Anisotropic shrinkage of insect air sacs revealed in vivo by X-ray microtomography

Abstract

Air sacs are thought to be the bellows for insect respiration. However, their exact mechanism of action as a bellows remains unclear. A direct way to investigate this problem is in vivo observation of the changes in their three-dimensional structures. Therefore, four-dimensional X-ray phase contrast microtomography is employed to solve this puzzle. Quantitative analysis of three-dimensional image series reveals that the compression of the air sac during respiration in bell crickets exhibits obvious anisotropic characteristics both longitudinally and transversely. Volumetric changes of the tracheal trunks in the prothorax further strengthen the evidence of this finding. As a result, we conclude that the shrinkage and expansion of the insect air sac is anisotropic, contrary to the hypothesis of isotropy, thereby providing new knowledge for further research on the insect respiratory system.

Figure 1. Picture of the bell cricket and its corresponding in vivo images.

(a) The photograph of the bell cricket used in experiments. The region of interest (ROI) for the imaging is noted with a blue rectangle frame; length of the scale bar is 2 mm. (b) The longitudinal section in the middle of the ROI reconstructed from three-dimensional microtomography, where the corresponding structures in (a) are labelled; length of the scale bar is 500 μm. (c) The tracheae extracted from the ROI, with the back of the bell cricket to the observers; length of the scale bar is 600 μm. (d) The air sac extracted individually from (c). A Z-axis is given to locate the position of each slice; length of the scale bar is 380 μm. 1—antennae; 2—head; 3—legs on the prothorax; 4—wings; 5—cerci; 6—air sac; 7—tracheal trunk 1 in the prothorax; 8—tracheal trunk 2 in the prothorax; 9—tracheal trunk 3 in the prothorax; 10—tracheal trunk 4 in the prothorax; 11—tracheae in the head; 12—the 110th slice of the air sac; 13—the 180th slice of the air sac; and 14—the 290th slice of the air sac.

Figure 2. Quantitative volumetric changes.

(a) Volume of the air sac as a function of time. In the first few seconds, the volumetric changes show an erratic pattern. Next, the volumetric changes come into a steady state and represent a periodic pattern within the seven full periods captured. The periods have small fluctuations with an average of 8.7 s. (b) Volumes of tracheal trunk 1–4 in the prothorax as a function of time. The volume of trachea 1 has a periodic change with a period of 11.5 s, while others show irregular change.

Figure 3. The longitudinal respiratory pattern of the air sac.

(a) The changing rate of every cross-sectional area of the air sac. Each pixel in this image gives the changing rate of the specified cross-sectional area at a specified time point. The X-axis represents the time, and the Y-axis represents the slice number of the air sac. The colour represents the changing rate of the cross-sectional area. (b) The segmented volumes of the air sac as a function of time. The volumetric change of slices 80–140 is in sinusoidal form, with an average period of 8.5 s. The volumetric change of slices 150–210 reaches a local minimum with a period of 11.8 s, and the volumetric change of slices 260–320 reaches a local minimum with a period of 22.5 s. The volumes between each two local minima change slightly.

Figure 4. The transverse velocity fields at the 110th, 180th and 290th slices of the air sac at the moment of the 25th second from the start of the experiment.

The maximum black regions in the middle of these three slices are the transverse sections of the air sac. The red arrow represents the direction of motion; the length represents the value of speed. (a) The transverse velocity fields of the 110th slice. The velocity vectors at the top left corner of the air sac show an outward trend, while velocity vectors at the bottom right corner are nearly zero. The other portions of this section show an expanding process with moderate speed. (b) The transverse velocity fields of the 180th slice. The compression portion is located at the bottom right corner, and the inflation portion is located at the top right corner. Other velocity vectors are nearly zero. (c) The transverse velocity fields of the 290th slice. The left edge of the air sac exhibits a shrinking trend, while other portions are nearly still.