The role of radiology in provenance research - experiences from the collaboration between radiology and anatomy at the University of Rostock and future perspectives.

Worldwide, the study and examination of human remains and the circumstances of their acquisition for anatomical collection have received great interest. As part of provenance research projects, a large number of collections are being investigated to determine whether the human remains have been acquired in a correct or unlawful way because the people could have been killed in order to be used as "anthropological objects" for research purposes and to become so-called "specimens". These topics have also been addressed by the Institute of Anatomy at the University Medical Center Rostock. The role of radiology in this interdisciplinary project will be presented using selected examples.The anatomical collection at the University of Rostock includes 40 human skulls, 14 plaster casts, 6 Egyptian mummy heads, and 1 full-body mummy. In addition to the examination by a historian, an anthropologist, and forensic pathologists, additional computed tomography was carried out on nine skulls and the full-body mummy. Micro-computed tomography was also carried out on seven skulls in order to enable a look behind the mummification material and tissue remains.(Micro-)computed tomography was able to close diagnostic gaps and the results presented some rather unexpected findings.Due to interdisciplinary collaboration, individual fates could be determined, which provided information about the individual's life and death circumstances. None of the examined individuals showed evidence of colonial-era injustice or the use of violence that would have led to their inclusion in the collection. (Micro-)computed tomography was a valuable addition to this provenance research project. · Computed tomography enhances interdisciplinary provenance research projects.. · Computed tomography enables a non-destructive examination of human remains.. · The future of research and presentation of human remains will increasingly be virtual.. · Steinhagen I, Brinker U, Kolbe V et al. The role of radiology in provenance research - experiences from the collaboration between radiology and anatomy at the University of Rostock and future perspectives. Fortschr Röntgenstr 2024; DOI 10.1055/a-2303-0312.

Ex Vivo Micro-CT in Ophthalmology: Preparation and Contrasting for Non-invasive 3D-Visualisation. / Ex-vivo-Mikro-CT in der Augenheilkunde: Probenbehandlung und -kontrastierung für die 3D-Darstellung.

X-ray-based micro-computed tomography (micro-CT) is a largely non-destructive imaging method for the visualisation and analysis of internal structures in the ex vivo eye and affords high resolution. In contrast to other high-resolution imaging methods, micro-CT enables spatial recording of larger and more complex tissue structures, such as the anterior chamber of the eye. Special contrasting methods help to enhance the absorption properties of soft tissue, that is otherwise only weakly radiopaque. Critical point drying (CPD), as primarily used in scanning electron microscopy, offers an additional tool for improving differential contrast properties in soft tissue. In the visualisation of intraosseous soft tissue, such as the efferent lacrimal ducts, sample treatment by decalcification with ethylenediaminetetraacetic acid and subsequent CPD provides good results for micro-CT. Micro-CT can be used for a wide range of questions in 1. basic research, 2. application-related studies in ophthalmology (e.g. evaluation of the preclinical application of microstents for glaucoma treatment or analysis of the positioning of intraocular lenses) but also 3. as a supplement to ophthalmological histopathology.

Morphology of the murine choroid plexus: Attachment regions and spatial relation to the subarachnoid space.

The choroid plexus has recently been identified as a possible migration route for peripheral immune cells into the central nervous system. For future investigation of this route, profound knowledge of the morphology of the murine choroid plexus is a prerequisite. We here present a detailed morphological description of the murine choroid plexus, its attachment regions as well as its spatial relation to the subarachnoid space. We used micro-computed tomography of immersion-contrasted fixated brains to generate three-dimensional models of the ventricle system and the choroid plexus and aligned micro-computed tomography-based sections with histological paraffin-embedded sections after immunohistochemical labeling of the basal lamina and choroid plexus epithelium marker proteins (laminin and aquaporin 1). The murine choroid plexus is located in all four ventricles and is attached to the brain parenchyma in narrow attachment regions with a specific morphology in each ventricle. While in the lateral and fourth ventricle, the attachment site is formed by thin tissue bridges, the choroid plexus attachment in the third ventricle has a more complex V-like shape. In all ventricles, the choroid plexus is in close spatial relationship with the subarachnoid space that extends from the brain surface along physiologic openings toward the choroid plexus. In summary, we here provide a description of the morphology of the murine ventricle system and choroid plexus, the attachment regions of the choroid plexus and its connection to the subarachnoid space, as well as a three-dimensional model of the ventricles, the choroid plexus, and the subarachnoid space to facilitate a spatial understanding of these complex structures.

Micro-CT in ophthalmology: ex vivo preparation and contrasting methods for detailed 3D-visualization of eye anatomy with special emphasis on critical point drying.

Background: Micro-computed tomography (micro-CT) provides detailed 3-dimensional (3D) visualization of anatomical structures and encourages morphological reinvestigation of organs with delicate features. The low radiodensity of soft tissues necessitates preceding sample preparation to conduct X-ray imaging with decent contrast between different tissues. In this study, we demonstrate the preparation with three radiopaque agents in combination with elimination of liquids by critical point drying (CPD) introduced for ocular samples. Methods: Enucleated porcine eyes were prepared with ethanolic iodine (EI), aqueous iodine-potassium iodide, or ethanolic phosphotungstic acid (EPTA). Micro-CT scans of the samples were conducted in a moist environment with an isotropic resolution of 9.2-12.5 µm voxel size. Subsequently, samples were chemically dehydrated and critical point (CP) dried to conduct a second scan in a dry environment with a resolution up to 4.7-5.4 µm in voxel size. The visualization effects were qualitatively and semi-quantitatively evaluated with regard to the generated contrast between different ocular tissues. Results: All three contrast agents accumulated well in most of the investigated ocular tissues and lead to an increased X-ray attenuation which allowed for differentiated visualization of ocular structures. Problematic agent penetration into the lens was obvious for iodine-potassium iodide and EPTA. Artificial damages of the lens and thickness reduction for the cornea and sclera due to CPD were noticed. The effects of the different contrasting treatments are described and compared with regard to the effects of CPD. Exclusively CP dried samples that were not treated with contrast agents could also be visualized excellently with a good distinction of different ocular structures from each other. Conclusions: All ocular structures can be visualized by micro-CT. To contrast moist samples, the best results were achieved with iodine potassium iodide (IPI). CPD improved the scan quality in all cases. Even without pretreatment with contrasting agents, the CP dried samples showed a contrast similar to the IPI treated samples.

Spontaneous Hind Limb Paralysis Due to Acute Precursor B Cell Leukemia in RAG1-deficient Mice.

RAG1-deficient mice are a frequently used immunodeficient mouse strain lacking mature lymphocytes. Apart from an elevated risk for infections, no predispositions for diseases of this strain have been described so far. We here report a high incidence of spontaneous pro B cell leukemia resulting in hind limb paralysis in our colony of RAG1-deficient mice. At an age of 7-13 months, animals developed hind limb paralysis and rapid decrease of the overall health condition leading to the need of euthanasia. Histological and flow cytometric analyses as well as micro-computed tomography (micro-CT) scans revealed CD45+ CD19+ IgM- cell infiltrates in the spleen, the bone marrow, and the spinal canal. Monthly blood sampling and screening for CD19+ blast frequency in the peripheral blood was successfully established for monitoring of leukemia development before symptom onset. We conclude that facilities that breed RAG1-deficient mice should be aware of the risk of leukemia development in this strain and recommend to implement regular blood sampling for aged RAG1-deficient animals.

Cuprizone Intoxication Results in Myelin Vacuole Formation.

Myelin damage is a histopathological hallmark of multiple sclerosis lesions. Results of post mortem studies suggest that impaired myelin-axon interaction characterized by focal myelin detachments is an early event during lesion genesis. In this study, we investigated the ultrastructural changes of the axon-myelin interface in the cuprizone model using serial block face scanning electron microscopy and immunohistochemistry. We show that non-inflammatory injury of oligodendrocytes by cuprizone intoxication results in myelin vacuole formation and axonal swellings, paralleled by early alterations of the node of Ranvier cytoarchitecture. This remarkable resemblance of ultrastructural myelin characteristics in multiple sclerosis and the cuprizone animal model suggests that the cuprizone model is a valuable tool to study early pathologies during lesion formation.

Comfrey (Symphytum spp.) as a feed supplement in pig nutrition contributes to regional resource cycles.

In smallholder agriculture, the fast-growing and perennial accumulator plant comfrey (Symphytum spp.) was used to supply pigs with protein and minerals. Comfrey leaves show similar values in dry matter as soybean or blue lupine in crude protein content, but much higher levels of calcium and phosphorus. However, in terms of increased efficiency in animal husbandry, comfrey has been displaced by mainly soybean and cereals. Due to its profile of macro- and micronutrients the use of comfrey could have the potential to re-establish local resource cycles and help remediate over-fertilized soils. The aim of the study was to evaluate whether a modern pig breed accepts a continuous feed supplement of dried comfrey leaves. After an initial adaptation period post-weaning, German Landrace piglets were subjected to either a standard control diet or a diet supplemented with 15% dried comfrey leaves for 4 weeks. Body weight was reduced in comfrey-supplemented piglets compared to controls, which might be attributed to reduced palatability in the experimental setting. Nevertheless, comfrey-supplemented piglets exhibited adequate bone mineralization and intestinal integrity. The microbiome profile in feces and digesta revealed higher diversity in comfrey-supplemented piglets compared to controls, with pronounced effects on the abundances of Treponema and Prevotella. This may be due to described bio-positive components of the comfrey plant, as data suggest that the use of comfrey leaves may promote intestinal health. Digestive tract phosphorus levels were reduced in piglets receiving comfrey supplementation, which may ultimately affect phosphorus levels in manure. Results indicate that comfrey leaves could serve as a feed component in integrated agricultural systems to establish regional nutrient cycles. The trial provides a basis for further work on comfrey as a regionally grown protein source and effective replacement for rock mineral supplements.

mRNA Profiles of Porcine Parathyroid Glands Following Variable Phosphorus Supplies throughout Fetal and Postnatal Life.

Knowledge of gene expression profiles reflecting functional features and specific responsiveness of parathyroid glands (PTGs) contributes to understanding mineral homeostasis and parathyroid function in healthy and diseased conditions. The study aims to reveal effector molecules driving the maintenance of phosphorus (P) homeostasis and parathyroid hormone (PTH) responsiveness to variable P supply throughout fetal and postnatal life. In this study, a long-term dietary intervention was performed by keeping pig offspring on distinct mineral P levels throughout fetal and postnatal life. Respective adaptation processes of P homeostasis were assessed in mRNA profiles of PTGs and serum minerals. RNA sequencing data and resulting molecular pathways of PTGs showed that the PTH abundance is very strictly controlled via e.g., PIN1, CaSR, MAfB, PLC and PKA signaling to regulate PTH expression, stability, and secretion. Additionally, the observed dietary effects on collagen expression indicate shifts in the ratio between connective tissue and parenchyma, thereby affecting cell-cell contacts as another line of PTH regulation. Taken together, the mRNA profiles of porcine PTGs reflect physiological responses in-vivo following variable dietary P supplies during fetal and postnatal life. The results serve to evaluate a long-term nutrition strategy with implications for improving the mineral balance in individuals with pathological disorders.

Quantitative and Qualitative Assessment of Adhesive Thrombo-Fibrotic Lead Encapsulations (TFLE) of Pacemaker and ICD Leads in Arrhythmia Patients-A Post Mortem Study.

The demand for cardiac implantable electronic devices for arrhythmia therapy is still unabated and rising. Despite onward optimizations, lead-related problems such as infections or fractures often necessitate lead extraction. Due to adhesive thrombo-fibrotic lead encapsulations (TFLE) transvenous lead extraction is challenging and risky. However, knowledge on TFLEs and possible correlations with technical lead parameters and dwelling time (DT) were hitherto insufficiently studied. Therefore, we analyzed TFLEs of 62 lead from 35 body donor corpses to gain information for a potential lead design optimization. We examined both TFLE topography on the basis on anatomical landmarks and histo-morphological TFLE characteristics by means of histological paraffin sections and scanning electron microscopy of decellularized samples. The macroscopic analysis revealed that all leads were affected by TFLEs, mainly in the lead bearing veins. Half (47.2%) of the right-ventricular leads possessed adhesions to the tricuspid valve. On average, 49.9 ± 21.8% of the intravascular lead length was covered by TFLE of which 82.8 ± 16.2% were adhesive wall bindings (WB). The discrete TFLEs with at least one WB portion had a mean length of 95.0 ± 64.3 mm and a maximum of 200 mm. Neither sex, DT nor certain technical lead parameters showed distinct tendencies to promote or prevent TFLE. TFLE formation seems to start early in the first 1-2 weeks after implantation. The degree of fibrotization of the TFLE, starting with a thrombus, was reflected by the amount of compacted collagenous fibers and likewise largely independent from DT. TFLE thickness often reached several hundred micrometers. Calcifications were occasionally seen and appeared irregularly along the TFLE sheath. Leadless pacemaker systems have the advantage to overcome the problem with TFLEs but hold their own specific risks and limitations which are not fully known yet.

Comfrey (Symphytum spp.) as an alternative field crop contributing to closed agricultural cycles in chicken feeding.

Local cultivars of comfrey (Symphytum spp.) have been used to cover protein and mineral requirements of farm animals in low-input systems. Due to its known health-promoting (e.g. allantoin), but also anti-nutritive ingredients (e.g. pyrrolizidine alkaloids), multidisciplinary approaches are essential in order to quantify the nutritional value and the potential of its use in poultry and farm animals in terms of meeting animal needs, using local resources as well as remediating over-fertilized soils. Focusing on animal effects, here one-day old sexed Cobb500 broiler chickens were subjected to either a standard control diet or a standard diet supplemented with 4% dried comfrey leaves for 32 days. Performance traits indicate good acceptance of supplementation with comfrey leaves. Parameters for liver function, mineral homeostasis, bone mineral density as well as intestinal microanatomy revealed no signs of impairment. Quantified pyrrolizidine alkaloids were below the detection limit in liver and breast muscle (<5 µg/kg tissue). Comfrey supplemented male broiler chickens showed higher ash content in breast muscle and revealed altered gene expression profiles for metabolic pathways in blood cells. In healthy broiler chickens, the transcriptome analyses revealed no aberrations in the immune-related pathways due to comfrey supplementation. The results imply that the use of comfrey leaves in a high-performance broiler line seems feasible and offers the potential for closed nutrient cycles in site-adapted local agricultural systems. Further analyses need to focus on possible growth-promoting and health-improving components of comfrey and the safe use of chicken products for human consumption.

Prevalence and Mortality of Venous Leg Diseases of the Deep Veins: An Observational Cohort Study Based on German Health Claims Data.

This study estimates the prevalence and mortality of diseases of the deep veins of the legs such as deep vein thrombosis (DVT), postthrombotic syndrome (PTS), and venous leg ulceration (VLU). We used a random sample of 250 000 patients at age 50+ years of the register of the Allgemeine Ortskrankenkasse from 2004 to 2015. Selected manifestations of venous diseases assumed as risk factors for mortality were analyzed using Cox models while adjusting for various basic demographic and health characteristics. The prevalence in 2004 was 0.05% for DVT of the femoral veins, 0.50% for DVT of any deep veins, 0.86% for PTS, and 0.91% for VLU. The mortality rate in 2004 to 2015 was 20.40 deaths/100 person-years for DVT of the femoral veins, 10.69 for DVT of any deep veins, 4.34 for PTS, and 7.02 for VLU. The model revealed a 35% higher risk (p < .001) in patients with any DVT, an 88% higher mortality (p < .001) for femoral DVT, a 23% higher risk (p < .001) for VLU, and no health disadvantage in persons with PTS. Our study revealed an increased mortality for patients with VLU and DVT. Even after adjustment for embolic events and infections of the venous ulcers mortality remained significantly higher.

Morphometric Characterization of Human Coronary Veins and Subvenous Epicardial Adipose Tissue-Implications for Cardiac Resynchronization Therapy Leads.

Subvenous epicardial fat tissue (SEAT), which acts as an electrical insulation, and the venous diameter (VD) both constitute histomorphological challenges for optimal application and lead design in cardiac synchronization therapy (CRT). In this study, we characterized the morphology of human coronary veins to improve the technical design of future CRT systems and to optimize the application of CRT leads. We retrospectively analyzed data from cardiac computed tomography (CT) of 53 patients and did studies of 14 human hearts using the postmortem freeze section technique and micro CT. Morphometric parameters (tributary distances, offspring angles, luminal VD, and SEAT thickness) were assessed. The left posterior ventricular vein (VVSP) had a mean proximal VD of 4.0 ± 1.4 mm, the left marginal vein (VMS) of 3.2 ± 1.5 mm and the anterior interventricular vein (VIA) of 3.9 ± 1.3 mm. More distally (5 cm), VDs decreased to 2.4 ± 0.6 mm, 2.3 ± 0.7 mm, and 2.4 ± 0.6 mm, respectively. In their proximal portions (15 mm), veins possessed mean SEAT thicknesses of 3.2 ± 2.4 (VVSP), 3.4 ± 2.4 mm (VMS), and 4.2 ± 2.8 mm (VIA), respectively. More distally (20-70 mm), mean SEAT thicknesses decreased to alternating low levels of 1.3 ± 1.1 mm (VVSP), 1.7 ± 1.1 mm (VMS), and 4.3 ± 2.6 mm (VIA), respectively. In contrast to the VD, SEAT thicknesses alternated along the further distal vein course and did not display a continuous decrease. Besides the CRT responsiveness of different areas of the LV myocardium, SEAT is a relevant electrophysiological factor in CRT, potentially interfering with sensing and pacing. A sufficient VD is crucial for successful CRT lead placement. Measurements revealed a trend toward greater SEAT thickness for the VIA compared to VVSP and VMS, suggesting a superior signal-to-noise-ratio in VVSP and VMS.

The femoral vein diameter and its correlation with sex, age and body mass index - An anatomical parameter with clinical relevance.

BACKGROUND: The femoral vein diameter is a critical factor when assessing endoprosthetic valve size for the treatment of chronic venous insufficiency. To examine the previously stated correlation between body mass index and femoral vein diameter and to re-assess the anatomical and physiological demands for a valve implant for chronic venous insufficiency treatment, we measured the femoral vein diameter in 82 subjects. METHOD: Femoral vein diameters (164 legs) were measured with B-mode sonography both in supine position at rest and in upright position during Valsalva maneuver. RESULT: The mean femoral vein diameter differed significantly between supine position (13.6 ± 3.0 mm) and upright position (16.4 ± 2.6 mm). Males possessed a significant bigger diameter than females. A significant positive correlation between femoral vein diameter and body mass index was observed. CONCLUSION: Assuming an increased femoral vein diameter due to obesity would further impair valve functionality by increasing distance between both valve cusps. For the development of artificial venous valves, it is crucial to consider patient- and condition-dependent vein dilation.

Human Femoral Vein Diameter and Topography of Valves and Tributaries: A Post Mortem Analysis.

The femoral vein (FV) is a clinically important vessel. Failure of its valves can lead to chronic venous insufficiency (CVI) with severe manifestations such as painful ulcers. Although they are crucial for identifying suitable implant sites for therapeutic valves, studies on the topography of FV tributaries and valves are rare. Moreover, the femoral vein diameter (FVD) must be known to assess the morphometric requirements for valve implants. To reassess the anatomical requirements for valve implants, 155 FVs from 82 human corpses were examined. FVDs and tributary and valve topographies were assessed using a laboratory straightedge. The FVD increased from 6 mm in the distal femoropopliteal vein to 11 mm in the iliofemoral vein proximal to the saphenofemoral junction (SFJ). Diameters were significantly bigger in males than females. Height correlated positively with FVD. Distal to the SFJ, within a distance of 38 cm, one to eight valves were present. Up to two valves were present within 10 cm proximal to the SFJ. Individual tributary and valve topography must be considered to ensure appropriate design and successful implantation of a venous valve for CVI therapy in the FV. A suitable implant site would be proximal to the SFJ via an infrainguinal transfemoral access. Clin. Anat. 31:1065-1076, 2018. © 2018 Wiley Periodicals, Inc.

Fast and reliable dissection of porcine parathyroid glands - A protocol for molecular and histological analyses.

As calcium and phosphorus are of vital importance for life, physiological activity of the parathyroid glands (PTGs) is crucial to maintain mineral homeostasis and bone mineralization. However, PTG-specific molecular routes in response to environmental factors and intrinsic hormonal responses are not yet fully understood. Since nutrient requirements, pathophysiology and functional genomics of pigs are similar to those of humans, pigs might be a suitable model to study the holistic gene expression and physiological aspects of the parathyroid gland, which could be used in both animal sciences and biomedical research. However, due to their small size and hidden location, the dissection of the PTGs, particularly in pigs, is difficult. Therefore, a protocol for untrained dissectors has been established that allows a fast and reliable identification of the PTGs in domestic pigs. Based on their localization within the cranial thymus near the carotid bifurcation, sampling was verified by histological staining and mRNA expression pattern. Analyses revealed the prominence of parathyroid hormone (PTH)-producing chief cells. Moreover, the copy numbers of PTH differed substantially between the PTGs and their surrounding thymus tissue, as PTH was expressed virtually exclusively in the PTGs. The developed protocol will substantially facilitate a fast and reliable dissection of porcine PTGs which is essential for studies characterizing the molecular mechanisms of parathyroid glands, e.g. when applying new feeding strategies in pigs.

Neointimal fibrotic lead encapsulation - Clinical challenges and demands for implantable cardiac electronic devices.

Every tenth patient with a cardiac pacemaker or implantable cardioverter-defibrillator implanted is expected to have at least one lead problem in his lifetime. However, transvenous leads are often difficult to remove due to thrombotic obstruction or extensive neointimal fibrotic ingrowth. Despite its clinical significance, knowledge on lead-induced vascular fibrosis and neointimal lead encapsulation is sparse. Although leadless pacemakers are already available, their clinical operating range is limited. Therefore, lead/tissue interactions must be further improved in order to improve lead removals in particular. The published data on the coherences and issues related to lead associated vascular fibrosis and neointimal lead encapsulation are reviewed and discussed in this paper.

The First Organ-Based Ontology for Arthropods (Ontology of Arthropod Circulatory Systems - OArCS) and its Integration into a Novel Formalization Scheme for Morphological Descriptions.

Morphology, the oldest discipline in the biosciences, is currently experiencing a renaissance in the field of comparative phenomics. However, morphological/phenotypic research still suffers on various levels from a lack of standards. This shortcoming, first highlighted as the "linguistic problem of morphology", concerns the usage of terminology and also the need for formalization of morphological descriptions themselves, something of paramount importance not only to the field of morphology but also when it comes to the use of phenotypic data in systematics and evolutionary biology. We therefore argue, that for morphological descriptions, the basis of all systematic and evolutionary interpretations, ontologies need to be utilized which are based exclusively on structural qualities/properties and which in no case include statements about homology and/or function. Statements about homology and function constitute interpretations on a different or higher level. Based on these "anatomy ontologies", further ontological dimensions (e.g., referring to functional properties or homology) may be exerted for a broad use in evolutionary phenomics. To this end we present the first organ-based ontology for the most species-rich animal group, the Arthropoda. Our Ontology of Arthropod Circulatory Systems (OArCS) contains a comprehensive collection of 383 terms (i.e., labels) tied to 296 concepts (i.e., definitions) collected from the literature on phenotypic aspects of circulatory organ features in arthropods. All of the concepts used in OArCS are based exclusively on structural features, and in the context of the ontology are independent of homology and functional assumptions. We cannot rule out that in some cases, terms are used which in traditional usage and previous accounts might have implied homology and/or function (e.g. heart, sternal artery). Concepts are composed of descriptive elements that are used to classify observed instances into the organizational framework of the ontology. That is, descriptions in ontologies are only descriptions of individuals if they are necessary/and or sufficient representations of attributes (independently) observed and recorded for an individual. In addition, we here present for the first time an entirely new approach to formalizing phenotypic research, a semantic model for the description of a complex organ system in a highly disparate taxon, the arthropods. We demonstrate this with a formalized morphological description of the hemolymph vascular system in one specimen of the European garden spider Araneus diadematus. Our description targets five categories of descriptive statement: "position", "spatial relationships", "shape", "constituents", and "connections", as the corresponding formalizations constitute exemplary patterns useful not only when talking about the circulatory system, but also in descriptions in general. The downstream applications of computer-parsable morphological descriptions are widespread, with their core utility being the fact that they make it possible to compare collective description sets in computational time, that is, very quickly. Among other things, this facilitates the identification of phenotypic plasticity and variation when single individuals are compared, the identification of those traits which correlate between and within taxa, and the identification of links between morphological traits and genetic (using GO, Gene Ontology) or environmental (using ENVO, Environmental Ontology) factors. [Arthropoda; concept; function; hemolymph vascular system; homology; terminology.].

Functional transformation series and the evolutionary origin of novel forms: evidence from a remarkable termite defensive organ.

The origins of evolutionary novelties are often deeply puzzling. They are generally associated with new functions that were absent in ancestors. The new functional configuration should arise via intermediate stages without any loss of function or impediment to the whole organism during the transitions. Therefore, understanding of the functional configurations of transitional states can shed light on how novel forms arise. Here we infer the evolutionary origin of a highly specialized termite defensive organ "nasus" where different functions overlap in different structural configurations at intermediate evolutionary stages to ensure that each phase is functional. Soldiers of a nasutitermitine termite use reconfigured mandibular muscles to squirt a viscous secretion from a nozzle-like head projection (the nasus). This contrasts sharply with the primitive defensive strategy where mandibles are used to bite. MicroCT observations of soldiers of Nasutitermes takasagoensis and of species with the ancestral state (Hodotermopsis sjostedti, Embiratermes neotenicus) revealed three different yet fully functional configurations in the transition from ancestral to novel state: (i) elevated hydrostatic pressure induced by contraction of mandibular muscles when biting gently oozes secretion from a gland; (ii) direct pressure on an enlarged gland arises from expansion of the mandibular muscles when biting; (iii) squirting in a piston-like manner by an inflated gland enveloped by highly modified mandibular muscles. Even a structure as exotic as the nasus therefore appears to have evolved with no loss of function at any stage. Such a functional approach, holds much promise for understanding the evolutionary origin of seemingly preposterous novel forms.

Evolutionary morphology of the organ systems in squat lobsters and porcelain crabs (crustacea: Decapoda: Anomala): an insight into carcinization.

Porcelain crabs (Porcellanidae) are one of three taxa within anomuran crustaceans (Anomala) which possess a crab-like body form. Curiously, these three lineages evolved this shape independently from true crabs (Brachyura) in the course of the evolutionary process termed carcinization. The entire pleon in porcelain crabs is flexed under the cephalothorax and the carapace is approximately as broad as long. Despite their crab-like habitus, porcelain crabs are phylogenetically nested within squat lobsters (Munidopsidae, Munididae, Galatheidae). With a pleon which is only partly flexed under the cephalothorax and a cephalothorax which is longer than it is broad, squat lobsters represent morphologically intermediate forms between lobster-like and crab-like body shapes. Carcinization has so far mostly been studied with respect to outer morphology; however, it is evident that internal anatomical features are influenced through this change of body shape too. In this paper, the situation in Galatheoidea is elucidated by adding more taxa to existing descriptions of the hemolymph vascular systems and associated structures and organs. Micro-computer tomography and 3D reconstruction provide new insights. Autapomorphic states of various internal anatomical characters are present in nearly all the studied species, also reflecting some degree of anatomical disparity found within Galatheoidea. The ventral vessel system of porcelain crabs differs distinctly from that of squat lobsters. The differences in question are coherent (i.e. structural dependent) with morphological transformations in the integument, such as the shortening of the sternal plastron, which evolved in the course of carcinization. Shifts in the gonads and the pleonal neuromeres are coherent with the loss of the caridoid escape reaction, which in turn is a consequence of carcinization. The arterial transformations, however, are minor compared to other instances of carcinization in anomuran crustaceans since the last common ancestor of squat lobsters and porcelain crabs was already "half carcinized".

Evolutionary morphology of the hemolymph vascular system in hermit and king crabs (Crustacea: Decapoda: Anomala).

The morphological transformation of hermit crabs into crab-like king crabs in the evolution of decapod crustaceans represents a remarkable case of carcinization or evolutionary shaping into a crab-like form. In this study, we focus on internal organs such as the hemolymph vascular system and adjacent anatomical structures of several Recent hermit crab (Paguridae) and king crab (Lithodidae) species. There are various correspondences in the morphology of the arterial systems in the dorsal cephalothorax of the two taxa, especially with regard to the anterior aorta, anterior lateral arteries, and hepatic arteries. In the pleon, the posterior aorta in both taxa displays a proximal bifurcation and follows an asymmetrical course. The ventral vessel system, on the other hand, which mainly supplies the limbs, differs significantly between the taxa, with pagurids displaying the plesiomorphic condition. The pattern of the ventral vessel system in Lithodidae is influenced by morphological transformations of integumental structures during carcinization. One of these transformations was the broadening of the sternites, which resulted in a widening of the space between the endosternites. In addition, changes in the morphology of the endophragmal skeleton in Lithodidae led to an increase in the potential for intraspecific variability and interspecific variation in the arterial branching pattern.