The Last Breath: A μCT-based method for investigating the tracheal system in Hexapoda.

In recent years, μCT-based studies of the insect tracheal system have become an increasingly important area of research. Nevertheless, the methods proposed in previous research for investigating the respiratory system in the three-dimensional space were described and tested based on a relatively small number of specimens. Additionally, the individuals studied in all these cases represented only a single post-embryonic stadium - pupa or imago - of a particular insect species. Therefore, in the current situation it is difficult to predict the reliability and possible limitations of these methods. To address this problem we conducted a methodological study, during which we used 65 individuals representing larvae, pupae and imagines of the mealworm beetle (Tenebrio molitor). In addition to the protocol previously described, which implicated freezing as a killing technique, we also tested a novel one, which was based on ethyl acetate fumigation of the specimens studied. We included step-by-step guides for the manual and semiautomatic approaches in order to facilitate the digital visualization of the tracheal system. Our investigations enabled us to generate multiple models of the tracheal system of all post-embryonic stages of the mealworm beetle. The methods used proved to be minimally invasive, thus allowing for the application of post-scanning manipulations, such as drying with critical point dryer (CPD). This approach enabled us to merge different three-dimensional models into a single picture and analyse the relationship of the tracheal system with other tissues (e.g., muscles, nervous system). We comprehensively discuss the advantages and possible limitations of the tested methods and provide practical suggestions for conducting the analyses on a wider scale. The visualizations presented in this publication are the first three-dimensional models of the respiratory system using a representative of the extremely diverse order Coleoptera.