Lung ventilation during treadmill locomotion in a terrestrial turtle, Terrapene carolina
1 Graduate Program in Organismic and Evolutionary Biology, University of
Massachusetts Amherst, 611 North Pleasant Street, Amherst, MA 01003,
USA
2 Biology Department, University of Massachusetts Amherst, 611 North
Pleasant Street, Amherst, MA 01003, USA
* Author for correspondence (e-mail: tobias{at}bio.umass.edu)
Accepted 23 June 2003
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Summary |
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Key words: Terrapene carolina triunguis, North American three-toed box turtle, Emydidae, breathing mechanism, respiration, functional morphology, exercise physiology, hypaxial musculature
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Introduction |
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The breathing mechanisms of turtles have been of enduring interest to
scientists for more than three centuries (e.g.
Malpighi, 1671; Townson,
reprinted in Mitchell and Morehouse,
1863
; Gans and Hughes,
1967
). A hyobranchial pumping mechanism was proposed (most notably
by Agassiz, 1857
) to function
like the buccal pump of fishes and amphibians, forcing air into the lungs
under positive pressure, but numerous experimental investigations have found
that the oscillatory throat movements do not contribute to lung ventilation in
turtles at rest (Mitchell and Morehouse,
1863
; Francois-Franck,
1908
; Hansen,
1941
; McCutcheon,
1943
; Gans and Hughes,
1967
; Gaunt and Gans,
1969
; Brainerd,
1999
; Druzisky and Brainerd,
2001
; Landberg et al.,
2001
,
2002a
). Experimental studies
have found two main breathing mechanisms in resting turtles: (1) the action of
sheet-like muscles such as the oblique and transverse abdominis,
diaphragmaticus, and striatum pulmonale muscles
(Fig. 1A;
Mitchell and Morehouse, 1863
;
Hansen, 1941
;
McCutcheon, 1943
; George and
Shaw, 1954
,
1955
,
1959
;
Shaw, 1962
;
Gans and Hughes, 1967
;
Gaunt and Gans, 1969
) and (2)
a limb-pump ventilation mechanism (Fig.
1B,C; Gans and Hughes,
1967
; Gaunt and Gans,
1969
).
The transverse abdominis (TA) and oblique abdominis (OA) muscles alternate
bilateral muscle activity to produce exhalation-inhalation breathing cycles in
turtles at rest (McCutcheon,
1943; Gans and Hughes,
1967
; Gaunt and Gans,
1969
; Currie,
2001
). These abdominal muscles are considered the primary
ventilation mechanism of turtles because they are present in all extant turtle
species (George and Shaw,
1959
; Shaw, 1962
)
and have been found to be active consistently during lung ventilation
(Gans and Hughes, 1967
;
Gaunt and Gans, 1969
). The OA
is a paired, thin, cup-shaped muscle attaching along the rear carapacial and
plastral margins in the inguinal limb pockets, anterior to each hindlimb and
just deep to the skin (Fig.
1A). At rest, this muscle curves into the body cavity; when
contracted, it flattens to move the flank postero-ventero-laterally, which
reduces intrapulmonary pressure and produces inhalation when the glottis is
open.
The paired transverse abdominis (TA) lies deep to the oblique abdominis (OA). It attaches to the inside of the carapace and is cupped around the posterior half of each lung (Fig. 1A). As the TA contracts, intrapulmonary pressure increases, producing exhalation when the glottis is open. The convex sides of the TA and OA face each other and are attached by connective tissue at their apexes. When one muscle (the agonist) contracts and flattens, the antagonist is stretched into a highly curved position from which it can contract to reverse the motion.
Despite close anatomical approximation to the pelvic girdle and hindlimbs,
the TA and OA are considered abdominal muscles because they are innervated by
spinal projections (from the 6th and 7th vertebrae of
the carapace) that branch off before the pelvic enlargement of the spinal cord
(Bojanus 1819, reprinted 1970;
Currie and Gonsalves, 1997
).
These muscles are often called `respiratory muscles', but we reject this term
because many vertebrate muscles perform more than one function, and the
functions of muscles may change during evolution
(Carrier and Farmer, 2000
;
Deban and Carrier, 2002
). The
importance of avoiding functional names for the abdominal muscles was
illustrated recently when they were found to be active in the absence of
breathing during underwater locomotion of the red-eared slider (Trachemys
scripta; Currie,
2001
).
In non-locomoting turtles, movements of the limbs and girdles have been
shown to contribute to ventilation as well as to the redistribution of air
into different parts of the lungs
(Francois-Franck, 1908;
Gans and Hughes, 1967
;
Gaunt and Gans, 1969
;
Spragg et al., 1980
). Authors
have variously speculated that this limp pump is the main ventilation
mechanism (e.g. Pope, 1939
),
that breathing is an obligatory consequence of locomotion
(Orenstein, 2001
), and even
that turtles must locomote to breathe at all
(Tauvery, 1701
; cited in
Gans and Hughes, 1967
).
Because the volume within the turtle shell is nearly constant, retraction of
the pectoral or pelvic limb and girdle elements into the shell drives air out
of the lungs while protraction of limb elements creates subatmospheric
pressures, which can produce inhalation
(Fig. 1; Gans and Hughes, 1967
;
Gaunt and Gans, 1969
). The
muscles of the pectoral (testoscapularis, testocoracoideus and pectoralis) and
pelvic (atrahens and retrahens pelvim) limbs and girdles that have been shown
to be active during ventilation in resting turtles are also recruited for limb
movement during locomotion (Gans and
Hughes, 1967
; Gaunt and Gans,
1969
). If these muscles are used for both breathing and
locomotion, might locomotion either interfere with or assist breathing?
Experimental evidence from adult female green sea turtles, Chelonia
mydas, suggests that locomotion may interfere with breathing performance
(Prange and Jackson, 1976;
Jackson and Prange, 1979
).
During terrestrial locomotion, C. mydas stops breathing during bouts
of locomotion and resumes breathing during pauses in locomotion. Jackson and
Prange (1979
) suggested that
the use of limb musculature for both locomotion and breathing prevents the two
behaviors from being performed at the same time.
Mechanical interactions between locomotion and breathing in extant
tetrapods are of particular interest because lung ventilation has been
hypothesized to conflict with locomotion in the common ancestor of amniotes
(Carrier, 1987a). The
primitive amniote locomotor pattern includes lateral undulation, which
requires unilateral activity of axial musculature. Locomotion and ventilation
come into mechanical conflict because costal ventilation requires bilateral
activity of those same muscles (Carrier,
1987b
,
1991
). Birds, mammals and
crocodilians have circumvented this constraint through the independent
evolution of body postures and/or ventilatory mechanisms that partially
decouple breathing from locomotion
(Carrier, 1987a
;
Farmer and Carrier, 2000
). In
some lizards, the gular pump serves as an accessory mechanism to supplement
lung ventilation while costal musculature is in use for locomotion (Owerkowicz
et al., 1999
,
2001
). If limb movements
interfere with breathing in turtles, alternative ventilation mechanisms such
as the gular pump might be employed during locomotion. Previous studies have
shown conclusively that gular oscillations do not contribute to lung
ventilation in resting turtles (e.g.
McCutcheon, 1943
;
Druzisky and Brainerd, 2001
),
but none of these studies measured ventilation during locomotion.
The respiratory and locomotor functions of vertebrates are often highly
integrated and many vertebrates couple breathing and locomotion
(Bramble and Carrier, 1983;
Bramble, 1989
;
Bramble and Jenkins, 1989
;
Simons, 1996
;
Boggs et al., 1997
;
Carrier and Farmer, 2000
;
Boggs, 2002
). During the
locomotor cycle of mammals and birds, there are moments of acceleration and
deceleration as well as sagittal flexion and/or movements of the sternum.
These mechanical consequences of locomotion can produce cyclic loading regimes
within the thoracic cavity (e.g. Boggs et
al., 1997
). Birds and mammals may breathe at a particular point in
the locomotor cycle so that the forces generated by locomotion can contribute
to (Alexander, 1989
;
Boggs et al., 1997
;
Bramble and Carrier, 1983
;
Suther et al., 1972
;
Young et al., 1992
) or avoid
negative interaction with (Funk et al.,
1993
) pressure changes necessary for ventilation. In
locomotor-respiratory coupling, components of the breathing cycle are
predicted to maintain a fixed phase relationship with the stride cycle
(Simons, 1999
).
The goals of this investigation were to determine whether the box turtle, Terrapene carolina breathes during locomotion, and if so: (1) does locomotion alter breathing performance (i.e. tidal volume, breath frequency and/or minute volume); (2) are ventilation and locomotion temporally coupled; (3) are airflow rates directly affected by the stride cycle; and (4) are lung ventilation mechanisms the same as in resting animals (limb-pump and abdominal muscles), or is locomotion the impetus for an accessory mechanism such as the gular pump? Additionally, information about breathing performance during locomotion in box turtles may help to interpret the evolution of lung ventilation mechanisms in relation to the turtle's unique morphology.
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Materials and methods |
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Experimental animals
Three individual Terrapene carolina triunguis
Agassiz 1857 were used in
treadmill locomotion experiments (304, 420 and 305 g and 11.4, 12.4 and 11.9
cm carapace length for individuals 01-03, respectively). Animals were housed
individually in
150 liter terraria containing deep sandy soil, structure
to hide under and a water dish for soaking. They were fed earthworms, crickets
and/or vegetables twice a week, and kept at 26±4°C on a 14 h:10 h
light:dark cycle. Several attempts were made to run experiments during winter
months but the animals were torpid and refused to locomote. Therefore, all
experiments analyzed for this study were conducted during the summer
months.
Mask construction
Turtles lack narial valves and can breathe through either the nares or
mouth, so both were included in the pneumotach mask
(Winokur, 1982). To avoid
interference with vision or hearing, the mask was trimmed back from the eyes
and tympanic membranes. During construction, attachment and removal of the
mask, a padded restraint collar was fitted snugly around the turtle's neck to
prevent withdrawal of the head into the shell. When complete, the mask was
attached to the animal with surgical adhesive (cyanoacrylate). The seal of the
mask was tested by gently blowing into the port after the adhesive was
applied. The mask was removed immediately after the experiment without
apparent harm to the underlying keratinized skin. The small mass of the mask
(
3 g) should not have affected locomotion
(Marvin and Lutterschmidt,
1997
; Wren et al.,
1998
).
The pneumotach masks were custom built for each experiment from high
viscosity, rubber-based dental impression material (Henry Shein Co., Port
Washington, NY, USA) and required two stages of construction before being
glued to the animal's head (Fig.
2). During the first stage, the mask covered the nares while the
animal breathed through the mouth. Modeling clay (0.2 ml) was placed over
the nares and rounded to the size of the breathing port
(Fig. 2A). When removed, this
clay created open space in the mask for air to flow through. Dental impression
material was applied over the clay and around the eyes
(Fig. 2B). When set, the mask
was removed, cured and trimmed back away from the eyes and mouth, and a
plastic port was inserted through a hole punched in the tip of the mask.
During the second stage of mask construction, the animal breathed through the
port while clay was placed over the area where the upper and lower beaks
(maxillary and mandibular tomia) meet, and from the apex of the maxillary beak
up to the nares (Fig. 2C).
Dental impression material was applied over the clay, jaws and entire head
(except the nares). The previously constructed mask was placed over the
uncured material and pressed into place ensuring solid contact at all points.
After the composite mask was removed, cured, trimmed and the clay removed, the
mask was ready to be glued to the animal on the day of the experiment.
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The pneumotach itself was constructed from 53 µm nylon mesh screen
secured between two cylinders made from 0.6 cm long pieces of 1 ml
syringe (Fig. 2C). The walls of
the cylinder on each side of the screen were pierced by short (0.5 cm) pieces
of metal tubing (an 18-gauge hypodermic needle). The pneumotach was inserted
into the plastic breathing port (6 mm inner diameter) embedded in the mask
(Fig. 2C), and could be removed
during experiments for inspection and cleaning. The deadspace created by the
mask (the sum of pneumotach, breathing port and channels in the mask left
after removing the clay) was approximately 0.5 ml. The mask was not ventilated
with flowing fresh air because the dead space inside the mask was smaller than
normal tidal volume. If the combined tracheal and bronchial dead space is
estimated as 0.61 ml kg-1
(Perry, 1978
), the anatomical
and dead space created by the mask add up to approx. 0.75ml. The pneumotach
was calibrated before each experiment using known airflow rates and volumes
and was found to produce linear responses to flow over the ranges recorded
from the animals (r2>0.99).
Data acquisition
Locomotion experiments were conducted in a Plexiglass chamber enclosing a
low-speed motorized treadmill. A mirror was placed above the treadmill at a
45° angle, so that experiments filmed from the side recorded both lateral
and dorsal views. The pneumotach was connected to a differential pressure
transducer (Validyne DP103-06, Northridge, CA, USA) via thin plastic
tubing (PE 160). Data from the pressure transducer passed through a carrier
demodulator (Validyne CD-15) and were recorded using SuperScope 2.1 software
on a Macintosh computer. A real-time image of the pressure trace from the
computer was displayed on a television screen with a simultaneous image of the
treadmill chamber from a video camera. The video and computer images were
synchronized with a video overlay device (TelevEyes Pro, Dedham, MA, USA) and
recorded at 30 frames s-1 on S-VHS videotapes for frame-by-frame
analysis.
A target experimental temperature of 30°C was chosen to maximize
voluntary locomotion (Adams et al.,
1989; Gatten,
1974
), and temperature was controlled by a small space heater
placed just outside the experimental chamber. When an animal stopped
locomotion, it would be carried backward on the treadmill belt and typically
resumed locomotion when it neared the heat source. Occasionally, however, the
animals would rest close to this heat source, causing rapid increases in
cloacal temperatures. Cloacal temperatures, checked at least once every hour,
varied between 25 and 35°C and probably fluctuated more than core body
temperature.
The treadmill experiments were organized into four parts: (1) acclimation to the mask, treadmill chamber and experimental temperature; (2) pre-exercise; (3) locomotion; and (4) recovery (post-exercise). Stages 1, 2 and 4 were periods of 1 h each, while the locomotion part of the experiment varied between 2 and 3 h, depending on the animals' performance.
Locomotion was voluntary during the experiments, thus locomotor speed and actual amount of time spent walking during the `locomotion' segments was variable. Treadmill speed was manually adjusted using a variable-speed control dial to match the animals' chosen locomotor speed. After a series of strides, the animals typically rested and then spontaneously resumed locomotion again after a few seconds (e.g. Fig. 3A). Otherwise, turtles were stimulated to resume locomotion by starting the treadmill belt underneath them, being carried on the treadmill belt back toward the heat source or finally having their shells gently tapped against the back wall.
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X-ray video recordings were made (separately from the previous experiments) to compare ventilatory airflow with movements of the inguinal flank. Ventilatory airflow was recorded simultaneously with lateral view X-ray and light video images at 30 frames s-1. In order to visualize movements of the inguinal flank for kinematic analysis, a small piece of metal wire (1 mm diameter x 5 mm long) was glued to the skin anterior to the right hindlimb, at the most dorso-cranial extension of the limb pocket, just superficial to the region where the apexes of the oblique and transverse abdominal muscles come together. When the abdominal muscles are at rest (during apnea), this marker was just medial to the carapacial margin at the 7th marginal scute. Inguinal limb-pocket kinematics were measured by digitizing movements of the metal marker in the X-ray video relative to a point on the rear margin of the carapace, and calibrated by measuring the carapace height (in pixels) on screen and setting that equal to the actual carapace height (in cm).
Data analysis
A `bout' of locomotion was defined as a sequence of continuous locomotion
containing at least ten strides. 54 locomotor bouts from individual 01 were
analyzed to quantify the relationship between locomotor speed and stride
length, stride frequency, tidal volume and breath frequency. Distance traveled
during a locomotor bout was calculated from video recordings (to the nearest
0.05 m) by counting the number of evenly spaced marks that the turtle passed
on the treadmill belt. Locomotor speed was calculated for each bout by
dividing this distance by the duration of the bout. The number of strides per
bout was counted to the nearest half stride and average stride length and
frequency were calculated by dividing the number of strides per bout by the
distance traveled or duration of the bout respectively.
For all three individuals, single 20 min periods of locomotion were selected for analysis on the basis of locomotor consistency and duration. Within these intervals, however, the turtles would spend variable amounts of time resting between bouts of locomotion. In order to determine if these pauses might be acting as very short periods of recovery, we categorized each breath as either occuring during a pause or during locomotion, and analyzed these categories separately. For comparison, 20 min periods of breathing immediately before and after the locomotion trials were analyzed as pre-exercise and recovery respectively. The same 20 min periods of pre-exercise, locomotion and recovery were used in analyses of minute volume, tidal volume, breath frequency and phase.
In our tidal volume analysis, every breath was individually measured to calculate a mean tidal volume (± S.D.) for four behaviors: pre-exercise, locomotion, pauses and recovery. A two-way analysis of variance (ANOVA; StatView 5.0.1) tested for differences in tidal volume by including all of the 3325 measured breaths while accounting for within- and between-individual variation. Tukey's post-hoc test was used to test for pairwise differences between the four behaviors and the three individuals.
Minute volume (ml min-1) and breath frequency (breaths min-1) were calculated for each of the four behaviors (pre-exercise, locomotion, pauses and recovery) by dividing the sum of exhaled volumes or number of breaths by the duration of the sample period (20 min exactly for pre-exercise and recovery and the proportion of the 20 min period spent locomoting or in pause during the locomotion segment). Because these variables are measured over one long time period, there is no variance associated with the values. Paired t-tests (StatView 5.0.1) were used to make comparisons between the four behaviors (paired by individual).
We used phase analysis to quantify the temporal relationship between
breathing and locomotion. For each individual, the first ten locomotor bouts
containing at least ten breaths were selected for phase analysis. Maximum left
hindlimb extension (MHE) was the kinematically distinct point in the stride
cycle chosen to anchor the time measurements of the stride cycle (0°), and
was defined as the video frame in which both knee and ankle extension were
greatest (this corresponds to the end of stance for that limb). The duration
of each stride was normalized to 360° and peak inhalatory and exhalatory
airflow from each breath in a locomotor bout were plotted relative to when
they occurred in the locomotor stride cycle
(Simons, 1999). Raleigh's test
of circular uniformity (Zar,
1996
) was used to determine whether breath peaks were randomly
distributed relative to the stride cycle. We analyzed each of the 30 bouts
separately and analyzed the combined breaths from the ten bouts of each
individual together.
Airflow rate analysis was designed to test whether the magnitude of peak exhalatory and inhalatory airflow rates vary with respect to the stride cycle. We determined that peak inhalatory and exhalatory airflow rates of turtles breathing at rest were not statistically different from each other (P>0.05 for all three individuals; three separate unpaired t-tests with 50 exhalations and 50 inhalations measured per individual during recovery). If the stride cycle had no effect on airflow rates, the magnitude of inhalatory and exhalatory airflow peaks during locomotion would also be expected to be the same. If, however, limb movements during locomotion caused pressure changes around the lungs, the magnitude of peak inhalatory and exhalatory airflow at different points in the stride cycle would be expected to differ. For example, positive pressures created by limb movements would be expected to add to exhalatory airflow rates and subtract from inhalatory airflow rates, making exhalations larger than inhalations. For each of the three individuals, the locomotor stride cycle was divided into 18 (20°) bins and the mean peak airflow rate was plotted for inhalations and exhalations occurring in each bin. The means were considered significantly different if the 95% confidence limits did not overlap within a bin.
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Results |
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Both the pectoral and pelvic girdles have highly mobile connections to the
shell that may permit a wide range of movement during locomotion (compare
Fig. 1B,C). The pelvic girdle
articulates with the spine on either side via two small unfused ribs
and has considerable freedom to rotate mediolaterally, dorsoventrally and even
to translate craniocaudally (Bramble,
1974). The epipubis occupies the venterocranialmost position of
the pelvis and can translate mediolaterally during locomotion and
dorso-ventrally during plastral adduction. Each triradiate half of the
pectoral girdle lies inside the shell as it does in all turtles. It
articulates with the carapace via two small sesamoid ossifications
within the suprascapular cartilage
(Walker, 1973
). These supra-
and episcapular bones are interconnected by ligaments that allow the pectoral
girdle to translate anteroposteriorly during plastral adduction and abduction
(Bramble, 1974
). The presence
of both these bones is unique to Terrapene and they have been
hypothesized to lock the scapula passively in place when the plastron is
abducted and the pectoral girdle is protracted
(Bramble, 1974
).
Right and left pairs of antagonistic abdominal muscles are present in Terrapene carolina (Fig. 1A). The thin, sheet-like domes of the oblique abdominis (OA) and transverse abdominis (TA) muscles are cupped in opposite directions, with the convex sides of each muscle juxtaposed at their apexes. The OA lies just under the skin of the inguinal leg pocket (cranial to the hindlimb). The OA has broad attachment to the edges of the shell from the 10th to the seventh peripheral bones of the carapace, ventrally over the bridge, and from the hypoplastron halfway to the caudal limit of the ziphiplastron. The origin of the TA describes an `L' shape on the inner surface of the carapace. It runs parasagittally near the neural bones from the seventh to the fourth costal plates, turning 90° to continue ventro-laterally down the length of the fourth costal plate to the seventh peripheral plate. From the origin, the fibers of the TA travel posteroventrally around the caudal portion of the lungs, and then in an anteroventral arc under the internal organs to insert on a broad connective tissue aponeurosis. This connective tissue sheet continues from the plastral hinge cranio-dorsally around the anterior extent of the viscera to insert posterior to the pectoral girdle. In other turtle species, there may be muscular investment (diaphragmaticus) of the anterior portion of this connective tissue sheet, but this was not found in any of the T. carolina specimens examined in this study. There was also no indication of a striatum pulmonale muscle.
Airflow measurements and locomotion
Lung ventilation occurs almost continuously during treadmill locomotion
(Fig. 3; to view video clips of
turtle breathing during locomotion, refer to the online version of this
article:
http://jeb.biologists.org/).
Small buccal oscillations (<0.4 ml) were recorded during locomotor and
non-locomotor behavior, and were distinguished from lung ventilations by
expansion and contraction of the throat region (visible in video recordings).
Gular pumping for lung ventilation would be evident in airflow traces as small
inhalations followed by little or no exhalatory airflow
(Owerkowicz et al., 2001;
Druzisky and Brainerd, 2001
).
No such airflow pattern was ever observed in Terrapene carolina,
indicating that gular pumping for lung ventilation does not occur in this
species (or in any other turtle studied to date;
Mitchell and Morehouse, 1863
;
Hansen, 1941
;
McCutcheon, 1943
;
Gans and Hughes, 1967
;
Gaunt and Gans, 1969
;
Druzisky and Brainerd,
2001
).
During treadmill experiments, the three individual Terrapene carolina all showed similar patterns of short (approximately 10-30 s) voluntary locomotor bouts interspersed with brief pauses (approximately 2-60 s). During the 20 min periods of locomotion selected for analysis, the animals spent between one half and two-thirds of the time actually locomoting (61, 48 and 66%, respectively, for individuals 01-03).
Analysis of 54 locomotor bouts from individual 01 revealed that mean voluntary locomotor speed on the treadmill was 0.10 m s-1 and the range (0.074-0.124 m s-1) was relatively narrow (Fig. 4). Only data from individual 01 are presented here, but results were similar from individuals 02 and 03 except that the range of speeds was even smaller. Stride frequency and stride length were both strongly and positively correlated with speed (Fig. 4A). The slope of the relationship between stride frequency and speed was 7.7 times greater than the slope of stride length versus speed, indicating that increases in stride frequency accounted for 88% of increases in speed while increases in stride length accounted for the remaining 12% (Fig. 4A). Breath frequency and tidal volume were only weakly correlated with speed (Fig. 4B).
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During locomotion, breathing frequency and stride frequency were negatively but only weakly correlated (y=-1.03x+1.96, r2=0.264, P<0.0001). Tidal volume and stride length (y=19.06x-0.232, r2=0.206, P<0.001) were also weakly but positively correlated (graphs not shown).
Expired volumes ranged from 1 ml to over 40 ml. The largest exhalations occurred when the animals were accidentally startled and the head and limbs were retracted into the shell. Mean tidal volumes during locomotion, pauses and recovery were small (range 1.0-4.3 ml breath-1; Fig. 5) and not significantly different from each other (two-way ANOVA; P>0.05). Tidal volumes during pre-exercise were relatively large and significantly different from locomotion, pause and recovery values (Fig. 5; two-way ANOVA with Tukey's post-hoc tests; P<0.0001 for all three pairwise comparisons).
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In all three individuals, the highest breath frequency (breaths min-1) was recorded during locomotion (Fig. 5), but no statistically significant differences between behaviors (pre-exercise, locomotion, pauses and recovery) were detected (paired t-tests; P>0.05).
Mean minute volumes (ml min-1) were high during all four behaviors (Fig. 5). However, minute volumes during locomotion were exceptionally high (range 75-102 ml min-1) and significantly different (paired t-test; P=0.0037) from recovery values (range 5-40 ml min-1).
Polar plots of the temporal distribution of peak inhalatory and exhalatory
airflow relative to the stride cycle show no fixed phase relationship
(Fig. 6). Inhalations and
exhalations were analyzed separately for each of the ten locomotor bouts from
three individuals. Raleigh's test of circular uniformity
(Zar, 1996) revealed that
breaths were uniformly distributed in 55 bouts (P>0.05) and five
sequences had statistically non-random distributions of breaths relative to
the stride cycle (P<0.05). However, when the ten sequences from
each individual were combined, inhalations were randomly distributed relative
to the stride cycle in all three individuals
(Fig. 6A). Exhalations were
uniformly distributed for individuals 02 and 03 and non-uniformly distributed
for individual 01 (P<0.001;
Fig. 6B).
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To determine whether limb movements affect airflow rates during locomotion, mean airflow rates (±95% confidence intervals) were calculated for inhalations and exhalations occurring within 20° intervals of the stride cycle (Fig. 7). If the confidence intervals overlapped within a bin, the means were considered statistically indistinguishable. Very few statistically significant differences were found between inhalatory and exhalatory peak airflow rates and the differences that we did find were not consistent across the three animals studied. In individual 03, peak exhalation and peak inhalation were not statistically different at any point in the stride cycle (Fig. 7C). In individual 02, peak exhalation was greater than peak inhalation in two bins between 270° and 360° (Fig. 7B) and in individual 01, peak exhalation was greater than peak inhalation in two bins between 180° and 270° (Fig. 7A).
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In order to determine whether the abdominal muscles could be the mechanism
responsible for breathing during locomotion, we used X-ray video recordings to
track the movement of the inguinal flanks during breathing. A small metal
marker was glued to the skin of the inguinal flank just superficial to the
oblique and transverse abdominis muscles on the right side of the body
(Fig. 8). The y
coordinate (dorso-ventral component of flank movement) was measured and
plotted with simultaneous recordings of ventilatory airflow from the
pneumotach mask (Fig. 9). When
the turtle was not locomoting, exhalation was accompanied by dorsal movement
of the marker, and the marker moved ventrally during inhalation. These
movements are not likely to be passive deflections of the inguinal flank;
acting passively, they would be expected to move down (and laterally) during
exhalation (when pressure is greatest inside the pleuroperitoneal cavity) and
up (and medially) during inhalation (when pressure is lowest inside the
pleuroperitoneal cavity). During locomotion, inguinal flank movements were
similar to those at rest (up during exhalation and down during inhalation);
however, kinematic analysis was obscured by motion artifact caused by pitch,
roll and yaw during locomotion. X-ray video clips of Terrapene
carolina breathing at rest and during locomotion can be viewed on line as
part of this article
(http://jeb.biologists.org/).
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Discussion |
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Most lizard species locomote intermittently with low tidal and minute
volumes during high-speed bursts of locomotion and high tidal and minute
volumes during pauses and recovery
(Carrier, 1987b). When lizards
are forced to locomote steadily, minute volume generally decreases as speed
increases, and the highest minute volumes are recorded during recovery from
exercise (Wang et al., 1997
).
Breathing performance declines with increasing speed because axial muscles
used for breathing require a bilateral motor pattern while those same muscles
must be activated unilaterally to bend the body during locomotion
(Carrier, 1991
). Monitor
lizards circumvent this mechanical conflict by using a gular pump to inflate
the lungs during locomotion (Owerkowicz et al.,
1999
,
2001
). In the present study,
we hypothesized that Terrapene carolina might use gular pumping for
lung ventilation during locomotion if it experiences the apparent mechanical
conflict observed during locomotion in Chelonia mydas. However, in
agreement with all previous experimental studies of turtle ventilation
mechanisms, we found no evidence for the use of a gular pump during locomotion
in T. carolina.
Because the thoracic cavity undergoes cycles of pressurization with each
stride and with each breath, breathing and locomotion are often coordinated in
mammals and birds (e.g. Simons,
1996; Boggs, 2002
).
The shell of Terrapene carolina contains a nearly fixed volume, and
therefore we hypothesized that a cyclic pressure regime may be imposed on the
lungs as the limbs are protracted and retracted during locomotion. Whether
limb and girdle movements comprise the main (limb-pump) lung ventilation
mechanism or whether another breathing mechanism is synchronized to its
rhythm, the breathing and stride cycles were predicted to show phase coupling.
However, our results show that peak inhalatory airflow for all three
individuals and peak exhalatory airflow for two out of three individuals were
randomly distributed with respect to the stride cycle
(Fig. 6). We conclude that
T. carolina does not couple breathing and locomotion and limb
movements do not contribute to lung ventilation during locomotion.
Even though the timing of breaths relative to the stride cycle was found to be random, the airflow rates could still be affected by limb movement during locomotion. When the turtles were at rest, inhalations and exhalations were symmetrical and did not differ statistically in peak airflow rates. During locomotion, net retraction of the limbs during a given part of the stride cycle might increase peak exhalatory airflow rates and decrease peak inhalatory rates of breaths that happen to fall in that part of the stride cycle. Contrary to this hypothesis, however, we found few statistical differences between mean peak inhalatory and exhalatory airflow rates; the observed differences occurred at different parts of the stride cycle in different individuals (Fig. 7). Furthermore, because Terrapene carolina uses an alternating (symmetrical) gait, effects of limb movement on intrapulmonary pressure would be expected to cycle twice with each stride (see Fig. 3B). Contrary to this prediction, we found no cases in which statistical differences within individuals were mirrored on the opposite side of the stride cycle. Together, these results on the timing and magnitude of breaths relative to the stride cycle indicate that locomotion has no consistent, measurable mechanical effect on breathing in T. carolina.
Given the apparent independence of the breathing and stride cycles of Terrapene carolina, the lung ventilation mechanism must be mechanically separate from the locomotor system. At rest, T. carolina uses the transverse and oblique abdominal muscles to breathe (see Figs 1, 8, 9 and supplemental video clips). Since we found neither diaphragmaticus nor striatum pulmonale muscles in this species and no evidence for the use of a limb or gular pump mechanism, the abdominal muscles are the most likely mechanism for breathing during locomotion. Turtles rotate about all three orthogonal axes during locomotion, thereby making quantitative measurements of flank movements from two-dimensional X-ray videos difficult. However, X-ray videos show clearly that, when our study animals breathed during locomotion, the inguinal flanks moved in phase with the ventilatory cycle and independently from the stride cycle (see supplemental video clip).
The kinematics of locomotion in Chelonia mydas and Terrapene
carolina differ substantially and may help explain differences in their
breathing performance. When locomoting on land, adult C. mydas lift
the body and push it forward by retracting both front limbs simultaneously
(Wyneken, 1997). As pointed
out by Jackson and Prange
(1979
), the bilaterally
synchronous motor pattern presumably needed to produce this gait is also used
during limb-pump lung ventilation (Gans
and Hughes, 1967
). Terrestrial locomotor movements in C.
mydas may therefore generate large intrapulmonary forces. If the glottis
were open during the support phase of the stride cycle, limb movement
otherwise producing forward thrust could instead be producing exhalation, and
deflation of the lungs could result in medial rotation of both halves of the
pectoral girdle. Chelonia mydas may therefore cease breathing during
locomotion because the pressurized lungs are used as a support platform to
stabilize limb movements during locomotion (pneumatic stabilization:
Simons, 1996
;
Kidd and Brainerd, 2000
). In
contrast to the bilaterally synchronous gait of C. mydas, T. carolina
employs the more typical lateral sequence diagonal couplet walk used by most
turtles (Walker, 1971
;
Zug, 1971
;
Fig. 3). In this alternating
(symmetrical) gait, one (slightly staggered) diagonal pair of limbs is
extended while the other (also staggered) pair is flexed and retracted. The
balanced effect of these paired limb movements on internal shell volume,
combined with the independence of the abdominal muscles from the locomotor
muscles, may sufficiently explain the absence of any consistent measurable
effect of locomotion on ventilation in box turtles.
Despite not measuring any consistent effect of locomotion on breathing, we
still consider it possible, even likely, that locomotion has momentary, net
effects on internal shell volume. It seems unlikely that locomotion is so
tightly regulated that every movement of the limbs on the left side is
accompanied by perfectly synchronized and exactly opposite counter-action on
the right side. Additionally, left and right limb pairs are 180° out of
phase, but each limb spends more time in contact with the ground and applying
a rearward-directed force than it does in recovery or forward-directed
movement (duty factor >0.5; Fig.
3B). There are therefore two moments in each stride cycle when
both right and left members of each limb pair are moving backwards. The
unilateral abdominal motor pattern that Currie found in swimming turtles
(Currie, 2001,
2003
) is one potential
mechanism that may counteract the effects that limb movements probably have on
the lungs.
The speeds observed in this study are slow - even for turtles. These speeds
are typical for Terrapene carolina
(Muegel and Claussen, 1994;
Marvin and Lutterschmidt,
1997
); however, the interactions that we hypothesized between
locomotion and breathing may be more apparent in faster turtles e.g.
Chrysemys picta (Zani and Claussen,
1994
,
1995
), Terrapene
ornata (Adams et al.,
1989
; Claussen et al.,
2002
; Wren et al.,
1998
) and Trachemys scripta
(Landberg et al., 2002b
).
Breathing patterns
Minute volume was substantially higher during locomotion than during
recovery from exercise and not significantly different from pauses during
locomotion, indicating that Terrapene carolina is meeting (if not
exceeding) its aerobic metabolic demands during locomotion. Surprisingly, the
high minute volumes during locomotion were achieved by reducing breath size
(and duration) while increasing breath frequency. Previous studies have found
that turtles increase breath frequency and decrease tidal volume with
increases of temperature and metabolic rate (Altland and Parker, 1955;
Glass et al., 1979). The
relatively small tidal volumes associated with locomotion could be a response
to increased metabolism, but they may also minimize the mechanical
interactions between limb movement and breathing.
Pre-exercise breathing values in this study were recorded shortly (1-2
h) after the pneumotach mask was attached to the animal and may not be
entirely characteristic of `rest' (Glass
and Wood, 1983
). Turtle breathing at rest is typically
characterized by several large breaths clustered into bouts that are separated
by variable length non-ventilatory periods
(Milsom and Jones, 1980
).
Tidal volume during pre-exercise was higher than during locomotion, pause and
recovery (two-way ANOVA; P<0.0001), but not as large as reported
in other studies of box turtles at rest (e.g. Altland and Parker, 1955). The
turtles in our study showed pre-exercise breath frequencies that were high
(8.4±9.0 breaths min-1; mean ± S.D. for
N=3 individuals) compared to published data showing that
Terrapene carolina breathes 4-5 times min-1 at around
30°C (Altland and Parker, 1955), T. ornata breathes 1.5 times
min-1 at 25°C (Glass et
al., 1979
) and Trachemys scripta breathes 1-2
times-1 at 30° (Jackson,
1971
; Jackson et al.,
1974
). We interpret the high frequency and small (relative to
other studies) pre-exercise tidal volumes to be due to the presumed stress
associated with the masking procedure or experimental conditions.
Evolutionary considerations
Extant lizards exhibit a mechanical conflict between simultaneous
ventilation and locomotion because axial muscles are used in a unilateral
activation pattern to bend the body from side to side during locomotion, while
many of those same muscles require a bilaterally synchronous motor pattern to
expand the thoracic cavity during breathing (Carrier,
1987a,b
,
1991
). Extant turtles would
not be subject to this constraint because their ribs are fused to form part of
the shell, and therefore do not contribute to either locomotion or
ventilation. However, the shell-less ancestor of turtles probably did rely on
axial bending during locomotion and rotation of the ribs during breathing. In
the absence of another breathing mechanism, this hypothetical ancestor of
turtles would have experienced a mechanical conflict between locomotion and
ventilation. We hypothesize that the specialized ventilatory functions of the
abdominal muscles in extant turtles were favored by natural selection because
they permitted breathing during locomotion in the lineage that led to turtles.
This accessory ventilation mechanism would then have become the primary lung
ventilation mechanism as the ribs abandoned their ventilatory function and
fused into the shell.
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Acknowledgments |
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Footnotes |
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References |
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