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.

Multielectrode array use in insect auditory neuroscience to unravel the spatio-temporal response pattern in the prothoracic ganglion of Mecopoda elongata.

Mechanoreceptors in hearing organs transduce sound-induced mechanical responses into neuronal signals, which are further processed and forwarded to the brain along a chain of neurons in the auditory pathway. Bushcrickets (katydids) have their ears in the front leg tibia, and the first synaptic integration of sound-induced neuronal signals takes place in the primary auditory neuropil of the prothoracic ganglion. By combining intracellular recordings of the receptor activity in the ear, extracellular multichannel array recordings on top of the prothoracic ganglion and hook electrode recordings at the neck connective, we mapped the timing of neuronal responses to tonal sound stimuli along the auditory pathway from the ears towards the brain. The use of the multielectrode array allows the observation of spatio-temporal patterns of neuronal responses within the prothoracic ganglion. By eliminating the sensory input from one ear, we investigated the impact of contralateral projecting interneurons in the prothoracic ganglion and added to previous research on the functional importance of contralateral inhibition for binaural processing. Furthermore, our data analysis demonstrates changes in the signal integration processes at the synaptic level indicated by a long-lasting increase in the local field potential amplitude. We hypothesize that this persistent increase of the local field potential amplitude is important for the processing of complex signals, such as the conspecific song.

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.

Micromotion measurement at the interfaces of cemented tibial endoprosthetic replacements: A new standardized in vitro model using open-cell rigid foam.

Early aseptic loosening following primary total knee arthroplasty related to several factors might appear at the interface implant-cement or cement-bone. A standardized in vitro model might provide information on the relevance of single variable parameter of cementation including technique and cement respectively bone structure on fixation strength. Micromotion measurement using different directions of load should detect the primary stability of the interfaces. An open-cell rigid foam model was used for cementation of PFC-Sigma tibial trays with Palacos®. Pins were applied to the model for continuous non-destructive measurement. Relative micromotions for rotation, valgus-varus and extension flexion stress were detected at the interfaces as well as cement penetration was measured. The reproducibility of the measurement could be shown for all interfaces in extension-flexion movements. For rotation a negative trend was shown for the interface cement-prosthesis and cement-bone concerning varus-valgus stress reflecting varying surgical cementation technique. More micromotion related to extension-flexion force might reflect the design of the implant. Measurement of relative micromotion and cement distribution appear accurate to detect small differences of movement at different interfaces of cemented tibial implants and the results are reproducible for most parameter. An increased number of specimens should achieve statistical relevance for all measurements.

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.

Tuned vibration modes in a miniature hearing organ: Insights from the bushcricket.

Bushcrickets (katydids) rely on only 20 to 120 sensory units located in their forelegs to sense sound. Situated in tiny hearing organs less than 1 mm long (40× shorter than the human cochlea), they cover a wide frequency range from 1 kHz up to ultrasounds, in tonotopic order. The underlying mechanisms of this miniaturized frequency-place map are unknown. Sensory dendrites in the hearing organ (crista acustica [CA]) are hypothesized to stretch, thereby driving mechanostransduction and frequency tuning. However, this has not been experimentally confirmed. Using optical coherence tomography (OCT) vibrometry, we measured the relative motion of structures within and adjacent to the CA of the bushcricket Mecopoda elongata We found different modes of nanovibration in the CA that have not been previously described. The two tympana and the adjacent septum of the foreleg that enclose the CA were recorded simultaneously, revealing an antiphasic lever motion strikingly reminiscent of vertebrate middle ears. Over the entire length of the CA, we were able to separate and compare vibrations of the top (cap cells) and base (dorsal wall) of the sensory tissue. The tuning of these two structures, only 15 to 60 µm (micrometer) apart, differed systematically in sharpness and best frequency, revealing a tuned periodic deformation of the CA. The relative motion of the two structures, a potential drive of transduction, demonstrated sharper tuning than either of them. The micromechanical complexity indicates that the bushcricket ear invokes multiple degrees of freedom to achieve frequency separation with a limited number of sensory cells.

Comparative micromechanics of bushcricket ears with and without a specialized auditory fovea region in the crista acustica.

In some insects and vertebrate species, the specific enlargement of sensory cell epithelium facilitates the perception of particular behaviourally relevant signals. The insect auditory fovea in the ear of the bushcricket Ancylecha fenestrata (Tettigoniidae: Phaneropterinae) is an example of such an expansion of sensory epithelium. Bushcricket ears developed in convergent evolution anatomical and functional similarities to mammal ears, such as travelling waves and auditory foveae, to process information by sound. As in vertebrate ears, sound induces a motion of this insect hearing organ (crista acustica), which can be characterized by its amplitude and phase response. However, detailed micromechanics in this bushcricket ear with an auditory fovea are yet unknown. Here, we fill this gap in knowledge for bushcricket, by analysing and comparing the ear micromechanics in Ancylecha fenestrata and a bushcricket species without auditory fovea (Mecopoda elongata, Tettigoniidae: Mecopodinae) using laser-Doppler vibrometry. We found that the increased size of the crista acustica, expanded by a foveal region in A. fenestrata, leads to higher mechanical amplitudes and longer phase delays in A. fenestrata male ears. Furthermore, area under curve analyses of the organ oscillations reveal that more sensory units are activated by the same stimuli in the males of the auditory fovea-possessing species A. fenestrata. The measured increase of phase delay in the region of the auditory fovea supports the conclusion that tilting of the transduction site is important for the effective opening of the involved transduction channels. Our detailed analysis of sound-induced micromechanics in this bushcricket ear demonstrates that an increase of sensory epithelium with foveal characteristics can enhance signal detection and may also improve the neuronal encoding.

Functional basis of the sexual dimorphism in the auditory fovea of the duetting bushcricket Ancylecha fenestrata.

From mammals to insects, acoustic communication is in many species crucial for successful reproduction. In the duetting bushcricket Ancylecha fenestrata, the mutual acoustic communication between males and females is asymmetrical. We investigated how those signalling disparities are reflected by sexual dimorphism of their ears. Both sexes have tympanic ears in their forelegs, but male ears possess a significantly longer crista acustica containing 35% more scolopidia. With more sensory cells to cover a similar hearing range, the male hearing organ shows a significantly expanded auditory fovea that is tuned to the dominant frequency of the female reply to facilitate phonotactic mate finding. This sex-specific auditory fovea is demonstrated in the mechanical and neuronal responses along the tonotopically organized crista acustica by laservibrometric and electrophysiological frequency mapping, respectively. Morphometric analysis of the crista acustica revealed an interrupted gradient in organ height solely within this auditory fovea region, whereas all other anatomical parameters decrease continuously from proximal to distal. Combining behavioural, anatomical, biomechanical and neurophysiological information, we demonstrate evidence of a pronounced auditory fovea as a sex-specific adaptation of an insect hearing organ for intraspecific acoustic communication.

Auditory fovea in the ear of a duetting katydid shows male-specific adaptation to the female call.

Convergent evolution has led to surprising functional and mechanistic similarities between the vertebrate cochlea and some katydid ears [1,2]. Here we report on an 'auditory fovea' (Figure 1A) in the duetting katydid Ancylecha fenestrata (Tettigoniidae). The auditory fovea is a specialized inner-ear region with a disproportionate number of receptor cells tuned to a narrow frequency range, and has been described in the cochlea of some vertebrates, such as bats and mole rats [3,4]. In tonotopically organized ears, the location in the hearing organ of the optimal neuronal response to a tone changes gradually with the frequency of the stimulation tone. However, in the ears of A. fenestrata, the sensory cells in the auditory fovea are tuned to the dominant frequency of the female call; this area of the hearing organ is extensively expanded in males to provide an overrepresentation of this behaviorally important auditory input. Vertebrates developed an auditory fovea for improved prey or predator detection. In A. fenestrata, however, the foveal region facilitates acoustic pair finding, and the sexual dimorphism of sound-producing and hearing organs reflects the asymmetry in the mutual communication system between the sexes (Figures 1B, S1).

Gating of Acoustic Transducer Channels Is Shaped by Biomechanical Filter Processes.

Mechanoelectrical transduction of acoustic signals is the fundamental process for hearing in all ears across the animal kingdom. Here, we performed in vivo laser-vibrometric and electrophysiological measurements at the transduction site in an insect ear (Mecopoda elongata) to relate the biomechanical tonotopy along the hearing organ to the frequency tuning of the corresponding sensory cells. Our mechanical and electrophysiological map revealed a biomechanical filter process that considerably sharpens the neuronal response. We demonstrate that the channel gating, which acts on chordotonal stretch receptor neurons, is based on a mechanical directionality of the sound-induced motion. Further, anatomical studies of the transduction site support our finding of a stimulus-relevant tilt. In conclusion, we were able to show, in an insect ear, that directionality of channel gating considerably sharpens the neuronal frequency selectivity at the peripheral level and have identified a mechanism that enhances frequency discrimination in tonotopically organized ears.

Position-dependent hearing in three species of bushcrickets (Tettigoniidae, Orthoptera).

A primary task of auditory systems is the localization of sound sources in space. Sound source localization in azimuth is usually based on temporal or intensity differences of sounds between the bilaterally arranged ears. In mammals, localization in elevation is possible by transfer functions at the ear, especially the pinnae. Although insects are able to locate sound sources, little attention is given to the mechanisms of acoustic orientation to elevated positions. Here we comparatively analyse the peripheral hearing thresholds of three species of bushcrickets in respect to sound source positions in space. The hearing thresholds across frequencies depend on the location of a sound source in the three-dimensional hearing space in front of the animal. Thresholds differ for different azimuthal positions and for different positions in elevation. This position-dependent frequency tuning is species specific. Largest differences in thresholds between positions are found in Ancylecha fenestrata. Correspondingly, A. fenestrata has a rather complex ear morphology including cuticular folds covering the anterior tympanal membrane. The position-dependent tuning might contribute to sound source localization in the habitats. Acoustic orientation might be a selective factor for the evolution of morphological structures at the bushcricket ear and, speculatively, even for frequency fractioning in the ear.