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Lung ventilation during treadmill locomotion in a terrestrial turtle, Terrapene carolina
Abstract and Figures
The limb girdles and lungs of turtles are both located within the bony shell, and therefore limb movements during locomotion could affect breathing performance. A mechanical conflict between locomotion and lung ventilation has been reported in adult green sea turtles, Chelonia mydas, in which breathing stops during terrestrial locomotion and resumes during pauses between bouts of locomotion. We measured lung ventilation during treadmill locomotion using pneumotach masks in three individual Terrapene carolina (mass 304-416 g) and found no consistent mechanical effects of locomotion on breathing performance. Relatively small tidal volumes (2.2+/-1.4 ml breath(-1); mean +/- S.D., N=3 individuals) coupled with high breath frequencies (36.6+/-26.4 breaths min(-1); mean +/- S.D., N=3 individuals) during locomotion yield mass-specific minute volumes that are higher than any previously reported for turtles (264+/-64 ml min kg(-1); mean +/- S.D., N=3 individuals). Minute volume was higher during locomotion than during recovery from exercise (P<0.01; paired t-test), and tidal volumes measured during locomotion were not significantly different from values measured during brief pauses between locomotor bouts or during recovery from exercise (P>0.05; two-way ANOVA). Since locomotion does not appear to conflict with breathing performance, the mechanism of lung ventilation must be either independent of, or coupled to, the stride cycle. The timing of peak airflow from breaths occurring during locomotion does not show any fixed phase relationship with the stride cycle. Additionally, the peak values of inhalatory and exhalatory airflow rates do not differ consistently with respect to the stride cycle. Together, these data indicate that T. carolina is not using respiratory-locomotor coupling and limb and girdle movements do not contribute to lung ventilation during locomotion. X-ray video recordings indicate that lung ventilation is achieved via bilateral activity of the transverse (exhalatory) and oblique (inhalatory) abdominal muscles. This specialized abdominal ventilation mechanism may have originally circumvented a mechanical conflict between breathing and locomotion in the ancestor of turtles and subsequently allowed the ribs to abandon their role in lung ventilation and to fuse to form the shell.
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... While terrestrial locomotion may constrain breathing in the green sea turtle, this does not appear to be the case for terrestrial chelonians. Landberg et al. (673) measured increased minute ventilation and breathing frequency during slow voluntary walking. The lack of conflict between locomotion and breathing was explained by exhalation being achieved via the transverse abdominal muscles whereas inhalation was achieved by the oblique abdominal muscles (673). ...
... Landberg et al. (673) measured increased minute ventilation and breathing frequency during slow voluntary walking. The lack of conflict between locomotion and breathing was explained by exhalation being achieved via the transverse abdominal muscles whereas inhalation was achieved by the oblique abdominal muscles (673). In a subsequent study, Landberg et al. (674) measured lung ventilation during treadmill locomotion in semi-aquatic turtles and found small tidal volumes during locomotion that increased during pauses, while overall minute ventilation was not affected. ...
The ectothermic vertebrates are a diverse group that includes the Fishes (Agnatha, Chondrichthyes, and Osteichthyes), and the stem Tetrapods (Amphibians and Reptiles). From an evolutionary perspective, it is within this group that we see the origin of air-breathing and the transition from the use of water to air as a respiratory medium. This is accompanied by a switch from gills to lungs as the major respiratory organ and from oxygen to carbon dioxide as the primary respiratory stimulant. This transition first required the evolution of bimodal breathing (gas exchange with both water and air), the differential regulation of O2 and CO2 at multiple sites, periodic or intermittent ventilation, and unsteady states with wide oscillations in arterial blood gases. It also required changes in respiratory pump muscles (from buccopharyngeal muscles innervated by cranial nerves to axial muscles innervated by spinal nerves). The question of the extent to which common mechanisms of respiratory control accompany this progression is an intriguing one. While the ventilatory control systems seen in all extant vertebrates have been derived from common ancestors, the trends seen in respiratory control in the living members of each vertebrate class reflect both shared-derived features (ancestral traits) as well as unique specializations. In this overview article, we provide a comprehensive survey of the diversity that is seen in the afferent inputs (chemo and mechanoreceptor), the central respiratory rhythm generators, and the efferent outputs (drive to the respiratory pumps and valves) in this group. © 2022 American Physiological Society. Compr Physiol 12: 1-120, 2022.
... Our pneumotach data were therefore not consistently calibrated for volume per minute but were sufficient to record pressure variation and estimates of volume change associated with exhalation and inhalation, which we visually confirmed in X-ray videos when possible. Following techniques used in similar lung ventilation studies, we fabricated a small lightweight mask from a 500 ml plastic bottle, with a PVC extension from the cap hole that was divided with an 88 μm screen (Landberg et al., 2003). A differential pressure transducer (Validyne DP103-06; Northridge, CA, USA) was connected to ports on either side of the mesh and used in series with a pneumotachograph to measure airflow (Fig. S1). ...
... A differential pressure transducer (Validyne DP103-06; Northridge, CA, USA) was connected to ports on either side of the mesh and used in series with a pneumotachograph to measure airflow (Fig. S1). All pneumotachography data were recorded with PowerLab data acquisition hardware and LabChart software (ADInstruments, Colorado Springs, CO, USA), calibrated following procedures in similar experiments, and synchronized with the X-ray trigger system (Landberg et al., 2003). ...
The evolution of constriction and of large prey ingestion within snakes are key innovations that may explain the remarkable diversity, distribution and ecological scope of this clade, relative to other elongate vertebrates. However, these behaviors may have simultaneously hindered lung ventilation such that early snakes may have had to circumvent these mechanical constraints before those behaviors could evolve. Here, we demonstrate that Boa constrictor can modulate which specific segments of ribs are used to ventilate the lung in response to physically hindered body wall motions. We show that the modular actuation of specific segments of ribs likely results from active recruitment or quiescence of derived accessory musculature. We hypothesize that constriction and large prey ingestion were unlikely to have evolved without modular lung ventilation because of their interference with lung ventilation, high metabolic demands and reliance on sustained lung convection. This study provides a new perspective on snake evolution and suggests that modular lung ventilation evolved during or prior to constriction and large prey ingestion, facilitating snakes' remarkable radiation relative to other elongate vertebrates.
... The present paper addresses such opportunities, as well as related constraints, by examining the convergent evolution of shell kinesis, i.e., the ability to actively move shell bones such that the head and extremities are better concealed ( Fig. 1) (Bramble, 1974;Bramble & Hutchison, 1981;Bramble et al., 1984). Musculoskeletal alterations will be reviewed to discuss how architectural challenges were mitigated in evolution: The head and limbs must be retracted deeply into a rigid body cavity that also houses the limb girdles and vital organs, including lungs that do not expand in conjunction with the thoracic region during inhalation, as in other vertebrates (Landberg et al., 2003). ...
Synopsis
Species groups that feature traits with a low number of potentially variable (evolvable) character states are more likely to repeatedly evolve similar phenotypes, that is, convergence. To evaluate this phenomenon, this present paper addresses anatomical alterations in turtles that convergently evolved shell kinesis, for example, the movement of shell bones to better shield the head and extremities. Kinesis constitutes a major departure from the evolutionarily conserved shell of modern turtles, yet it has arisen independently at least 8 times. The hallmark signature of kinesis is the presence of shell bone articulations or “hinges,” which arise via similar skeletal remodeling processes in species that do not share a recent common ancestor. Still, the internal biomechanical components that power kinesis may differ in such distantly related species. Complex diarthrodial joints and modified muscle connections expand the functional boundaries of the limb girdles and neck in a lineage-specific manner. Some lineages even exhibit mobility of thoracic and sacral vertebrae to facilitate shell closure. Depending on historical contingency and structural correlation, a myriad of anatomical alterations has yielded similar functional outcomes, that is, many-to-one mapping, during the convergent evolution of shell kinesis. The various iterations of this intricate phenotype illustrate the potential for the vertebrate musculoskeletal system to undergo evolutionary change, even when constraints are imposed by the development and structural complexity of a shelled body plan. Based on observations in turtles and comparisons to other vertebrates, a hypothetical framework that implicates functional interactions in the origination of novel musculoskeletal traits is presented.
... The rigid shell is a relatively fixed volume, which means that anything that alters the space available inside the shell could restrict lung volume and ultimately influence breathing. Despite having the pelvic and pectoral girdles inside the shell, through their specialised abdominal ventilatory mechanism, terrestrial Testudines locomotion performance does not appear to conflict with breathing performance 29 . However, our data indicate that self-righting incurs a significant energetic expense, relative to walking, that suggests there may be an as yet unconsidered novel constraints associated with having evolved a shell. ...
Armoured, rigid bodied animals, such as Testudines, must self-right should they find themselves in an inverted position. The ability to self-right is an essential biomechanical and physiological process that influences survival and ultimately fitness. Traits that enhance righting ability may consequently offer an evolutionary advantage. However, the energetic requirements of self-righting are unknown. Using respirometry and kinematic video analysis, we examined the metabolic cost of self-righting in the terrestrial Mediterranean spur-thighed tortoise and compared this to the metabolic cost of locomotion at a moderate, easily sustainable speed. We found that self-righting is, relatively, metabolically expensive and costs around two times the mass-specific power required to walk. Rapid movements of the limbs and head facilitate successful righting however, combined with the constraints of breathing whilst upside down, contribute a significant metabolic cost. Consequently, in the wild, these animals should favour environments or behaviours where the risk of becoming inverted is reduced.
... A pseudodiaphragm is present, which separates the lungs from the celomic cavity, and limb movements stretch this septum downward resulting in lung expansion. This relationship between breathing and locomotion has been studied by several authors 17,18 and should be considered when immobilization will lead to ventilatory depression. For this reason, positive pressure ventilation (PPV) is generally recommended in anesthetized chelonians. ...
Anesthetic management of chelonians represents a unique challenge; the order Chelonia includes numerous species that display diverse anatomic features, habitats, body sizes, temperaments, and metabolic rates. Owing to their peculiar characteristics, safe and effective sedation and anesthesia may be more complicated than in other animals. For example, gas inductions are not indicated, and intravenous catheterization requires practice. The pharmacology of anesthetic drugs is severely impacted by body/environmental temperature, site of administration, and organ function. This review will summarize the current knowledge in terms of anatomy, physiology, and drug metabolism in chelonians, before discussing practical aspects of anesthesia.
... These anesthetic agents were used in this population of tortoises, and likely contributed to hypoventilation and apnea. However, there are other factors influencing ventilation requirements in anesthetized tortoises, including impairment of muscular motion of the limbs that normally facilitates movement of gases through the respiratory system and the use of high concentrations of oxygen as a carrier gas during inhalant anesthesia, which may suppress the ventilatory drive of chelonians [5,7,[19][20][21]. Body temperature and positioning also have a profound effect on ventilation in reptiles [22,23]. ...
Tortoises belong to the taxonomic family Testudinidae, which is considered one of the most imperiled families of the order Testudines. Anesthesia is often required for the medical and surgical management of large tortoises. The objectives of this retrospective study were to review drug regimens used to successfully anesthetize Galapagos (Chelonoidis nigra), Aldabra (Aldabrachelys gigantea) and African spurred (Centrochelys sulcata) tortoises, and to compare the times to effect and to extubation in tortoises administered different premedication protocols. Anesthetic records of giant tortoises admitted to the University of Florida College of Veterinary Medicine between January 2009 and December 2019 were reviewed. A total of 34 tortoises (six Aldabra, 23 Galapagos, and five African spurred) were included, resulting in 64 anesthetic events. Frequently used premedication protocols included an α-adrenergic agonist and ketamine combined with either midazolam (group αadrenergic agonist, midazolam, ketamine, AMK; n = 34), a μ-opioid receptor agonist (group αadrenergic agonist, μ-opioid receptor agonist, ketamine, AOK; n = 13), or a μopioid receptor agonist and midazolam (group αadrenergic agonist, midazolam, μ-opioid receptor agonist, ketamine, AMOK; n = 10). Inhalant anesthetics (isoflurane, n = 21; sevoflurane, n = 23) were frequently used for maintenance of anesthesia following premedication. Out of the 34 total tortoises, 22 had only one anesthetic event, five had two anesthetic events, three had three anesthetic events, and four had four or more anesthetic events. Few adverse effects were observed and there was no mortality reported during the peri-anesthetic period. Sedation and general anesthesia of giant tortoises can be successfully performed with a combination of an α-adrenergic agonist and ketamine in combination with midazolam and/or a μopioid receptor agonist.
... In most turtles, keratin scutes overlay the carapace and plastron, which increase the shell's stiffness (Wyneken 2001). Furthermore, shell bones store water and fat besides acting as a pH buffer (Gilbert et al. 2001) and also play a structural role in other behaviours such as support the muscles attached to produce force during locomotion and ventilatory movements during breathing (Landberg 2003, Lyson et al. 2016. and Jannello et al. (2020) attributed a correlation between shell microanatomy and lifestyle, establishing histological categories to determine crucial features in shell microstructure of Testudines with terrestrial, aquatic non-marine or marine habits. ...
In this study we provide a comprehensive investigation of the microanatomical and microstructural aspects of the carapace and limb bones of the Early Cretaceous side-necked turtle, Araripemys barretoi, from the Araripe Basin, Brazil. Inter-elemental histovariability reveals different secondary remodelling of the skeletal elements within the same individual. The vascularisation is scarce and mainly longitudinal, also it ceases towards the bone surface, forming an avascular parallel-fibred bone with closely spaced LAGs. These traits indicate a late ontogenetic stage and a slow growth rate for one of the two A. barretoi specimens. The high cortical thickness of the costal plate suggests an increase of the shell stiffness. The elevated relative bone wall thickness of the ulna compared to other limb bones indicates a case of local pachyosteosclerosis, possibly to improve body stability in the aquatic environment.
... In sea turtles, the brachial plexus has been reported to provide innervation to the abdominis oblique and transversus abdominis muscles that are involved in changing coelomic volume during ventilation (Wyneken 2001;Harms et al. 2017). In T. carolina, lung ventilation is similarly achieved via bilateral activity of the transverse and oblique abdominal muscles, and a unilateral brachial plexus block is unlikely to severely decrease ventilation (Landberg et al. 2003). Further studies are needed to understand the potential influence of brachial plexus blockade on ventilation in the eastern box turtle. ...
Objective
To describe the anatomy of the brachial plexus in eastern box turtles (Terrapene carolina carolina), develop a blind perineural injection technique for brachial plexus blockade and evaluate the distribution of three volumes of new methylene blue dye for injection in cadavers.
Study design
Prospective, randomized, blinded cadaveric study.
Animals
A total of 24 frozen-thawed box turtle cadavers; two turtles identified with shoulder injuries were subsequently exluded from the study. The remaining 22 turtles weighed 397 (190–581) g, median (range).
Methods
The brachial plexus and regional anatomy were identified by dissection of seven cadavers to determine anatomic landmarks for a perineural injection technique. This technique was tested by randomizing 15 cadavers into one of three groups to be injected bilaterally with one of three volumes (0.1 mL, 0.2 mL, 0.3 mL) of methylene blue dye 1% aqueous solution. Investigators blinded to the assigned group dissected cadavers 15 minutes after injection and used staining of the four cervical spinal nerves (C5–C8; 25% for each nerve) to record a staining score of the brachial plexus (0–100%).
Results
Based on descriptions of the anatomy of the brachial plexus, an injection technique was designed. Injections of 0.1 mL methylene blue dye resulted in 9/10 injections with 100% nerve staining, and one injection with 2 (50%) nerves stained. All injections of 0.2 mL or 0.3 mL of methylene blue dye resulted in 100% nerves stained.
Conclusions and clinical relevance
Perineural injection of the brachial plexus with 0.1, 0.2 or 0.3 mL methylene blue dye was successful in 29/30 injections in box turtle cadavers weighing 190–581 g. Further studies are needed to determine the minimum volume of injectate that can be successfully used for this technique, and to evaluate its application and efficacy in live turtles.
... Lung ventilation in box turtles is achieved by both the movement of the transverse and oblique abdominal muscles at rest and coupling of the stride cycle. 30 Multiple studies also demonstrate that chelonian lactate concentrations increase because of forced submergence, which prohibits respiration. 6,22,48 The current study also documented a positive association between blood lactate concentrations and PCV. ...
Studies to assess wildlife health commonly evaluate clinical pathology changes, immune responses, pathogen presence, and contaminant exposure, but novel modalities are needed to characterize the unique physiologic responses of reptiles. Lactate is an indicator of hypoperfusion and/or anaerobic respiration and can be quickly and easily measured using a point-of-care analyzer. This study evaluated baseline blood lactate concentrations in free-living eastern box turtles (Terrapene carolina carolina, n = 116) using a point of care analyzer and then determined the effect of handling time, physical examination (PE) abnormalities, and quantitative polymerase chain reaction pathogen detection (Terrapene herpesvirus 1, Mycoplasma sp., Terrapene adenovirus) on lactate concentrations. Blood lactate concentrations were higher in turtles with Terrapene herpesvirus 1 (n = 11), quiet mentation, and increased packed cell volume (P < 0.05). Lactate concentrations increased between initial capture and PE, with peak values reaching 129 min after capture. Lactate at PE was positively associated with baseline lactate concentrations. Turtles with Terrapene herpesvirus 1 may have alterations in blood flow, oxygen delivery, or activity patterns, driving increases in baseline lactate. Increased handling time likely leads to more escape behaviors and/or breath holding, causing turtles to undergo anaerobic metabolism and raising lactate concentrations. Overall, lactate measured by a point of care analyzer shows variability caused by capture and health factors in eastern box turtles and may be a useful adjunctive diagnostic test in this species after full methodologic validation.
