IS1
The Function and Metabolism of Certain Insect Muscles in
Relation to their Structure
By GEORGE A. EDWARDS AND HELMUT RUSKA
(From the Departamento de Fisiologia Geral e Animal, Universidade de Sao Paulo; Division of
Laboratories and Research, New York State Department of Health, Albany, New York; and
SecfSo de Virus, Institute Butantan, Sao Paulo, Brazil)
With 4 plates (figs. 2, 3, 6, and 7)
SUMMARY
Electron microscopic observations on ultrathin sections of the red thoracic flightmuscles and white leg muscles of Hydrophilus and Dytiscus are reported.
In red muscle-fibres with high values in frequency of contraction, oxygen consumption, and dehydrogenase activity, the single fibrils are completely surrounded by huge
mitochondria. Tracheoles penetrate the sarcolemma and supply the mitochondria with
oxygen by intracellular branches. In the less active white muscle fibres, mitochondria
are found irregularly scattered between the fibrils or along the I band. The intracellular tracheolization is sparse but an endoplasmic reticulum is widely spread between
the synfibrillar contractile material. The same muscles of the two insects differ considerably in detail.
INTRODUCTION
T
HE highly specialized functions of insect muscles make these the best
models for the study of the relationships between function, metabolism,
and structure. This had been noted by the earlier microscopists such as
Knoll (1889) and Holmgren (1909, 1913), but in recent years has not received
sufficient attention. Marked metabolic differences are to be found among the
various muscles of various insects, these differences being closely related to
the function of the muscles and the phylogenetic position of the insect involved
(see Perez Gonzalez and Edwards, 1954, for summary of the literature).
Physiological studies of insect flight (Roeder, 1951) have also brought to
light considerable differences in nerve-muscle relationships between the higher
and lower insects. Structural differences in the tracheation of muscle fibres,
differences in colour of fibres, and variations of ultrastructure of isolated fibrils
also occur from muscle to muscle in the insect (Edwards and others, 1954a,
19546). In mammalian muscle (Ruska, 1954) and fowl-breast muscle (Bennett
and Porter, 1953), electron microscope studies have suggested that the endoplasmic reticular and mitochondrial systems may be closely related to the
process of contraction within the fibril and further related to specialization
in muscle function.
With these facts in mind we have begun to study ultrathin sections of
certain insect muscles, with the electron microscope, to determine the
relationship of metabolism and specialization in function to principles of
structure. Particularly we have been interested in the relationship of the
tracheoles to the mitochondria, and the roles played by the mitochondria
[Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp. 151-159, 1955.]
152 Edwards and Ruska—Function and Structure of Insect Muscles
and reticular systems in widely differing types of muscular functions, such
as flying and walking.
MATERIAL AND METHODS
The muscles used were the dorso-longitudinal, indirect, flight (red), and
the coxal levator (white) muscles of adult Hydrophilus ater and Dytiscus spp.
The muscles were observed in both the contracted and stretched states. They
FIG. I. Red fibre of Hydrophilus. Many tracheoles outside and inside the thin sarcolemma,
intermingled with the mitochondria between the contracted myofibrils and located close to
one nucleus.
were fixed in i per cent, buffered osmium tetroxide solution, according to
the method of Palade (1952), and embedded in methacrylate by the conventional method for electron microscopy. The sections were cut with a
microtome built in the Rockefeller Institute for Medical Research. The
microscope used was the Siemens UM-ioob, of the Instituto Butantan in
Sao Paulo. From several micrographs, drawings were composed to show the
overall construction and to reduce the number of illustrations (see figs, i, 4,
5, and 8). The originals were exhibited at the International Conference of
the Joint Commission on Electron Microscopy, held at London University,
16-21 July 1954.
FIG. 2 (plate). Red fibre of Hydrophilus. Electron micrograph. Four contracted fibril
segments; m, mitochondria; t, tracheoles.
G. A. EDWARDS and H. RUSKA
G. A. EDWARDS and H. RUSKA
Edwards and Ruska—Function and Structure of Insect Muscles 153
RESULTS
Oxygen supply
The tracheoles have previously been thought to terminate on the surface
of the muscle fibre. The red flight-muscle fibres have a large external tracheal
supply; the white leg-muscle fibres have few tracheal branches. In our
preparations we have seen that the tracheoles actually penetrate all muscle
fibres, but the number and distribution of penetrating tracheoles vary with
the type of muscle.
FIG. 4. Red fibre of Dytiscus. Few tracheoles outside and inside the thin sarcolemma; they
are attached to the closely packed mitochondria between the stretched myofibrils.
In the sections of Hydrophilus red fibres (figs. 1 and 2) it can be seen that
as the trachea approaches the fibre surface it gives off numerous fine branches
(tracheoles) which penetrate the sarcolemma at various points. Just beneath
the sarcolemma may be seen many branches in cross, tangential, and longitudinal section. Between the fibrils, and closely associated with the mitochondria, are found cross-sections of very fine, intracellular tracheoles
throughout the entire fibre. It is interesting to note that well in the interior
FIG. 3 (plate). Red fibre of Dytiscus. Electron micrograph. One stretched fibril segment;
m, mitochondria; t, tracheoles.
154 Edwards and Ruska—Function and Structure of Insect Muscles
of the fibre the tracheoles.appear to be uniform in diameter; they are often
in clusters, and always near or actually in contact with the mitochondria.
The space between the fibrils in this muscle is considerable and appears to
be filled principally with the fine tracheoles and large mitochondria.
In the sections of the red flight-muscle of Dytiscus (figs. 3 and 4) fewer
tracheoles are observed. The general tracheal picture is similar to that of
FIG. 5. White fibre of Hydrophilus, Few tracheoles in cross-section beneath the sarcolemma and between the mitochondria. The endoplasmic reticulum between the myofibrils
spreads along the Z bands, which are attached to the sarcolemma.
Hydrophilus, in the branching of the trachea at the surface and the penetration
by the finer tracheoles into the fibre. Differences appear in that there are
fewer tracheoles within the fibre and they generally run longitudinally between
the fibrils, together with the mitochondria. Interestingly enough the tracheole
diameter in this muscle is greater in relation to the fibril diameter than in
the Hydrophilus muscle. The mitochondria of this muscle are much more
closely packed and this may well influence the tracheole distribution. Thus,
it appears that in the red flight-muscle fibres of both insects the tracheoles
FIG. 6 (plate). White fibre of Hydrophilus. Electron micrograph. Fibrils; m, mitochondria; r, endoplasmic reticulum.
G. A. EDWARDS and H. RUSKA
G. A. EDWARDS and H. RUSKA
Edwards and Ruska—Function and Structure of Insect Muscles 155
and mitochondria form a continuous system in which lie the discontinuous
fibrils.
In the sections of the white muscles (figs. 5-8) few tracheoles were observed.
They penetrated the sarcolemma, thus entering the fibres, at relatively few
FIG. 8. White fibre of Dytiscus. One tracheole beneath the sarcolemma. Endoplasmic
reticulum between the myofibrils, accumulating in the I region, where small mitochondria
can be seen.
legions along its length. In the white muscle of Hydrophilus relatively few
cross-sections of tracheoles were seen among the mitochondria, which sometimes occurred in rows between the myofibrils. In the white muscle of
Dytiscus only subsarcolemmal tracheoles were observed.
FIG. 7 (plate). White fibre of Dytiscus. Electron micrograph. Fibrils; m, mitochondria,
close to the Z bands; r, endoplasmic reticulum.
156 Edwards and Ruska—Function and Structure of Insect Muscles
In no muscle were tracheoles seen to enter the myofibrils or to have a
definite relation to the muscle bands.
Loci of oxidations
The outstanding characteristic of the red flight-muscle of these two insects
is the number and size of the sarcosomes, i.e. the mitochondria of insect
muscles.
In the Hydrophilus flight-muscle the mitochondria are found in rows
parallel to and between the myofibrils and closely associated with the numerous
tracheole branches throughout the entire fibre. Just beneath the sarcolemma
there are few mitochondria, relatively speaking, scattered among the larger
tracheolar branches. Within the body of the fibre, however, internal to the
peripheral fibrils, the mitochondria are extremely numerous, one usually
touching upon the next, separated from the fibrils in most cases by some
distance. In number they average about one mitochondrion to each fibril
segment. The Hydrophilus mitochondria are largely ovoid or spherical,
averaging 2-2 X 1*5/x. The internal structure of the mitochondria appears
like a pile of irregular corrugated cardboard. When cut normal to the planes
one sees straight to wavy lines, usually oriented at right angles to the alignment
of the myofilaments. This pattern is interrupted by lacunae, sometimes
opening to the surface. When cut with the planes, the mitochondria more
often present the lacunar picture. It may be possible that the tracheoles in
contact with the mitochondria open into the lacunar system.
In the red flight-muscle fibre of Dytiscus the mitochondria appear to be
more numerous, much more closely packed, of the same size generally
(averaging 2-5X1-5/A), and often compressed into polygonal shapes with
clearly marked limiting membranes. As in Hydrophilus a row of mitochondria
is found between the sarcolemma and the peripheral fibrils, and thereafter
there are single or double rows between and parallel to the succeeding
myofibrils. In number they appear to be about 2-3 per segment, tightly
squeezed one against the other. Inasmuch as the tracheoles in this muscle
are larger in diameter, fewer in number, and longitudinally arranged between
the fibrils, the picture of a mitochondria-tracheole continuous system is less
clearly seen here than in the Hydrophilus muscle fibre. However, the tracheoles
again appear to be more correlated with the mitochondria than with the
myofibrils. The internal structure of the mitochondria of the red muscle of
Dytiscus, although fundamentally similar to that of Hydrophilus, is much
finer and is visible only in exceptionally thin sections with good resolution.
Characteristic of the Dytiscus flight muscle, and giving the sections a
checker-board appearance, are numerous spherical bodies of uniform size,
slightly smaller than the mitochondria described above and scattered among
them. The bodies seem to be more often associated with the I than the A
region of the fibril, and occur on the average as one such body to each fibril
segment. A definite limiting membrane is visible between any two of these
bodies. Internally the structure is similar to that of the other mitochondria,
Edwards and Ruska—Function and Structure of Insect Muscles 157
but shows a looser and thinner arrangement. These bodies can be interpreted
as either mitochondria which have undergone a physiological change, or else
a different type of mitochondria. We are more inclined to the former view,
believing that this could represent a reversible metabolic state or irreversible
ageing.
The white coxal muscles characteristically have very few mitochondria and
these are of small size. The outstanding characteristic of the white muscles
is the endoplasmic reticulum. The Hydrophilus white muscle shows great
similarity to the mammalian muscle structure shown by Ruska (1954). The
mitochondria are aligned in longitudinal rows separating bundles of myofibrils,
the fibrils themselves being separated by the endoplasmic reticulum. In
Dytiscus the mitochondria are arranged singly in transverse rows, i.e. always
at the level of the I band between the individual fibrils. In the white muscles
of both insects the internal structure of the mitochondria appears to be
similar to but less distinct than that of the mitochondria of the red muscle
fibres. In form they are roughly oval, averaging 0-4 X I-O/A.
Endoplasmic reticulum
The endoplasmic reticulum is peculiar to the white muscles but apparently
lacking or much less developed in the flight-muscle fibres. It appears to be
a continuous system located between the myofibrils and connected to them
at the level of the I region. Thus in low magnifications a longitudinal section
through a white fibre shows an apparently continuous Z line. On amplification, however, one can see that the Z is actually restricted to the myofibril
but that connexion of one fibril to the next is made by the reticular system.
In Hydrophilus the system appears to be represented by dark threads between
the fibrils, sending very fine branches between the myofilaments predominantly at the level of the I region. In Dytiscus, on the other hand, the system
is definitely tubular and appears rather to surround the fibrils than to enter
them. The greatest concentration of tubes is at the Z line, but a secondary
accumulation occurs near the middle of each segment. In the red muscle
of Hydrophilus faint traces of endoplasmic material may be seen in the
vicinity of Z.
Myofibrils
The sections of the fibrils confirm the older findings concerning the
differences between red and white insect muscles. The red fibrils in Hydrophilus are more uniform in diameter and are more clearly separated than the
white fibrils. Actually in the white muscle fibres the fibrils form a synfibrillar
continuous system. In all fibrils of both insects the myofilaments are clearly
visible; they are continuous, at least in the contracted state, throughout
both A and I regions. The filaments of the white muscle of Hydrophilus
appear to be more loosely packed and in less orderly arrangement than in
the red and the white muscles of Dytiscus. In both types of muscle it could
be seen that the fibrils are certainly composed of more than just filaments.
158 Edwards and Ruska—Function and Structure of Insect Muscles
Further details of their structure will be discussed elsewhere. It should be
noted here, however, that only in the Hydrophilus white muscle was the Z
line seen to be attached to the sarcolemma, giving it a scalloped form.
DISCUSSION
The results have shown very clearly that the specialized functions of the
muscles studied are based upon specialization in structure. The flight-muscle,
with its high velocity of movement and its high oxygen consumption, possesses
an intracellular tracheole system closely linked to large numbers of huge
mitochondria, thus providing the carriers of the oxidative enzymes with an
ample oxygen supply. The flight-muscle is therefore essentially a fast-acting
energy-producing machine. The oxidative mechanism is very close to the
contractile mechanism. The loci of oxidations completely surround each
fibril. This is a logical mechanism inasmuch as the flight-muscle fibril must
have energy available to it, must have end products removed, and must get
back resynthesized carbohydrates and phosphates as rapidly as possible for
repetitive contractions. To this end strength has been sacrificed, most of the
fibre space being occupied by the mitochondria-tracheole system.
The white muscle is slower, capable of continuous tension, has a lower
oxygen consumption, but needs a continuous low energy supply. The greater
part of the fibrillar areas is filled with the contractile substance, the mitochondria occupying a very small fraction of the space. Actually this muscle
has less mitochondria and tracheolization than would be expected on the
basis of its metabolic activity, but we musf keep in mind that the central
part of the white Dytiscus muscle contains more cytoplasmic material. The
remaining space between the fibrils is occupied by the endoplasmic reticulum.
The white muscles apparently are constructed for strength rather than fast
action and have therefore sacrificed oxidative capacity.
The presence of the endoplasmic reticulum in the white muscle and the
mitochondria-tracheole system in the red muscle raises the question of their
respective roles in the metabolism and functions of these muscles. The
mitochondrial system is predominant in mammalian diaphragm (Ruska, 1954),
in vertebrate heart-muscle (Kisch and Philpott, 1953), and insect flightmuscle. The endoplasmic reticulum has been found to occur in the breast
muscle of the hen (Bennett and Porter, 1953), mammalian leg muscles
(Ruska, 1954), and in insect white muscles. Thus the mitochondrial system
appears to be linked to the need for repeated bursts of energy for fast and
strong action, whereas the basiphil reticular system is more suitable for the
maintenance of tension where more time is available for resynthesis.
We are very grateful to Dr. Aristides Vallejo-Freire, of the Seccao de Virus
of the Instituto Butantan, who placed at our disposal so many of the facilities
necessary for the work. We wish to thank also Dr. Keith R. Porter and
Edwards and Ruska—Function and Structure of Insect Muscles 159
Mr. G. Gilcher, of the Rockefeller Institute for Medical Research, who made
available the microtome used in this study. The work was supported in part
by a grant from the Conselho Nacional de Pesquisas, Rio de Janeiro, Brazil.
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