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TISSUE & CELL 198214 (2) 319-328
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IAN A. JOHNSTON
QUANTITA
TIVE ANAL YSES OF
UL TRASTRUCTURE
AND VASCULARIZA
OF THE SLOW MUSCLE
FIBRES OF THE
ANCHOVY
Key words: Muscle, fish, capillary development, ultrastructure,
TION
stereology.
ABSTRACT. A quantitative study has been made of the ultrastructure and vascularization of slow fibres in the lateral muscles of the European anchovy (Engraulis encrasicolus). Mitochondria and myofibrils occupy 45.5 and 44.3% of total fibre volume
respectively. More than 95% of all myofibrils are adjacent to mitchondria. A total of
51% of the sarcolemma is in direct contact with capillaries with a mean of 12.9 capillaries
per fibre. In transverse sections anchovy slow fibres are considerably flattened (long
to short axis 12: I) such that the surface to volume ratio is more than twice that of a
cylindrical fibre of the same area (1115 [J.m2).The capillary surface required to supply
I [J.msof mitochondria is 0.18 [J.m2and the maximum distance between any capillary
and mitochondrion
8 [J.m. T -system and sarcoplasmic reticulum occupy 0.43 and
2.7 % of fibre volume respectively. Adaptations for increasing the capacity of skeletal
mll.cle for aerohic work are discussed.
Johnston, 1980,1981).Whereasslow fibres in
frog are principally concerned with maintaining posture those in fish are actively
concerned with locomotion.
An unusual slow fibre structure has recently been reported among fish of the
family Engraulidae (Greer-Walker et at.,
1980). Anchovy species are highly active
planktonic feeders which are widespread in
temperate and tropical seas. In transverse
section anchovy slow fibres are highly
flattened and are characterized by an
extensivenetwork of capillaries. rhe present
study makes a quantitative analysis of the
ultrastructure and vascular supply of slow
fibres from the European anchovy (Engraulis encrasicolus). Adaptations for increasing the aerobic capacity and decreasing
diffusion distances in skeletal muscle are
di~cu~~ed.
Introduction
THE lateral trunk muscles of most fish
contain a superficial wedge of slow fibres.
The proportion of this fibre type varies both
along the length of the body and with the
form of body movements and mode of life
of each species. For example, slow fibres
comprise around 26 % of the total in actively
migrating open ocean species such as tuna
and only 2% in the more sedentary wrasses
(Greer-Walker and Pull, 1975).
In common with amphiqian slow muscle
fibres those in fish are multiply innervated
and are considered to be incapable of
generating a propagated action potential
(Barets, 1961; Stanfield, 1972). However,
they differ from frog tonic fibres in having a
well-developed T -system, and sarcoplasmic
reticulum, numerous mitochondria and an
abundant vascular supply (Peachey, 1965;
Flitney, 1971; Kryvi, 1977; Bone, 1978a;
Materials and Methods
Department of Physiology, University of St
Andrews, St Andrews, Fife, Scotland, Great Britain.
Received4 December1980.
R"vi",rl 2 Octnher 1981.
319
Six adult anchovy (Engraulis encrasicolus L.)
were obtained from local fisherman at
Pouzzuoli. neaf Naples. Italy during early
.
1
~,o
JOHNSTON
June 1980. All specimens were between 12
and 14 cm in length. Although dead on
arrival at port muscle tissues retained local
excitability at the time of fixation. Samples
were dissected from the posterior lateral
trunk muscles (Fig. I). Small strips of muscle
attached to skin ( -3 mm2 diameter) were
fixed at their resting length by pinning to
cork strips and fixed by immersion in 3 %
glutaraldehyde,
0.15 M phosphate buffer
pH 7.4 at 20°C.
Subsequently small fibre bundles were
dissected using a binocular microscope, and
post-fixed in 1 % osmium tetroxide in 0.1 M
phosphate pH 7.4, dehydrated in a series of
alcohols up to 100% and embedded in
Araldite r...~in { Jltrathin secti(ln~ were cut
on a Reichart OMU2 Ultramicrotome
and
double stained with uranyl acetate and lead
citrate. Determinations
of fibre area and
capillary counts were made from 1 p-m
sections stained with either toluidine blue or
1.5% p-phenylene diamine in 1: 1 isopropanol:methonal
(Hollander
and Vaaland,
1968)
Morphometric methods
Fibre and capillary areas and perimeters
were determined directly from micrographs
(light micrographs x 480, low power electron
micrographs x 1900) of transverse sections
using a summagraphic digitizer in conjunction with a minicomputer
(Walesby and
Johnston.
1980). The fractional
volume
~
~
~
A
@(@@00
'I, Slow
r~s
6
II
16
')9
]8
B
10~
Fig. 1. (a) Tracing of the trunk and arrangement of slow fibres in the lateral muscles
of Engraulis encrasicolus. Estimates of percentage of slow fibres at different points
along trunk were obtained directly from micrographs using a digitizer and minicomputer. Point of sampling of lateral muscles in the present study is indicated by an
arrow. (b) A tracing of several overlapping low power micrographs to show the degree
of vascularization of a large area of slow muscle. Note the flattened appearance of
muscle fibres in transverse section (long short fibre axes -12: 1) and the extensive
network of capillaries (C). A layer of lipid droplets (LC) is present between the skin
and underlvinl! slow fibres.
SLOW MUSCLE
V ASCULARIZA
TION
occupied by sarcoplasmic reticulum and
T -system was determined
in a similar
fashion from micrographs of longitudinal
sections at higher magnification ( x 15,000).
All other quantitative analyses of electron
micrographs
(magnification ~ 2500-3400 x)
was carried out from transverse sections
using a point-grid method (Weibel, 1969),
as previously
described (Egginton
and
Johnston, 1982). Good agreement was found
between the stereological methods of Weibel
and direct estimates of cell component fractional volumes from the same micrographs
using digitizer and minicomputer. Measurements were made from around 125 micrographs taken at random from 36 blocks cut
out of a total of 104 prepared.
Results
Slow fibres from the lateral trunk muscles of
anchovy are flattened in transverse section
(Fig. 2a, b). In general, the ratio of long to
short axis in this plane is 12: 1. Myofibrils
which comprise 44.3% of fibre volume are
irregularly packed and are almost always in
direct contact with a mitochondrion
(Figs.
2a, b, 3a). Mean mitochondrial
density is
45.5% (Table 1) with a range from 30 to 60%
of total fibre volume (Fig. 3b). Slow fibre
mitochondria
have a complex and highly
developed cristae structure (Figs. 2c, 4a, b).
A layer of large lipid droplets ( ~ 10-15 p.m)
occurred between the skin and the most
superficial slow muscle but is not observed
within the fibres themselves (Figs. 2, 4). The
sarcotubular system (Table 1) is relatively
poorly developed compared to other fishes
(Johnston, 1980a). T-tubules occur at the
level of the Z-disc (Fig. 4a, b) and a dis-
321
tinctive M-Iine is visible in longitudinal
section (Fig. 4b).
The anatomical separation of fibre types
in fish greatly facilitates quantitative studies
of the vascular bed. A large number of
indices are available with which to express
capillary supply (Table 2). The parameters
measured in the present study have been
chosen to allow a direct comparison with
data on other aquatic vertebrates (Flood,
1979; Totland et al., 1980). Most of the
derived parameters are dependent on a
knowledge of capillary diameter. This is
likely to vary not only with the physiological
state of the fish prior to fixation but also
with the precise method of tissue preparation. The presence of red cells almost filling
the capillary lumen in around 45% of
capillaries in the present study suggests that
the measured diameters are likely to be
within the range experienced in life.
Anchovy slow muscle fibres are extensively
capillarized with an average of 12.9 capillaries per fibre (Table 2). The range of values
obtained for 100 fibres is presented in Fig. 5.
On average around 51% of the total fibre
surface is in contact with a capillary (Table 2).
Other data using various methods to express
vascular supply are presented in Table 2.
The capillary surface required to supply
1 fLm3 of mitochondria is 0.18 fLm2 which is
somewhat higher than for less active fish
species with lower aerobic capacities (see
Totland et al.. 1980).
Discussion
Quantitative ultrastructural
studies of fish
skeletal muscle have recently been reviewed
(Johnston, 1980, 1981). Both fast and slow
.
.
SLOW MUSCLE
V ASCULARIZA
TION
fibre types have been distinguished using
histochemical
and ultrastructural
criteria
(Patterson and Goldspink, 1972; Johnston
et al., 1975; Mosse and Hudson, 1977; Bone,
1978a, b). A wide variation of fine structure
of homologous
fibre types is observed
between species related both to different
modes of locomotion and adaptations to
different physical environments (e.g, temperature,
pressure, oxygen
availability)
(Kryvi and Totland, 1978; Bone, 1978b;
Johnston and Maitland, 1980; Walesby and
Johnston, 1980). There are also quantitative
differences in ultrastructure between fibres
from different regions of the trunk musculature although these are far less pronounced
for slow than fast fibre types (Egginton
and Johnston, 1982; Johnston and Moon,
1980a).
Anchovy slow fibres constitute a relatively
uniform population with respect to fibre size
(Fig. 1). In transverse section fibres are
flattened with short axes in the range
6-11 p.m across (Figs, 1, 2, 4). The fraction
of fibre 'volume occupied by mitochondria
(45'5%) is the highest so far reported for any
fish slow fibre (see Johnston, 1980b) and is
reminiscent of micrographs of hummingbird
flight muscles (Grinyer and George, 1969),
There is a reasonable correlation between
the mitochondrial
content of fish slow
muscle/and sustained swimming performance.
For example, the fraction of slow fibre
volume occupied by mitochondria is 18-24 %
in Scycliorhinus canicula (Totland et al.,
1981) and 34% in Etmopterus spinax (Kryvi,
1977), a sedentary, bottom living and active
mid-water elasmobranchs respectively. The
lowest value reported is that for Chimera
montrosa where mitochondria only occupy
around 5% of total fibre volume (Kryvi and
Totland, 1978). In this species the function
323
60
50
40
X,Fibres
30
20
10
JO
40
%
60
50
Myofibrils
40
30
% Fibres
20
10
20
30
40
50
60
% Mitochondria
Fig. 3. Frequency histograms showing the fraction
of total fibre volume ( %) occupied by (a) myofibrils
and (b) mitochondria in 50 anchovy slow fibres.
of the trunk in slow speed swimming is
transferred to enlarged rectoral fins. Recruitment of myotomal slow fibres is
probably restricted to producing rudder-like
Fig. 2. (a) Transverse section of anchovy slow fibres showing abundant
mitochondria (MT), irregularly packed myofibrils (MY) and capillaries (C), some of
which contain red cells (R). Note that almost all myofibrils are in direct contact with
mitochondria. x 3100. (b) Transverse section through part of an anchovy slow fibre
illustrating the high proportion of sarcolemmal surface in contact with capillaries (C).
x 6500. (c) Longitudinal section through a capillary showing endothelial cell 1ining
(Fr) "nd .uh.arcolemmal mitochondria zone (SM). x 22.000.
.
~
.
SLOW
MUSCLE
VASCULARIZATION
Table 2. Quantitative
325
analyses of the vascularization
of slow muscle fibres from
the European anchovy
(EnJ!raulis encrasicolus)
Units
Parameter
Fibre area (A)
Fibre perimeter (B)
Number capillaries per muscle fibre (C)
Perimeter supplied by each capillary (D)
Capillary contact length per fibre (p-m) (E)
Mean vascularized fibre surface as % of total fibre
surface (EID)
Mean fibre cross-sectional area per capillary
Mean capillary contact (p-m) supplying 1 p-m2of fibre
cross-sectional area (El A = F)
Capillary surface (p-m2)supplying 1 p-m3of mitochondria
(
F x 100
Fractional volume mitochondria
40
30
Fibres
10
3
9
15
JLm2
1115:t52
JLm
180:t7
12.9:tO.5
p.m
p.m
%
p.m2
p.m2
p.m2
14.0:t3.8
91.6:t5.1
50.9:t2.1
86.4:t3.1
0.082
0.18
)
movements of the trunk associated with
changesin direction. Thus these fibres may
have a largely postural function similar to
that found in amphibian tonic fibres (Kryvi
and Totland, 1978).
In addition to occupying almost half total
fibre volume anchovy slow fibres have a
;110.Capillaries/
Mean:t SE 100 fibres
21
27
Fibre
Fig. 5. Frequency histogram
showing the number
of capillaries
per muscle fibre surrounding
100
anchovy slow fibres.
densely packed and highly complex cristae
structure (Figs. 2, 4). More than 95% of
myofibrils are adjacent to mitochondria
(Fig. 2a, b), suggestinga high dependenceon
aerobic metabolism. This is supported by
measurementsof the degree of vascularization of the fibres (Table 2). Although such
measurementsgive no indication of physiological blood flow they do provide a measure
of the potential size of the capillary bed. It
should be noted, however, that the rate of
utilization of oxygen by mitochondria will be
as important as diffusion distances in
establishing the size of capillary bed and
blood flow necessaryto sustain a given level
of aerobic metabolism.
Unfortunately, comparisons of the present
data with that of other animals is complicated by the different indices of vascularization employed by previous workers. However, compatible data on the vascularization
of aquatic vertebrates is available for hagfish
(Flood, 1979), a chondrostean (Acipencer
stellatus) (Kryvi et al., 1980),severalelasmo-
Fig. 4. (a) Longitudinal section through an anchovy slow fibre showing the high
proportion of mitochondria (MT). x 3100. (b) Longitudinal
section through an
anchovy slow fibre illustrating T -tubules (T) at the junction of the Z-line (Z), a
relatively sparse sarcoplasmic reticulum (SR), distinctive M-Iines (M) and highly
complex internal structure of mitochondria (CS). x 18,000.
-
326
branchs (Totland et at., 1981) and some
teleosts (Mosse, 1978, 1979). The percentage
of fibre surface in direct contact with
capillaries is 31% in hagfish (Flood, 1979),
23% in the velvet belly shark, 16% in
dogfish (Totland et at., 1981) and 51% in
anchovy (Table 2).
The capillary surface (.um2) required to
supply 1 .um3 of mitochondria is 0.06 in
Etmopterus spinax, 0.06 in Scyliorhirus
canicula (Totland et at., 1981) and 0.18 in
Engraulis encrasicolus(Table 2).
In anchovy slow fibres no myofibril is
more than about 8 .urn from the nearest
capillary (Figs. 1, 2). Estimates of the
maximum hypothetical diffusion distancesin
other slow fibres are 47.5 .urn for the velvet
belly shark, 27.4.um for Scyliorhinus and
52 .urnfor Chimera montrosa (Totland et at.,
1981).
An interesting feature of anchovy slow
fibres is their flattened structure in transverse
section (Greer-Walker and Pull, 1975; GreerWalker et at., 1980) (Figs. 1, 2). Flattened
muscle fibres are also found in the cephalochordates (Peachey, 1961; Flood, 1968).
In amphioxus (Branchiostoma lanceolatum)
the trunk muscle is made of lamellae about
1 .urn thick consisting of a single myofibril
(Flood, 1968, 1977). These fibres lack
transverse tubules and the sarcoplasmic
reticulum is represented by Ca2+-accumulating subsarcolemmalvesicleslocated adjacent to the Z an4 I bands (Flood, 1977).
Since the myofilaments are no more than
0.5-1 .urn from the plasma membrane a
specialized structure for the inward spread
of depolarizing current is unnecessary
(Peachey,1961; Flood, 1968, 1977).
It seems likely that the development of
flattened fibres represents an adaptation to
reduce diffusion distances.Compared with a
cylindrical muscle fibre of the same area
anchovy fibres have around 2.1 times the surface/volume ratio. Thus the capillary contact
length supplying 1 .um2of fibre cross-sectional
area is 0.018 .um2 for Etmopterus spinax,
0.007 .um2 for Chimaera and 0.033 .um2for
Scyliorhinus (Totland et at., 1980) and 0.082
for anchovy (Table 2). Interestingly, Greer-
JOHNSTON
Walker and co-workers have calculated that
diffusion distances are ihdependent of fibre
size for anchovy slow fibres. Thus as body
sizeincreasesthe cross-sectionalarea of fibres
increasesby elongation of the long fibre axis
so that distances between capillaries and
central mitochondria remains approximately
constant. (Greer-Walker et al., 1980). In
contrast fibre diameter increases around
four times in the cylindrical slow fibres of
cod (Gadus morhua) from aroung 12 ILm in
5 cm fish to 50 ILm in 100cm fish (GreerWalker, 1970). Unfortunately, there are no
data available on diffusion distances in
cylindrical fibres in fish of different sizes.
Anchovies along with a number of other
more primitive teleost groups have focally
innervated fast muscles (Bone, 1970). There
is some electromyographical evidencethat in
such fish the slow motor system is almost
entirely responsible for sustained swimming
activity. For example, in a herring species
Clupea harenguspallasi (order Clupeiformes)
it has been shown that 15 cm fish can
maintain speedsof up to 4 bodylengths/sec
by recruiting only slow fibres (Bone et al.,
1978). In order to swim at higher speeds
(~ 5 bodylengths/sec)fast fibres are recruited
and the fish fatigues following a further
1-2 min swimming (Bone et al., 1978).
Anchovies are highly active pelagic fishes
which are primarily filter-feeders of plankton. During feeding the gap of the mouth is
greatly expanded increasing the crosssectional area by around four times. The
continuous activity and high drag imposed
on the body by this method of feeding
require a high and sustained power output
from the slow motor system. As GreerWalker et al. (1980)suggestthis has probably
been a major factor in the evolution of
flattened slow muscle fibres in these.fishes.
Acknowledgements
I wish to thank Dr Bruno Tota for his
hospitality during my stay in Naples and for
his help in obtaining samples.The receipt of
a grant from the Science Research Council
is gratefully acknowledged.
.
SLOW
MUSCLE
VASCULARIZATION
327
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