Lung ventilation during treadmill locomotion in a terrestrial turtle, Terrapene carolina

1. Lung ventilation during treadmill locomotion in a terrestrial turtle, Terrapene carolina Landberg, T., Mailhot, J.D., Brainerd, E.L. “Lung ventilation during treadmill locomotion in a terrestrial turtle, Terrapene carolina.” Journal of Experimental Biology. 206 (2003): 3391-3404.

2. Background Information • Anatomy with regard to breathing mechanisms • Limb girdles (pectoral and pelvic) and lungs both located within bony shell • Rigid shell contains fixed volume • Air in lungs displaced when axial/appendicular elements move within the shell • Retraction of pectoral/pelvic limb and girdle elements into shell drives air out of lungs • Protraction of limb elements creates subatmospheric pressures, producing inhalation

3. Mechanical interactions between locomotion and breathing in extant tetrapods are of particular interest because lung ventilation is hypothesized to conflict with locomotion in the common ancestor of amniotes • In primitive amniotes, locomotion and ventilation come into mechanical conflict because locomotion requires unilateral activity of axial musculature while costal ventilation requires bilateral activity of those same muscles • Some vertebrates have independently evolved to cope with this constraint by developing body postures or alternative ventilatory mechanisms that partially decouple breathing from locomotion • i.e. mammals, birds, crocodilians • Lizards – gular pump to supplement lung ventilation while costal musculature is used for locomotion • Alternative breathing mechanisms such as gular pump may be employed during locomotion for turtles, if limb movements interfere with their breathing; however, previous studies show that gular oscillations do not contribute to lung ventilation of resting turtles

4. Two main breathing mechanisms 1. Action of oblique (OA) and transverse abdominis (TA), diaphragmaticus, and striatum pulmonale muscles TA and OA alternate bilateral muscle activity to produce exhalation-inhalation breathing cycles at rest TA and OA considered primary ventilation mechansim for turtles present in all extant species active consistently during lung ventilation 2. Limb-pump ventilation mechanism Limbs and girdles contribute to ventilation and redistribution of air into lungs Muscles of pectoral/pelvic limbs and girdles are active during ventilation at rest as well as during limb movement during locomotion

5. If these muscles are used for both breathing and locomotion, then locomotion may either interfere or assist breathing. Respiratory and locomotor functions of vertebrates often highly integrated Many vertebrates couple breathing and locomotion Goals of this study were to determine whether T. carolina breathes during locomotion Does locomotion alter breathing performance Are ventilation and locomotion temporally coupled Are airflow rates directly affected by stride cycle Are lung ventilation mechanisms the same as in resting animals Information about breathing performance during locomotion may help interpret evolution of lung ventilation mechanisms in relation to turtle’s unique morphology Hypothesis

6. Methods • Three individual Terrapene carolina triunguis • Controlled temperature of 30 deg C to maximize voluntary locomotion • Constructed pneumotach masks that did not interfere with vision, hearing, or breathing • Holes made for nares and mouth • Connected to a differential pressure transducer • Ventilatory airflow recorded simulataneously with lateral view x-ray and light video images • Four part experiment: • Acclimation to mask, treadmill chamber, and temperature • Pre-exercise • Locomotion • Recovery

7. Results • Airflow recordings show large exhalations accompany head and limb retraction and plastral adduction; lung ventilation still occurs in fully retracted position • Front and back halves of plastron connect to each other and the carapace by ligamentous connective tissue • Lungs are located in the dorsal region of the carapace with large neck retractor muscles situated between them • high domed shape of carapace allows for large residual lung volume • Lung ventilation occurs continuously during treadmill locomotion • Evidence that gular pump is not employed because airflow recordings would show small inhalations followed by little or no exhalation • Highest breath frequency recorded during period of locomotion • An average airflow rate was calculated for inhalations and exahalations to determine whether limb movements affect airflow rates during locomotion • Results not consistent • Few statistically significant differences between inhalatory and exhalatory peak airflow rates

8. X-ray recordings tracked movement of inguinal flanks during breathing Determine whether abdominal muscles are mechanism for breathing during locomotion At rest and locomotion: exhalation accompanied by dorsal movement of the marker; inhalation accompanied by ventral movement of the marker Inguinal flanks move in phase with ventilatory cycle and indepently from stride cycle

9. Discussion • Green sea turtles exhibit breathing that ceases during locomotion, whereas box turtles have been found to breathe continuously • Findings did not support the hypotheses • 1. T. carolina does not couple breathing with locomotion • Mammals and birds: breathing and locomotion are coupled due to pressurization cycles of stride and breath in thoracic cavity • 2. Limb movements do not contribute to lung ventilation during locomotion • Lizards: breathing performance is impeded with increasing locomotive speed because axial muscles are needed at times for breathing and locomotion simultaneously

10. Timing of breaths relative to stride cycle • At rest and during locomotion: • Little difference between peak inhalatory and exhalatory airflow rates • Indicates that locomotion has no mechanical effect on breathing • Breathing and stride cycle are independent of each other • Lung ventilation mechanism must be separate from locomotor system • TA and OA muscles used to breathe when at rest and most likely are the mechanism for breathing during locomotion • No evidence for gular pump or limb mechanism • Species lacks diaphragmaticus and striatum pulmonale muscles

11. Chelonia mydas vs. Terrapene carolina • Kinematics of locomotion • C. mydas: front limbs retract simultaneously to push body forward, a bilateral synchronous motor pattern which is also used during limb-pump lung ventilation • May cease breathing during locomotion because pressurized lungs are used to stabilize limb movements • T. carolina: alternating gait with one diagonal pair of limbs extended while other pair is flexed • A more balanced effect on internal shell volume, in addition to abdominal muscles separate from the locomotor muscles, provides explanation for absence of effect of locomotion on ventilation

12. Conclusion • Specialized ventilatory functions of abdominal muscles were favored by natural selection since they permit breathing during locomotion • Shell-less ancestor of turtle most likely relied on mechanism similar to that of extant lizards (axial bending during locomotion, rotation of ribs during ventilation) and would have experienced a mechanical conflict as do lizards • Hypothetical ancestor gave rise to turtles as ribs abandoned ventilatory function and fused into the shell • Thus, extant turtles are not subject to the constraint experienced by shell-less ancestor or extant lizards because their ribs do not contribute to either locomotion or ventilation