Comparative studies of high performance swimming in sharks I. Red muscle morphometrics, vascularization and ultrastructure
1 Center for Marine Biotechnology and Biomedicine and Marine Biology
Research Division, Scripps Institution of Oceanography, University of
California San Diego, La Jolla, CA 92093-0204, USA
2 Department of Medicine University of California San Diego, La Jolla, CA
92093-0623, USA
* Author for correspondence at present address: Department of Zoology, Weber State University, Ogden, UT 84408-2505, USA (e-mail: dbernal{at}weber.edu)
Accepted 8 May 2003
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Summary |
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Key words: lamnid shark, tuna, myotome, red muscle, aerobic capacity, myoglobin, muscle ultrastructure, scaling, allometry, Lamnidae, Scombridae, Isurus, Lamna, Alopias, Mustelus, Triakis, Prionace
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Introduction |
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The `high-performance swimming adaptations' of tunas and lamnids include
features enhancing tissue O2 transfer at the gills (i.e. a large
gill-surface area), the capacity to deliver a large quantity of O2
to the RM (i.e. a large heart with a thick compact myocardial layer, a large
stroke volume and well-developed coronary circulation and both a high blood
hemoglobin concentration [Hb] and hematocrit) and regional endothermy
(Dickson, 1996;
Lai et al., 1997
;
Bernal et al., 2001a
;
Brill and Bushnell, 2001
;
Korsmeyer and Dewar, 2001
).
Although the presence of these high-performance adaptations in tunas and
lamnids suggests that both groups are capable of sustaining a higher aerobic
metabolism during swimming relative to that of other fishes
(Bernal et al., 2001a
), there
is no direct experimental evidence that these adaptations increase swimming
efficiency (Katz, 2002
).
Relative to the RM of other fishes, tuna RM receives a large percentage of
cardiac output (White et al.,
1988) and has a greater capacity for mitochondrial oxidative
phosphorylation (i.e. ATP production; Dickson,
1995
,
1996
). Consistent with the
heightened aerobic capacity of tuna RM are structural and biochemical features
favoring O2 transfer from the RM capillaries to the fiber
mitochondria. These include a relatively small RM fiber diameter, high
capillary densities, the presence of capillary manifolds, which increase
capillary surface area to fiber volume ratio, and a higher muscle myoglobin
concentration [Mb], which enhances the diffusion of O2 from the
blood into the muscle cells (Wittenberg,
1970
; Bone, 1978b
;
Mathieu-Costello et al., 1992
,
1995
,
1996
; Dickson,
1995
,
1996
;
Sidell, 1998
;
Suzuki and Imai, 1998
).
The objective of this study is to provide comparative data on the position
and quantity of lamnid RM and on this tissue's structural properties related
to high-performance swimming. Although lamnid sharks are thought to have RM
specializations for enhanced O2 transfer that are comparable to
those of tunas, this has not been documented. Also, data showing the position
of the maximal RM cross-sectional area of makos and other lamnids presented by
Carey et al. (1985) are
suggestive of a tuna-like RM distribution pattern but indicate a much smaller
RM quantity in lamnids relative to tunas. Using mako sharks and other lamnids,
we have quantified RM position and developed an algorithm to extract more
quantitative RM data from the findings of Carey et al.
(1985
). We also report
preliminary RM ultrastructural and biochemical findings relating to lamnid RM
aerobic capacity and compare these features with those of other non-lamnid
sharks and tunas.
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Materials and methods |
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RM distribution and quantification
The longitudinal distribution of RM was measured in two lamnid and three
non-lamnid shark species. The lamnid sharks are the shortfin mako shark
(Isurus oxyrinchus Rafinesque 1810; body mass, 550 kg;
N=8) and the salmon shark (Lamna ditropis Hubbs and Follet
1947; 15.9148 kg; N=2). The three non-lamnid sharks are the
common thresher shark (Alopias vulpinus Bonnaterre 1788;
9.170.4 kg; N=6) and two species in the order
Carcharhiniformes, the blue shark (Prionace glauca L.; family
Carcharhinidae; 2.122.2 kg; N=4) and the leopard shark
(Triakis semifasciata Girard 1855; family Triakidae; 1.415 kg;
N=3).
In all of the species studied, measurable quantities of RM did not occur anterior to 2325% FL. Beginning at this position, whole frozen sharks were cut into approximately 23 cm-thick transverse sections to the caudal peduncle (8595% FL). In the common thresher shark, where the RM extends far into the very long upper caudal fin lobe (Fig. 1), sections were extended to 140% FL.
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The TRMM and RM linear distribution determinations for mako sharks in this
study enabled us to convert the RM data presented by Carey et al.
(1985) into additional
estimates of TRMM. These workers had originally reported the maximal RM
cross-sectional area of nine makos (575 kg) and expressed TRMM as a
percentage of total axial muscle mass. By combining the RM data for seven of
those makos with our mako RM distribution analysis, we were able to express
them in terms of percentage body mass.
Myoglobin analysis
General
Red muscle myoglobin concentration [Mb] was quantified using a modification
of the high-performance liquid chromatography (HPLC) method of Kryvi et al.
(1981). Fresh or frozen
(-80°C) RM samples were obtained from I. oxyrinchus
(5.946.7 kg; N=10), L. ditropis (91.2114 kg;
N=2) and A. vulpinus (6.3545.5 kg; N=7). To
compare our methods with those of Kryvi et al.
(1981
), we measured the RM
[Mb] in one specimen of the gray smoothhound (Mustelus californicus;
order Carcharhiniformes; family Triakidae; 0.8 kg), which belongs to the same
order and has an RM distribution pattern similar to that of the species
studied by those investigators.
Approximately 0.2 g of RM was homogenized in a 15 ml tissue grinder (Kontes Duall 23) using 9x the tissue mass of running buffer (30 mmol l-1 KH2PO4, pH 7.2 at 20°C). Solid particulates were separated by centrifugation at 12 000 g for 10 min at 4°C and the supernatant containing the dissolved Mb was removed and diluted 2x using running buffer. Sufficient Na2S2O4 (approximately 1 mg) was added to each sample supernatant to ensure that Mb was in the reduced state (cherry red in color) prior to passing the sample through a 0.45 µm syringe filter (Gelman Acrodisc LC 13 PVDF).
HPLC parameters
Separation of tissue Mb and Hb is based on their different molecular masses
(Mb, 16 kDa; Hb,
64 kDa) and thus their different elution times
through a silica-based size exclusion column [Alltech PEEK Macrosphere
GPC (60 Å; 7 µm; 4.6 mm diameter x 250 mm length), protected by
a guard column (Alltech MF Guard; 60 Å; 6 µm)]. The gel permeation
column (approximately 1.7 ml exclusion volume) was equilibrated with at least
150 ml of running buffer prior to the injection of the first sample. A 200
µl injection loop was used to load 25 µl of filtered and reduced tissue
homogenates. Myoglobin was quantified by flowing degassed running buffer (0.3
ml min-1) through a diode array detector (Beckman Gold 168) at 413
nm.
Myoglobin quantification
A linear relationship (r=0.99) between known quantities of
purified Mb (Sigma M 0630) and the integrated area under the curve was used to
quantify RM [Mb]. The lower [Mb] detection limit was 17 pmol Mb (0.28 mg Mb
g-1 tissue wet mass). Verification of an adequate size-based
separation was established by the injection of mixed Mb and Hb (Sigma H 4632)
standards and the resulting time-separated maximum absorbance peaks.
RM vascularization and ultrastructure
The tissues of two mako sharks (95 cm FL, 9 kg; 100 cm
FL, 12 kg) were fixed in situ via perfusion of
glutaraldehyde following methods detailed in Mathieu-Costello et al.
(1992). Sharks were attracted
to the boat, dip netted and returned to the laboratory alive (see
Bernal et al., 2001b
). Once in
the laboratory, sharks were secured ventral-side up in a restraining V-board,
and a 2.5 cm-diameter hose was inserted into the mouth to ensure that
well-oxygenated running seawater flowed over the gills during the entire
procedure. A dose of anesthetic (1:5000; MS-222) was mixed with seawater and
the fish was ventilated for an additional 510 min to allow for complete
sedation before surgery.
A midline incision exposed the heart, and a cannula was inserted into the
conus arteriosus. All systemic blood returning to the heart was drained by
cutting away the sinus venosus. Perfusion with heparinized saline solution
[574 mmol l-1 NaCl (approximately 1100 mosmol l-1)
containing 20 ml l-1 1000 sodium heparin USP; Elkins-Sinn Inc.,
Cherry Hill, NJ, USA] preceded the fixative solution [6.25% glutaraldehyde in
0.1 mol l-1 sodium cacodylate (Polysciences Inc., Warrington, PA,
USA) buffer, pH 7.4 at 20°C, 132 mmol l-1 NaCl (approximately
1100 mosmol l-1)]. Both the saline solution and the fixative buffer
were administered at an in vivo non-pulsatile blood pressure of
approximately 9.3 kPa (Lai et al.,
1997).
Following perfusion, RM samples (1 cmx4 mmx1 mm; approximately
0.04 g) were taken at approximately 45% FL (under the first dorsal
fin) and cut into longitudinal strips for storage in the fixative solution.
Samples were minced into small blocks (1 mmx1 mmx2 mm) for
subsequent transverse (=0°, angle between normal section and muscle
fiber axis) and longitudinal (
=
/2) section orientations and
postfixed with osmium tetroxide solution prior to being embedded in Araldite
for morphometric analyses using light microscopy and transmission electron
microscopy, as described in Mathieu-Costello et al.
(1992
).
Sections (1 µm) from each mako shark were cut in transverse (46
blocks per shark) and longitudinal (2 blocks) orientations and stained with
0.1% aqueous Toluidine Blue solution. Morphometric analyses followed the
methods in Mathieu-Costello et al.
(1992) for tuna locomotor
muscle. The mean sarcomere length (lo) in each mako was
estimated from longitudinal sections by direct measurements of 40 muscle cells
at 1000x magnification. A section angle closest to
/2 was determined
by rotating the tissue block on the microtome in 1° increments until the
shortest lo was measured in the sections. Mean fiber
cross-sectional area [
(f)], and capillary density per
mm2 sectional area of muscle fiber [transverse,
QA(0); longitudinal, QA(
/2)] were
estimated by point-counting, at 400x magnification, one transverse
section from each block (total 10 blocks). Capillary density was calculated
from the longitudinal sections (2 blocks) in the 9 kg mako but not in the 12
kg specimen because the capillaries were partially collapsed as a result of an
incomplete perfusion fixation.
The ratio QA(0)/QA(/2) was used
to calculate the capillary anisotropy concentration parameter, K (from tables
in Mathieu et al., 1983
), and
then to estimate the orientation coefficient c(K,0), which relates
the capillary counts per unit area of fiber in a transverse orientation and is
used to estimate the relative increase in capillary length per volume of
muscle fiber. A c(K,0) of 1 indicates that capillaries run straight
and parallel to the muscle fiber axis and are unbranched, while the maximum
c(K,0) value of 2 indicates that there is no preferential orientation
relative to the muscle fiber axis (i.e. it is random;
Mathieu et al., 1983
). The
product of QA(0) and c(K,0) was used to estimate
total capillary length per volume of muscle fiber, JV(c,f)
[i.e. this term includes the contributions of capillary tortuosity (vessel
convolutions that increase the fiber contact area) and branching;
Mathieu-Costello et al.,
1992
]. The mean number of capillaries surrounding each muscle
fiber (NCAF) were estimated by counting directly at a
magnification of 400x (n=
200 fibers per muscle sample), and
the mean fiber diameter [
(f)] was
estimated by 2·[
(f)/
]0.5, assuming a
circular fiber cross-sectional area. Mean capillary diameter
[
(c)] was measured (n=21)
using a 1 µm scale eyepiece grid at a magnification of 400x.
Measurement of
(c) was limited to
circle-shaped capillary sections in which the ratio between the smaller and
larger diameters did not exceed 1.2 (i.e. 20%).
Mitochondrial density was measured using a total of eight tissue blocks
(four from each mako) from which ultrathin (5070 nm) transverse
sections were obtained and contrasted with uranyl acetate and bismuth
subnitrate (Mathieu-Costello et al.,
1992). The volume densities of mitochondria and myofibrils were
each measured by point-counting on 70 mm film micrographs (3038 for
each muscle sample) taken by systematic random sampling of one transverse
section from each block examined at a final magnification of 9208x using
a Zeiss 10 transmission electron microscope
(Mathieu-Costello et al.,
1992
).
Statistical analysis
All statistical analyses were performed at a significance level of
=0.05. The allometric relationship
aMb±95%C.I. (where a is a constant and
M is body mass) was used to obtain the scaling coefficient b
for TRMM in the different shark species, and a Student's t-test was
used to determine if b=1 (i.e. isometric scaling). The mean values
for different data sets were compared using a Student's t-test.
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Results |
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RM scaling
The image analysis (IA) and gravimetric (GD) techniques for TRMM
determination are in good agreement. Respective values for the three makos
studied are given in terms of body mass (kg) and TRMM (kg) for IA/GD
techniques: 5.3, 0.099/0.095; 14.0, 0.255/0.260; 16.6, 0.337/0.355.
Table 1shows the TRMM
determined for each shark studied and also includes values for seven of the
makos for which TRRM mass was estimated from the maximum RM cross-sectional
area data reported by Carey et al.
(1985). There is no
significant difference in the RM scaling coefficients for the shark species
studied and there is considerable overlap in the percentage of RM found for
all species (Table 1).
Moreover, the scaling coefficients and the relative amount of RM are not
different, even when the shark species are separated into two distinct groups
based on similarities in RM position [i.e. in a more posterior and lateral
position (blue sharks and leopard sharks) vs a more anterior and
central position (mako sharks, salmon sharks and common thresher sharks);
Table 1]. Thus, the entire RM
data set for the five species was combined to form a single scaling function
(0.018M1.05±0.08; r2=0.96,
N=30; Fig. 2). This
function adequately describes RM scaling in all of the sharks studied,
irrespective of the marked differences in RM position documented in
Fig. 1. The scaling coefficient
in this equation is not different from 1.0 (indicating isometric RM scaling)
and there is no significant difference (one-way ANOVA, P'0.05)
in the mean quantity of RM estimated for the different species, which ranges
from 2.01% to 2.65% of body mass.
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RM myoglobin
RM [Mb] data for seven shark species are shown in
Table 2. Similarities between
the Kryvi et al. (1981) [Mb]
values reported for the velvet belly lantern shark (Etmopterus
spinax; family Dalatiidae), small spotted catshark (Scyliorhinus
canicula; family Scyliorhinidae) and blackmouth catshark (Galeus
melanopterus; family Scyliorhinidae) and our estimates for a single gray
smoothhound (7.5 mg Mb g-1 tissue) validate our HPLC [Mb]
methodology. On the other hand, our RM [Mb] values for the salmon shark (mean,
35 mg Mb g-1 tissue; range 3139 mg Mb g-1
tissue), shortfin mako shark (mean ± S.E.M., 21±2.4
mg Mb g-1 tissue) and common thresher shark (16.3±1.6 mg Mb
g-1 tissue) range from 3x to 12x higher than values
reported by Kryvi et al.
(1981
) for sharks in
Table 2 and are among the
highest reported to date for any fish species
(Dickson, 1996
). The slopes of
the scaling equations determined for both the mako and common thresher shark
[Mb] per g RM tissue do not change with body mass (i.e. slopes are not
significantly different from zero).
|
RM vascularization and ultrastructure
Mako RM vascularization and ultrastructure details are shown in Figs
3,
4.
Table 3 provides additional
morphometric data (based on a mean lo of 2.06 µm) for
mako RM and comparative data for other species. We found little variation in
the RM ultrastructure and vascularization of the two relatively small
(9.712 kg) makos studied. We did, however, note some collapsed
capillaries and a few remaining erythrocytes
(Fig. 3B,C) in some of the RM
transverse blocks, which suggests an incomplete in vivo perfusion
fixation and thus an underestimation of QA(0),
QA(/2) and NCAF.
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Longitudinal and transverse RM fiber sections
(Fig. 3AC) detail
capillary distributions and suggest the presence of capillary manifolds. Mako
RM capillary density [QA(0)=743918 mm-2]
appears to be the highest measured to date for any shark, ranging from
1.7x to 5.9x higher than in the other species (i.e. the blackmouth
catshark and velvet belly lantern shark) and is also 7x higher than in
the Chimaera monstrosa (rabbit fish)
(Table 3). Relative to the
other sharks, RM NCAF is significantly higher in both the
shortfin mako shark and blackmouth catshark, which are not significantly
different from each other. The capillary-to-fiber ratio
[NN(c,f)] is 1.410.8x greater in mako RM
relative to other sharks and the rabbit fish
(Table 3). Mako shark RM fiber
cross-sectional area [(f); 1437 µm2] is not
statistically different from the values for the blackmouth catshark and rabbit
fish but is smaller than in the velvet belly lantern shark
(Table 3). An electron
micrograph of mako RM (Fig. 4) shows the relationship between myofibrils, subsarcolemmal and intrafibrillar
mitochondria and other cellular structures. The total volume density of
mitochondria [VV(mt,f)] in mako RM (mean, 27.4%) is in the
range of values given for the RM of other sharks (30.434.1%) and tunas
(28.5%) (Table 3).
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Discussion |
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RM distribution and scaling
Biomechanical implications of RM position
Aerobically functioning RM powers the sustained swimming of most fishes. In
the majority of sharks and bony fishes, RM occurs mainly in the posterior half
of the body along the lateral midline, directly under the skin
(Greer-Walker and Pull, 1975;
Bone, 1978a
;
Fig. 1). RM in this position is
linked mechanically to the skin as well as to the adjacent myotomal white
muscle (WM). Force transmission from the RM to the caudal fin therefore occurs
via the skin and also involves the local bending of body segments
remote from the caudal fin (i.e. the bending waves seen in most fish swimming
modes, including the anguilliform swimming of sharks;
Lindsey, 1978
;
Sfakiotakis et al., 1999
).
In occurring both more anterior in the body and more central (i.e. nearer
the vertebral column; Fig. 1),
the lamnid RM distribution (and that of the common thresher shark) is
different from that of most sharks and similar to that of tunas
(Graham et al., 1983;
Carey et al., 1985
;
Bernal et al., 2001a
). Also,
and in contrast to most sharks, lamnid RM is neither connected to the adjacent
WM or the skin. Rather, it extends, via connective myocomata,
directly into the thick skin of the caudal keel
(Reif and Weishampel, 1986
;
fig. 5 in Bernal et al.,
2001a
). Force from the RM is thus transmitted directly to the
caudal fin and does not impose strain on either the surrounding WM or the
adjacent skin.
Similarities in lamnid and tuna RM position have been postulated to reflect
convergence in both body shape and the development of a more rigid swimming
mode. With respect to body shape, an anterior shift in RM position reduces
posterior-body height, thereby increasing both posterior-body taper and
streamlining (Graham and Dickson,
2000). Biomechanical studies of skipjack tuna (Katsuwonus
pelamis) and yellowfin tuna (Thunnus albacares)
(Shadwick et al., 1999
;
Katz et al., 2001
;
Katz, 2002
) and work in
progress with the mako shark (J. Donley and R. Shadwick, personal
communication) indicate that the anterior and central RM position imparts a
mechanical benefit during sustained swimming. This benefit, the reduction of
hydrodynamic drag through a lessening of the extent of lateral displacement of
more anterior body segments during force production, is derived from the
decoupling of RM contraction from local body bending (this would occur if RM
fibers were connected to either the adjacent WM or skin). In other words, with
a direct link between the remotely positioned RM and the caudal fin, both
tunas and lamnids swim with more rigid bodies, which reduces induced drag
(Shadwick et al., 1999
;
Altringham and Shadwick, 2001
;
Bernal et al., 2001a
;
Katz, 2002
).
RM position and endothermy
In addition to its biomechanical importance, the central and anterior RM
position of tunas and lamnids is closely linked to another unique feature
shared by these groups, the capacity to maintain elevated temperatures in RM
and other tissues (regional endothermy;
Carey et al., 1971; Bernal et
al.,
2001a
,b
).
Endothermy increases total aerobic metabolic biochemical capacity (see
Bernal et al., 2003
) and power
output of tunas and lamnids and has also contributed to the adaptive radiation
of both groups (Carey and Teal,
1966
; Johnston and Brill,
1984
; Dickson,
1995
,
1996
;
Brill, 1996
;
Altringham and Block, 1997
;
Graham and Dickson, 2001
). The
functional basis of endothermy in both groups is the capacity of the RM
vascular supply to conserve, by counter-current heat exchange, metabolic heat
generated by the continuous action of the highly oxidative RM (Carey and Teal,
1966
,
1969a
,b
;
Carey et al., 1971
,
1985
;
Anderson and Goldman, 2001
;
Bernal et al.,
2001a
,b
).
Although the Alopiidae resemble lamnids in having a central and anterior RM
position (Fig. 1) that is
served by a lateral circulation and a small putative heat exchanger
(Bone and Chubb, 1983
;
Block and Finnerty, 1994
; D.
Bernal, C. Sepulveda and K. Dickson, personal observations), there are no
published descriptions conclusively documenting RM endothermy in the thresher
sharks (Carey et al.,
1971
).
Interspecific differences in RM position
Although we found the RM position of the two lamnids and the common
thresher shark to be generally similar to that of tunas, there are noteworthy
specific differences among the sharks (Fig.
1). In makos, RM ends at the caudal peduncle (approximately 90%
FL). In the salmon shark, RM ends well in advance of the peduncle
(approximately 61% FL) and, in the thresher, RM reaches far into the
caudal fin's upper lobe (140% FL).
How might these various RM positions relate to the locomotion and biology
of the different species? Assuming the RMcaudal fin linkage of the
salmon shark is similar to that of the mako, a shorter RM section requires a
longer force-transmitting connection between RM and the caudal fin. This would
in turn imply a more rigid (less undulatory) swimming mode for the salmon
shark relative to the mako. By this standard, extension of RM well into the
upper caudal lobe of the common thresher's caudal fin implies greater tail
flexibility (i.e. both maneuverability and mobility), which is consistent with
the tail's importance in feeding (Gubanov,
1972). During feeding, we have observed the long caudal lobe of
threshers being used to herd small schooling fishes (e.g. sardines and
anchovies) into a tight group and then `clubbing' and stunning the prey prior
to feeding. Also, many of the thresher sharks we captured for this study were
hooked by the upper lobe of the caudal tail, a capture scenario also reported
by Gruber and Compagno
(1981
).
Comparative aspects of RM scaling
Findings for sharks in this study indicate TRMM values of 23% of
total body mass with no interspecific differences
(Table 1). Thus, neither RM
position nor presumed differences (based on morphology and behavior) in
high-performance swimming capacity (i.e. lamnids vs less mobile
forms) correlate with TRMM.
This contrasts with what is known for tunas, in which TRMM ranges from 4%
to 13% of total body mass (Graham et al.,
1983). While the relative amount of RM in some tuna species is
higher than in any other fish species (e.g. black skipjack tuna Euthynnus
lineatus 11%; frigate tuna Auxis thazard 13%), the TRMM of most
tunas is similar to values for other fishes
(Graham et al., 1983
; Graham
and Dickson, 2000
,
2001
). Furthermore,
comparisons within the family Scombridae show that the scaling coefficient for
tuna RM is less than or equal to 1.0, while the RM scaling coefficient for
non-tunas is significantly greater than 1.0
(Graham et al., 1983
;
Goolish, 1989
). Thus, while
tuna TRMM is directly proportional to body size, or even declines with size in
some tuna species, TRMM in non-tuna scombrids increases at a
disproportionately greater rate than body size.
The RM scaling coefficient determined for lamnids in this study is not
different from that of non-lamnids, and the combined scaling equation for all
sharks examined has a slope of 1. As reviewed by Webb
(1978) and Videler
(1993
), drag on a swimming
fish is determined mainly by velocity and wetted surface area. Considering
that the wetted surface area of the shark (i.e. the skin) scales with total
body surface area, the principal effect of body growth on swimming power
requirements are cruising speed (usually a function of FL) and
surface area (M0.67). Therefore, the most conservative
interpretation to make of an RM scaling coefficient of M1
is that, over the size range of makos examined, mass-proportional increases in
TRMM would be more than adequate to power cruise swimming. However, we do not
know how RM scaling or the scaling of several morphological features (e.g.
body cross-sectional area, paired-fin lift area, caudal fin area) affecting
drag, lift or the minimum sustainable velocity (i.e. the minimum velocity
required for hydrostatic equilibrium and for ram gill ventilation) might vary
across the entire size range of the mako [maximum total length, 400 cm
(Compagno, 1998
); 2001
International Game Fish Association recorded maximum mass, 554 kg] or that of
other species we studied. We therefore cannot rule out the possibility of a
change in RM scaling in larger makos or other lamnids. Carey et al.
(1985
), for example, reported
a TRMM of approximately 3% of body mass for a small (approximately 200 kg)
white shark (Carcharodon carcharias) but a TRMM of 6% of body mass in
a much larger (1256 kg) specimen (Table
1). [Carey et al.
(1985
) expressed their TRMM
data as a percentage of total axial muscle mass, which is here converted to
percent total body mass.]
Thus, more RM scaling data and information about the scaling of factors
influencing the biomechanics and physiology of high performance are needed to
determine whether RM scaling differs in sharks with different RM distribution
patterns. RM scaling differences in tuna and non-tuna scombrids have been
attributed to the positive effect of temperature on RM function
(Graham et al., 1983). Studies
of mako endothermy have also raised the possibility of a physiological
influence on RM scaling based on the finding that, by having a warmer RM,
larger makos achieve a disproportionately greater power production per unit
tissue mass (Bernal et al.,
2001b
,
2003
).
Oxygen delivery to RM
Animals having a high aerobic scope usually also possess cardiorespiratory
adaptations favoring high O2 delivery to the working tissues.
Compared with other fishes, tunas have both a high metabolic rate and a high
O2 transport capacity (Lowe et
al., 2000; Brill and Bushnell,
2001
; Korsmeyer and Dewar,
2001
). Tuna RM is also relatively specialized for a high
O2 flux rate, having small diameter RM fibers with a high [Mb] and
a rich supply of capillaries characterized by structural modifications (e.g.
manifolds) that optimize O2 transfer by maximizing the
fibercapillary contact area and extending red cell residence time
(Mathieu-Costello et al.,
1992
,
1995
,
1996
;
Dickson, 1996
).
The mako shark also has numerous morphological and physiological attributes
consistent with a high rate of O2 delivery to its tissues
(Bernal et al., 2001a), and
initial studies indicate a high metabolic rate and high tissue aerobic
capacity compared with other sharks
(Graham et al., 1990
;
Dickson et al., 1993
). Our
study of the mako has also confirmed specializations related to a greater RM
O2 flux.
RM [Mb]
Myoglobin facilitates the diffusion of O2 from a capillary to
its site of utilization within the mitochondria; a larger [Mb] is thus
indicative of a greater potential for O2 flux
(Wittenberg, 1970;
Sidell, 1998
;
Suzuki and Imai, 1998
). The
finding of a high RM [Mb] in the mako, salmon and thresher sharks indicates
that RM in all three species is poised for elevated O2 transfer.
Moreover, the RM [Mb] of these sharks is much higher than reported for other
sharks (Table 2) and exceeds
values reported for most other fishes except tunas
(Dickson, 1996
;
Table 2). Our data do not show
a significant scaling for RM [Mb] in either the mako or thresher sharks
(Table 2) and we therefore have
no new insight concerning the postulated role of intracellular [Mb] in
compensating for size-related changes in blood circulation time (reviewed in
Kayar et al., 1994
;
Goolish, 1995
).
RM ultrastructure
RM ultrastructure was examined in only two relatively small (9.712
kg) mako sharks. Even though there was little difference between these sharks,
our data are not adequate to fully describe mako RM ultrastructural properties
or to make definitive comparisons with other species. The finding of collapsed
capillaries and remaining erythrocytes in some of the RM transverse blocks
moreover indicates an incomplete in vivo perfusion-fixation in some
cases, meaning that QA(0), QA(/2)
and NCAF were probably underestimated. Nevertheless,
because this is the first ultrastructure information reported for a lamnid
shark, a general comparison of mako RM with that of other species is
warranted. Only limited comparisons are possible because of the paucity of
comparative information and because important details such as specimen body
mass and the sarcomere length (lo) at which fiber data
were obtained are usually not reported. It is critically important to indicate
lo because the state of muscle contraction affects both
fiber cross-sectional area and fiber diameter estimates
(Mathieu-Costello and Hepple,
2002
).
Mako RM fiber cross-sectional areas (at lo=2.06 µm)
are similar to those reported for other sharks
(Table 3). However, mako RM
capillary density [QA(0)=831 mm-2] and
capillary-to-fiber ratio [NN(c,f)=1.19], which are the
highest measured for any shark species
(Table 3), do indicate a
greater O2 diffusion capacity. Also, the mako RM capillary
orientation coefficient [c(K,0)=1.19] indicates a 19% increase in
capillary length of contact per volume of muscle fiber
[JV(c,f)=1092 mm-2] than would occur if the
capillaries were straight and unbranched
(Mathieu-Costello et al.,
1992). While our data for mako RM ultrastructure identify features
related to an increased O2 flux capacity relative to other sharks,
Table 3 indicates that these
vascular specializations are much less extensive than those in tunas.
Mathieu-Costello et al.
(1992) reported the presence
of capillary manifolds in tuna locomotor muscle and suggested that these
facilitated O2 diffusion by increasing the capillaryfiber
contact area. Capillary manifolds, which are most frequently found at the
venular end of the vessel bed, also occur in the locomotor muscle of active
birds (Mathieu-Costello et al.,
1992
). Birds and most fishes are similar in having nucleated
erythrocytes and, because these are both larger and less deformable that
non-nucleated erythrocytes, a possible role for capillary manifolds in
enhancing red cell flow was suggested
(Mathieu-Costello et al.,
1992
).
In view of the proposed role of manifolds in augmenting circulation, we
expected to find large numbers of these structures in mako RM. In addition,
with shark erythrocyte diameters averaging about 4x higher than those of
tunas (Emery, 1986;
Bernal et al., 2001a
) and
because our study (Table 3)
indicates that mako RM capillary diameter is about 2x larger than in
tunas, we predicted that mako manifolds would be larger.
Capillary manifolds were neither obvious nor abundant in mako RM.
Fig. 3A shows what appears to
be a capillary manifold in a longitudinal RM section, but this structure is
much smaller than tuna manifolds
(Mathieu-Costello et al.,
1992), which is inconsistent with the supposed function of
facilitating the flow of larger, less compliant red cells. However, we studied
a limited number of longitudinal RM sections from which manifolds could be
documented, and additional studies are needed to verify the presence of
manifolds in makos and to search for them in larger specimens, other lamnids
and other active sharks. It could be that differences between tunas and makos
insofar as manifolds are concerned reflect differences in absolute RM
O2 demand; while the metabolic rate of a swimming mako is higher
than that of other sharks, it is about 4x less than that of a tuna
(Graham et al., 1990
;
Bernal et al., 2001a
;
Korsmeyer and Dewar,
2001
).
Mako RM total mitochondrial volume density is 2529%, which is in the
range of other sharks and tunas (Table
3). As reviewed by Mathieu-Costello et al.
(1992), conformance in
mitochondrial volume density among most fishes is generally regarded as
indicating the maximum amount of non-contractile elements that can be
contained within the myofibril without affecting muscle contractility
(Block, 1991
;
Ballantyne, 1995
).
In summary, lamnidtuna evolutionary convergence in specializations for high-performance swimming extends to similarities in RM position but not in RM amount, which for tunas is larger, more variable and scales negatively with body mass in some species. All sharks in this study had 23% TRMM and, irrespective of RM position, had an RM scaling coefficient of 1. Similarities in tuna and lamnid RM position have a basis in similar swimming biomechanics and may also relate to the presence of regional endothermy. The common thresher shark has an RM distribution similar to that of the mako. Tuna and lamnid RM is similar in having specializations enhancing O2 delivery to the mitochondria, including a high [Mb], large capillary-to-fiber ratios and structural modifications increasing capillaryfiber contact, and both groups have similar myofibrillar mitochondrial densities. Tuna RM, however, appears to have a greater degree of `specializations for O2 delivery', as evidenced, for example, by higher capillary density, a more extensive capillary manifold system and higher capillary tortuosity. Nonetheless, relative to other sharks, lamnids have many of the adaptations that may allow for a higher O2 flux to the RM, which can potentially increase the aerobic capacity of this tissue. Additional studies comparing the RM morphology, vascularization and ultrastructure of other actively swimming ectothermic sharks are needed to understand the degree of lamnid RM morphological specializations that support their categorization as high-performance swimmers.
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