Respiratory Physiology & Neurobiology 173S (2010) S65–S73 Contents lists available at ScienceDirect Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol Review Issues of convection in insect respiration: Insights from synchrotron X-ray imaging and beyondଝ,ଝଝ John J. Socha a,∗, Thomas D. Förster b, Kendra J. Greenlee c a Virginia Tech, Engineering Science and Mechanics, 332 Norris Hall (MC 0219), Blacksburg, VA 24061, United States b Humboldt Universität zu Berlin, Animal Physiology Group, Philippstrasse 13, 10115 Berlin, Germany c North Dakota State University, Biological Sciences, 317 Stevens Hall, PO Box 6050, Fargo, ND 58108, United States article info abstract Article history: While it has long been known that in small animals, such as insects, sufficient gas transport could be pro- Accepted 11 March 2010 vided by diffusion, it is now recognized that animals generate and control convective flows to improve oxygen delivery across a range of body sizes and taxa. However, size-based methodological limitations Keywords: have constrained our understanding of the mechanisms that underlie the production of these convective Insect gas exchange flows. Recently, new techniques have enabled the elucidation of the anatomical structures and physio- Development logical processes that contribute to creating and maintaining bulk flow in small animals. In particular, Synchrotron imaging synchrotron X-ray imaging provides unprecedented spatial and temporal resolution of internal functional Convection Tracheal compression morphology and is changing the way we understand gas exchange in insects. This symposium highlights Passive suction ventilation recent efforts towards understanding the relationship between form, function, and control in the insect respiratory system. © 2010 Elsevier B.V. All rights reserved. 1. Introduction flation of the tracheal system itself (Herford, 1938; Westneat et al., 2003; Socha et al., 2008), including both tracheal tubes and air sacs The template of the insect gas exchange system is simple. Respi- (Greenlee et al., 2009a,b). This combination of morphological and ratory gases enter and exit the body through spiracular valves, the behavioural variations results in a great variety of respiratory pat- gases are transported through air-filled tracheal tubes that ram- terns and an overall general flexibility in the gas exchange system ify throughout the body, and diffusion occurs directly to and from that may play a large role in the ecological and evolutionary success the tissues. Although this basic pattern is conserved throughout of insects. But despite over a century of study, our understanding of the insects, tremendous variation exists in tracheal morphology insect gas exchange systems—both pattern and process—can still be and respiratory dynamics (e.g., Buck, 1962; Keister and Buck, 1974; considered immature, largely due to this immense variation. Fun- Wasserthal, 1996; Harrison, 1997; Chapman, 1998; Bradley, 2007). damental questions, such as the relative roles of convection and Anatomically, variation is manifest in such features as spiracle diffusion as a function of body size, phylogeny, development, and number and location; tracheal size, shape, wall thickness, and life history, remain unanswered. branching pattern; density of tracheoles; and presence or absence Most research on gas exchange dynamics has focused on of air sacs. Dynamically, time-dependent variation occurs due to measurements made externally to the animal. These include mea- coordinated opening and closing of the spiracles (e.g., Mill, 1985; surements of CO2 and H2O emission (Lighton, 2008), airflow Lehmann and Heymann, 2005; Wasserthal, 2001), movement of the patterns at the spiracles (Slama, 1988), frequency and duration fluid level at the terminal ends of the tracheoles (Wigglesworth, of spiracle opening and closing (Lighton and Garrigan, 1995; 1935; Woods et al., 2009) and, in some species, collapse and rein- Terblanche et al., 2008), and magnitude of exoskeleton movements (Slama, 2008). These techniques have been and still serve as an invaluable source of information on insect respiration, but they do not provide direct knowledge of internal gas exchange pro- ଝ This paper is part of the ‘ICRS Supplement’, guest-edited by Dr. Steven F. Perry. cesses. Studies of morphology include description by dissection ଝଝ From the symposium: Insights from synchrotron X-ray imaging and beyond: (Hartung et al., 2004; Harrison et al., 2005), endocasting using Mechanisms and regulation of flow generation in tracheal systems, Organized by low-viscosity polymers (Meyer, 1988), and stereology (Schmitz John J. Socha, Virginia Polytechnic Institute and State University, USA. ∗ and Perry, 1999; Hartung et al., 2004), which involves sectioning Corresponding author. Tel.: +1 540 231 6188; fax: +1 540 231 0696. E-mail addresses: [email protected] (J.J. Socha), [email protected] of the animal. These techniques are effective for determining tra- (T.D. Förster), [email protected] (K.J. Greenlee). cheal system morphology, but they are relatively time consuming, 1569-9048/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2010.03.013 S66 J.J. Socha et al. / Respiratory Physiology & Neurobiology 173S (2010) S65–S73 require skillful preparation, or involve altering the system in some 1.1. Synchrotron imaging of insects way, either by breaching the exoskeleton and relieving the native hemolymph pressure around the tracheal system, or by cutting the Synchrotron imaging has changed the way that we understand system itself. Perhaps resulting from these difficulties, it is not sur- insect respiration. Although a few isolated X-ray images of insects prising that there are relatively few quantitative descriptions of appeared in the physical literature in the early 2000s (e.g., Matsui entire tracheal systems (e.g., Clarke, 1957; Lease et al., 2006), and et al., 2000; Hwu et al., 2002), synchrotron X-ray imaging of insects little is known about the variation of tracheal morphology across began in earnest in 2001 when Wah-Keat Lee, a physicist at Argonne phylogeny. National Laboratory’s Advanced Photon Source, became curious This symposium was inspired by a new tool used to study insect about what an insect would look like when viewed at his X-ray physiology, synchrotron X-ray imaging, which is opening up a beamline (1-ID). Lee took an X-ray snapshot of the head of a carpen- whole new window into the world of insect respiration. This tool ter ant (Fig. 1a) caught outdoors and, with it, sought the interest of provides the ability to visualize the tracheal system in an intact, liv- biologists. He contacted Mark Westneat, a biomechanist at the Field ing animal, and is motivating new work that revisits long-standing Museum and University of Chicago, and they teamed together to questions of insect respiratory physiology. Because the tracheal explore the diversity of internal movements and morphology that system can be observed in real time, this resource is particularly can be seen with this powerful tool. As a necessary first step, their powerful for understanding internal dynamics involved in respira- experimental approach was exploratory—they captured insects tion. from the woods just beyond the doors of the synchrotron source Fig. 1. Samples of synchrotron X-ray images of insects. (A) Demonstration of the capability of phase-contrast imaging with synchrotron X-rays. The two images show the head of a carpenter ant (Camponotus pennsylvanicus) in standard absorption-only configuration (left) and in phase-contrast configuration (right). Images were recorded in 2001 at beamline 1-ID-C at the Advanced Photon Source, Argonne National Laboratory. Images courtesy of Wah-Keat Lee. (B) and (C) Depict high-quality projection images of an ant worker (Camponotus pennsylvanicus) and carabid beetle (Notiophilus spp.), respectively. Large-scale differences in tracheal system architecture between the two insects can be easily discerned, including the ant’s abdominal and head air-sacs and sac-like main tracheal trunks in the thorax. The round structures in the ant’s abdomen are air bubbles. J.J. Socha et al. / Respiratory Physiology & Neurobiology 173S (2010) S65–S73 S67 and observed what could be seen inside their living bodies. Quite in insect respiration. Broad questions that are under current inves- quickly, it became evident that the internal insect was far more tigation include: dynamic than expected. Indeed, many of the videos they created What explains the diversity of tracheal architecture and organi- were puzzling, because what they were seeing had not been previ- zation? ously documented. Within two years, they published data showing How does the respiratory system respond to changing metabolic massive collapsing movements in the tracheal system of beetles, needs during development and the transition from rest to activity? crickets and ants (Westneat et al., 2003), movements that either What are the rules of convection—under what circumstances is had not been described or were under-appreciated. Since then, it required or employed? synchrotron imaging of insects has been improved and refined to How are convective airflows created, and what airflow patterns address more sophisticated and specific questions of insect respi- are used? ration (Lee, 2009). Below (in Sections 1.2–1.6), we highlight symposium contri- The power of synchrotron imaging is twofold: (1) it enables the butions that aimed to answer some of