ABSTRACT
Certain Muscid eggs are shown to change their shape almost simultaneously with changes in the relative humidity of the air in their immediate vicinity. Evidence is produced that these shape changes are caused by the effect of humidity on the chorion. The net effect of the shape changes appears to be the setting up of increasing strain in the chorion, making it progressively easier for the larvae to rupture their shells, with increasing humidity. These shape changes therefore provide an explanation for certain well-known humidity effects on the hatching of Muscid eggs.
Introduction
It is well known that the hatching of eggs in many insects, particularly Muscid flies, is markedly affected by humidity. This is well illustrated by the observation of Davies & Hobson (1935) that Lucilia sericata (Mg.) eggs which had been incubated under moist conditions and normally would have hatched in a few minutes, completely failed to do so when transferred to an atmosphere of 50% R.H. It has been assumed that delay or prevention of hatching by low humidity is largely a result of the chorion hardening under dry conditions.
The following observations are intended to throw light on the precise mode of action of humidity on hatching in the eggs of Muscid flies. In the course of the work, eggs of the following species were used, and the observations apply to them all: L. sericata (Mg.), L. caesar (L.), L. illustris (Mg.), L. ampullacea (VilL), Protophormia terra-novae (R.-D.), Calliphora erythrocephala (Mg.) and C. vomitoria (L.).
Humidity-dependent shape changes in eggs
Egg measurements were made by means of a microscope with × 6 eye-piece and 16 mm. (× 10) objective and fitted with an eye-piece micrometer scale, so that the total magnification was × 60 (1 micrometer division = 16 · 47 μ). The above apparatus was found to permit fairly accurate measurements of the length changes of eggs, with a range or error of about ± 6 μ. For the observation of width changes and the dimensions of certain smaller topographical features of Muscid eggs, attempts were made to use a higher magnification (× 400). The low accuracy of these latter measurements do not permit a numerical evaluation of them but this limitation in the observations does not invalidate the hypothesis put forward in the following pages, linking these egg-shape changes with the hatching process.
Solid watch-glasses, sealed with flat plates vaselined at the edges, were used as humidity chambers, humidities being controlled within them by sulphuric acid/water mixtures (Wilson, 1921), 100% R.H. being maintained with distilled water. The cavity of each watch-glass was filled with the appropriate liquid to within 5 mm. of the underside of the lid. Eggs were attached to the underside of the cover plates, so that they lay within 5 mm. of the surface of the acid, and their dimensions could then be measured through the lid when the whole watch-glass was placed on the mechanical stage of the microscope. Care was taken to use uniform-sized watchglasses and similar thickness cover-glasses in all observations. Successive measurements on several eggs at various humidities were made by quickly transferring the cover-glass with its attached eggs on to another watch-glass containing the appropriate humidity solution, repeating the transfer for each R.H. All vessels were kept covered and airtight except during the short period of a few seconds when a transfer was being made. A period of 5 min. was allowed to elapse after eggs had been transferred to a new humidity, before measurements were taken, in order to allow the egg shells to reach equilibrium with the air of the chamber, and for the correct R.H. to be restored after the disturbance. Eggs were found to have different lengths at different humidities (see below). Successive length measurements made on eggs, from immediately after they had been transferred to a new humidity, until 30 min. later, showed that they reached their final length within 3 min. of the transfer. The length changes during humidity changes are thus seen to be very rapid.
Before describing in detail the shape changes observed, the external morphology of the Muscid egg must briefly be described. The eggs are roughly sausage-shaped, about five times as long as wide, the anterior end being occupied by a flatish circular area with the micropyle in its centre, and termed in this paper ‘the micropylar plate’. On the dorsal surface of the egg, the chorion bears a pair of roughly parallel pleats (termed the ‘hatching pleats ‘hereafter) running posteriorly from the edge of the micropylar plate for about four-fifths of the egg length. The area of the chorion between the hatching pleats is thickened (Davies, 1948) and is termed the ‘hatchings-trip ‘in the following account.
The shape changes observed may be summarized as follows :
- With increasing R.H. from o to 100% the eggs of all species gradually increased in length. Length measurements of eggs of three species at different humidities are given in Fig. 1. Differences in the amount of length change in the three species will be noted. The variation between individual eggs of one species in the percentage increase of length at 100% R.H. over the length at 0% (ten eggs in each case) is shown in Table 1. The points at each humidity in Fig. 1 show the amount of error to be expected when measuring a distance of about 1200 μ in units of 16·5 μ. When eggs were transferred through the humidity series 0–100% R.H. several times upwards and downwards, the length values obtained at each humidity always coincided to within 4 μ.
Fig. 1.Variation of egg length with humidity. Each curve is the average of 5 eggs. A, Calliphora erythrocephala ; B, Lucilia sericata ; C, Protophormia terra-novae.
- Accompanying the elongation described above, a reduction in the cross-sectional area of eggs occurred. The amount of this reduction was too small to be accurately measured with a magnification of × 60. All that can be said is that it appeared to involve a reduction in the diameter of eggs of the order of 5%. A feature of this reduction in cross-sectional area was observable qualitatively. It occurred mainly by an inward movement of the dorsal side of the egg, the hatching pleat area. It is likely that the cross-section remained approximately circular during the shape changes (Fig. 2B).Fig. 2.
A, dorsal view of egg showing length increase only; B, cross-section of egg showing reduction in diameter by inward movement of dorsal side. The amount of the diameter reduction is greatly exaggerated—., outline at o% R.H.;----, outline at 100% R.H.
Simultaneously with the above shape changes, and probably forming an integral part of them, the hatching strip appeared to narrow and elongate slightly. Even with 400 × magnification, the hatching strip narrowing was too small to be accurately measured, since the distance involved is about 50 μ,.
These shape changes are represented diagrammatically in Fig. 2, and were further investigated, the results being given below as numbered observations for the sake of clarity:
Observation 1. When eggs were subjected to large humidity changes by transferring them from o to 100% R.H. directly, a proportion of the chorions split by a longitudinal fissure running closely along the outer margin of either of the hatching pleats. This fissure did not originate at any fixed point along the length of the pleats. Eggs which did not split during the first 0–100% R.H. change could be induced to do so by repeating the process 2–5 times. This shows that sudden large humidity changes caused strains in the chorion, making it split along one or other of two fairly constant lines. Only eggs in an early stage of development were used, so that splitting was not due to larval activity within the eggs. With smaller humidity changes of 50–100% R.H. or 0–50% R.H., no rupture of chorions occurred, even after ten repeated changes. Thus with smaller changes in R.H., the changes in length were also smaller and the strains set up not sufficient to rupture the chorion.
Observation 2. The accessory gland secretions covering the outside of the chorion of the laid egg were removed by washing eggs in 1 % sodium sulphide solution for i hr. Such eggs underwent shape changes in the same way and to the same extent as eggs with the secretion covering intact. This shows that the latter played no part in governing the shape changes.
Observation 3. Chorions were removed from eggs (cf. Evans, 1934) which retained their general shape since they were still enclosed by the intact vitelline membrane underlying the chorion. Such chorion-less eggs when subjected to successive humidities from o to 100% R.H. and vice versa, underwent no detectable shape changes. It is concluded, therefore, that the chorion alone was responsible for the humidity-dependent shape changes. The tendency of the Muscid chorion to alter shape at different humidities would be resisted by the egg it invests, thus causing strains to be set up in the former. The amount of strain will be humidity dependent because the shape of the chorion is humidity dependent.
Observation 4. That the shape changes caused by the chorion were not due to the effects of humidity on a small specialized area of the shell, such as the hatching strip and pleats, was demonstrated in the following way. The lengths of ten eggs at o and 100% R.H. were measured. The hatching strip and pleats of six of these eggs were carefully covered with waterproof cellulose paint. On the other four eggs a similar longitudinal strip of chorion was painted, but this time on the ventral side of the egg, on unspecialized chorion. Only in the latter four eggs would the hatching strip be exposed to humidity changes. The lengths of the eggs at o and 100% R.H. were re-measured. It will be seen from the results in Table 2 that both groups of eggs elongated with rise in humidity, but to a smaller extent than before treatment, there being no significant difference between the two groups. The smaller length increase in both groups after treatment was probably due to the stiffening effect of the paint itself. It is considered therefore that the shape changes were not due to the properties of the hatching pleat region alone, but rather to the properties of the chorion as a whole.
Observation 5. It was earlier mentioned that the decrease in cross-sectional area of eggs with increase in humidity appeared to occur mainly by inward movement of the dorsal side of each egg—the side bearing the hatching pleats. This may have been due to the fact that the contraction across the hatching strip was relatively greater than that of the unspecialized chorion surrounding the rest of the short-axis circumference of the egg. Measurements of the width of the hatching strip were obtained, but the accuracy of the results was so low (the distance involved is but 50 /x or so) that the above possibility could not be verified. The idea is given support, however, by the following observation. Imperfect eggs of Lucilia caesar were observed, in which the hatching strip and pleats were very short and extended for only one-tenth of the egg length from the anterior end, leaving the rest of the egg covered entirely by unspecialized chorion of uniform thickness. It was found that in dorsal and lateral views the reduction in diameter with rise in R.H. appeared to occur in these eggs by an inward movement of the whole of the circumference to about the same extent all round the egg.
Observation 6. At 0% R.H. it was observed that the hatching pleats stood nearly upright from the egg surface, and that with increasing humidity they leaned progressively inwards towards each other, so that in saturated air they lay at an angle of 30-40° with the egg surface. These changes are shown in Fig. 3 as diagrammatic sections across the hatching pleats. This movement of the pleats increased the difficulty of measuring the width of the hatching strip with any accuracy, but the movements would appear to involve a decrease (at high humidity) in the distance between the outer edge of the two pleats (distance AB in Fig. 3). If this is so, movement of pleats provides a possible mechanism whereby the short axis circumference of the egg may become reduced with increasing humidity, with only a fairly small contraction of the unspecialized chorion, since pleat movements would ‘take up the slack’.
The movements of the pleats with humidity may also explain why the chorion usually ruptures on hatching, along the outer margin of either of the pleats, without the necessity of postulating the existence of lines of weakness there. With such movements, the strain would be expected to be greatest at these positions.
The anterior localization of the chorion split during hatching, in contrast to its haphazard occurrence along the length of the pleats when eggs were subjected to sudden large humidity changes (p. 439), is certainly due to the fact that the effects of larval movement are confined to the front end of the shell. When eggs in humid air were observed under the microscope, immediately prior to hatching, the larva could be seen to hammer on the inside of the micropylar plate. This caused the whole of the anterior end of the egg to elongate slightly, thus reinforcing, at the front end only, the strains set up in the shell by elongation of the egg due to high humidity.
Observation 7. When a small perforation was made through the chorion and the vitelline membrane of an otherwise intact egg, a small drop of yolk appeared over the hole. This showed that at room humidity the yolk contents were held under slight pressure by the egg membranes. If the egg was then exposed to increased humidity, the drop of yolk grew larger, but when the humidity fell to its original level, it decreased to its former size by flow of liquid yolk back into the egg. A drop of yolk on a slide did not change size when measured quickly at the two humidities employed above. Thus the egg volume must have been slightly reduced as the humidity increased. It follows therefore that in an intact egg an increase in humidity results in an increase in internal pressure.
Observation 8. If the hatching pleats and strip were entirely removed from an egg at room humidity, the egg became slightly shorter than before, while the gap between the free edges of the chorion became slightly wider than the total width of the hatching pleats and strip removed. This indicated that the chorion was under some transverse (i.e. around the short axis of the egg) tension at room humidity. When such eggs were exposed to saturated air, the gape in the chorion became narrower. This latter phenomenon is equivalent to the curling movements of chorion fragments with humidity changes, described by Davies (1948). It has since been observed that these curling movements of chorion fragments occur predominantly around the short axis circumference of the egg, and only to a negligible degree along the long axis. This fits in with the above observation of the closing of the longitudinal gape in the chorion with increasing humidity, and with the following observation that a split in the chorion running part of the way round the short axis of an egg, did not show pronounced opening and shutting with changing humidity.
Observation 9. The lengths of eggs were determined at o and 90% R.H. at both 17 and 37° C. The increase in length at the high humidity over that at the low was found to be the same at both temperatures, variations being within the range of error (± 6μ) of the measuring technique. It seems, therefore, that the shape changes are little influenced by temperature, humidity alone being the governing factor.
Observation 10. The Muscid egg chorion was seen to be stiffer at all humidities below saturation than in saturated air itself. Vacated shells in saturated air collapsed and became closely applied to the substratum. At 90% R.H. and lower, vacated shells did not collapse. In saturated air it was observed that the chorion was limp and that after initial splitting the two sides of the split could readily be forced apart by the hatching larva. In the hatching at 30° C., larvae usually escaped from the shell within 2 min. of initial chorion rupture. At 80% R.H. at the same temperature, larvae were seen to have difficulty in forcing the two sides of the split apart, and took 3-20 min. to escape entirely after initial rupture of the shell. The greater stiffness of the chorion at humidities below saturation does not however account for the effects of humidity on hatching. Examination of large numbers of eggs containing fully developed larvae which had failed to hatch at 70-90% R.H. showed that in no case had the chorion been ruptured. The limiting factor appears to have been the difficulty of initially rupturing the chorion, and not the difficulty of escaping from the shell after rupture had been accomplished at these humidities.
Discussion
The above observations show that the chorion alone is responsible for the humiditydependent shape changes in Muscid eggs, and further analysis of the mechanism whereby they are accomplished depends on a knowledge of its structure. Davies (1948) showed that chorion fragments underwent curling movements dependent on humidity and ascribed them to the two-layered nature of that membrane indicated by the fact that the outer part of its thickness could be tanned by p-benzoquinone, while its inner part could not. The two layers are probably differentially affected by water absorption, leading to expansion of the outer layer around the short axis of the egg with rising humidity. That water absorption is involved is supported by the fact that the shape changes were little affected by temperature. The almost instantaneous nature of the curling movements of chorion fragments, and of the egg shape changes, with humidity change, means that the chorion must contain extremely hydrophilic protein, with a large surface area in relation to volume. The latter condition is fulfilled by the outer chorion layer whose thickness is but 2-3 μ (Davies, 1948) and with an external surface area of about 8600 μ2 in L. sericata eggs. Since oily materials do not spread over it (Davies, unpublished observations) it is also hydrophilic.
Since the two layers appear to behave differentially in changing humidities, the layer of dark bodies embedded between them described by Davies (1948), possibly counteracts shear strain at the interface. The outer layer may be constructed of long protein molecules, producing a structure affected by humidity in a way comparable with mammalian hair-and wool-keratin as described by Astbury & Woods (1931, 1933). No data by X-ray methods are available for the Muscid egg chorion. When pieces of chorion are viewed in polarized light, they are seen to be anisotropic, suggesting that there is no marked parallel orientation of long molecules through most of its thickness. Evidence exists (Davies, unpublished observations) that the inner chorion layer is of a spongy nature, the cavities of the sponge forming a continuous gas space comparable to that shown to exist in the Rhodnius egg by Tuft (1950). With this spongy structure, the inner layer may readily ‘give’ under stress from the outer layer when the latter changes its length and curvature with humidity.
These observations on shape changes may be linked with the hatching process of Muscid eggs as follows: with increasing R.H. from 0%, the cross-sectional area is reduced while the egg elongates. Whether cross-sectional area reduction and egg elongation are respectively cause and effect or vice versa is difficult to say. However, the former process will set up strains in the chorion, liable to result in longitudinal rupture and not rupture around the short axis of the egg. That this was so, was shown by the observation that in eggs subjected to sudden transfer from o to 100% R.H., the chorion ruptured along the outer margin of either hatching pleat. Since the amount of elongation of eggs is almost certainly a measure of the cross-sectional area reduction, this amount is also a measure of the strain on the chorion. The curves in Fig. 1 (p. 439) are thus indicative of the progressive increase in strain on the chorion with increasing humidity. They show therefore an increasing probability of successful rupture of the chorion by the enclosed larva, with increasing R.H. From Fig. 1 it is to be expected that at 90-100% R.H. the chorion is under maximum strain owing to the shape changes, and blows by the larva on the inside of the anterior end of the egg are likely to cause rupture fairly quickly. With lower R.H. the strain owing to the shape changes will be less, and many more blows by the larva will be required to produce rupture. Exhaustion or death by desiccation frequently supervenes before hatching has been achieved. This is perhaps the explanation of how at certain humidities below saturation, Muscid eggs show a reduced percentage hatch and a greater dispersal of hatching times in groups of eggs, recorded for Lucilia sericata eggs by Davies (1948) and for the eggs of certain dung-breeding Muscids by Larsen (1943). At these humidities all the eggs of the various species may contain fully developed larvae, showing that failure or delay in hatching is caused by some mechanical effect on the shell.
ACKNOWLEDGEMENTS
I wish to thank Mr J. B. Cragg for his encouragement and for much useful discussion. The work was wholly financed by the Agricultural Research Council.
