An enteric ultrastructural surface atlas of the model insect Manducasexta.

The tobacco hornworm is a laboratory model that is particularly suitable for analyzing gut inflammation, but a physiological reference standard is currently unavailable. Here, we present a surface atlas of the healthy hornworm gut generated by scanning electron microscopy and nano-computed tomography. This comprehensive overview of the gut surface reveals morphological differences between the anterior, middle, and posterior midgut, allowing the screening of aberrant gut phenotypes while accommodating normal physiological variations. We estimated a total resorptive midgut surface of 0.42 m2 for L5d6 larvae, revealing its remarkable size. Our data will support allometric scaling and dose conversion from Manduca sexta to mammals in preclinical research, embracing the 3R principles. We also observed non-uniform gut colonization by enterococci, characterized by dense biofilms in the pyloric cone and downstream of the pylorus associated with pore and spine structures in the hindgut intima, indicating a putative immunosurveillance function in the lepidopteran hindgut.

Evaluation of Mechanical Versus Manual Root Canal Preparation in Primary Molars-A Comparative In Vitro Study.

The endodontic treatment of primary teeth is to maintain the function of the tooth free of symptoms until its physiological exfoliation. A critical factor for success is how quickly and effectively the root canal preparation can be performed. Therefore, the aim of this comparative in vitro study was to analyze the efficiency of two mechanical root canal preparation systems FM (FlexMaster) and HF (HyFlex EDM) to manual KF (K-file) on extracted primary molars. A total of 45 teeth were divided into three groups (n = 15): KF (#15-35), FM (04#30) and HF (25/~ OneFile). Root canal preparation was performed, and the preparation time was measured. All root canals were non-destructively analyzed by micro-computed tomography in the cervical, middle and apical thirds before and after preparation with regard to the parameters of canal transport (in µm) and centering ratio (0-1). Statistical analysis was performed at a 5% significance level using non-parametric tests. HF caused the lowest canal transport in the apical third (p = 0.008). The centering ratio value of HF was significantly higher in the middle third of the root canals than in the other two groups (p < 0.01). The mean instrumentation time was significantly higher for KF (6.67 min) than for FM (4.69 min) and HF (4.03 min, p < 0.01). HF can be recommended for primary molar root canal treatment.

Dextran sulfate sodium and uracil induce inflammatory effects and disrupt the chitinous peritrophic matrix in the midgut of Tribolium castaneum.

Dextran sulfate sodium is used in inflammatory bowel disease (IBD) mice models to trigger chronic intestinal inflammation. In this study, we have analyzed DSS effects in the genetic model and pest beetle, Tribolium castaneum, which can be easily and cost-effectively cultivated and examined in very large quantities compensating for individual variations. We fed the larvae with DSS and uracil, which is known to induce the production of reactive oxygen species by activating DUOX, a member of the NADPH oxidase family. Both chemicals induced IBD-like phenotypes, including impaired growth and development, midgut thickening, epithelial swelling, and a loss of epithelial barrier function. RNAi mediated knockdown of DUOX expression enhanced the effects of DSS and uracil on mortality. Finally, we showed that both treatments result in an altered activity of the intestinal microbiome, similar as observed in IBD patients. Our findings suggest that both chemicals impair the epithelial barrier by increasing the permeability of the peritrophic matrix. The loss of the barrier function may facilitate the entry of midgut bacteria triggering innate immune responses that also affect the intestinal microbiome. As the observed effects resemble those induced by DSS treatment in mice, T. castaneum might be suitable high-throughput invertebrate model for IBD research and preclinical studies.

Biodegradable polyphosphoester micelles act as both background-free 31P magnetic resonance imaging agents and drug nanocarriers.

In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, assembling polymers into colloids, and tailoring the polymers' microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta. Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications.

A quantitative micro-tomographic gut atlas of the lepidopteran model insect Manduca sexta.

The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

Analysis of fixation materials in micro-CT: It doesn't always have to be styrofoam.

Good fixation of filigree specimens for micro-CT examinations is often a challenge. Movement artefacts, over-radiation or even crushing of the specimen can easily occur. Since different specimens have different requirements, we scanned, analysed and compared 19 possible fixation materials under the same conditions in the micro-CT. We focused on radiodensity, porosity and reversibility of these fixation materials. Furthermore, we have made sure that all materials are cheap and easily available. The scans were performed with a SkyScan 1173 micro-CT. All dry fixation materials tested were punched into 5 mm diameter cylinders and clamped into 0.2 ml reaction vessels. A voxel size of 5.33 µm was achieved in a 180° scan in 0.3° steps. Ideally, fixation materials should not be visible in the reconstructed image, i.e., barely binarised. Besides common micro-CT fixation materials such as styrofoam (-935 Hounsfield Units) or Basotect foam (-943 Hounsfield Units), polyethylene air cushions (-944 Hounsfield Units), Micropor foam (-926 Hounsfield Units) and polyurethane foam, (-960 Hounsfield Units to -470 Hounsfield Units) have proved to be attractive alternatives. Furthermore, more radiopaque materials such as paraffin wax granulate (-640 Hounsfield Units) and epoxy resin (-190 Hounsfield Units) are also suitable as fixation materials. These materials often can be removed in the reconstructed image by segmentation. Sample fixations in the studies of recent years are almost all limited to fixation in Parafilm, Styrofoam, or Basotect foam if the fixation type is mentioned at all. However, these are not always useful, as styrofoam, for example, dissolves in some common media such as methylsalicylate. We show that micro-CT laboratories should be equipped with various fixation materials to achieve high-level image quality.

High-throughput screening of caterpillars as a platform to study host-microbe interactions and enteric immunity.

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

Dynamic monitoring of vital functions and tissue re-organization in Saturnia pavonia (Lepidoptera, Saturniidae) during final metamorphosis by non-invasive MRI.

Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis.