Elsevier

Journal of Insect Physiology

Volume 57, Issue 9, September 2011, Pages 1312-1316
Journal of Insect Physiology

Moulting of insect tracheae captured by light and electron-microscopy in the metathoracic femur of a third instar locust Locusta migratoria

https://doi.org/10.1016/j.jinsphys.2011.06.006Get rights and content

Abstract

The insect tracheal system is an air-filled branching network of internal tubing that functions to exchange respiratory gases between the tissues and the environment. The light and electron-micrographs presented in this study show tracheae in the process of moulting, captured from the metathoracic hopping femur of a juvenile third instar locust (Locusta migratoria). The images provide evidence for the detachment of the cuticular intima from the tracheal epithelial cells, the presence of moulting fluid between the new and old cuticle layers, and the withdrawal of the shed cuticular lining through larger upstream regions of the tracheal system during moulting. The micrographs also reveal that the cuticular intima of the fine terminal branches of the tracheal system is cast at ecdysis. Therefore, the hypothesis that tracheoles retain their cuticle lining at each moult may not apply to all insect species or developmental stages.

Highlights

► Light and electron-micrographs of moulting insect tracheae are presented. ► Both tracheae and tracheoles increase in size and shed their cuticle lining. ► Retention of cuticle lining is therefore not a defining feature of all tracheoles.

Introduction

The insect tracheal system functions to exchange respiratory gases between the tissues and the environment (Wigglesworth, 1965). The pathway for oxygen begins with its diffusion into the spiracle openings, where it is then delivered in the gas-phase along an internal system of successively smaller tracheal tubes (Fig. 1), the terminal branches of which are commonly referred to as tracheoles. Oxygen diffuses across the tracheole walls and into the surrounding tissue where it is consumed by mitochondria during aerobic respiration (Whitten, 1972, Wigglesworth, 1965, Wigglesworth, 1983).

The maximum rate that oxygen can be delivered along the tracheal system is at least equal to the maximum rate it is required for aerobic metabolism, because even during heavy exercise there is little accumulation of anaerobic products (Beenakkers et al., 1981, Sacktor, 1976, Worm and Beenakkers, 1980). The tracheal system also keeps pace with increasing oxygen demands of the insect throughout ontogeny, and we now have a good understanding of the developmental processes involved. The basic plan of the tracheal system is laid out during embryogenesis when major tracheae arise from epithelial sacs composed of tracheal precursor cells (Ghabrial et al., 2003, Manning and Krasnow, 1993). In Drosophila at least, the embryonic growth of major tracheae occurs solely by cell migration and elongation (Samakovlis et al., 1996), and it is not until egg hatching that the terminal tracheoles really begin to develop in the juvenile instar (Ghabrial et al., 2003, Manning and Krasnow, 1993). Impressively, the tracheoles react and grow toward signals produced by oxygen-starved cells (Guillemin et al., 1996, Jarecki et al., 1999, Wigglesworth, 1954). Experiments performed mostly on Rhodnius have shown that when a major trachea is severed, or if a metabolically active organ is implanted into the insect body, nearby tracheoles are pulled by epidermal filaments to service the hypoxic tissue, and if the insect is then given the opportunity to moult, the intrusion and proliferation of tracheoles is even more extensive (Locke, 1958a, Manning and Krasnow, 1993, Wigglesworth, 1953, Wigglesworth, 1954, Wigglesworth, 1959, Wigglesworth, 1977).

The growth and proliferation of hypoxic-seeking tracheoles is not the only mechanism insects use to enhance oxygen delivery capacities during development. Insects also significantly increase the size of the tracheal system with each moult. This is achieved through the synthesis of new tracheal tubes that are cemented onto the system, and by a substantial increase in the volume of existing tracheae (Keister, 1948, Locke, 1958b, Manning and Krasnow, 1993, Wigglesworth, 1954, Wigglesworth, 1973, Wigglesworth, 1981). Existing tracheae increase in size as the epithelial cells divide and multiply (Ryerse and Locke, 1978, Wigglesworth, 1981), although in Drosophila larvae it might solely be due to changes in cell shape and size (Madhavan and Schneiderman, 1977, Manning and Krasnow, 1993). Either way, the diameter of the tracheal wall increases, and as this occurs the cuticular intima becomes detached from the epithelial layer in a process known as apolysis (Wigglesworth, 1973). The exuvial space between the new wall and the shed cuticular intima fills with moulting fluid (Jungreis, 1974, Passonneau and Williams, 1953), and a new cuticle begins to line the new and larger tracheae (Ryerse and Locke, 1978). The old cuticular intima is then withdrawn through the tracheal system towards the spiracles where it is shed along with the rest of the exuviae at ecdysis. To complete the moulting process, moulting fluid is resorbed by the epithelial layer (Lensky and Rakover, 1972) and the lumen of the new and more extensive tracheal system fills with air (Keister, 1948, Locke, 1958b, Ryerse and Locke, 1978, Wigglesworth, 1981).

Although the process of tracheal apolysis and ecdysis is fairly well established, there is confusion over whether or not the cuticular intima of the tracheoles is shed along with the rest of the tracheal system at moulting. Apparently, tracheoles in Rhodnius remain unchanged throughout their lifecycle (Wigglesworth, 1973), and in Bombyx, the tracheoles also appear to retain their cuticle with each moult, at least in the first three larval stages (Matsuura and Tashiro, 1976). However, earlier studies on Sciara (Keister, 1948) and Drosophila (Whitten, 1957) using phase-contrast optical microscopy found that the cuticular intima of tracheoles is shed in the early larval stages, but not in the final larval or pupal moults. Keister (1948) suggested that partial moulting might take place in complex tracheal systems, especially when tracheoles penetrate the surface of other cells, and complete moulting might only occur in insects with relatively simple tracheal systems.

High magnification electron micrographs of tracheoles in the process of moulting have never been captured and published, and so the early claims made by Keister (1948) and Whitten (1957), that in some insects at least, the tracheoles do in fact shed their cuticular lining remain unsubstantiated. The purpose of the current paper is to present a series of light and electron-micrographs captured from the metathoracic femur of a third instar migratory locust Locusta migratoria that provide unequivocal evidence that some tracheoles shed their cuticle lining at moulting.

Section snippets

Materials and methods

The locust reported in the current study was a single third instar gregarious-phase nymph, 3–4 days post-moult, of the species L. migratoria. It was sourced from a breeding colony at the University of Adelaide, Australia, where locusts were reared in a large terrarium at 33 ± 1 °C, with a relative humidity of ∼30%, under a 12:12 h light–dark cycle, with ad libitum access to seedling wheatgrass and wheat germ. When the locust was taken from the colony, it displayed no apparent visual indication that

Description of moulting tracheae

The moulting of the tracheal system begins with the formation of a new and larger tracheal wall around the existing wall (Fig. 2A), and the detachment of the old cuticular intima from the epithelial layer (Fig. 2B) (Ryerse and Locke, 1978, Wigglesworth, 1973). The large trachea depicted in Fig. 2A for instance, appears to have increased nearly 2-fold in diameter, while the smaller tracheae (tracheoles) in Fig. 3 also appear to have increased in size as the fourth instar tracheal system

Acknowledgements

This research was supported by the Australian Research Council, project no. DP0879605. Mr Kerry Gascoigne and Dr Mike Teo of Flinders Microscopy and Ms Ruth Williams and Ms Lyn Waterhouse of Adelaide Microscopy shared their expertise in the field of electron microscopy, which was greatly appreciated. The authors also thank Professor Stephen Simpson and Mr Tim Dodgson from the University of Sydney for technical advice and providing the founding locust colony. We also thank Professor Jon Harrison

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