It is always interesting when a seemingly minor study actually contributes significantly to our understanding of a key concept in animal biology. A key concept in the field of insect respiratory biology is understanding the structure, function and formation of the insect tracheal system. Not using lungs (for gas exchange) nor blood circulation (for oxygen and carbon dioxide transport and exchange) is a characteristic that sets insects apart from vertebrates and even some invertebrates. The insect tracheal system consists of a ramifying system of successively finer tubes – entering the insect body at pairs of openings (spiracles) on certain body segments, infiltrating tissues and terminating in blind-ending tubes between or even within cells. Air moves down these tubes, called tracheae, transporting respiratory gasses toward the very fine blind ends, the tracheoles, where aerobic gas exchange occurs. Thus, the tracheal system dually transports and exchanges respiratory gasses directly between tissues and air.
A uniquely curious thing about the tracheal system is that it is part of the insect cuticle, or exoskeleton. Consequently, developmentally during each moult, not only is the external skeleton being replaced but also the respiratory system. Prior to moulting, the existing tracheal cuticle layers (also the exoskeleton) detach from the epithelial layer, a moulting fluid – containing enzymes that partially break down old cuticle – is secreted between the old cuticle and epithelial cells, new cuticle lines the epithelium, and the old cuticle is discarded along with old tracheal cuticular tubes withdrawing from the newer larger tubular system.
Three common ‘understandings’ distinguished tracheae from tracheoles: (1) tracheae, similar to exoskeletal cuticle, consist of an epicuticle layer and a chitin–protein matrix layer while the smallest blind-ending tracheoles lack the chitin–protein layer; (2) the tracheolar epicuticle remains intact during moulting (no detachment); and (3) the tracheoles do not have taenidia (the structural support rings similar to vacuum cleaner tubes and human windpipes). Earlier studies on true bugs reinforced the idea that the tracheolar epicuticle is not shed during moulting. However, subsequent studies challenged these ‘understandings’. Studies on moths showed tracheolar epicuticle being shed after the third larval instar whereas in flies tracheolar epicuticle is shed during early moults but remains intact later on. These data introduced ambivalence into how to define tracheoles, at least in a developmental context.
Edward Snelling and colleagues, from the University of Adelaide, prepared several juvenile third-instar locusts, Locusta migratoria, for light and electron microscopy. Unbeknown to them, one individual, giving no external indications, entered the early stages of moulting. This was clear in the way the cuticle layers of the tracheal system in the jumping leg detached and retracted from newly laid-down layers. What was most significant in this serendipitous observation was that the epicuticular layer of the terminal ends – the tracheoles – detached and withdrew from the newly formed layers and showed microtaenidia analogous to tracheae. This discovery, in effect a fortuitous study with an N=1, provided confirmatory evidence, along with supporting information from recent studies on tracheole structure and development, and it improves our understanding of the insect respiratory system by allowing for a more functional definition of tracheoles: tracheoles are the blind-ending terminal tubes, with a <2 μm diameter, arising along cytoplasmic extensions from terminal tracheolar cells, differing structurally from tracheae, and provide the primary sites of gas exchange in the insect tracheal system.