The minnow Phoxinus lumaireul (Leuciscidae) shifts the Adriatic-Black Sea basin divide in the north-western Dinaric Karst region.

Karst landscapes are characterized by intermittent and sinking streams. The most common method used to study underground hydrological connections in karst is tracing tests. However, a more biologically oriented approach has been suggested: analysis of the genetic structure of aquatic organisms. Biological tracers can be sought among trogloxenes, that is, surface species that occasionally enter caves and groundwater. One such example is the fish genus Phoxinus, which exhibits high genetic diversity and complex phylogeography in the Balkan Peninsula. In the north-western Dinaric Karst, the complex hydrological network was digitalized in 2020. Contemporaneously, Phoxinus lumaireul populations in the Slovenian Dinaric Karst were intensively sampled and analysed for fragments of two mitochondrial genes and one nuclear gene. The derived phylogeographic structure and data on hydrological connections were compared to evaluate support for three alternative scenarios: The genetic structure (1) is a consequence of the ongoing geneflow through underground connections, (2) reflects a previous hydrological network or (3) is an outcome of anthropogenic translocations. The results suggest that the first two scenarios seem to have played a major role, while the third has not had profound effects on the genetic composition. Comparison between the genetic structure of Slovenian Dinaric Karst sampling sites and that of hydrologically isolated reference sampling sites indicated a greater genetic connectivity in the former. Moreover, the range of Adriatic (1a) and Black Sea (1c) haplotypes does not correspond to the Adriatic-Black Sea basin divide but is shifted northwards.

High-Resolution Episcopic Microscopy (HREM) in Multimodal Imaging Approaches.

High-resolution episcopic microscopy (HREM) is a three-dimensional (3D) episcopic imaging modality based on the acquisition of two-dimensional (2D) images from the cut surface of a block of tissue embedded in resin. Such images, acquired serially through the entire length/depth of the tissue block, are aligned and stacked for 3D reconstruction. HREM has proven to be specifically advantageous when integrated in correlative multimodal imaging (CMI) pipelines. CMI creates a composite and zoomable view of exactly the same specimen and region of interest by (sequentially) correlating two or more modalities. CMI combines complementary modalities to gain holistic structural, functional, and chemical information of the entire sample and place molecular details into their overall spatiotemporal multiscale context. HREM has an advantage over in vivo 3D imaging techniques on account of better histomorphologic resolution while simultaneously providing volume data. HREM also has certain advantages over ex vivo light microscopy modalities. The latter can provide better cellular resolution but usually covers a limited area or volume of tissue, with limited 3D structural context. HREM has predominantly filled a niche in the phenotyping of embryos and characterisation of anatomic developmental abnormalities in various species. Under the umbrella of CMI, when combined with histopathology in a mutually complementary manner, HREM could find wider application in additional nonclinical and translational areas. HREM, being a modified histology technique, could also be incorporated into specialised preclinical pathology workflows. This review will highlight HREM as a versatile imaging platform in CMI approaches and present its benefits and limitations.

First Record of Trichobilharzia physellae (Talbot, 1936) in Europe, a Possible Causative Agent of Cercarial Dermatitis.

Several species of avian schistosomes are known to cause dermatitis in humans worldwide. In Europe, this applies above all to species of the genus Trichobilharzia. For Austria, a lot of data are available on cercarial dermatitis and on the occurrence of Trichobilharzia, yet species identification of trematodes in most cases is doubtful due to the challenging morphological determination of cercariae. During a survey of trematodes in freshwater snails, we were able to detect a species in the snail Physella acuta (Draparnaud, 1805) hitherto unknown for Austria, Trichobilharzia physellae; this is also the first time this species has been reported in Europe. Species identification was performed by integrative taxonomy combining morphological investigations with molecular genetic analyses. The results show a very close relationship between the parasite found in Austria and North American specimens (similarity found in CO1 ≥99.57%). Therefore, a recent introduction of T. physellae into Europe can be assumed.

Cross-modality imaging of bisphosphonate-treated murine jawbones.

In this proof-of-principle study, we established and implemented a cross-modality imaging (CMI) pipeline to characterize and compare bisphosphonate (BIS)-treated jawbones of Sprague-Dawley rats after tooth extraction after physical therapies (photobiomodulation and extracorporeal shockwave therapy (PBMT and ESWT)). We showcase the feasibility of such a CMI approach and its compatibility across imaging modalities to probe the same region of interest (ROI) of the same jawbone. Jawbones were imaged in toto in 3D using micro-Computed Tomography to identify ROIs for subsequent sequential 2D analysis using well-established technologies such as Atomic Force Microscopy and Scanning Electron Microscopy, and recent imaging approaches in biomedical settings, such as micro-X-Ray Fluorescence Spectroscopy. By combining these four modalities, multiscale information on the morphology, topography, mechanical stiffness (Young's modulus), and calcium, zinc and phosphorus concentrations of the bone was collected. Based on the CMI pipeline, we characterized and compared the jawbones of a previously published clinically relevant rat model of BIS-related osteonecrosis of the jawbone (BRONJ) before and after treatment with BISs, PBMT and ESWT. While we did not find that physical therapies altered the mechanical and elemental jawbone parameters with significance (probably due to the small sample size of only up to 5 samples per group), both ESWT and PBMT reduced pore thicknesses and bone-to-enamel distances significantly compared to the controls. Although focused on BIS-treated jawbones, the established CMI platform can be beneficial in the study of bone-related diseases in general (such as osteoarthritis or -porosis) to acquire complementary hallmarks and better characterize disease status and alleviation potentials.

Cross-Modality Imaging of Murine Tumor Vasculature-a Feasibility Study.

Tumor vasculature and angiogenesis play a crucial role in tumor progression. Their visualization is therefore of utmost importance to the community. In this proof-of-principle study, we have established a novel cross-modality imaging (CMI) pipeline to characterize exactly the same murine tumors across scales and penetration depths, using orthotopic models of melanoma cancer. This allowed the acquisition of a comprehensive set of vascular parameters for a single tumor. The workflow visualizes capillaries at different length scales, puts them into the context of the overall tumor vessel network and allows quantification and comparison of vessel densities and morphologies by different modalities. The workflow adds information about hypoxia and blood flow rates. The CMI approach includes well-established technologies such as magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and ultrasound (US), and modalities that are recent entrants into preclinical discovery such as optical coherence tomography (OCT) and high-resolution episcopic microscopy (HREM). This novel CMI platform establishes the feasibility of combining these technologies using an extensive image processing pipeline. Despite the challenges pertaining to the integration of microscopic and macroscopic data across spatial resolutions, we also established an open-source pipeline for the semi-automated co-registration of the diverse multiscale datasets, which enables truly correlative vascular imaging. Although focused on tumor vasculature, our CMI platform can be used to tackle a multitude of research questions in cancer biology.

Multimodality imaging beyond CLEM: Showcases of combined in-vivo preclinical imaging and ex-vivo microscopy to detect murine mural vascular lesions.

In imaging, penetration depth comes at the expense of lateral resolution, which restricts the scope of 3D in-vivo imaging of small animals at micrometer resolution. Bioimaging will need to expand beyond correlative light and electron microscopy (CLEM) approaches to combine insights about in-vivo dynamics in a physiologically relevant 3D environment with ex-vivo information at micrometer resolution (or beyond) within the spatial, structural and biochemical contexts. Our report demonstrates the immense potential for biomedical discovery and diagnosis made available by bridging preclinical in-vivo imaging with ex-vivo biological microscopy to zoom in from the whole organism to individual structures and by adding localized spectroscopic information to structural and functional information. We showcase the use of two novel imaging pipelines to zoom into mural lesions (occlusions/hyperplasia and micro-calcifications) in murine vasculature in a truly correlative manner, that is using exactly the same animal for all integrated imaging modalities. This correlated multimodality imaging (CMI) approach includes well-established technologies such as Positron Emission Tomography (microPET), Autoradiography, Magnetic Resonance Imaging (microMRI) and Computed Tomography (microCT), and imaging approaches that are more novel in the biomedical setting, such as X-Ray Fluorescence Spectroscopy (microXRF) and High Resolution Episcopic Microscopy (HREM). Although the current pipelines are focused on mural lesions, they would also be beneficial in preclinical and clinical investigations of vascular diseases in general.

First confirmed record of Trichobilharzia franki Müller & Kimmig, 1994, from Radix auricularia (Linnaeus, 1758) for Austria.

Avian schistosomes are of medical and veterinary importance as they are responsible for the annually occurring cercarial dermatitis outbreaks. For Austria, so far, only Trichobilharzia szidati Neuhaus 1952 was confirmed on species level as causative agent of cercarial dermatitis. Here we present the first record of Trichobilharzia franki Müller & Kimmig 1994 in Austria. The species was detected during a survey of digenean trematodes in Upper Austrian water bodies. Furthermore, we provide DNA barcodes of T. franki as well as measurements of several parasite individuals to indicate the intraspecific diversity. We also recommend the usage of an alternative primer pair, since the "standard COI primer pair" previously used for Schistosomatidae amplified an aberrant fragment in the sequence of T. franki. Overall, our study shows how limited our knowledge about occurrence and distribution of avian schistosomes in Austria is and how important it is to acquire such a knowledge to estimate ecological and epidemiological risks in the future.

Unravelling the hidden biodiversity - the establishment of DNA barcodes of fish-parasitizing Acanthocephala Koehlreuther, 1771 in view of taxonomic misidentifications, intraspecific variability and possible cryptic species.

Acanthocephalans are obligate parasites of vertebrates, mostly of fish. There is limited knowledge about the diversity of fish-parasitizing Acanthocephala in Austria. Seven determined species and an undetermined species are recorded for Austrian waters. Morphological identification of acanthocephalans remains challenging due to their sparse morphological characters and their high intraspecific variations. DNA barcoding is an effective tool for taxonomic assignment at the species level. In this study, we provide new DNA barcoding data for three genera of Acanthocephala (Pomphorhynchus Monticelli, 1905, Echinorhynchus Zoega in Müller, 1776 and Acanthocephalus Koelreuter, 1771) obtained from different fish species in Austria and provide an important contribution to acanthocephalan taxonomy and distribution in Austrian fish. Nevertheless, the taxonomic assignment of one species must remain open. We found indications for cryptic species within Echinorhynchus cinctulus Porta, 1905. Our study underlines the difficulties in processing reliable DNA barcodes and highlights the importance of the establishment of such DNA barcodes to overcome these. To achieve this goal, it is necessary to collect and compare material across Europe allowing a comprehensive revision of the phylum in Europe.

An integrative taxonomic approach to reveal the status of the genus Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) in Austria.

Species of the genus Pomphorhynchus Monticelli, 1905 (Acanthocephala: Pomphorhynchidae) are obligate endoparasites infesting mostly freshwater fish. Morphological identification is challenging due to high intraspecific variations. The use of molecular analyses enabled new insights into the diversity and revealed high cryptic presence and unknown distribution patterns for various European species. In Austria only one species, Pomphorhynchus laevis (Müller, 1776), has been reported so far. We conduct an integrative analysis of Pomphorhynchus in Austria with a combination of morphological and molecular methods. Our results revealed the presence of three species of Pomphorhynchus in Austrian waters: Pomphorhynchus laevis, Pomphorhynchus tereticollis (Rudolphi, 1809) and Pomphorhynchus bosniacus Kiskároly and Cankovic, 1967. While P. bosniacus was the predominant species in the Danube, P. laevis was recorded exclusively in Styria. Pomphorhynchus tereticollis occurred mainly in rivers of Styria except for one individual found in the Danube. We document the first occurrence of P. bosniacus and P. tereticollis in Austria. We found a high intraspecific haplotype variation in P. bosniacus suggesting that the species has a longer history in Central and Western Europe. It was previously misidentified as P. laevis, which is also true for P. tereticollis. A large number of hosts examined were infected with only juvenile and cystacanth stages suggesting paratenic infections. Our study highlights the importance of using an integrative taxonomic approach in the identification of species of Pomphorhynchus.