The Imaginal Ecdysis of Blowflies. Evidence for A Change in the Mechanical Properties of the Cuticle at Expansion

The characteristic digging movements and the expansion process in newly emerged blowflies both involve the production of internal hydrostatic pressures of approximately the same magnitude (Cottrell, 1962c). The peak pressures achieved during expansion are maintained for several minutes while those involved in a digging cycle are transitory, but digging movements may be continued for several hours (Cottrell, 1962 a) so that it is by no means clear why the soft cuticle of a digging fly does not undergo some expansion or distortion in the course of this activity.

The present paper describes certain experiments in which flies were subjected to artificially produced internal hydrostatic pressures at various times after their emergence. The maximum pressure attained during the normal expansion of Calliphora erythrocephala (Meigen) varies from 4·0 to 7·3 cm. of mercury (Cottrell, 1962c) and the artificial pressure usually employed in the present experiments was 7-0 cm. of mercury. In normal flies the production of maintained pressures of this value is dependent on the ability to swallow air so that in a fly with its proboscis blocked there is no increase in the basic haemolymph pressure and the only changes observable are the rhythmic pressure pulses (about 2 cm. in height) which result from the performance of ‘muscular efforts’ (Cottrell, 1962c ). In such flies there is no detectable expansion, and one can be reasonably certain that any which is observed to follow the application of an artificial pressure has been caused by that pressure.

Newly emerged individuals of C. erythrocephala (Meigen) were used throughout. Each fly was anaesthetized with carbon dioxide at the moment of its emergence and its proboscis and anus were blocked with wax to prevent volume changes due to airswallowing or defaecation. The fly was then suspended by inserting the glass cannula of the pressure apparatus into its scutellum and sealing it in place with wax as previously described (Cottrell, 1962c). After recovery from anaesthesia and being given a cottonwool ball to hold such an insect would soon begin to make air-swallowing movements and then go through the whole air-pumping cycle, without of course being able to take in any air. The apparatus used to apply the internal hydrostatic pressures was filled with insect Ringer solution (Ephrussi & Beadle, 1936) and is shown in Text-fig. 1. Tap A was normally positioned so as to cut off the reservoir (R) thus enabling the mercury manometer (M) to be set to any chosen height by a micrometer-driven ‘Agla’ syringe (S). With tap B positioned so as to cut off the cannula (C), the volume of fluid displaced in raising the mercury column to a specified height was easily obtained from the reading of the micrometer head. For a pressure of 7 cm. of mercury this was 90 μl. When tap B was included in the system, the fly would be distended by inflow of saline and the difference between the micrometer reading under these conditions and that obtained with B closed gave the increase in volume of the fly. Subsequently, by screwing back the micrometer head the manometer could be returned to atmospheric pressure and then any permanent change in the volume of the system due to expansion of the fly was indicated by the difference between the original and final readings of the micrometer head.

The air-pumping cycle was followed by means of a suitably placed mirror viewed through a binocular microscope. An eyepiece micrometer (with 50 divisions) was used to take measurements of the distance between two sets of thoracic bristles before, during and after the application of a particular pressure. The measurements made were the distance between the most anterior bristles of the right and left rows of dorsocentrals and the distance between the most anterior acrostichial bristle on mesoprescutum and the third acrostichial bristle (anterior on mesoscutum) of the right row.

In the first series of experiments newly emerged flies (with proboscis and anus blocked with wax) were subjected to an internal pressure excess of 7·0 cm. of mercury for two periods of 10 min. each. The first period of increased pressure (P₁) was begun within 3–4 min. of attachment to the cannula and the second (P₂) when the rate of pulsation of the pharyngeal muscle had exceeded about 180 per minute, that is, at a time some 1–3 min. before the wings would be expected to have been fully extended in a fly capable of swallowing air (Cottrell, 1962 a). Under the conditions of the experiment this moment was reached some 40 min. after attachment to the cannula.

The results obtained from nine flies are shown in Tables 1–3, and individual trials are illustrated by Text-fig. 2 (fly no. 5) and Pl. 1 (an unrecorded fly). Tables 1 and 2 show that after the application of P₁ there was only a slight increase (means : 4·1 and 3·4 % ) in the distance between the pairs of bristles measured and that after return to atmospheric pressure about half this increase disappeared. The position was quite different at P₂ where an average increase of 41 % and 30 % took place, this increase being diminished by only about one-tenth when the pressure excess was removed.

These observations suggest that some change in the mechanical properties of the cuticle must have taken place between the application of P₁ and P₂, and this idea is further supported by the changes in volume of the system as indicated by the micrometer head of the ‘Agla’ syringe. Distension and expansion of a fly subjected to a given pressure was not immediate and it was necessary continually to adjust the micrometer head in order to keep the mercury column at 7 cm. A plot of the volume changes in the system during a typical experiment is shown in Text-fig. 2, which also includes a plot of the rate of pulsation of the pharyngeal muscle up to the time of application of P₂. The horizontal line at 90 μl. indicates the volume necessary to set the mercury column to 7 cm. without any other changes in the system. It can be seen that when P₁ was applied the volume of the fly was immediately increased by 19 μl. Comparison of Pl. 1 a and b shows that this was due largely to the simultaneous distension of the ptilinum as well as to distension of the intersegmental membrane of the abdomen and other membranous areas, but that there was little or no expansion of the presumptive sclerite areas of the cuticle. Over the 10 min. during which P₁ was applied the volume of the fly increased by another 16μl. giving a total increase of 35 μl. by the end of P_1. This was partly due to further distension and partly to the gradual unfolding of the wing membranes. However, the costal elbows did not open, so that the result of the unfolding of the membrane was to force the tips of the wings upwards (Pl 1c). On returning the mercury column to atmospheric pressure the membranous areas collapsed and, except for the unfolded wing membranes, the fly returned to its newly emerged state. (Note that in comparing Pl. 1 a with 1 d allowance should be made for the fact that in Pl. 1 d the ptilinum is retracted). Moreover, the reading on the micrometer head indicated that there had been no permanent change in the volume of the system (Fig. 2).

The situation was quite different when P₂ was applied. The fly immediately increased in volume by 39 μl. (Text-fig. 2) while the presumptive sclerite areas of the thorax, abdomen and legs expanded to the typical adult form and the costal elbows opened out (Pl. re). Over the next 10 min. the volume increased to 62μl. apparently by further expansion of the cuticle (Pl. 1f). The wings straightened out and became quite stiff and flat and the fly assumed the fully developed adult form. There was some distension of membranous areas particularly of the abdomen and in the cervical, ptilinal and rostral regions. When the pressure was again returned to atmospheric, the typical adult form was retained, and the wings remained extended, but the micrometer head indicated that there had been a permanent change in volume of 31 μl. This value presumably represents the true expansion while the value of 62 μl. obtained for injection of fluid represents both expansion and distension. Values for the volume changes induced in the remaining eight flies of the series are given in Tables 3 a and b. All behaved in essentially the same manner as the fly described above.

It is worth mentioning that the rapid and normal expansion produced by a pressure applied at, or shortly before, the peak rate of air-pumping is not dependent on there having been a previous exposure to pressure (P₁). Thus flies which are subjected to a single pressure increase after the pumping rate has reached about 180 pulsations/ minute expand just as well as those which receive an additional pressure increase shortly after being attached to the cannula. However, in the former group there is, of course, no unfolding of the wing membranes prior to the application of the expanding pressure.

In a second series of experiments involving eight flies (with proboscis and anus blocked) the application of pressure was delayed until 1–3 min. after the cessation of air-pumping. The results are shown in Tables 4 and 5. It can be seen that a pressure of 7 cm. of mercury applied at this time produced very little expansion (mean values 1·4 and 4·0%). The flies themselves showed a distension of the membranous areas (rather like that shown in 1 c) and there was no clear alteration towards the adult body form. The wing membranes unfolded, and in addition in six out of eight specimens the upper and lower lamellae became separated from one another, allowing large quantities of blood and saline to flow between them, converting the wings into fluid-filled sacs. Nevertheless, as in flies subjected to pressure before the start of air-pumping, the costal elbows did not open. In the two flies in which obvious ‘bladdering’ of the wings did not occur the maximum volume injected during the pressure increase was 23 and 26 μl. while the permanent change in volume of the system was 8 and 12μl Clearly, at the end of the air-pumping cycle the presumptive sclerite cuticle is just as incapable of expansion as it is before air-pumping begins.

The volumes of groups of ten normal flies were measured by displacement of 80 % ethyl alcohol. Flies taken at emergence gave values ranging from about 45 to 50 μl./ fly, those shortly after expansion gave values of 100–110 μl./fly and those after the completion of hardening and darkening values of 80–90 μl./fly.

The experiments described above clearly show that normal expansion can take place only in the very restricted period between the initiation and the cessation of air-pumping movements. Apparently there is a change in the mechanical properties of the cuticle which is confined to the presumptive sclerites and leaves the membranous areas unaffected. These latter areas (the ptilinum, intersegmental and articular membranes) allow the distension of different parts of the body (by bulk movements of blood) during the digging of newly emerged flies. When flies are subjected to artificial pressures simultaneous distension of all these regions allows a considerable increase in volume, which disappears on removal of the pressure excess. The estimates (obtained by displacement) of the volumes of flies before expansion, at expansion and after darkening indicate that there is also a temporary increase during normal expansion. Thus the approximate increase of 55–60 μl. shown by flies taken shortly after completing expansion agrees roughly with the figure (column 4, Table 3 a) for the increase in volume produced by artificial pressure P₂, while the increase of 35–40 μ l. shown by darkened flies is more in line with the permanent change in volume after P₂ (Table 3 A). This is because in hardened and darkened flies the swallowed air which filled the gut during expansion has diffused away, eliminating the distension of membranous areas. In agreement with the figure for flies taken shortly after expansion, Fraenkel (1935) records an increase of 128 % for unsexed groups of C. erythrocephala.

The wing membranes appear to represent a special type of membranous area. Prior to expansion they are completely folded and although they may become very partially unfolded during prolonged digging they do not normally do so to any marked extent until the fly begins to swallow air. Despite this, unfolding seems to be possible at any time before or after air-pumping, provided the fly is subjected to a maintained pressure of sufficient magnitude. On the other hand attainment of definitive wing form is not possible unless the expansion of the wing veins (which later become sclerotized) accompanies the unfolding of the membrane.

There can be little doubt that at a normal expansion muscles play a considerable part in the production of the definitive body form. Thus it is evident from Pl. I that at P₁ the ptilinum is greatly distended while at P₂ it is only slightly so. The most probable explanation for this is that the appropriate ptilinal muscles are maintained in a contracted state during the period when air-pumping occurs. However, it is just possible that expansion of the various cephalic areas destined to undergo sclerotic-zation could so alter the spatial relations of the head as to make the expansion of the ptilinum relatively more difficult.

Despite the likelihood that the muscles play a role in the production of the definitive adult body form it does not seem possible to interpret the inability of presumptive sclerite cuticle to expand before air-pumping begins in terms of muscle action. In the region of the cuticle where measurements were made the muscle attachments are normal to the surface and it seems highly unlikely that they could affect its expansion.

The timing of the changes in the mechanical properties of the cuticle becomes of increased interest when it is considered in relation to what is known of the control of cuticular hardening and darkening and the mechanism of expansion (Cottrell, 1962 a−c). Expansion can only occur during the period of continuous air-swallowing. At 22 ° C., in conditions suitable for expansion, air-pumping begins about 10 min. after emergence but does not become continuous until about 20 min. In normal flies it ceases between 40 and 45 min. but in flies with their probosces blocked, it continues up to 45 or 55 min. Normal hardening and darkening is initiated by the release into the blood of an active factor. This release takes place between 3 and 15 min. after emergence and the first signs of darkening appear at about 65 min. There is thus a lag period of from 45 to 50 min. before the action of the darkening factor becomes apparent

In the present paper it has been shown that an artificial pressure applied at the end of air-pumping (i.e. at 45–55 min.) will not produce expansion even though no cuti-cular darkening is detectable at this time. Since the cuticle is known to be sufficiently plastic at the time of the peak rate of air-pumping (i.e. at 30-35 min.) to expand under the same pressure, it seems reasonable to suppose that by 45–55 min. the cuticular proteins have undergone sufficient cross-linking to prevent expansion. This idea is supported by previous experiments (Cottrell, 1962b) in which it was shown that anticipation of the natural release of the darkening factor by transfusing an experimental fly with active blood prevented expansion, although at the time when expansion was attempted no colouring had yet appeared in the cuticle.

From what has been said above it is clear that ‘plasticization’ is closely associated with sclerotization both temporally and (since the phenomenon is confined to presumptive sclerites) spatially. This suggests that plasticization may actually be a stage in the process of sclerotization.

According to current views (Hackman, 1959; Richards, 1951, 1958; Mason, 1955; Wigglesworth, 1948, 1951) at sclerotization dihydroxyphenols diffuse outwards through the cuticle and on reaching the epicuticle become oxidized to o-quinones by a phenol oxidase. The quinones then diffuse inwards and as was first recognized by Pryor (1940a, b) tan the cuticular proteins forming the hard epicuticle and exocuticle. The peptide effect between amines and quinones (Mason, 1955) makes it probable that the quinones first react with the terminal amino groups of the protein chains to form N-dihydroxyphenyl proteins which, in the presence of excess quinone, are then converted to N-quinonoid proteins. The N-quinonoid proteins react with the N-terminal amino groups of other proteins or with the e-amino groups of lysine which occur along the chains and in this way a cross-linked structure is formed.

Hackman & Goldberg (1958) have shown that in the soft larval cuticle of Agrianome spinicollis (Coleoptera) 56% of the protein is bound to other components (especially chitin) by covalent bonds, 14% is not bound to other components, 2% is bound by van der Waal’s forces, 3 % by salt linkages and/or double covalent bonds and 25 % by hydrogen bonds. If some of these types of bonds are responsible for the inexpansibility of the sclerite cuticle at emergence, it seems possible that the formation of N- catechol proteins (which is the first step in sclerotization) could, by interfering with the bonding, result in a plasticization. If so, since the A-catechol proteins can only be converted to the reactive N-quinonoid proteins by excess of free quinone, it seems possible that there would be a stage of maximum plasticity before the excess free quinones build up sufficiently to bring about sclerotization.

It is worth while to draw attention to another change in mechanical properties revealed by the present experiments. Prior to the cessation of air-pumping the wing membrane will unfold in response to an adequate pressure without separation of the upper and lower lamellae. However, after cessation an increasing proportion of individuals exhibit ‘bladdering’ until at the first signs of darkening all do. Apparently, associated with sclerotization, there is a change in the mechanical properties of the components responsible for maintaining the spatial relationships of the upper and lower lamellae of the wings.

The present experiments owe something to those of H. C. Bennett-Clark who discovered an analogous phenomenon during feeding in nymphal Rhodnius protixus Stàl. I am grateful to Prof. V. B. Wigglesworth for encouragement and laboratory facilities and to Dr E. Bursell who read the manuscript The work was performed while the author was Gulbenkian Research Fellow of Churchill College, Cambridge.

Effect of subjecting a specimen of CalUphora erythrocephala to pressure excesses of 7 cm. of mercury (P₁ and P₂) at different times after emergence, a, After attachment to cannula and before P₁ ; b, immediately after application of P₁ ; c, 9 min. after application of P₁ ; d, immediately after removal of P₂ ; e, immediately after application of P₂; g 9 min, after application of P₂ ; g, immediately after removal of P₂.