A Study on the Fibre Diameter and Certain Enzyme Concentrations in the Flight Muscles of Some Butterflies

Recent studies in insect metabolism indicate that, in certain insects like the beet-leaf hopper, locust and some Lepidoptera which indulge in sustained flight, fat forms the chief fuel (Fulton & Romney, 1940; Krogh & Weis-Fogh, 1951; Weis-Fogh, 1952; Zebe, 1954). In some cases it has been found that a respiratory quotient around 0·75, which is indicative of fat utilization, was obtained even at rest and in the presence of considerable amounts of glucose (Zebe, 1954, 1959). Flying vertebrates like birds and bats are also believed to utilize fat as the chief source of energy during sustained muscular activity (George & Jyoti, 1955, 1957, 1958; Odum & Connel, 1956). In this context the discovery of high concentrations of a fat-splitting enzyme (lipase) in muscles capable of such prolonged activity such as pigeon and bat breast muscles (George & Scaria, 1956; George, Susheela & Scaria, 1958a), locust and dragon fly flight muscles (George, Vallyathan & Scaria, 1958) and also cardiac muscle (George & Scaria, 1957) is of special significance.

The results of the recent studies conducted in our laboratories on the flight muscles of pigeon and bat have indicated a close relationship between structure and function. It has been shown that in both pigeon and bat breast muscles there exist two different types of fibres, dissimilar in size, colour, content of mitochondria, enzymes and metabolites (George & Naik, 1957, 1958, 1959; George & Scaria, 1958a, c; George, Susheela & Scaria, 1958a, b). In these muscles a correlation could be established between the concentration of the different enzymes and their fibre diameter (George & Scaria, 1958a, b, c; George, Susheela & Scaria, 1958a, b). A similar correlation was also arrived at in the skeletal muscles of the rat by Nachmias & Padykula (1958). Oxidative enzymes are concentrated more in fibres with lesser diameter and as a rule highly active muscles have more narrow fibres. Tiegs (1955) studied the anatomy and histology of the flight muscles of a large number of insects. However, he did not include in his study the fibres of the flight muscles of butterflies. A study on the flight muscles of butterflies should be of considerable interest since some butterflies fly short distances while others indulge in long and sustained migratory flights. Here we report our observations on the fibre diameter and the concentration of enzymes, especially lipase, in the flight muscles of a few butterflies in an attempt to correlate structure and function at the cellular level.

The flight muscles of the butterflies belonging to the following four families were studied :

They were captured during flight and used immediately, after killing by decapitation. The thorax was opened on the ventral side, and after pulling out the alimentary canal the muscles were carefully removed from their attachment by means of a pair of clean forceps and used for the various studies.

Thin hand sections of fresh frozen muscles, cut according to the method of George & Scaria (1958 a) and mounted in glycerine jelly were used for the measurement of fibre diameter. A microscope with an occular micrometer was used for the measurement.

The lipase concentration in the flight muscles of all the above butterflies was studied quantitatively in a manometric system in a bicarbonate-carbon dioxide buffer of pH 7·4 at 37° C., using tributyrin as substrate as described in an earlier publication (George, Vallyathan & Scaria, 1958). Lipase activity is expressed as the amount of μl. CO₂/mg. protein/hr. Protein was estimated according to the micro-Kjeldahl steam distillation method (Hawk, Oser & Summerson, 1954).

The presence and localization of the following enzymes were studied histo-chemically: lipase, acid and alkaline phosphatases, adenosine triphosphatase (ATPase) and succinic dehydrogenase.

For histochemical study of the enzymes fresh frozen sections of the muscles, prepared according to the method described by George & Scaria (1958 a), were used. Lipase was studied according to the method of Gomori (1953) using ‘Tween 80’ as substrate at pH 8·4 (George & Scaria, 1958a). The revised method of Gomori, using sodium glycerophosphate as substrate, was employed in the study of acid and alkaline phosphatases (George, Nair & Scaria, 1958) at pH 5·0 and 9·2 respectively. Sections kept in boiling water for 10 min. and incubated along with the samples under investigation were taken as control. The sections were incubated for 6–8 hr. for lipase and for 6 and 24 hr. respectively for acid and alkaline phosphatases at 40° C. The method of Pearse and Reis (Pearse, 1954) was adopted for the demonstration of ATPase. Sections kept in the incubation media for alkaline phosphatase at pH 7·4 and 9·2 were used as control. The period of incubation was 3 hr.

Succinic dehydrogenase activity in the muscle was determined by the tetrazolium chloride reduction method exactly as described in an ealier paper (George & Scaria, 1958c). 2:3:5-triphenyl tetrazolium chloride was used as the electron acceptor.

The average diameter of the fibres in the different species is given in Table 1. It was observed that the diameter of the fibres in the muscle was more or less uniform for the species. Fibres with the largest diameter (130-178 μ) were found in the PapiHonidae and the smallest in the Danaidae (80-98 μ). The Nymphalidae and Pieridae have fibres ranging from 84 to 105 μ in diameter. The figures given are the average of several hundred fibres from thirty to forty animals.

The results of the quantitative determination of lipase activity in the flight muscles of the different species is presented in Table 1. It can be seen that the greatest activity of 22·92 μl. CO₂/mg. protein/hr. is in the danaid butterfly, Danais chrysippus, which has the narrowest fibres and the least (4·52 μl. CO₂/mg. protein/hr.) in the papilionid, Zetidus agamemnon, with the largest muscle fibres.

The flight muscles of all the butterflies studied except the papilionids (Papilio polytes and Zetidus agamemnon) gave positive staining reaction for lipase. The distribution of the enzyme was uniform throughout the muscle fasciculi. However, from the histochemical observations no definite conclusions could be arrived at regarding the quantitative differences in the flight muscles of the different butterflies (Pls. 4, 5, figs. 1–8).

All the phosphatases tested for, viz. acid and alkaline phosphatases and adenosine triphosphatase, could be demonstrated in the flight muscles of all these butterflies. The distribution of these enzymes was also uniform in the fibres. Very strong positive reaction, especially for ATPase, was obtained in all the cases.

The colour developed due to formazan was uniform in the fibres of the same muscle. It was not possible to decide whether there were any quantitative differences in the enzyme content of the muscle of the different butterflies studied.

The peculiar type of striated muscle in insects has been of special interest to physiologists and biochemists ever since Von-Siebold, in 1848, revealed the histological features of the striated muscles of insects. Yet not much is known about its physiology. This prompted Chadwick (1953a) to make the remark that ‘the flight muscles of insects are among the most specialized contractile tissues in the animal kingdom and at the same time among the least investigated from the physiological point of view’. The recent studies of Tiegs (1955) on the anatomy and histology of the flight muscles of a number of insects have been a valuable contribution, but no information is given about the flight muscles of butterflies. It has been reported that some butterflies cover hundreds of miles at a stretch during flight, and they are in the air for much longer periods than most other insects observed (Chadwick, 1953 b). Butterflies therefore seem to be excellent material for studies directed towards a clearer understanding of the physiology of sustained muscular activity.

Insect muscle as a rule is devoid of myoglobin, and its absence is compensated for by the copious supply of oxygen from the tracheal system, the tracheoles penetrating even the individual muscle fibres. As such there is no differentiation into red and white fibres as seen in some vertebrate skeletal muscles. Red fibres in vertebrate muscles are usually narrower than the white ones, and are equipped with a more efficient system of oxidative enzymes. Highly active muscles such as the flight muscles are made up mostly if not completely of narrow fibres. Although no such differentiation of the fibres into red and white occurs in the insect flight muscles it seems that the fibres in the flight muscles of the more active insects are narrower than those of the less active forms. The danaid butterflies are very good fliers. Though they fly slowly and in a relaxed way, they fly for hours continuously without stop. The three danaid butterflies we have studied are all reported to be migratory (Wynter-Blyth, 1957). Among the nymphalids, Hypolimnas bolina is a migratory form. Nymphalids are almost similar in their mode of flight to the danaids. In these forms the diameter of the muscle fibre is decidedly less than that in the papilionids (Table 1), which are supposed to fly fast but only for a short time, indicating clearly that the decrease in diameter of the fibres has some relation to the ability to maintain continuous flight.

The data presented in Table 1 lend further support to the view that a muscle to become more active and efficient should possess fibres smaller in diameter. This view seems to be gaining ground, particularly in the case of vertebrate skeletal muscles indulging in sustained activity. Why it is so in the vertebrate skeletal muscles could be easily explained when we consider that the oxygen supply from the blood to the interior of the fibres is greater and faster when the volume of the individual fibre is less, and thereby the overall surface area of the fibres comparatively much greater. But why it should be so in the case of the insect muscle in which the oxygen supply is by diffusion across the tracheal capillaries which penetrate the fibre itself is not understood. A study of the distribution of the intracellular tracheae in these fibres having different diameters should therefore throw some light on this aspect.

It has been shown by George & Scaria (1956, 1957, 1959) that the amount of lipase present in a muscle is an indirect indication of the extent to which fat could be metabolized in the muscle. In vertebrates the correlation between the concentration of lipase in a muscle and its activity is evident from their data. The same could hold good for the insect flight muscles also (George, Vallyathan & Scaria, 1958). The data presented in this paper is further evidence in favour of their suggestion. Thus the highest value of 22· 92 μl. CO₂/mg. protein/hr. is obtained for the (migratory form) Danais chrysippus and the lowest of 4·52 μl. CO₂/mg. protein/hr. for the pafiilionid, Zetidus agamemnon, which is a comparatively poor flier. Further, it can also be seen that in the other butterflies studied the lipase value varies more or less according to the diameter of the muscle fibre. These observations speak unmistakably in favour of the postulated relationship between the diameter of the fibres in muscle and their enzyme concentration and activity. Again, the higher concentration of lipase in the more active muscle suggests the utilization of fat for energy during flight. As already pointed out, in the desert locust (Schistocera gregaria) it has been shown that fat is the chief source of energy for flight. Beall’s study (1948) on the fat content of the monarch butterfly (Danais plexippus) during migration, indicates the utilization of fat. The studies of Zebe (1954) on the R.Q. of butterflies during rest and during flight also indicate the utilization of fat, especially during flight. It may therefore be concluded that the higher concentration of lipase in the flight muscles of some of the butterflies we have studied is an indication of fat utilization in them. Further evidence for this view is provided by the fact that an appreciable difference in the concentration could be observed only in the case of the enzyme lipase. The failure to demonstrate lipase in the muscles of the papilionid butterflies may be due to an extremely small amount of the enzyme being present which might have been completely destroyed in the process of fixing the sections in formalin.

Another important observation is that the fibres in the muscles of the same butterfly are more or less uniform with regard to their structural and chemical organization. Just as there is no difference in the fibre diameter, so also there is no difference in the enzymic make-up of the fibres. This was found to be the case in all the butterflies examined. The significance of this is realized more when it is taken into account that in most vertebrate skeletal muscles hitherto studied, in spite of apparent resemblances between individual fibres in a particular muscle, there are deep-seated physiological differences which become recognizable only on histochemical examination of the various enzymes contained in them (George & Scaria, 1956; George, Nair & Scaria, 1958; George, Susheela & Scaria, 1958b) In none of the insect muscles which Tiegs studied could there be seen any appreciable difference in structure among the individual fibres. The absence of clear individual variations in the structure and physiology of the fibres within a muscle, though there be such differences between different muscles in the same animal, may be regarded as major feature in which the insect muscle differs from the vertebrate skeletal muscle. This organizational difference may be directly associated with the tracheal mode of respiration which is so characteristic of insects.

One of us (N. M. G. B.) is indebted to the Ministry of Education, Government of India, for the award of a Senior Research Scholarship which made this work possible.

Photomicrographs of transverse sections of the flight muscle of butterflies, lipase activity demonstrated histochemically.

Fig. 1. Danais chrysippus. Note the abundance of the precipitate (maximum lipase activity).

Fig. 2. Euploea core.

Fig. 3. Danais limniace.

Fig. 4. Delias eucaris.

Fig. 5. Zetidus agamemnon. Note the large size of the fibres and very little precipitate present (minimum lipase activity).

Fig. 6. Papilio polytes.

Fig. 7. Precise lemonias.

Fig. 8. Hypolimnas bolina.