Distribution of the Two Kinds of Myofilaments in Insect Muscles

AM. ZOOLOCIST, 7:451-456 (1967). Distribution of the Two Kinds of Myofilaments in Insect Muscles JACQUES AUBER Laboratoire de Cytologie, Faculte des Sciences, Paris, et Laboratoire de Microscopie Electronique Appliquee a la Biologie, C.N.R.S., Paris 5, France SYNOPSIS. Different insect muscles have been studied with the electron microscope and the distribution of the two kinds of myofilaments compared. In muscles other than those of flight, each thick filament is surrounded by 9-12 thin filaments, whereas, in the flight muscles, the contraction frequency of which is much higher, there are only 6 thin filaments surrounding each thick one; nevertheless, in the flight muscles of some butterflies, the wing stroke frequency of which is particularly low, there are 7-9 thin filaments. It seems then that there may be a relation between the ratio of the two kinds of myofilaments and the frequency of muscular contraction. In the muscles which have more than 6 thin filaments surrounding each thick one, the structure of the 7. line appears to be different from that which was described in dipteran flight muscles. A peculiar aspect of the M line is observed in lepidopteran flight muscles. In insects, the arrangement of myofilaments was first described in the flight muscles of the blow-fly, Calliphora erythrocephala Meig (Huxley and Hanson, 1957). At the level of the A band of these muscles, each thin filament lies mid-way between two thick filaments, and as these thick filaments lie in hexagonal array, each of them is surrounded by 6 thin filaments. This arrangement (Fig. 1), found again in the flight muscles of many insects and in some muscles of copepods (Bouligand, 1962; Fahrenbach, 1963), is nevertheless not common for all arthropodan muscles. Research on scorpion muscles (Auber- Thomay, 1963), crustacean muscles (Bouligand, 1964; Swan, 1963) and insect muscles other than those of flight (Toselli, 1965; Hagopian, 1966; Smith, 1966; Smith, Gupta, and Smith, 1966; Auber, 1966), has revealed that the hexagonal array remains constant for the thick filaments, but the number of thin filaments surrounding each of them may vary from 9 to 12. Observation of many Calliphora muscles, in adults and larvae, shows that, except in the flight muscles and in the haltere [i.e., the mainflight muscles (indirect flying muscles) but not the wing adjustor muscles] muscles, there are always more than 6 thin filaments surrounding each thick one; this number is variable according to the muscle under consideration; for example, it is (451) from 10 to 12 in the abdominal segmental muscle and from 9 to 11 in the tergotrochanteral muscle (Figs. 2 and 3). In all the muscles studied, the same ratio of thick and thin filaments was found at every level of the A band, except in the H band where there were thick filaments only; the greater number of thin filaments may, therefore, not result from any possible phenomena of supercontraction with double overlap of the two sets of thin filaments of each sarcomere. In insects, the distribution of myofilaments described in the flight muscles seems, therefore, to be peculiar to this kind of muscle. The flight muscles happen to have a faster work rhythm than that of all the other muscles, and in these other muscles, the ratio of thin filaments is always higher. In the flight muscles of some moths (Phytometra, Agrotis, Minucia, Abraxas), as in those of Diptera, each thick filament is surrounded by 6 thin filaments. But, when the frequency of muscular contraction is low enough, as in some butterflies (Pieris, Vanessa) which have a wing-beat of not more than 10/sec (Sotavalta, 1947), that is to say 5 to 6 times less than the moths, each thick filament is no longer surrounded by 6, but by 7-9 thin filaments (Figs. 4 and 5) (Auber, 1967). It should be noted that in the flight muscles of all these Lepidoptera, the M

452 JACQUES AUBER Explanation of Figures Electron micrographs of insect muscle (cross sections taken through the A band). Glutaraldehyde fixation with osmium tetroxide postfixation. Sections stained by uranyl acetate followed by lead citrate. 1'late ) HC. 1. Cross section of a fibril in a flight muscle of Diptera (Bombylius). Each thick filament is surrounded by 6 thin filaments. X 65,000.

DISTRIBUTION OF MYOFILAMENTS IN INSECTS 453 Plate II edge of the H band on the left part of the micro- FIG. 2. Cross section through the tergo-trochanteral graph, which excludes the hypothesis of a supermuscle of a fly (Calliphora). Each thick filament is contraction phenomena. X 95,000. surrounded by 9 to 11 thin filaments. Note the FIG. 3. Higher magnification. X 135,000.

454 JACQUES AUBER Plate III ot the I band. Each thick filament is surrounded FIG. 4. Flight muscle of a butterfly (Vanessa). by 7 to 10 thin filaments. X 85,000. Slightly oblique cross section of a myofibril showing FIG. 5. Higher magnification. X 150,000. the edge of the A band, and below, the beginning

DISTRIBUTION OF MYOFILAMENTS IN INSECTS 455 line presents a particular aspect: at this level, the thick filaments become flattened and appear generally to be composed of two subunits placed side by side. A similar structure of the M line was also observed in the flight muscles of an hemipteroid insect (Reedy, 1966). In addition, in the flight muscles of moths, the sarcoplasmic reticulum penetrates into the myofibrils themselves (Auber, 1967). With regard to the Z line, its structure appears to be related to the number of thin filaments: in the flight muscles of moths, as in those of Diptera (Auber and Couteaux, 1963), the thin filaments are grouped in threes and participate in tubular formations, which -give the Z line the aspect of a perforated plate. On the other hand, in the flight muscles of butterflies, as in other muscles with more than 6 thin filaments around each thick one, the Z line exhibits only, in an electron dense substance, filaments of about 50 A in diameter, distributed without apparent order, which seem to be prolongations of the actin-containing filaments. An examination of the frequency of contraction of flight muscle and of the number of thin filaments in various insects, shows that in the Calliphora with a rate of wing-beat of 200 per second, as in the Phytometra, with a rate of wing-beat of 50/sec, there are 6 thin filaments surrounding each thick one. The arrangement of myofilaments seems, therefore, to be independent of the manner of working of the flight muscles, "asynchronous" (see Pringle, 1957) in the case of Diptera, or "synchronous" in the case of Lepidoptera. However, in Aesclma mixta (Odonata), whose wing-beats average 30/sec, there are 6 to 8 thin filaments; in butterflies with a rate of wing-beat of only 10/sec, this number rises to 9 and for muscles other than the flight muscles, the rhythm of which is even slower, it may reach 12. It seems then, that there may be a relation between the ratio of the two kinds of myofilaments and the frequency of muscular contraction when this frequency falls below a certain level. With regard to the arrangement of myofilaments, it is possible to observe in the flight muscles of the blow-fly, during the first stages of myofibrillogenesis, isolated thick filaments surrounded by 6 thin filaments. This suggests, as in the hypothesis previously put forward by Swan (1963) concerning crayfish muscles which have a high ratio of thin filaments, that each thick filament may interact with its own set of 6 thin filaments. As a result of this, in a myofibril, each thick filament would be surrounded by 12 thin ones. When each thick filament is surrounded by 7-11 thin filaments, some of these would be shared between two thick filaments next to each other. In the flight muscles, where each thick filament is only surrounded by 6 thin ones, each of these filaments is shared between two thick ones. The number of cross-bridges issuing from each thick filament seems to be constant whatever may be the number of thin filaments and, when there are more than 6 thin filaments surrounding each thick one, the bridges seem to link at random. It is in the muscles which vary most from the hexagonal pattern, with 6 thin filaments, that the contraction-rhythm would seem to be the lowest. REFERENCES Auber, J. 1966. Distribution des deux types de myofilaments dans divers muscles de Dipteres. J. Microscopie 5:28a. Auber, J. 1967. Particularites ultrastructurales des myofibrilles des muscles du vol des Lepidopteres. Comp. Rend. Acad. Sci., Paris 264:621-624. Auber, J., and R. Couteaux. 1963. infrastructure de la strie Z dans des muscles de Dipteres. J. Microscopie 2:309-324. Auber-Thomay, M. 1963. Remarques sur l'ultrastructure des myofibrilles chez des scorpions. J. Microscopie 2:233-236. Bouligand, Y. 1962. Les ultrastructures du muscle stri<5 et de ses attaches au squelette chez les Cyclops (Crustaces Copepodes). J. Microscopie 1:377-394. Bouligand, Y. 1964. Les ultrastructures musculaires des Copepodes. III. Nature de la bande de contraction CM des sarcomfcres. J. Microscopie 3:697-710. Fahrenbach, W. H. 1963. The sarcoplasmic reticulum of striated muscle of a cyclopoid copepod. J. Cell Biol. 17:629-640.

456 JACQUES AUBER Hagopian, M. 1966. The myofilament arrangement in the femoral muscle of the cockroach, Leucophaea maderae Fabricius, J. Cell Biol. 28:545-562. Huxley, H. E., and J. Hanson. 1957. Preliminary observations on the structure of insect flight muscle. Proc. Stockholm Conf. on Electron Microscopy, 1956 (Uppsala):202-204. Academic Press, New York. Pringle, J. W. S. 1957. Insect flight. Cambridge Univ. Press, London and New York. Reedy, M. 1966. Personal communication. Smith, D. S. 1966. The structure oe intersegmental muscle fibers in an insect, Periplaneta americana L. J. Cell Biol. 29:449-459. Smith, D. S., B. L. Gupta, and U. Smith. 1966. The organisation and myofilament array of insect visceral muscles. J. Cell Sci. 1:49-57. Sotavalta, O. 1947. The flight tone (wing-stroke frequency of insects. Acta Entomol. Fennica. 4: 1-119. Swan, R. C. 1963. The structure of crayfish sarcomeres. J. Cell Biol. 19:68A. Toselli, P. A. 1965. The fine structure of the fully developed intersegmental abdominal muscles of Rhodnius prolixus. Anat. Rec. 151:427.