The terrestrial dominance of insects is attributed mainly to their ability, amongst other adaptations, to resist desiccation through their respiratory systems (Villani et al., 1999). However, despite many years of investigation (Wigglesworth, 1965), there is still uncertaintity as to exactly how gas and water move through this system of spiracular openings and tracheae (Wasserthal, 1996). Cockroaches and locusts pass inspired air from the anterior spiracles, through longitudinal trunks that connect the segmental tracheae and out through the more posterior abdominal spiracles, generating a flow of fresh air through the body (Miller, 1982). Lepidopteran pupae show similar patterns of respiration (Schneiderman, 1960), and some species may be able to control the actions of individual spiracles (Slama, 1988, 1999). Retrograde respiratory airflow is presumed to be typical for all insects (Hadley, 1994; Lighton, 1996), and expelling air into a sealed space below the elytra is assumed to be an adaptation, found in many species of wingless beetle (Cloudsley-Thompson, 1964; Ahearn, 1970; Draney, 1993), to an arid habitat. Because the cavity maintains its air at a high humidity, water loss during respiration should be reduced because the tracheae will not be exposed to dry air. However, the validity of this hypothesis requires an anterior-to-posterior flow of respiratory gases through the body and differential control of the spiracles, neither of which has previously been demonstrated in these insects (Hadley, 1994). Spiracular control appears to be most precise in insects that inhabit dry environments, where they limit water loss by opening their spiracles for only limited periods during a discontinuous gas-exchange cycle (DGC) (for reviews, see Kestler, 1985; Lighton, 1994, 1996; Wasserthal, 1996). The DGC is a cyclic discontinuity in external gas exchange that typically consists of three periods (Miller, 1981; Kestler, 1985). There is a closed (C) period, during which the spiracles are shut, preventing both respiratory water loss and gas exchange. Oxygen levels in the tracheae drop, while CO 2 is largely buffered in the tissues and haemolymph. This is followed by the flutter (F) period, during which slight, intermittent opening of the spiracles allows some normoxic O 2 uptake through the spiracles by diffusion and convection, but little CO 2 or water vapour is lost. The final period, the CO 2 burst (B) period, is triggered when the accumulation of CO 2 from respiring tissues causes some or all of the spiracles to open widely. Rapid unloading of CO 2 should minimise the time that the spiracles are open and therefore reduce water vapour loss. However, the role of the DGC as a water-saving 2489 The Journal of Experimental Biology 205, 2489–2497 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 JEB4059 The sealed subelytral cavity of many flightless beetle species is widely acknowledged to be an adaptation to water saving in arid-habitat species. However, this hypothesis relies on the acceptance of two largely untested assumptions: (i) that the movement of respiratory gases is unidirectional from anterior to posterior and (ii) that the coordinated action of the spiracles directs this flow. We tested these assumptions by simultaneously measuring CO 2 and O 2 exchange at the anterior mesothorax, independently of gas exchange in the posterior body, which included the subelytral cavity, of a large apterous beetle, Circellium bacchus. Flow-through respirometry revealed a marked discontinuous gas-exchange cycle (DGC) pattern from the anterior half of the body. Very little CO 2 was released from the posterior body, where the DGC was not apparent. Labelled air was shown to flow forwards from the posterior to the anterior body. Individual sampling from the mesothoracic spiracles revealed that the right mesothoracic spiracle, lying outside the elytral cavity, is the primary route for respiratory gas exchange in C. bacchus at rest. This discovery necessitates a reassessment of the currently assumed role of the subelytral cavity in water conservation and is, to our knowledge, the first demonstration of forward airflow associated with the unilateral use of a single thoracic spiracle for respiration in an insect. Key words: Circellium bacchus, discontinuous gas exchange cycle, respiration, spiracle, Scarabaeidae, dung beetle, subelytral cavity. Summary Introduction Respiratory airflow in a wingless dung beetle Frances D. Duncan 1, * and Marcus J. Byrne 2 1 School of Physiology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa and 2 Ecophysiological Studies Research Programme, School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, Wits 2050, South Africa *e-mail: duncanfd@physiology.wits.ac.za Accepted 8 May 2002