Preliminary Observations on the Structure of Insect Flight Muscle 



H. E. Huxley and Jean Hanson 



Medical Research Council. Department of Biophysics, University College, London, and 

 Medical Research Council, Biophysics Research Unit, Wheatstone Laboratory, King's College, London 



The structure of striated muscle from vertebrates 

 has been analysed in considerable detail in recent 

 years, particularly by the techniques of x-ray diffrac- 

 tion, electron microscopy, and phase-contrast and 

 interference light microscopy. A theory has been 

 developed which accounts for all the observed fea- 

 tures of the structure, including the pattern of cross- 

 striations and the changes in that pattern during 

 contraction, in terms of a series of overlapping, 

 interdigitating arrays of longitudinal filaments which 

 slide into each other when the muscle shortens (2, 3, 

 5, 6). Two types of filament appear to be present; 

 they differ in their location, diameter, and protein 

 composition. The thicker filaments extend from end 

 to end of the A-bands and account for the high 

 density and birefringence of that band; they consist 

 largely of myosin. The thinner filaments extend from 

 the Z-line, through the I-band, into the A-band, up 

 to the edge of the H-zone; these filaments contain 

 actin. When the muscle shortens, these "secondary"' 

 filaments are apparently drawn further into the array 

 of "primary" filaments which form the A-band. 



In the electron microscope, cross-sections of 

 muscle show a double hexagonal array of filaments 

 in the A-bands where the two arrays of filaments 

 overlap (Huxley, 1953). Each secondary filament is 

 located symmetrically between three primary fila- 

 ments, so that each primary filament has six sec- 

 ondary filaments around it which it shares with its 

 six neighbouring primary filaments. 



Insect night muscle exhibits a pattern of cross- 

 striations which is very similar in many respects to 

 that of vertebrate striated muscle, but differs from 

 it in that the I-bands are usually very short or 

 entirely absent. However, such muscles undergo 

 only very small changes in length (of the order of a 

 few per cent) during activity, and it could be argued 

 that if they contract by the same sort of process as 

 has been suggested for vertebrate striated muscle, 

 they do not need to have very long I-bands. 



That the process of contraction should be similar 

 in all types of striated muscle, would seem likely, 

 but remains to be proven. Recently, studies of insect 

 flight muscle have been made by Hodge (1955) and 

 by Hanson (I ). Hodge's observations on the effect of 

 ATP on glycerinated flight muscle seemed to show a 

 migration of A-substance to the Z-lines. Hanson, 

 on the other hand, observed ATP-induced contrac- 

 tions of insect flight muscle which seemed very 

 analogous to those given by vertebrate striated 

 muscle, and she has been unable to repeat Hodge's 

 observations. She has also made many other obser- 



vations on insect flight muscle which show a very 

 high degree of similarity to corresponding observa- 

 tions on vertebrate striated muscle. 



On the basis of his electron-microscope and light- 

 microscope observations, Hodge came to the con- 

 clusion that a double array of filaments is not present 

 in insect flight muscle, but that, instead, a system of 

 cross-bridges exists between the filaments. He has 

 demonstrated the existence of these bridges by ex- 

 tremely elegant electron micrographs of cross-sections 

 of flight muscle. The bridges appeared rather less 

 well-organised in longitudinal sections, but no con- 

 tinuous axial structure was visible in his sections in 

 between the main axial filaments. 



We have now carried out the first stages of an 

 electron microscope investigation of insect flight 

 muscle. On the basic issue of whether or not two 

 sets of filaments are present, the results are very clear- 

 cut, and are in conflict with Hodge's conclusions; 

 they appear to show that insect flight muscle has a 

 structure which is highly analogous to that of verte- 

 brate striated muscle, and it is these results which 

 we shall now describe. 



Materials and methods. — Insect flight muscle from Cal- 

 liphora was fixed in osmic acid, dehydrated in alcohol, 

 embedded in methacrylate, and sectioned on a modified 

 version of the Hodge-Huxley-Spiro (4) microtome. 

 Sections were mounted on carbon films and examined 

 in the Siemens Elmiskop I. Additional staining was usu- 

 ally provided by 1 % phosphotungstic acid. 



Results. — Longitudinal sections of flight muscle 

 fixed fresh usually show fibrils with virtually no 

 I-bands. Z-lines are visible, and between them stretch 

 longitudinal filaments (fig. I ). in cross-sections, these 

 may be seen to form a very regular hexagonal array. 

 Longitudinal sections often display a pattern of 

 pseudo-striations when the plane of sectioning is 

 not quite parallel to the layers of the lattice, and 

 successive layers of filaments are seen. 



Examination of very thin longitudinal sections at 

 high magnification and high resolution shows that 

 mid-way between each two primary filaments, a 

 secondary filament is present (fig. 2). The secondary 

 filaments are connected to the primary filaments on 

 either side by cross-bridges which occur at mode- 

 rately regular intervals of about 100 A. This structure 

 is observed with great consistency. The cross-bridges 

 often appear to occur alternately on either side of 

 the secondary filaments. 



In cross-sections of these fibrils, the same structure 

 is very clearly visible (fig. 3). The predominating 

 feature of the secondary material in such cross-sec- 

 tions is the set of six secondary filaments around 



