondrial genes. The Mare-MAGE database contains quality-checked sequences of the mitochondrial 12S ribosomal RNA and Cytochrome c Oxidase I gene. All sequences were obtained from the National Center for Biotechnology Information- GenBank (NBCI-GenBank), the European Nucleotide Archive (ENA), AquaGene Database and BOLD database, and have undergone intensive processing. They were checked for false annotations and non-target anomalies, according to the Integrated Taxonomic Information System (ITIS) and FishBase. The dataset is compiled in ARB-Home, FASTA and Qiime2 formats, and is publicly available from the Mare-MAGE database website ( http://mare-mage.weebly.com/ ). It includes altogether 231,333 COI and 12S rRNA gene sequences of fish, covering 19,506 species of 4,058 genera and 586 families.

Behavioural and fitness effects of translocation to a novel environment: Whole-lake experiments in two aquatic top predators.

Translocation into a novel environment through common fisheries management practices, such as fish stocking, provides opportunities to study behavioural and fitness impacts of translocations at realistic ecological scales. The process of stocking, as well as the unfamiliarity with novel ecological conditions and the interactions with resident fish may affect translocated individuals, leading to alterations of behaviours and causing fitness impacts. Our objectives were to investigate how aquatic top predators behaviourally establish themselves and compete with resident individuals following introduction in a novel lake environment and to investigate the resulting fitness consequences. Using high-resolution acoustic telemetry, we conducted whole-lake experiments and compared the activity, activity-space size and fate of translocated and resident individuals in two model top predators, the northern pike Esox lucius (n = 160) and European catfish Silurus glanis (n = 33). Additionally, we compared the reproductive success of translocated and resident northern pike. The experiment was conducted with large (adult) individuals of different origins, resilient to predation, but subject to agonistic interactions and competition with resident fish. Over a period of several months, the translocated catfish exhibited consistently larger activity-space sizes than resident catfish, but did not differ from residents in activity and survival. The pike from one of the two translocated origins we tested also showed elevated space-use, and both translocated origins revealed higher mortality rates than their resident conspecifics, indicating maladjustment to their novel environment. When non-resident pike reproduced, they overwhelmingly produced hybrid offspring with resident fish, indicating that introductions fostered gene flow of non-native genes. Our study indicates that fish introductions result in behavioural and fitness impacts even in large-bodied top predators that experience low levels of natural predation risk.

Design of a user-friendly and rapid DNA microarray assay for the authentication of ten important food fish species.

Seafood is particularly susceptible to the substitution of species. In order to guarantee authentic seafood products, seafood processors and traders must perform self-checks on the authenticity of imported and purchased goods. However, the conventional Sanger sequencing of PCR products for the authentication of seafood species is time-consuming and requires advanced infrastructure. DNA microarrays (DNA chips) with species-specific oligonucleotide probes represent a rapid alternative to sequencing-based species authentication. So far, though, only DNA microarrays for the authentication of land vertebrate species have achieved market success. In this work, a user-friendly DNA microarray assay was developed for the authentication of ten important food fish species that can be performed in four to five hours from start to end. The assay was tested with authenticated specimens from 67 different fish species, and by comparing the probe signal patterns all target species and even closely related non-target species could be distinguished.

Improved DNA extraction and purification with magnetic nanoparticles for the detection of methicillin-resistant Staphylococcus aureus.

Molecular methods offer fast, safe and cost-efficient detection of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). These tests depend on a rapid extraction of bacterial DNA. The aim of this study was to compare an optimized DNA extraction and purification protocol for MRSA using magnetic nanoparticles with the original method. The purity of the extracted DNA was assessed by photometric measurements and the amount of DNA was determined by real-time PCR. Three MRSA reference strains (S. aureus ATCC® 70699, S. aureus ATCC® 43300; S. aureus ATCC® 33592) and eleven MRSA field strains, which include SCCmec elements of types I to XI, were used in this study. The optimized protocol can save approximately 20 min time compared to the original method and the DNA yield was higher with the new protocol. Therefore, this new protocol allows a faster DNA extraction from MRSA cultures.

Development of a nucleic acid lateral flow immunoassay (NALFIA) for reliable, simple and rapid detection of the methicillin resistance genes mecA and mecC.

The gene mecA and its homologue mecC confer methicillin resistance in Staphylococcus aureus and other staphylococci. Methicillin-resistant staphylococci (MRS) are considered resistant to all ß-lactam antibiotics. To avoid the use of ß-lactam antibiotics for the control of MRS infections, there is an urgent need for a fast and reliable screening assay for mecA and mecC that can easily be integrated in routine laboratory diagnostics. The aim of this study was the development of such a rapid detection method for methicillin resistance based on nucleic acid lateral flow immunoassay (NALFIA) technology. In NALFIA, the target sequences are PCR-amplified, immobilized via antigen-antibody interaction and finally visualized as distinct black bars resulting from neutravidin-labeled carbon particles via biotin-neutravidin interaction. A screening of 60 defined strains (MRS and non-target bacteria) and 28 methicillin-resistant S. aureus (MRSA) isolates from clinical samples was performed with PCR-NALFIA in comparison to PCR with subsequent gel electrophoresis (PCR-GE) and real-time PCR. While all samples were correctly identified with all assays, PCR-NALFIA was superior with respect to limits of detection. Moreover, this assay allowed for differentiation between mecA and mecC by visualizing the two alleles at different positions on NALFIA test stripes. However, since this test system only targets the mecA and mecC genes, it does not allow to determine in which staphylococcal species the mec gene is included. Requiring only a fraction of the time needed for cultural methods (i.e. the gold standard), the PCR-NALFIA presented here is easy to handle and can be readily integrated into laboratory diagnostics.

Population differentiation of zander (Sander lucioperca) across native and newly colonized ranges suggests increasing admixture in the course of an invasion.

In addition to ecological factors, evolutionary processes can determine the invasion success of a species. In particular, genetic admixture has the potential to induce rapid evolutionary change, which can result from natural or human-assisted secondary contact between differentiated populations. We studied the recent range expansion of zander in Germany focusing on the interplay between invasion and genetic admixture. Historically, the rivers Elbe and Danube harboured the most north-western source populations from which a north-westward range expansion occurred. This was initiated by introducing zander outside its native range into rivers and lakes, and was fostered by migration through artificial canals and stocking from various sources. We analysed zander populations of the native and invaded ranges using nuclear and mitochondrial genetic markers. Three genetic lineages were identified, which were traced to ancestral ranges. Increased genetic diversity and admixture in the invaded region highlighted asymmetric gene flow towards this area. We suppose that the adaptive potential of the invading populations was promoted by genetic admixture, whereas competitive exclusion in the native areas provided a buffer against introgression by novel genotypes. These explanations would be in line with evidence that hybridization can drive evolutionary change under conditions when new niches can be exploited.

"Candidatus Paraholospora nucleivisitans", an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host.

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological-botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.

Microstructured scaffolds for liver tissue cultures of high cell density: morphological and biochemical characterization of tissue aggregates.

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ-specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver-specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10-13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver-specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.