The supply of oxygen to the flight muscles of insects: A Theory of tracheole physiology

0040-8166/82/00420501

TISSUE & CELL 1982 14 (3) 501-518 i‘ 1982 Longman Group Ltd

V. B. WIGGLESWORTH

THE SUPPLY OF OXYGEN TO THE MUSCLES OF INSECTS: A THEORY TRACHEOLE PHYSIOLOGY Key words:

Insect, tracheoles,

respiration,

$02.00

and W. M. LEE

FLIGHT OF

muscle, flight.

ABSTRACT. In the flight muscles of insects, virtually every mitochondrion is in contact with or is encircled by terminal tracheoles which reach them by following the channels formed by the invaginated plasma membrane of the muscle fibres, the Tsystem tubules. In Musca, Calliphora and Drosophila (Diptera), Apis (Hymenoptera) and Tenebrio (Coleoptera) the terminal tracheoles are smooth-surfaced tubes with a lumen of about 50 nm. In Pieris (Lepidoptera) the terminal tracheoles occupy the regular transverse tubular system which runs between the mitochondria and across the fibrils on either side of the H zone. They are smooth tubules of 80-200 nm diameter. Preliminary observations suggest the same arrangement in Zschnura (Odonata). In Rhodnius and other Hemiptera the transverse T-tubule system forms large cavities among the mitochondria: these cavities in Rhodnius are occupied by smooth-walled tracheole endings. In the mature adult of Schistocerca (Orthoptera) T-tubules of varying size are utilized by terminal tracheoles (diameter 50-100 nm). The terminal tracheoles of the flight muscles are highly permeable to myrcene and kerosine. They commonly fill with liquid during rest and this liquid is resorbed during activity. It is suggested that these adaptations increase the efficiency of respiration in the flight muscles by ensuring that, when it is most needed, gaseous oxygen extends to the surface of the mitochondria, from which it is separated by a very permeable barrier.

The contractile fibres contain only hydrolytic enxymes, notably ATPase (Tice and Smith, 1965; Zebe, 1966). Enzymes of the oxidizing system (dehydrogenases, cytochromes and cytochrome oxidase) are confined to the mitochondria (Levenbook and Williams, 1956; Walker and Birt, 1969). The mitochondria constitute perhaps 30-40 % of the mass of the muscle, so that the consumption of oxygen by the mitochondria at normal temperature and pressure will be some 6 ml Oa/ml/min or 0.1 ml Oa/ml/sec. In other words a single mitochondrion will consume about one-tenth of its volume of oxygen per second. In a study of a number of insects WeisFogh (1964a, b) concluded that there was ‘no reason to suggest other mechanisms (of oxygen supply) than a simple, normal diffusion’ combined of course with active ventilation of the larger tracheae. But the ultimate terminations of the tracheal system

Introduction

objectives of this paper are two-fold: (1) to describe the supply of tracheoles to the mitochondria of the flight muscles of insects; (2) to develop a general theory of the physiology of tracheoles. The metabolism of the flight muscles in the most active insects, such as the honey-bee or the blowfly, amounts to some 2200 kcal/ kg muscle/hr, equivalent to about 4 ml 02/g muscle/min (Weis-Fogh, 1961)-and this without the accumulation of any detectable oxygen debt, except during a brief period at the commencement of flight (Sacktor and Wormser-Shavit, 1966). THE

* Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ. t ARC Unit of Invertebrate Chemistry and Physiology, Cambridge. Received 27 May 1981 Revised 10 March 1982. 501

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were not seen. In an earlier paper WeisFogh (1961) wrote: ‘it is to be expected that evolution has proceeded so that every possible mechanism has been tested for intensifying work production and metabolism of wing muscle.’ The supply of tracheoles to the muscle mitochondria and the properties of these tracheoles are clearly of paramount importance. Indeed the richness of the oxygen supply to the ‘sarcosomes’ of insect flight muscles has often been pointed out (Edwards andRuska, 1955; Smith 1961a). The development of improved methods for injection of the tracheoles (more efficient than the cobalt sulphide method (Wigglesworth, 1950) which was used by Weis-Fogh) has provided the opportunity for the present study.

Material and Methods Material

The observations have been made on a series of insects representative of the main orders: Zschnura elegans (Odonata : Zygoptera) ; Schistocerca gregaria (Orthoptera); Rhodnius prolixus (Hemiptera); Pieris brassicae (LepiTenebrio molitor (Coleoptera); doptera); Apis mellifera (Hymenoptera); Musca domestica, Calhphora vicina and Drosophila melanoguster (Diptera). These species cover a broad spectrum of flight activity. Injection method

The method of injection was that already described (Wigglesworth, 1950) consisting of evacuation at oil-pump pressure in an atmosphere of hydrogen; immersion in the injection mixture; followed by slow admission of air to restore the pressure to atmospheric. Injection fluid

The mixture used at the outset was that developed for injection of tracheoles in the epidermis and central nervous system in Rhodnius (Wigglesworth, 1977) and successfully used also in the injection of tracheoles in the cryptonephridial system of Tenebrio and the aeroscopic chorion of a number of insects (unpublished). This mixture consists of equal volumes of myrcene (technical) and light petroleum (Shell odourless kerosine). New injection mixtures are described below.

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Dissection andfixation

After injection the thorax is dissected by cutting along the mid-dorsal and midventral lines and separating into two halves in 15% sucrose and then fixing for 1-2 hr in 2 % paraformaldehyde and 0.5 % glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 with the addition of 10% sucrose and 0.2% CaCla. The muscles, particularly the dorsal longitudinal indirect flight muscle, are then isolated and fixed for l-2 hr in 1% osmium tetroxide in 0.5 M cacodylate buffer at pH 7-3. Section for light and eIectron microscopes

The muscles are embedded in Spurr’s medium (1969) and cut for the light microscope at graded thicknesses of 0.5-4 pm and stained by mounting in Farrants’ medium containing 2 % ethyl gallate (Progallin A of Nipa Laboratories Ltd, Cardiff, U.K.) (Wigglesworth, 1957). Sections for the electron microscope are usually given no further staining. This ensures maximum contrast between the injected myrcene which is highly osmiophilic and the background staining of the mitochondria, etc., which shows only low contrast. The contrast in the mitochondria and other lipid containing structures can be increased by the procedures already described for lipid staining (Wigglesworth, 1981). Whole mounts

A method has been devised for viewing the distribution of the injected tracheoles in whole mounts of the muscles. After injection (preferably with arachis oil alone) and fixation as described above, the muscles are teased in Spurr’s medium to separate the fibres and are then mounted under a coverslip and stored under pressure at room temperature until the medium is partially hardened. Provided the preparations are not heated or stained with ethyl gallate the fibres remain pale with only the tracheal contents blackened. Such preparations show the general layout of the secondary and tertiary systems of tracheoles but the ultimate terminations are usually beyond resolution in the light microscope. Freeze-fkacture

Material for freeze-fracture was dissected out, placed immediately in an appropriate

THEORY

OF TRACHEOLE

PHYSIOLOGY

Ringer containing 25 % glycerol as cryoprotectant, and incubated for 2@35 min. It was then mounted in Balzer’s specimen holders and frozen in Freon 22 cooled with liquid nitrogen. Fracturing was carried out at - loO”C, 10-6 Torr, in a Balzer’s (BA 360M) freeze-etching device, shadowed with a tungsten/tantalum mixture, and backed with carbon. The replicas were cleaned in a sodium hypochlorite solution and viewed in a Philips EM300. Tracheal supply to flight muscles The tracheal supply to the flight muscles is conveniently divided by Weis-Fogh into three parts. A primary tracheal supply consists of abundant large tracheae, often expanded into air sacs and subject to active ventilation by pumping movements of the thorax or of the abdomen (Weis-Fogh, 1964a). The detailed anatomy of this system which varies greatly in different insects has been well described. The primary supply ends in more or less flattened tracheae, sometimes forming flattened air sacs, which are wrapped around the muscle fibres. They break up into multiple small tracheae or large tracheoles (l-2 pm diameter) which dip into the surface of the muscle and follow a more or less radial course branching as they proceed towards the central axis. It is these conspicuous tracheoles within the muscle fibres which constitute the secondary tracheal supply. The terminal tracheoles coming off these branching radial tracheoles and conveying oxygen to the mitochondria constitute the tertiary tracheal supply, the details of which have been incompletely described and are the sole concern of the present work. As is well known the mitochondria of flight muscles are ranged in continuous rows between the contractile fibrils. In transverse sections each muscle fibril is seen to be invested on all sides by mitochondria. The general appearance of the flight muscles as seen on longitudinal section is very similar in all flying insects but the arrangement and the properties of the terminal tracheoles differ widely in different groups. New trials of the injection mixture When developing

the cobalt sulphide

method

503

of injection it was found that the tar-like cobalt naphthenate could be induced to fill the fine tracheoles of the flea only when dissolved in very mobile petroleum; ‘white spirit’ (B.P. 150-200°C) was used (Wigglesworth, 1950). For this reason light petroleum (Shell odourless kerosine) was used as a solvent for myrcene. As recorded above, this filled the extremities of the tracheoles in Rhodnius epidermis and nervous system and Tenebrio cryptonephridial system with no sign of escape through the tracheole walls. When this mixture was used for the flight muscles of Musca, Calliphora and Drosophila it was found that the terminal tracheoles formed bands encircling the mitochondria, at least one band and often two around each mitochondrion. These bands (0.060.2 pm in diameter) commonly showed enlargements (up to 0.7 pm in diameter) resembling minute air sacs, which often appeared to be invaginated deeply into the surface of the mitochondria (Figs. 1, 2). One curious feature of these bands, which often lie at the junction between adjacent mitochondria, was that they were nearly always complete, even when the sections cut deeply through the mitochondria and would be expected to miss a superficial band. There was no sign of escape of the injected fluid through the walls of the small annulated tracheoles (0.220.4 pm internal diameter) approaching the mitochondria. When the same injection procedure was extended to the flight muscles of other insects it was found that in some of these the injected fluid was undoubtedly escaping through the walls of the terminal tracheoles on the surface of, and indeed appearing within, the mitochondria. Fig. 7 shows a transverse section of the flight muscle of the honey-bee with obvious escape of the injection fluid. But no such escape was visible in tracheoles (diameter 0.3-0.5 pm at some distance from the mitochondria. In Tenebrio (Fig. 21), Pieris (Fig. 14) and Rhodnius escapes of the myrcene/kerosene mixture occurred in varying degrees. Indeed, in these insects there was some escape even from the larger tracheoles (diameter 0.45 pm) of the tertiary system in the flight muscles. These observations show that there are differences in the permeability of the tracheole walls in different insects, and they strongly suggest that the supposed air sacs on the

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mitochondria of Musca flight muscles result from a well-localized escape of the injection fluid through the walls of the tracheoles and into the mitochondria. There are clearly differences in permeability in different parts of the tracheole system. In the search for an injection fluid which would not escape from the more permeable tracheoles the medicinal paraffin ‘Nujol’ was used, and rendered osmiophilic by partition in 10 y0 farnesol in 70 ‘A ethanol, after fixation but before a renewed treatment with osmium (Wigglesworth, 1981). Then it was found, remarkably enough, that even without partition in farnesol the Nujol had become osmiophilic. The same happens with injected odourless kerosine, which likewise is non-reactive with osmium tetroxide. It appears that unsaturated lipids are extracted from the inner walls of the tracheal system, particularly from the tracheoles, during the injection of these solvents. This recalls the fact that during the nineteenth century exposure to the vapour of osmium tetroxide, which blackens the walls of the tracheoles, was used as a method for demonstrating their terminations (Schultze, 1865). Triglyceride oils such as arachis oil likewise did not escape through the tracheole walls. Moreover these unsaturated oils react strongly with osmium tetroxide without further treatment and have proved satisfactory in all the insects tested. Arachis oil has two disadvantages. (I) Like Nujol it always leaves some branches of the fine tracheoles uninjected; (2) the amount of osmium taken up, especially after treatment with ethyl gallate, increases the volume of the tracheole contents and induces convolutions in the tubules. Injection of myrcene alone or of myrcene in an equal volume of odourless kerosine have been used to indicate the site of the most permeable tracheoles. Terminal In whole

tracheoles

in higher Diptera

mounts of the flight muscles of after injection with arachis oil, mounted in Spurr’s medium and kept at room temperature, the flattened tracheae coming from the air sacs can be seen to divide into as many as a dozen tracheoles of 1.0-1.5 pm diameter which spread fanwise and run inwards between the contractile Musca

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fibrils, tapering to about 0.5 pm. As they proceed these secondary tracheoles give off branches of deeply ringed tracheoles (maximum internal diameter O-3-0.4 pm). These branches form the greater part of the tertiary system. They can be followed in the light microscope but since they twist around contractile fibrils and mitochondria they are not conspicuous in whole mounts or in sections. Fig. 3 shows a transverse section of the flight muscle of Musca after injection with a mixture of Nujol and arachis oil. Almost every mitochondrion is in contact with and presumably encircled by injected vessels of about 0.07-0.08 pm. In some places the vessels appear to be even finer than this. Fig. 4 is a longitudinal section of the same material. It shows a 1.1 pm tracheole, one of the main radial tracheoles of the secondary system, and alongside it two of the tertiary branches have been cut (0.3 pm maximum internal diameter). Tracheoles of this size-range run to the mitochondria and may encircle them. But as indicated by arrows many mitochondria are invested by injected tracheoles 0.07 pm thick or less. When these fine tubes are seen in longitudinal section they do not show the deep annular folding of the larger tracheoles. There are commonly about two of these encircling tracheoles per sarcomere. The origin of these fine tubules from the annulated tertiary tracheoles is seldom seen in sections and they are too fine for resolution in the light microscope. The presence of the injection fluid provides the evidence that they are indeed the terminations of the respiratory system. It occurred to one of US (W.M.L.) that freeze-fracture might provide visual evidence of this continuity. Fig. 5 shows a freezefracture preparation in which the terminal vessels are seen as hollow tubes between the mitochondria, some with a lumen of about 0.06 pm diameter. These tubules are enclosed within a cytoplasmic sheath; often with more than one branch within a single sheath. The extensions of the lining cuticle of these tubes have proved unduly resistant to solution in the sodium hypochlorite with which the unfixed tissue is removed from the replica. Consequently the inner wall of the tubule, hanging down from the point of fracture, comes to lie on the surface of the

THEORY

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replica; and since the substance of the wall shows considerable electron density the persisting residues appear as dark tracks on the repiica. These tracks (Fig. 5) provide useful evidence that these structures really are the continuations of the tracheoles, which have similar opaque linings. The terminal tubules do not show the deep annulation of the larger tertiary tubules; Fig. 6 shows a weakly beaded tubule in longitudinal section with internal diameter oscillating from 0.06 to 0.15 pm, but many tubules have smooth walls (Fig. 28). The other higher Diptera studied, Culliphora and Drosophila, show the same encircling of the mitochondria by the fine terminations of the tracheal system. The diameter of the muscle fibrils and the size of the mitochondria and the terminal tracheoles is very similar in Musca, Calliphora and Drosophila. As will be discussed later in this paper, an important factor in the function of the tracheoles is the presence of liquid in their terminations during rest and its replacement by air during activity, as happens in many insects. Mature adult Musca (8 days after emergence) were exposed to air at a pressure of -690 mmHg. They were very active at first but soon became completely inert and were then exposed for 10 min in hydrogen at the same pressure and the flight muscles dissected out in 12.5% sucrose. As seen by transmitted light in the light microscope under the oil immersion lens the tracheoles were filled with air as far as they could be resolved. When flies from the same group were kept at rest by placing at 4°C for 2-3 hr and then dissecting and mounting as before, the column of air in many of the tracheoles ended abruptly; below this point the lumen was filled with liquid. This would indicate that during rest the tracheole endings do fill with liquid-but the resuIts are not completely unequivocal because even in the asphyxiated flies there are always some abrupt endings of the air of the tracheolesusually where the muscles have suffered damage during dissection. Terminal

tracheoles

in A&

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PHYSIOLOGY

mefriferu

As observed in the flight muscles of the honey-bee examined fresh in 12.5% sucrose

under a coverslip, or in whole mounts of muscle fibres injected with arachis oil, abundant short tracheae from air sacs between the muscles immediately break up into a great number of tracheoles which enter the muscle and branch repeatedly. The orientation of these tracheoles is transverse; as in Musca there is no tendency for the tracheoles to run longitudinally between the contractile fibrils. On focusing up and down on a well-injected fibre, at every level, the tracheoles, right down to the finest that can be resolved, run transversely (about two per sarcomere) and dip down into the cleft between the fibrils; that is between the mitochondria. At the time of injection the whole system is clearly filled throughout with air. Fig. 7 shows a transverse section of Apis flight muscle after injection with odourless kerosine and myrcene. There has been a widespread escape of the injection fluid through the walls of the narrow terminal tracheoles with accumulation within the mitochondria. There was no obvious escape from the larger tracheoles (diameter 0.30.5 pm). Fig. 8 shows a transverse section after injection of Nujol and arachis oil. Injected tracheoles ranging from 0.2 to 0.07 pm internal diameter run between the mitochondria. Figs. 9 and 10 are longitudinal sections after injection of Nujol only showing the same size range in the terminal tracheoles. Although the course of tracheoles within the muscle is quite different in Apis and in Muscn the terminal supply to the mitochondria is very similar. There are abundant tracheoles in the O-l-O-2 [Lrn range and in the 0.05-0.08 pm range applied to the mitochondria. Virtually every mitochondrion is in contact with and probably encircled by tracheoles. In Musca and Apis these terminal tracheoles are readily permeable to myrcene and odourless kerosine. Terminal

tracheoles

in Pieris brassicae

The Cabbage White butterfly, Pieris brassicae, spends most of its adult life on the wing and it is a species which makes long migratory flights across Europe. It has powerful flight mr scles. But a fresh preparation of the muscles from a resting specimen of Pieris shows rows of tracheae dividing into tracheoles which run mainly transversely across

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the muscles and a rather small number of tracheole branches which run in the long axis of the muscle between the fibrils (Fig. 11). It seems likely that there must be other tracheoles which become air filled when flight begins. An adult male Pieris was removed from storage at 4°C to air at room temperature. It became active immediately and was transferred to the evacuation flask, slowly evacuated to -680 mmHg. It fluttered vigorously as the pressure fell and remained active for 10 min at the reduced pressure; it then become completely immobile and was held for a further 10 min at the low pressure. The dorsal longitudinal indirect flight muscle was then dissected out in 12.5 % sucrose and examined under a coverslip. The muscle now had a silvery appearance which results from a transverse array of air-filled vessels from the surface of which light is

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reflected. This array is repeated in successive layers of the fibre; the separation of the vessels being only half that of the Z-lines. After injection with arachis oil, whole mounts of the muscle fibres sometimes show tracheole branches running transversely across the fibre at all levels. These transverse tubules vary in thickness from about 0.1 to 0.2 pm and are readily visible in the light microscope. They are precisely positioned, with two parallel vessels to each sarcomere (Figs. 15, 17). They run chiefly between the mitochondria and turn upwards or downwards as they reach a contractile fibril. As a result an optical section of the fibre shows a rectangular network with black points at the corners, where the tracheoles are seen in transverse optical section (Fig. 12). The appearance of a network is of course an optical illusion: the tracheoles are almost solely transverse; the longitudinal sides of

Fig. 1. Longitudinal section of Musca flight muscle after injection odourless kerosine, showing apparent ‘vesicles’ on narrow tracheoles mitochondria. x 12,000. Fig. 2. Mitochondria of Drosophila vesicles among the cristae. x 38,600.

after

injection

as Fig.

of myrcene in encircling the

1, showing

apparent

Fig. 3. A4usca muscle in transverse section after injection of Nujol and arachis oil showing tracheoles (finest about 0.06 pm) in cross-section investing the mitochondria. x 13,000. Fig. 4. The same in longitudinal section, showing radial tracheoles of the secondary system (s), annulated branches of the tertiary system (r) and the fine terminal tracheoles of this system (arrows) applied to the mitochondria. x 13,000. Fig. 5. Freeze-fraction preparation showing five plasma membrane sheaths each enclosing one or more terminal tracheoles, lying between the mitochondria (nz); the irregular shadows (c) represent the undissolved residues of the cuticular lining of the tracheoles. x 13,000. Fig. 6. Freeze-fracture showing a weakly beaded terminal tracheole, in its double plasma membrane sheath, between muscle fibres below and mitochondria above. x 24,200. Fig. 7. Transverse section of flight muscle of Apis after injection of myrcene in odourless kerosine showing extensive escape of the injection fluid into the mitochondria. x 13,000. Fig. 8. Transverse section of Apis muscle after injection Injected tracheoles between the mitochondria. x 13,000.

of Nujol and arachis

oil.

Fig. 9. Longitudinal section of Apis muscle after injection of Nujol only. Tracheoles in longitudinal section to left and mainly transverse section to right: smallest diameter about 0.04 pm. x 13,000. Fig. 10. As Fig. 9.

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the rectangles represent the margins of the contractile fibrils. The pattern often includes a darker shade between alternate pairs of transverse tracheoles, which gives an appearance of broader bands across the fibre. This again is an optical illusion due to the presence of the Z-line between the alternate pairs of tracheoles (see Fig. 15). Fig. 13 shows a longitudinal section, 1 cLmthick, of such an injected preparation stained with ethyl gallate after osmium tetroxide. In freshly isolated muscle fibres from a resting butterfly this transverse system, which exists throughout the substance of the fibre, cannot be seen; presumably because it is filled with liquid. Fig. 14 shows a transverse section of Pieris flight muscle after injection with odourless kerosine and myrcene. The injection fluid is escaping through the walls of a longitudinal tracheole (arrow) and accumulating in irregular deposits in the mitochondria. Fig. 15 shows a longitudinal section with the transverse system very incompletely in-

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jetted with arachis oil, but with the uninjetted channels conspicuous. Fig. 17 at a higher magnification shows the passage of the injected vessels between the mitochondria, of which there are three to each sarcomere (Smith, 1962). In transverse section the injected vessels encircle the fibrils (Fig. 16). Fig. 19 shows this in a transverse freeze-fracture preparation. The transverse tubules are bounded by plasma membrane. From the character of their walls and their regular position, crossing the fibrils on either side of the H zone, they are clearly the transverse T-tubes as found in many insects with synchronous flight muscles (Smith, 1966). But in Lepidoptera these T-tubes have been taken over as channels for the terminal tracheoles. The invading tracheoles have smooth thin walls (0.08-0.2 pm diameter) that do not always make contact with the walls of the tubular system (Fig. 18). Some of these terminal tracheoles may well be derived from the system branching within the muscle fibre (Fig. 11) but most of them

Fig. 11. Pieris muscle from resting insect injected with arachis oil, showing secondary and tertiary branches within the fibre but no injection of the transverse system. x 1200. Fig. 12. Optical section of whole mount of Pieh muscle fibre; tracheoles injected with arachis oil. Note the apparent rectangular tracheoles seen in optical transverse section at the corners showing x 1200. Fig. 13. 0.5 pm section of muscle The injected transverse tracheoles are Z-lines can be seen between alternate dark and pale bars across the fibre in

the transverse network with as dark dots.

fibre (like Fig. 12) stained with ethyl gallate. stained and in some places the faintly stained pairs of transverse tracheoles (cf. the alternate Fig. 12). x 1200.

Fig. 14. Transverse section ofPieris muscle after injection with myrcene in odourless distillate, showing widespread escape of the injection fluid into the mitochondria. Some escape is seen around the 0.25 pm tracheole (arrow). x 38,000. Fig. 15. Longitudinal section of Pieris muscle incompletely injected oil. The uninjected transverse tubules are conspicuous. x 13,000. Fig. 16. Same specimen as Fig. l$, in transverse by injected tracheoles. x 13,000.

section.

with arachis

Many fibrils surrounded

Fig. 17. The same at higher magnification showing the relation of the injected transverse tubules to the mitochondria and to the H-zone and Z-lines of the fibrils. x 20,000. Fig. 18. The same, showing that the injected smooth-walled tracheoles are not always in close contact with the walls of the transverse tubules (arrows). Below on the left an uninjected tracheole (0.08 pm) is seen in transverse section (small arrow). x 38.000.

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probably arise directly from small tracheoles on the surface of the fibre. Freeze-fracture preparations of the plasma membrane of the muscle fibre in surface view show the points of invagination of the transverse tubules in parallel rows. Many of these tubules contain a tracheole in the lumen (Fig. 20). Terminal tracheoles of Tenebrio molifor

Flight

muscles of the mealworm beetle dissected out in 12.5% sucrose show large tracheae at intervals along the muscle fibres breaking up into smaller branches which enter the fibre at many points and then run inwards giving off fine branches Tenebrio

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(0.154~4 pm diameter) which run longitudinally (cf. Smith, 1961a). These tracheoles branch still further to give rise to very fine tracheoles which can be seen in contact with the mitochondria-but any terminations between the mitochondria cannot be distinguished in the light microscope from the membranes of the mitochondria seen in optical section. At no point can abrupt terminations of the air columns, indicating the presence of liquid in the endings, be seen (except in areas injured during dissection). Fig. 21 shows mitochondria of Tenebrio flight muscle after injection of myrcene in odourless kerosine. The injection mixture is

Fig. 19. Transverse freeze-fracture of Pieris muscle showing the channels transverse system surrounding the contractile fibrils (cf. Fig. 16). x 24,000.

of the

Fig. 20. Freeze-fracture of Pieris muscle showing surface view of the muscle plasma membrane with the sites of invagination of the transverse tubules seen in parallel rows. Some of these invaginations contain tracheoles (arrows). x 13,000. Fig. 21. Transverse section of Tenebrio flight muscle after injection of myrcene in odourless distillate showing escape of the mixture from a 0.26 pm tracheole (arrow) and from the narrow terminal tracheoles (0.05 pm) on the mitochondria. x 38,000. Fig. 22. Transverse section of muscle from newly moulted adult Tenebrio injected with arachis oil; showing numerous small mitochondria and injected 0.3 pm tracheoles. x 13,000. Fig. 23. Longitudinal section of mature Tenebrio showing 0.25 pm tracheole top right and small terminal chondria (arrows). x 13,000.

muscle injected with Nujol, tracheoles investing the mito-

Fig. 24. Section of mature flight muscle of Tenebrio after injection with arachis oil, with two injected tracheoles at right and an uninjected tracheole at left which shows clearly the outer dual plasma membrane of the invaginated tubule, the inner dual membrane of the tracheoblast, and inside this the cytoplasm and cuticular lining of the tracheole. x 39,000. Fig. 25. Freeze-fracture of Tenebrio muscle showing two 0.15 pm tracheoles. Each is enclosed by (1) the outer plasma membrane of the invaginated tubule system, (2) the fluid contents of the invagination, continuous with the haemolymph and often containing minute membrane bound vesicles, and (3) the plasma membrane of the tracheoblast covering the cytoplasm of the tracheole. The cuticular wall of the tracheole lumen is continuous with the residual lining membrane (c). x 13,000. Fig. 26. Freeze-fracture from Tenebrio muscle showing the fractured end of a 0.12 pm tracheole lying in the plasma membrane of the tracheoblast (a) and separated by fluid contents from the invaginated muscle plasma membrane (b) bounding the T-tubule. x 24,000. Fig. 27. Freeze-fracture of muscle surface tions (arrows) without tracheoles. x 24,000.

in Tenebrio showing

T-tubule

Fig. 28. Freeze-fracture of Muscn muscle showing smooth walled tracheoles 0.09 pm diameter) encircling a mitochondrion (m). x 38,000.

invagina(about

512 escaping through the walls of a O-45 pm tracheole (arrow) and escaping also from fine tubules (about 0.05 pm) forming bands on the mitochondria, and accumulating in deposits within the mitochondria. Fig. 22 shows perhaps rather less than the usual separation of longitudinal tracheoles (about 0.3 pm diameter) injected with arachis oil. This is a section from a newly moulted adult with abundant small mitochondria; and the finer terminal tracheoles have not been injected. The figure illustrates the distance over which oxygen must diffuse through the tissues if, as is usually supposed, these tracheoles represent the terminal source of gaseous oxygen. Fig. 23 is a longitudinal section of a mature muscle after injection of Nujol. It shows a 0.25 pm tracheole in longitudinal section and sections of small terminal tracheoles (some indicated by arrows) applied to the mitochondria, some of which have diameters going down to 0.05-0.06 pm. Fig. 25 is an example of a freeze-fracture preparation from a mature adult Tenebrio flight muscle which illustrates the similarity in the terminal tracheoles to those already described in Musca and shows very clearly the concentric dual membranes which may enclose single tracheoles or several branches. The outermost paired membrane represents the invaginated plasma membrane of the muscle; inside this is the intrinsic plasma membrane of each tracheoblast enclosing the thin cytoplasmic layer that coats the electron-opaque lining membrane of the tracheole. The outer plasma membrane, forming the wall of the T-tube system (Smith, 1961a), doubtless extends beyond the terminations of the tracheoles at many points. But in the freeze-fracture preparations these tubules are much less conspicuous than those which contain tracheoles (Fig. 27). Fig. 24 is a longitudinal section of a mature flight muscle after injection of arachis oil. It shows two injected tracheoles (0.08 and 0.12 pm diameter) on the left, and to the right a slightly larger tracheole (uninjected) which illustrates very clearly the two dual plasma membranes that enclose the tracheoblast. Terminal tracheoles in Rhodnius prolixus In its natural haunts in Venezuela the bloodsucking bug Rhodnius feeds upon the blood

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of pigeons in the tree tops and they regularly fly into houses. The present culture has been maintained in the incubator for more than 50 years without an opportunity for flight. The fibres of the flight muscles are invested by abundant small tracheae and tracheoles which give off short branches that enter the muscles and at once break up into tracheoles of about 0.5 pm diameter. The fibres are filled with longitudinal tracheoles running a straight or slightly wavy course along the lines of the mitochondria often separated by only a single contractile fibril. No side branches or terminal branches can be seen (Fig. 29). These longitudinal tracheoles can be traced at least 150 pm to the point where they end abruptly-that is, there is a sudden end to the air column. The terminal region still contains liquid. After retention for 2 or 3 hr in the sucrose many of the air columns in the tracheoles are interrupted by sections filled with liquid, with air both proximal and distal. The walls are evidently very permeable-in contrast to Musca, Calliphora or Apis in which tne muscles can be kept in the sucrose solution for hours without the columns of air being interrupted by liquid. Injection with myrcene in odourless distillate confirms the high permeability of the tracheoles in Rhodnius. Fig. 30 shows a transverse section of the flight muscle of a young adult Rhodnius. There are abundant small mitochondria between which are extensive spaces lined by plasma membrane. These cavities are clearly a part of the T-tubule system. This preparation had been injected with Nujol followed by farnesol partition. It shows terminal extensions of the tracheoles invading the cavities and in some places almost filling them, in other places well separated from their walls. Extensive cavities of this kind were shown by Smith (1965) to be characteristic of the T-system in Hemiptera: he demonstrated them in Megoura (Aphidae) and in a Pentatomid and a Cercopid. Large cavities were described by Ashhurst (1967) among the mitochondria of Belostomatidae. There was no suggestion by these authors that the cavities might be utilized by the tracheole endings, but they do in fact bring the airfilled terminal tracheoles into close contact with the mitochondria. It seems likely that

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these terminations are filled with liquid during rest. In longitudinal sections of Rhodnius flight muscles it can be seen that the cavities form part of a transverse T-system in which a single tubule, somewhat variable in diameter, crosses the middle of the H zone of each sarcomere (Figs. 31, 32). In these preparations the system has been very incompletely filled with the injection fluid (cf. Fig. 18 of Pieris, in which there are two tubules per sarcomere). It is uncertain whether these airfilled cavities are connected with the longitudinal tracheoles of the muscles or whether, as is more probable, they are derived directly from the abundant tracheoles which fill the space between the muscle fibres (cf. Fig. 20 of Pieris). Terminal tracheoles in Sc/zistocerca gregaria The desert locust Schistocerca is a powerful flier and a migratory insect with an exceedingly rich thoracic tracheal supply described by Weis-Fogh (1964a). The entry of tracheoles into the muscle was described by Brosemer et al. (1963) in Locusta. These abundant tracheoles run longitudinally and are usually separated by no more than two or three contractile flbrils. The earlier injections of the tracheal system were made in adult locusts about 6 days after moulting. Injection with arachis oil showed no fine extensions of tracheoles around the mitochondria (Fig. 34). Injection with myrcene in odourless kerosine showed no obvious leakage from the tracheoles but osmiophil material accumulated in deposits in the mitochondria, so that some leakage was occurring. The situation was like that described in Pieris: the trachea1 supply to the mitochondria appeared inadequate for an active flier when compared with the other insects studied in this paper. An adult male locust at 1 month after moulting was exposed to evacuation in air. It was stimulated to activity by the reduction in oxygen and even spread its wings and fluttered vigorously for short periods of 15 set or so. When finally it ceased to move it was exposed to evacuation in hydrogen and injected with arachis oil. Figs. 35 and 36 show the results: fine tracheoles of diameter ranging from 0.05 to O-2 pm are everywhere applied to the surface of the mitochondria.

These observations have been confirmed in freeze-fracture preparations (Fig. 37). Locusts are unable to fly immediately after moulting. Within 3 or 4 days they will vibrate their wings and make short flights. Full flight power comes later. By 8 days after moulting the mitochondria and contractile fibrils of the adult Locusta appear fully formed (Brosemer et al., 1963). But at 8 days the activity of the Krebs cycle in the mitochondria is still rising steeply (Beenaker rt al., 1975). The invagination of the tracheoles into the flight muscle begins only in the last day or so before the moult to the adult (Brosemer et a/., 1963) and the present observations on Schistocerca suggest that the fine terminations around the mitochondria are developed later and that these terminations become filled with air only after flight activity. Terminal tracheoles in Iscirnuru eleguns

It was shown by Smith (1966) that the flight muscle fibres of Odonata are invested by tracheae and tracheoles but there are no tracheoles inside the fibres. Yet these fibres may have a radius of more than 10 pm and many dragon-flies are active fliers. The lack of internal tracheoles is surprising. Preliminary observations were made on the damsel fly ischnura but their season had gone by before the work could be completed. Fig. 38 shows a longitudinal section of the wing muscles after injection with Nujol and arathis oil. As Smith (1961b) pointed out, the flight muscles contain abundant lipid droplets which can confuse the interpretation of the osmium staining: but Fig. 38 recalls the observations on Pieris (Fig. 15) by the presence of injected vessels inside some of the transverse T-tubules, which have the same location on either side of the H-zone of the sarcomeres. Discussion Terminal tracheoles

It has long been recognized that tracheoles which appear to enter the cytoplasm of other cells invaginate the plasma membrane and this forms an additional sheath outside the intrinsic cytoplasmic tracheoblast (Edwards et al., 1958). The outcome of this process was described in detail in the flight muscles

0

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of Tenebrio by Smith (1961a). In Tenebrio the respiratory supply was described as consisting of numerous tracheoles running longitudinally between the contractile fibrils and thus closely applied to the mitochondria. These longitudinal tracheoles with the lumen ranging from 0.15 to 0.4 pm diameter were regarded as the immediate source for the supply of oxygen. The plasma membrane sheath covering these tracheoles was shown by Smith to form an extensive system of subsidiary tubules which branch and spread over the surface of mitochondria and fibrils. These tubules become associated with the vesicles of the sarcoplasmic reticulum to form the two components of a ‘dyad’. In the present work it has been found that these plasma membrane tubules contain the abundant terminal tracheoles which convey oxygen to the surfaces of the mitochondria. In Musca the secondary tracheoles which

enter the muscle have an annulated lumen of 0.5-1.0 pm. These give off tertiary tubules with lumen 0.1-0.4 pm which likewise have a deeply annulated lining membrane. As well revealed in freeze-fracture preparations they finally give rise to branching terminal tubules of lumen 0.05-0.08 pm with a lining membrane smooth or only slightly beaded. At what dimensions the terminal tracheoles end is not known but it is clear that, as described by Smith, the plasma membrane tubules extend further than these tracheoles. The terminal tracheoles are distinguished from the extensions of the tubular system (in freeze-fracture preparations) by the resistance of the lining membrane to solution in hypochlorite. The terminal tracheoles in Tenebrio are similar in form and dimensions; and terminal tracheoles of similar distribution, and lumen of 0.050.06 pm, are abundant also between and around the mitochondria of Apis.

Fig. 29. Whole mount of muscle fibre of Rhodnius injected with arachis elongated tracheoles giving off no side branches. x 750. Fig. 30. Transverse with Nujol occupying x 38.000.

section of Rhodnius flight muscle showing plasma membrane lined cavities among

oil showing

tracheoles injected the mitochondria.

Fig. 31. Longitudinal section of flight muscle of Rhodnius showing uninjected tubules crossing the fibres at the mid-point of the sarcomere. x 13,000.

T-

Fig. 32. A similar preparation showing partially injected tracheoles occupying the transverse tubule system; with one tubule per sarcomere (cf. Pieris, Figs. 15 and 17). x 13,000. Fig. 33. Freeze-fracture of Rhodnius muscle showing a tracheole with 0.23 pm lumen and the layout of membranes as described in Fig. 25. x 38,000. Fig. 34. Transverse section of young Schistocerca muscle with 0.24 pm tracheole injected with arachis oil but no injection of fine tracheoles. (The dark lipid rich inclusions in the mitochondria are present in uninjected locusts.) x 38,000. Fig. 35. Transverse section of muscle of mature (I month old) Schistocerca after injection of arachis oil; abundant tracheoles, down to 0.04 pm lumen diameter, applied to the mitochondria. x 13,000. Fig. 36. The same at higher magnification.

x 38,000.

Fig. 37. Freeze-fracture of Schistocerca muscle showing seven or eight ruptured terminal tracheoles; also two tubules without an included tracheole (arrows). x 13.000. Fig. 38. Longitudinal section of flight muscle of Ischnura after injection with Nujol and arachis oil showing the system of transverse tubules (two to each sarcomere) many of which contain an injected tracheole. Two lipid droplets are visible above. x 13,000.

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A paper by Smith and Sacktor (1970) on Phormia regina describes the disposition of the invaginated plasma membrane and the T-system derived from it along with the sarcoplasmic reticulum. The distribution of tracheoles was not discussed in detail by these authors but the illustrations show sections of tracheoles within the muscle fibres in the same size range as described in Musca in the present paper; that is from 1.0 pm down to 0.07 pm. In Pieris the major component of the terminal tracheoles is a system of smooth walled vessels (0.08-0.2 pm diameter) which run transversely and are regularly placed on either side of the H-zone of the sarcomeres to form a regular air-filled network in contact with the mitochondria throughout the muscle fibre. This system appears to be a derivative of the transverse tubular system which is commonly found crossing the sarcomeres on either side of the H-zone, in most insect muscles with synchronous innervation (Smith, 1962) and is here occupied by the tracheole endings. Preliminary observations suggest that a similar air-filled system may exist in the damsel fly Zschnura. In Rhodnias, as in other Hemiptera (Smith, 1965) there are extensive cavities derived from the T-system among the mitochondria and these are filled by smooth terminal tracheole tubules in close association with the mitochondria. The cavities form a transverse T-system as seen in longitudinal sections. And in the locust Schistocerca likewise the tracheole endings occupy T-tubule extensions among the mitochondria. It is interesting to note that Holmgren (1908) believed his nutritive ‘trophospongium’ to be continuous with a terminal network of the tracheal system in many insect flight muscles. This network as he figures it closely resembles that described in Pieris in the present paper (Fig. 12). He supposed that the trophospongium was formed by the invagination of the cell surface by processes from special cells. The modern T-tubule system has much in common with the trophospongium and the present work has shown that this system does often ensheath the terminal branches of the tracheoles. It is usual to regard the tracheal system as invaginating the plasma membrane of the cells to form the T-tubule system ‘like a finger pushed into the surface of a balloon’

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(Edwards et al., 1958) but since this system exists in the cells of animals without a tracheal system it seems reasonable to regard the tracheae merely as making use of invaginations produced by other causes. This suggestion was made by Beinbrech (1969) and supported by observations on Phormia; and it was shown by Bienz-Isler (1968) that in Antheraea pernyi (Lepidoptera), invagination of the plasma membrane of the flight muscles takes place in the pupa 4 days before the tracheoblasts begin to enter these preformed invaginations. The question was raised by Weis-Fogh (1964b) whether the rate of diffusion would diminish markedly if the diameter of the tracheoles should fall below the length of the mean free path of gaseous oxygen molecules. Pickard (1974), in a mathematical analysis of this problem, showed that diffusion will begin to be impeded only when the effective radius of the tracheole (a) falls to a point where the value of 2a/X (where X is the mean free path of the oxygen molecule, roughly 0.072 pm) is no longer greater than 1. Using the information obtainable from published electron micrographs, Pickard pointed out that the minimum tracheole diameter in insects, which ranged from 0.16 to 0.28 pm, gives an average value of 2alA of roughly 3+. In the present paper, however, it has been found that extensive terminal regions in the tracheoles of many insect flight muscles are in the range of 0*05-0.08 pm which brings the value of 2a/h into the critical region. The main conclusion from these new observations is that the air-containing channels in the flight muscles of insects follow the tubules of the invaginated plasma membrane, or T-system, much further than previously supposed. Indeed they utilize this system to extend right up to the surface of virtually all the mitochondria in the flight muscles. Thus one of the major factors impeding the access of oxygen to the active muscle-the gap between the tracheole endings and the mitochondria, over which diffusion through the body fluid was postulated (Weis-Fogh, 1964b)-has been largely eliminated. Theory of tracheole respiration From the examination of living insects by transmitted light under the microscope it was shown that the endings of the tracheoles supplying the muscles of the body wall or

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the wall of the gut, commonly contain liquid during rest. That was found in mosquito larvae, in Tenebrio, Aeschna, the flea Xenopsylla, the bed bug Cimex and other insects. During activity, particularly in reduced oxygen, the liquid is absorbed and gas extends to the finest terminations (Wigglesworth, 1930, 1931). On the other hand, in the epidermis of Rhodnius, once the fluid contents have been resorbed at the time of moulting, the tracheoles always contain air up to their extremities at about 0.1 pm (Wigglesworth, 1954). The physiological significance of this movement of fluid has remained unknown. We now suggest, however, that it should be viewed as an example of the compromise between the need to obtain oxygen and the need to conserve water which faces all terrestrial animals. It can be argued that the integument of Rhodnius can be adequately supplied with oxygen by tracheoles which are sufficiently impermeable to remain ‘dry’ to their extremities. Whereas it will be more efficient for tissues like muscle with high oxygen requirements during activity but low requirements during rest, to have highly permeable tracheal endings which will fill with liquid during periods of rest. For during activity the liberation of unoxidized metabolites in the tissues will increase the swelling pressure or colloidal osmotic pressure in the body fluid and cytoplasm around the tracheole endings, and thus resorb the liquid and allow

air to extend to the highly permeable extremities (Wigglesworth, 1938, 1953). The present observations have shown the variable permeability (to myrcene and kerosine) of the tracheoles in the flight muscles in different insects, and they have demonstrated the resorption of liquid during flight, notably in Musca and in Pieris. In the first few seconds of flight in blowflies the pyruvate in the thorax increases four-fold, shortly followed by increases in alanine and acetyl carnitene. But very rapidly the level of these metabolites falls to their initial level (Sacktor and Wormser-Shavit, 1966). Clearly on the commencement of flight, pyruvate is not metabolized in the Krebs cycle as fast as it is formed by glycolysis. Sacktor (1970) suggests various biochemical factors which may bring about the required adaptation; but it seems likely that improved oxygen supply to the mitochondria by the removal of liquid from the tracheole endings must be an important element. Acknowledgements

This work has been supported by a grant from the Agricultural Research Council to V.B.W. who thanks Professor G. Horn for facilities in the Department of Zoology; Dr J. E. Treherne and Dr Nancy J. Lane for the use of the electron microscope and for advice; and Dr S. A. Corbet, Mr B. 0. C. Gardiner and Mr R. Northfield for the supply of living material.

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