John J. Socha

Advances in Biological Structure, Function, and Physiology Using Synchrotron X-Ray Imaging*

Studies of the physiology and biomechanics of small ( approximately 1 cm) organisms are often limited by the inability to see inside the animal during a behavior or process of interest and by a lack of three-dimensional morphology at the submillimeter scale. These constraints can be overcome by an imaging probe that has sensitivity to soft tissue, the ability to penetrate opaque surfaces, and high spatial and temporal resolution. Synchrotron X-ray imaging has been successfully used to visualize millimeter-centimeter-sized organisms with micrometer-range spatial resolutions in fixed and living specimens. Synchrotron imaging of small organisms has been the key to recent novel insights into structure and function, particularly in the area of respiratory physiology and function of insects. X-ray imaging has been effectively used to examine the morphology of tracheal systems, the mechanisms of tracheal and air sac compression in insects, and the function of both chewing and sucking mouthparts in insects. Synchrotron X-ray imaging provides an exciting new window into the internal workings of small animals, with future promise to contribute to a range of physiological and biomechanical questions in comparative biology.

Increase in tracheal investment with beetle size supports hypothesis of oxygen limitation on insect gigantism

Recent studies have suggested that Paleozoic hyperoxia enabled animal gigantism, and the subsequent hypoxia drove a reduction in animal size. This evolutionary hypothesis depends on the argument that gas exchange in many invertebrates and skin-breathing vertebrates becomes compromised at large sizes because of distance effects on diffusion. In contrast to vertebrates, which use respiratory and circulatory systems in series, gas exchange in insects is almost exclusively determined by the tracheal system, providing a particularly suitable model to investigate possible limitations of oxygen delivery on size. In this study, we used synchrotron x-ray phase–contrast imaging to visualize the tracheal system and quantify its dimensions in four species of darkling beetles varying in mass by 3 orders of magnitude. We document that, in striking contrast to the pattern observed in vertebrates, larger insects devote a greater fraction of their body to the respiratory system, as tracheal volume scaled with mass1.29. The trend is greatest in the legs; the cross-sectional area of the trachea penetrating the leg orifice scaled with mass1.02, whereas the cross-sectional area of the leg orifice scaled with mass0.77. These trends suggest the space available for tracheae within the leg may ultimately limit the maximum size of extant beetles. Because the size of the tracheal system can be reduced when oxygen supply is increased, hyperoxia, as occurred during late Carboniferous and early Permian, may have facilitated the evolution of giant insects by allowing limbs to reach larger sizes before the tracheal system became limited by spatial constraints.

Real-time phase-contrast x-ray imaging: a new technique for the study of animal form and function

BackgroundDespite advances in imaging techniques, real-time visualization of the structure and dynamics of tissues and organs inside small living animals has remained elusive. Recently, we have been using synchrotron x-rays to visualize the internal anatomy of millimeter-sized opaque, living animals. This technique takes advantage of partially-coherent x-rays and diffraction to enable clear visualization of internal soft tissue not viewable via conventional absorption radiography. However, because higher quality images require greater x-ray fluxes, there exists an inherent tradeoff between image quality and tissue damage.ResultsWe evaluated the tradeoff between image quality and harm to the animal by determining the impact of targeted synchrotron x-rays on insect physiology, behavior and survival. Using 25 keV x-rays at a flux density of 80 μW/mm-2, high quality video-rate images can be obtained without major detrimental effects on the insects for multiple minutes, a duration sufficient for many physiological studies. At this setting, insects do not heat up. Additionally, we demonstrate the range of uses of synchrotron phase-contrast imaging by showing high-resolution images of internal anatomy and observations of labeled food movement during ingestion and digestion.ConclusionSynchrotron x-ray phase contrast imaging has the potential to revolutionize the study of physiology and internal biomechanics in small animals. This is the only generally applicable technique that has the necessary spatial and temporal resolutions, penetrating power, and sensitivity to soft tissue that is required to visualize the internal physiology of living animals on the scale from millimeters to microns.

Correlated patterns of tracheal compression and convective gas exchange in a carabid beetle

SUMMARY Rhythmic tracheal compression is a prominent feature of internal dynamics in multiple orders of insects. During compression parts of the tracheal system collapse, effecting a large change in volume, but the ultimate physiological significance of this phenomenon in gas exchange has not been determined. Possible functions of this mechanism include to convectively transport air within or out of the body, to increase the local pressure within the tracheae, or some combination thereof. To determine whether tracheal compressions are associated with excurrent gas exchange in the ground beetle Pterostichus stygicus, we used flow-through respirometry and synchrotron x-ray phase-contrast imaging to simultaneously record CO2 emission and observe morphological changes in the major tracheae. Each observed tracheal compression (which occurred at a mean frequency and duration of 15.6±4.2 min–1 and 2.5±0.8 s, respectively) was associated with a local peak in CO2 emission, with the start of each compression occurring simultaneously with the start of the rise in CO2 emission. No such pulses were observed during inter-compression periods. Most pulses occurred on top of an existing level of CO2 release, indicating that at least one spiracle was open when compression began. This evidence demonstrates that tracheal compressions convectively pushed air out of the body with each stroke. The volume of CO2 emitted per pulse was 14±4 nl, representing approximately 20% of the average CO2 emission volume during x-ray irradiation, and 13% prior to it. CO2 pulses with similar volume, duration and frequency were observed both prior to and after x-ray beam exposure, indicating that rhythmic tracheal compression was not a response to x-ray irradiation per se. This study suggests that intra-tracheal and trans-spiracular convection of air driven by active tracheal compression may be a major component of ventilation for many insects.

Issues of convection in insect respiration: Insights from synchrotron X-ray imaging and beyond☆☆☆

While it has long been known that in small animals, such as insects, sufficient gas transport could be provided 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 have constrained our understanding of the mechanisms that underlie the production of these convective flows. Recently, new techniques have enabled the elucidation of the anatomical structures and physiological processes that contribute to creating and maintaining bulk flow in small animals. In particular, synchrotron X-ray imaging provides unprecedented spatial and temporal resolution of internal functional morphology and is changing the way we understand gas exchange in insects. This symposium highlights recent efforts towards understanding the relationship between form, function, and control in the insect respiratory system.

How Locusts Breathe

Insect tracheal-respiratory systems achieve high fluxes and great dynamic range with low energy requirements and could be important models for bioengineers interested in developing microfluidic systems. Recent advances suggest that insect cardiorespiratory systems have functional valves that permit compartmentalization with segment-specific pressures and flows and that system anatomy allows regional flows. Convection dominates over diffusion as a transport mechanism in the major tracheae, but Reynolds numbers suggest viscous effects remain important.

Effects of size and behavior on aerial performance of two species of flying snakes (Chrysopelea).

SUMMARY Aerial locomotion in snakes (genus Chrysopelea) is kinematically distinct from any other type of gliding or powered flight, with prominent, high amplitude body undulations visually dominating the behavior. Because it is not known how flying snakes produce aerodynamic forces in flight, the factors that determine snake flight performance are not clear. In this study, the effects of size and behavior on aerial performance were examined both within a species (C. paradisi) and between two species (C. paradisi and C. ornata), using stepwise multiple regressions to identify relevant variables. Smaller C. paradisi traveled farther than larger snakes at lower sinking speeds, with trajectories that shallowed more quickly and reached lower minimum glide angles. Although wing loading increased faster than expected for isometric size increase, wing loading per se was not responsible for performance differences between large and small snakes. Snakes with higher interactions between relative undulation amplitude and body size transitioned out of the initial acceleration phase at higher airspeeds and sinking speeds, and attained higher maximum airspeeds and horizontal speeds; snakes that used higher average relative amplitudes transitioned out of the initial acceleration phase at higher horizontal speeds. Undulation frequency was not significantly related to any performance variable within C. paradisi and was not significantly different between the two species, suggesting that this variable (in contrast to relative undulation amplitude) may have a minor influence on the aerodynamic mechanism of force production in snake flight. C. paradisi and C. ornata differed significantly in most performance comparisons. C. ornata were more massive than C. paradisi at any given body length and in general exhibited poorer gliding performance than C. paradisi. This study contributes towards understanding how an unconventional body form and kinematics can produce a novel mode of aerial locomotion in a vertebrate glider.

Effects of VR System Fidelity on Analyzing Isosurface Visualization of Volume Datasets

Volume visualization is an important technique for analyzing datasets from a variety of different scientific domains. Volume data analysis is inherently difficult because volumes are three-dimensional, dense, and unfamiliar, requiring scientists to precisely control the viewpoint and to make precise spatial judgments. Researchers have proposed that more immersive (higher fidelity) VR systems might improve task performance with volume datasets, and significant results tied to different components of display fidelity have been reported. However, more information is needed to generalize these results to different task types, domains, and rendering styles. We visualized isosurfaces extracted from synchrotron microscopic computed tomography (SR-μCT) scans of beetles, in a CAVE-like display. We ran a controlled experiment evaluating the effects of three components of system fidelity (field of regard, stereoscopy, and head tracking) on a variety of abstract task categories that are applicable to various scientific domains, and also compared our results with those from our prior experiment using 3D texture-based rendering. We report many significant findings. For example, for search and spatial judgment tasks with isosurface visualization, a stereoscopic display provides better performance, but for tasks with 3D texture-based rendering, displays with higher field of regard were more effective, independent of the levels of the other display components. We also found that systems with high field of regard and head tracking improve performance in spatial judgment tasks. Our results extend existing knowledge and produce new guidelines for designing VR systems to improve the effectiveness of volume data analysis.

Hypoxia-induced compression in the tracheal system of the tobacco hornworm caterpillar, Manduca sexta

SUMMARY Abdominal pumping in caterpillars has only been documented during molting. Using synchrotron X-ray imaging in conjunction with high-speed flow-through respirometry, we show that Manduca sexta caterpillars cyclically contract their bodies in response to hypoxia, resulting in significant compressions of the tracheal system. Compression of tracheae induced by abdominal pumping drives external gas exchange, as evidenced by the high correlation between CO2 emission peaks and body movements. During abdominal pumping, both the compression frequency and fractional change in diameter of tracheae increased with body mass. However, abdominal pumping and tracheal compression were only observed in larger, older caterpillars (>0.2 g body mass), suggesting that this hypoxic response increases during ontogeny. The diameters of major tracheae in the thorax increased isometrically with body mass. However, tracheae in the head did not scale with mass, suggesting that there is a large safety margin for oxygen delivery in the head in the youngest animals. Together, these results highlight the need for more studies of tracheal system scaling and suggest that patterns of tracheal investment vary regionally in the body.

A Plesiosaur Containing an Ichthyosaur Embryo as Stomach Contents from the Sundance Formation of the Bighorn Basin, Wyoming

Herein we report the discovery of an ichthyosaur embryo from the Upper Member of the Sundance Formation (Oxfordian) of the Bighorn Basin, Wyoming. The specimen is the first known ichthyosaur embryo from the Upper Jurassic, and is the first Jurassic ichthyosaur embryo from North America. The embryo was discovered in close association with the abdomen of an articulated partial plesiosaur skeleton, and several lines of evidence support the interpretation of the embryo as plesiosaur stomach contents. The small size and extremely poor ossification of the embryo indicate that the animal was probably not a neonate. Although the taxonomic affinities of the fossil are unknown, the large ichthyosaurian (sensu stricto) Opthalmosaurus natans is the only known ichthyosaur from the Sundance Formation, and the embryo may belong to that taxon. The ichthyosaurs and plesiosaurs of the Sundance Formation of Wyoming (Oxfordian) have been known for over 100 years, having been first mentioned by Marsh (1891, 1893, 1895). The single currently recognized ichthyosaur taxon from the formation, Ophthalmosaurus natans, was described in a monograph by Gilmore (1906), but subsequently received little attention until recently (Massare and Young, 2004; Massare et al., 2006). Plesiosaurs are also known from the formation, comprising the large pliosaur taxon Megalneusaurus rex (Knight, 1898; Wahl et al., 2007) and the cryptocleidoid taxa Pantosaurus striatus (O’Keefe and Wahl, 2003a) and Tatenectes laramiensis (O’Keefe and Wahl 2003b; Mehl, 1912). These plesiosaur taxa have also received little attention in the scientific literature, despite their phylogenetic and biogeographic relevance to Jurassic plesiosaur evolution, and the context they may provide for the diverse and well-known plesiosaur fauna of the nearly coeval Oxford Clay of the United Kingdom (Andrews, 1910, 1913). In an effort to increase knowledge of this important fauna, the Sundance Plesiosaur Project has performed extensive fieldwork in the Upper Member of the Sundance Formation. Here we report on one discovery from this field program, a partial skeleton referable to Pantosaurus striatus. The skeleton is remarkable in preserving a gastric mass containing the partially digested remains of an embryonic ichthyosaur, and is the first evidence of the consumption of ichthyosaurs by plesiosaurs. Institutional Abbreviation—USNM, Smithsonian Institution, Washington, D.C. USA.