Background
Live imaging of whole invertebrates can be accomplished with X-ray micro-computed tomography (micro-CT) at 10-100 μm spatial resolution. However, image quality could be compromised by the movement of live subjects, producing image artefacts. We tested the feasibility of using CO2 gas to induce temporary full-immobilization of sufficient duration to image live insects based on their ability to tolerate hypoxic conditions. Additionally, we investigated the effects of these prolonged hypoxic conditions on several life history traits of a lepidopteran species.
Methods
Live Colorado potato beetle (CPB) and true armyworm (TAW) adults were immobilized under a constant CO2 gas flow (0.5 L/min), and scanned using micro-CT (80 kVp; 450 μA). An L8 (24) orthogonal array (OA) was used to evaluate the effects of prolonged CO2-induced anesthesia on the recovery, longevity, and incidence of mating of TAW adults. The variable factors were age (immature and mature), sex (female and male), exposure time (3 and 7 h), and exposure regime (single and repeated).
Results
With this method, successful 3D reconstruction and visualizations of CPB and TAW adults were produced at 20 micron voxel spacing at an acceptable radiation dose and image noise level. From the inverse-square relationship found between the radiation doses and image noise levels, the optimal scanning protocol produced an entrance dose of 6.2 ± 0.04 Gy with images of 129.6 ± 5.1 HU noise level during a 2.7 h scan.
Independent OA experiments indicated that CO2 gas did not result in death of exposed TAW adults, except when older males were exposed for longer durations. Exposure time and sex were more influential factors affecting recovery, longevity, and mating success than age and exposure regime following CO2 exposure.
Conclusion
We have demonstrated that using CO2 gas during micro-CT imaging effectively induces safe, repeatable, whole-body, and temporary immobilization of live insects for 3D visualizations without motion artefacts. Moreover, we have shown that exposed TAW individuals made a full recovery with very little impact on subsequent longevity, and mating success post hypoxia. This method is applicable to other imaging modalities and could be used for routine exploratory and time-course studies, for repeated scanning of live and intact individuals.
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Fig. 2 3D-CT reconstruction of live anesthetized 1 week-old female (♀) and male (♂) Colorado potato beetle adults. Sagittal views of (a) 3D-volume of insect body exoskeleton at 20 μm isotropic voxel spacing; (b) 3D-MPR (multi-planar reformatted) views of the insect body illustrating internal structures; and (c) insect body depicting the tracheal system. These 3D images for Protocol 1 (highest noise; lowest X-ray dose), 3 (optimal), and 5 (lowest noise; highest X-ray dose) allow qualitative comparison of image quality (improving from left to right), noise (decreasing from left to right), and dose (increasing from left to right; see Additional files 7: Videos S1, Additional files 8: Videos S1 and Additional files 9: Videos S2, Additional files 10: Videos S2 for female and male adults, respectively). While the images from Protocol 5 are of the highest quality, the conspicuity of internal structures from the Protocol 3 images are sufficient and spare the individual the higher anesthetic and radiation dose |
