In Vivo Time- Resolved Microtomography Reveals the Mechanics of the Blowfly Flight Motor

Dipteran flies are amongst the smallest and most agile of flying animals. Their wings are driven indirectly by large power muscles, which cause cyclical deformations of the thorax that are amplified through the intricate wing hinge. Asymmetric flight manoeuvres are controlled by 13 pairs of steering muscles acting directly on the wing articulations. Collectively the steering muscles account for <3% of total flight muscle mass, raising the question of how they can modulate the vastly greater output of the power muscles during manoeuvres. Here we present the results of a synchrotron-based study performing micrometre-resolution, time-resolved microtomography on the 145 Hz wingbeat of blowflies. These data represent the first four-dimensional visualizations of an organism's internal movements on sub-millisecond and micrometre scales. This technique allows us to visualize and measure the three-dimensional movements of five of the largest steering muscles, and to place these in the context of the deforming thoracic mechanism that the muscles actuate. Our visualizations show that the steering muscles operate through a diverse range of nonlinear mechanisms, revealing several unexpected features that could not have been identified using any other technique. The tendons of some steering muscles buckle on every wingbeat to accommodate high amplitude movements of the wing hinge. Other steering muscles absorb kinetic energy from an oscillating control linkage, which rotates at low wingbeat amplitude but translates at high wingbeat amplitude. Kinetic energy is distributed differently in these two modes of oscillation, which may play a role in asymmetric power management during flight control. Structural flexibility is known to be important to the aerodynamic efficiency of insect wings, and to the function of their indirect power muscles. We show that it is integral also to the operation of the steering muscles, and so to the functional flexibility of the insect flight motor

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography

© The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The attached file is the published version of the article

The cave-like sense organ in the antennae of Triatominae bugs

In the second segment of the antennae of haematophagous reduviids an unusual cave-like organ is found the function os which was investigated in Triatoma infestans. the morphology of the organ makes it difficult to ascribe it to a mechno- or chemoreceptive function, but shows some characteristics shared with thermoreceptors of other animals. The electrical activity of sense cells was recorded in the presence of stimuli that evoke behavioural responses in this species, i.e. warm, CO2, lactic and butyric acids at different concentrations. The three compounds tested failed to evoke a response at all concentrations assayed. Only thermal stimulation evinced a clear modification in the electrical activity of the sense cells.Both the morphological and electrophysiological findings support a thermoreceptive finding, habitat selection and circadian synchronization

A quantitative comparison of micro-CT preparations in Dipteran flies

X-ray-based 3D-imaging techniques have gained fundamental significance in research areas ranging from taxonomy to bioengineering. There is demand for the characterisation of species-specific morphological adaptations, micro-CT (μCT) being the method of choice in small-scale animals. This has driven the development of suitable staining techniques to improve absorption-based tissue contrast. A quantitative account on the limits of current staining protocols for preparing μCT specimen, however, is still missing. Here we present a study that quantifies results obtained by combining a variety of different contrast agents and fixative treatments that provides general guidance for μCT applications, particularly suitable for insect species. Using a blowfly model system (Calliphora), we enhanced effective spatial resolution and, in particular, optimised tissue contrast enabling semi-automated segmentation of soft and hard tissue from μCT data. We introduce a novel probabilistic measure of the contrast between tissues: PTC. Our results show that a strong iodine solution provides the greatest overall increase in tissue contrast, however phosphotungstic acid offers better inter-tissue discriminability. We further show that using paraformaldehyde as a fixative as opposed to ethanol, slows down the uptake of a staining solution by approximately a factor of two

Four-dimensional in vivo X-ray microscopy with projection-guided gating

Visualizing fast micrometer scale internal movements of small animals is a key challenge for functional anatomy, physiology and biomechanics. We combine phase contrast tomographic microscopy (down to 3.3 μm voxel size) with retrospective, projection-based gating (in the order of hundreds of microseconds) to improve the spatiotemporal resolution by an order of magnitude over previous studies. We demonstrate our method by visualizing 20 three-dimensional snapshots through the 150 Hz oscillations of the blowfly flight motor.ISSN:2045-232

Edge detail of two parts of the thorax.

<p>(A) Tomogram showing transverse section of the thorax, with the mount visible in the upper right of the image. The blue line cuts through the scutum, which is a rigid part of the thorax that did not move measurably during recordings. The red line cuts through the steering muscles, which oscillate at wingbeat frequency. (B) Pixel intensities along the two lines indicated in (A). Edge sharpness, as measured by the steepness of the change in pixel intensity along each line, is essentially identical for the scutum and the steering muscles. This indicates that the position of the steering muscles must have been consistent between wingbeats, at the phase of the wingbeat shown here, which indicates that the steering muscle kinematics did not vary measurably between wingbeats.</p

Statistical analysis of steering muscle asymmetries.

<p>95% confidence intervals computed for: (A) amplitude of oscillatory muscle strain, (B) phase of oscillatory muscle strain, relative to start of the downstroke; (C) mean muscle strain. Vertical bars denote 95% confidence intervals; points denote actual parameter estimate. Non-overlapping 95% confidence intervals are starred to indicate the statistically significant differences between the high-amplitude (blue) and low-amplitude (red) wings.</p

Overview.

<p>(A) Mean (red/blue lines) and standard deviation (red/blue shading) of wing tip position through all of the wingbeats of all four flies, showing differences in wing tip path between the left, high-amplitude (blue) and right, low-amplitude (red) wings. The arrows indicate the direction of the wings' movement. (B) External visualization of the thorax, covering the region outlined in (A). (C) Cutaway visualization of the thorax showing the five steering muscles analysed (green to blue) and the power muscles (yellow to red). <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001823#pbio.1001823.s001" target="_blank">Movie S1</a> provides an animated overview of the movements of these muscles (view <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001823#pbio.1001823.s001" target="_blank">Movie S1 </a><a href="http://youtu.be/P6lBkK3J9wg" target="_blank">here</a>).</p

Buckling of the <i>I1</i> tendon.

<p>(A, B) Visualizations of the <i>I1</i> muscle at the start and end of the downstroke, respectively. The tendon is buckled on both wings at the start of the downstroke (A) but has been pulled straight by the end of the downstroke (B). Each panel compares the state of <i>I1</i> on the high-amplitude (blue) and low-amplitude (red) wing. (C) effective strain measured along the straight line joining the attachment points of <i>I1</i> (dashed line). Comparing the amplitude of this effective strain with the amplitude of the actual <i>I1</i> muscle strain in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001823#pbio-1001823-g006" target="_blank">Figure 6F</a> shows that the buckling tendon accommodates a 4-fold enhancement in the range of movement of the first axillary sclerite on the high-amplitude wing.</p

Measurements of muscle strains.

<p>(A) Three-dimensional surface rendering showing the internal view of the left steering muscles (high-amplitude wing). The steering muscles are viewed from the inside of the thorax looking out toward the wing hinge. The green circles indicate the endpoints of the muscles that were tracked. The blue circle shows the ventral base of <i>b2</i>, which is hidden from view behind the <i>I1</i> and <i>III1</i> muscles. A clipping plane was used to remove these muscles so that the base of <i>b2</i> was visible and could be tracked. (B) Schematic showing approximate shape of the five steering muscles (black lines) and the lines along which the muscle lengths were calculated (red lines). The grey shaded regions of <i>I1</i> and <i>b3</i> show where 3D skeletonization was used to find the centre line of the tendons to take buckling into account. (C) Diagram showing the movements of the endpoints of the steering muscles for the high-amplitude (blue orbits) and low-amplitude (red orbits) wings, averaged across flies. The view shown here corresponds to that in (C), with data for the other wing mirrored about the sagittal plane and overlain. The schematic representations of the muscles (shaded grey) and tendons (black lines) indicate the mean posture of the muscles at the start of the downstroke. <i>b</i>., basalare sclerite (filled black).</p