Contractile activity of the pectoralis in the zebra finch according to mode and velocity of flap-bounding flight
Department of Biology, University of Portland, 5000 N. Willamette Boulevard, Portland, OR 97203, USA
* Author for correspondence (e-mail: tobalske{at}up.edu)
Accepted 6 June 2005
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Summary |
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Key words: zebra finch, Taeniopygia guttata, intermittent, flight, muscle
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Introduction |
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An alternative, long-standing explanation for the use of flap-bounding has
been previously described as a `fixed-gear' hypothesis (Rayner,
1977,
1985
;
Rayner et al., 2001
;
Tobalske, 2001
). Small birds
tend to either have a single fiber type or single myosin isoform in the fibers
of their primary downstroke muscle, the pectoralis
(Rosser and George, 1986
;
Rosser et al., 1996
). As a
given fiber type is expected to have an optimum velocity for efficient
contraction, work and power output (Hill,
1950
; Nelson et al.,
2004
), the fixed-gear hypothesis predicts that a physiological
constraint of a single fiber type limits the range of efficient contractile
velocities available to small birds. Furthermore, early work
(Aulie, 1970
) suggested that
small birds have only a single motor unit in their pectoralis, and this
condition should preclude variation in motor-unit recruitment as a mechanism
for modulating force among wingbeats (Rayner,
1977
, 1984). Thus, the
pectoralis of small birds is predicted to be `geared' for maximal mechanical
power output during activities such as acceleration, ascent or hovering.
According to the fixed-gear hypothesis, intermittent non-flapping phases
represent the only way to vary average power below maximal value without
incurring a loss of efficiency due to variation in contractile velocity
(Rayner, 1985
).
Indirect evidence from flap-bounding zebra finch (Taeniopygia
guttata Vieillot) casts doubt upon the validity of the fixed-gear
hypothesis because the birds exhibit variation in angular velocity of their
wing when flying at different velocities
(Tobalske et al., 1999). Also,
other small birds that use intermittent flight, such as the budgerigar
(Melopsittacus undulatus), vary motor-unit recruitment in a manner
that appears to be associated with variation in force production in their
pectoralis (Tobalske and Dial,
1994
; Tobalske,
2001
; Hedrick et al.,
2003
). In the present study, we seek to directly test the
fixed-gear hypothesis using in vivo measures of muscle length change
and neuromuscular recruitment. Explicitly, if the fixed-gear hypothesis is
correct, the zebra finch should exhibit no significant variation in two
variables in the pectoralis: (1) strain rate (L s1)
and (2) relative electromyographic (EMG) amplitude.
Although commonly used for studies of animal flight in the laboratory, wind
tunnels represent an artificial environment, and relatively few data are
available for comparing wind tunnel and free flight (e.g.
Tobalske et al., 1997;
Liechti and Bruderer, 2002
).
Moreover, virtually nothing is reported of the effects of surgical
implantation of transducers and electrodes upon flight performance. Thus, we
also compare flight performance among modes of flight with performance across
flight speeds and include tests for effects of surgery upon intermittent
flight kinematics.
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Materials and methods |
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A subset of the birds (N=7) were trained to fly in a flight cage, 1 m wide x 2 m long x 2 m high, constructed of 2.5 cm plastic pipes and covered with 1.36 cm nylon mesh. Four 650 W halogen lights (Lowel Tota-light, Lowel-Light Manufacturing, Inc., Brooklyn, NY, USA) were distributed around the cage to continuously illuminate the field for video recording. The birds were trained to fly between two perches in response to a hand signal. For level flight, these perches were 1.5 m apart and 1 m in height. Alternatively, the perches were configured to provide an average flight path of 1.5 m in length and ascending at +60° or descending at 60°.
A different subset of the birds (N=5) was trained to fly in a
variable-speed wind tunnel over the full range of flight speeds for which they
were willing to fly (014 m s1 prior to surgery and
012 m s1 after surgery). Training protocols followed
Tobalske et al. (1999). Three
650 W halogen lights (Lowel Tota-light) were used to illuminate the flight
chamber.
Wind tunnel
The wind tunnel was designed for studies of avian flight at the University
of Portland. It is generally similar in design to the Harvard-CFS tunnel
described in Hedrick et al.
(2003); Barlow et al.
(1999
) and original sources
therein were used in the design of the tunnel. The tunnel is an open circuit
with a closed jet, featuring a 6:1 contraction ratio. Total length is 6.1 m.
The working section in which the bird flies is square in cross-section,
60x60x85 cm inner diameter at the inlet, with clear lexan walls, 6
mm thick, used to provide views inside the working section. The flight chamber
increases to a 61.5x61.5 cm outlet to accommodate boundary-layer
thickening. Air is drawn through the tunnel using a 7.5 kW (10 horsepower)
direct current motor and a 0.75 m-diameter fan assembly (AFS-75 Series; SMJ
Incorporated, Grand Junction, CO, USA). During experiments, velocity is
selected as equivalent air velocity rather than true air velocity, as
recommended by Pennycuick et al.
(1997
).
To describe the general quality of airflow within the flight chamber, we
sampled profiles with the tunnel nominally set at equivalent airspeeds of 6,
10 and 18 m s1. The profiles were obtained in the mid-plane
of the working section using a pitot-static probe placed at 36 locations in a
10 cm spaced grid pattern (Hedrick et al.,
2003). Mean equivalent velocities
(±S.D.) at the three settings were:
5.9±0.2, 9.9±0.3 and 18.1±0.5 m s1.
Maximum absolute deviations from the mean were always less than 10% of the
nominal equivalent velocity (8.6%, 9.1% and 7.0% at 6, 10 and 18 m
s1). A velocity traverse in the mid-plane, mid-height
indicated that the local boundary layer thickness was <1 cm. Measurements
in the mid-plane, mid-traverse using a 30 cm turbulence sphere indicated a
percent turbulence of 1.2% (Barlow et al.,
1999
).
Kinematic analysis
We measured wing and body kinematics during non-implanted and implanted
flights using digital video. A Redlake PCI-2000 (San Diego, CA, USA) provided
a lateral view (250 Hz, PCI-2000, stored using PCI-R v.2.18 software), and a
Redlake Motionscope 250 (250 Hz) provided a dorsal view for correction of
lateral-view parallax and to confirm wing posture during non-flapping phases.
Analog output from the Motionscope was imported to computer and stored using
Quicktime v.3.5 software (Apple, Inc.). For both cameras, we used a shutter
speed of 1/1000 s.
The birds were marked with dots of non-toxic black ink to identify anatomical landmarks when viewing digital images: shoulder, base of tail and distal tip of wing at the 9th primary. Approximate center of mass was calculated as a point halfway between the shoulder and the base of the tail. We digitized these anatomical landmarks using Didge v.2.07 (A. Cullum, Creighton University, Omaha, NE, USA).
From the video, we measured wingbeat frequency (Hz) as the number of wingbeats within a flapping phase divided by the duration of the flapping phase (in s). Non-flapping intervals consisted of bounds during which the wings were held flexed against the body for periods of 8 ms or more (two instances, 0.2% of total, featured glides). From the observed duration of flapping and non-flapping phases, we calculated the percentage of time spent flapping. We analyzed 111 flights within the flight cage (37 for each flight mode: level, ascending and descending) and 868 flapping/non-flapping phases in wind tunnel flight.
Sonomicrometry and electromyography
Following unimplanted experiments, we surgically implanted electromyography
(EMG) electrodes and sonomicrometry transducers into the left pectoralis of
the zebra finch and repeated the experiments
(Hedrick et al., 2003;
Tobalske et al., 2003
). To
accomplish the implantation, birds were anesthetized using isoflurane inhalant
and maintained at a surgical plane. Feathers were removed from the upper back
as well as the left pectoralis. An alcohol solution was used to cleanse the
skin. A 1 cm incision was made on the ventral surface over the pectoralis, and
a 0.5 cm incision was made along the back. Sonomicrometry transducers
(Sonometrics, London, Ontario, Canada; omnidirectional, 1.0 mm, 37 Gauge
copper wire) and electromyography electrodes (twisted pair, bipolar 0.5 mm
exposed tips, 100 µm silver wire) were moved underneath the skin from the
dorsal incision to the incision over the pectoralis. Two holes, 8 mm apart,
were placed parallel to the longest muscle fascicles of the sternobrachialis
portion of the pectoralis (Tobalske and
Dial, 2000
), and the sonomicrometry crystals were placed 3 mm deep
into the holes. The crystals were sutured in place using 6-0 braided silk. A
25 Gauge hypodermic needle was used to implant the electromyography electrodes
immediately between the sonomicrometry crystals. All wires were sutured to the
intervertebral ligaments, and then all incisions were sutured closed. The
total mass of all recording equipment carried by the bird was 1.05 g (7.5% of
body mass). The animal was allowed to fully recover from surgery, with
experiments beginning within 4 h and lasting approximately 2 h. Following the
experiments, a recovery surgery was performed in which all recording equipment
was removed from the bird. All of the experimental animals recovered from the
experiments were later donated to private individuals.
Electromyographic signals were amplified (1000x) and filtered (60 Hz notch, 100 Hz low pass, 5000 Hz high pass) using an AM Systems (Carlsborg, WA, USA) Model 1700 Differential AC amplifier. Sonomicrometry signals were created and recorded using a Sonometrics TRX Series 4 Digital Ultrasonic Measurement System and Sonosoft 3.2.1 software. Synchronization between the video and pectoralis data was obtained by sending a trigger pulse from the video camera to a separate channel on the Sonometrics A/D converter.
Subsequent analysis of the recorded signals was accomplished using IGOR 3.6 (Wavemetrics, Inc., Lake Oswego, OR, USA). EMG signals remained as recorded in volts, while sonomicrometry data were calibrated to strain (L/Lrest) using the resting, perched values for crystal separation as Lrest. EMG bursts were identified as continuous sequences of spikes with rectified amplitude at least twice the amplitude of baseline electrical noise. For each contractile cycle (= wingbeat cycle), we measured the duration of EMG activity in the pectoralis (in ms) from onset to offset and also calculated the percentage of the wingbeat cycle in which the muscle was active. Start of downstroke was defined using the sonomicrometry trace at the onset of pectoralis shortening. Fractional lengthening (%) and fractional shortening (%) were the proportions of pectoralis strain in which the muscle was longer than or shorter than resting length, respectively. Strain rate (muscle L s1) was pectoralis strain divided by the duration of time between maximum and minimum length as the muscle shortened during a contractile cycle. We also measured shortening duration (%), the time interval from maximum to minimum muscle length, relative to total cycle time. We analyzed 704 wingbeats including at least 10 from each speed for each bird.
Statistical analysis
For each variable, we computed the mean value within each bird at each mode
or speed. We then tested for a significant effect of flight mode or speed upon
each variable using a univariate repeated-measures analysis of variance. To
evaluate the effect of implanting recording equipment upon flight kinematics,
we analyzed wingbeat frequency and the percent time spent flapping using
repeated-measures ANOVA with experimental condition (implanted versus
non-implanted) as a between-subjects factor. Values are presented as means
± S.D.
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Results |
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There was a significant effect of surgical treatment upon wingbeat frequency and percent time flapping (P<0.002 for both variables; Fig. 4). Surgery decreased wingbeat frequency by an average of 2.0±0.1 Hz among flight modes. By contrast, among flight velocities, post-surgery wingbeat frequencies were 3.4±0.7 Hz greater than pre-surgery flights. Post-surgery flights always featured a higher percentage of time spent flapping; the mean increase was 20.4±9.8 Hz among flight modes and 17.0±8.0 Hz among flight velocities. Wingbeat frequency was highest during ascending flight prior to surgery (29.5±1.3 Hz) and lowest (20.7±2.2 Hz) during pre-surgery wind tunnel flight at 4 m s1 (Fig. 4A,B). By contrast, the variation in percentage of time spent flapping was greatest among velocities in the wind tunnel, with a minimum of 62.6±9.6% during pre-surgery flights at 8 m s1 and a maximum of 98.7±1.1% during post-surgery flight at 0 m s1 (Fig. 4C,D).
The percentage of a wingbeat cycle that the pectoralis spent shortening (66.9±2.9%) did not change significantly among flight modes (P=0.2) or velocities (P=0.3; Fig. 5; Table 2), but mode and velocity caused significant variation in the timing of electrical activity within the wingbeat cycle. Burst duration of the EMG signal was greatest at intermediate velocities (19.7±3.7 ms) and during level free flight (14.5±1.6 ms) and was least during flight at 12 m s1 (16.0±1.8 ms) and during ascending free flight (11.0±1.8 ms). As may be observed in Fig. 5, the relative offset of EMG activity varied among flight modes (P=0.03) and velocities (P=0.03). Relative onset also appeared to change among flight modes (P=0.04) but not flight velocities (P=0.1).
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Discussion |
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In a comparative, biological context, variation in strain rate in the
pectoralis of zebra finch was more substantial than variation in motor unit
recruitment. Data for strain rate across flight velocities are available from
the cockatiel (Hedrick et al.,
2003), a species that does not use flap-bounding flight. As in the
finch, the cockatiel exhibits a U-shaped curve of strain rate as a function of
flight speed, ranging from 5.19 L s1 during flight
at 5 m s1 to 6.73 L s1 at 13 m
s1. The coefficient of variation (CV, expressed as
a percentage) for strain rate of the pectoralis in the cockatiel is 9.3%
whereas it was 15.3% among flight velocities in the zebra finch. Likewise,
among flight modes, pectoralis strain rate in pigeons (Columba livia;
649 g; B. W. Tobalske, R. J. Hicks, D. C. Stark and A. A. Biewener,
unpublished) exhibits a CV of 12.8% versus the 16.1% we
observed in the zebra finch. By contrast, at 7.7%, the CV for
relative amplitude of EMG in the zebra finch across velocities is less than
the CV of 50.0% in black-billed magpies (Pica hudsonica; 150
g; Tobalske et al., 1997
) and
27% in the cockatiel (Hedrick et al.,
2003
).
The zebra finch is presently the smallest bird species for which in
vivo pectoralis strain data are available. Average strain rates in this
species during flight in the wind tunnel (7.9±1.2 L
s1) are slightly higher than those exhibited by the
cockatiel (5.8±0.5 L s1;
Hedrick et al., 2003), and the
strain rate exhibited by the finch during ascent (11.3 L
s1) is well above values exhibited by galliform birds
engaged in ascent (4.36.3 L s1;
0.0435.2 kg; Tobalske and Dial,
2000
; Askew and Marsh,
2001
). Also, the peak strain rates observed in the zebra finch
pectoralis are near 90% of Vmax on forcevelocity
curves for the lateral gastrocnemius and peroneus longus of turkeys
(Meleagris gallopavo; Nelson et
al., 2004
). These data suggest that strain rate for a given
activity may scale negatively with increasing body mass in birds. This
prediction awaits further comparative study, as Vmax for
muscles from a wide size range of vertebrates (0.034.5 kg) is
independent of body size (Nelson et al.,
2004
).
Peak strains of 23.7% in the pectoralis
(Fig. 3) areconsiderably less
than the 3235% strains reported for pigeons
(Biewener et al., 1998),
black-billed magpies (Warrick et al.,
2001
), mallards (1 kg; Anas platyrhynchos;
Williamson et al., 2001
) and
cockatiels (Hedrick et al.,
2003
). The zebra finch strains are more similar to those exhibited
by small galliform birds (0.0431 kg) engaged in take-off
(Tobalske and Dial, 2000
;
Askew and Marsh, 2001
). Thus,
it appears that smaller birds may utilize less strain and higher strain rate
to develop power for flight compared with larger birds. Doubtless, these
patterns are related to phylogeny and ecology, with, for example, mallards and
chukars (Alectoris chukar) having similar body mass (1 kg) but quite
different strain rates during ascending flight
(Tobalske and Dial, 2000
;
Williamson et al., 2001
).
The variation we observed for the relative timing of the shortening phase
of the pectoralis contraction cycle, as well as the duration, onset and offset
of EMG activity, may signal additional mechanisms for modulating work and
power output per wingbeat (Hedrick et al.,
2003). Askew and Marsh
(1998
) provide important
insight into how strain trajectories and muscle activationdeactivation
modulate power output in mammalian muscle. A similar in vitro study
of muscle contractile dynamics of zebra finch pectoralis would aid greatly in
understanding the functional consequences of the variation we have reported in
this study.
Few studies have compared animal flight performance in wind tunnels with
performance in free flight (Tobalske et
al., 1997; Liechti and
Bruderer, 2002
), and no study, to our knowledge, has evaluated the
effect of surgical implantation on flight performance. Liechti and Bruderer
(2002
) report that effective
wingbeat frequency (wingbeat s1; including non-flapping
phases) was less in barn swallows (Hirundo rustica) and house martins
(Delichon urbica) during flight outdoors compared with in a wind
tunnel, and it appears that our observations of pre-surgery percentage time
spent flapping (Fig. 4C) are
consistent with their study. Although wingbeat frequency in zebra finch during
the short-duration flights in the flight corridor was higher than wingbeat
frequency at a comparable speed (2 m s1) in the wind tunnel,
the percentage time spent flapping was considerably less.
Our observation that surgery and the transport of recording transducers and electrodes increased wingbeat frequency and percent time flapping is perhaps not surprising, but it does provide new and sobering insight that confirms that the process of experimenting with flying animals alters their flight performance. In light of the change in performance pre- and post-surgery (Fig. 4A,B), our data must be interpreted with caution. Future in vivo experiments with flying birds should also seek to evaluate the effects of the experimental procedure on locomotor performance; in this way, we might refine experimental design to minimize `observer-induced' error.
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Acknowledgments |
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