Laboratory Studies on the Egg of the Blowfly Lucilia Sericata (Mg.)

The laboratory studies of Evans (1934) on the humidity and temperature relations of Lucilia sericata (Mg.) eggs showed that they were ill adapted to survive at low humidities. Yet, Davies & Hobson (1935) and Macleod (1940) have since demonstrated that low humidities are of general occurrence in sheep fleeces where L. sericata eggs may be laid. Further, Davies & Hobson re-emphasized Evans’s conclusion and expressed the opinion that a steady humidity of over 90 % R.H. for 14 hr. was necessary to ensure the hatching of eggs and the establishment of myiasis. In none of these investigations was the effect of humidity fluctuations studied. The present investigation was therefore undertaken to determine the effects on egg development and hatching of humidity fluctuations of a kind likely to occur under natural conditions. A description of humidity conditions in the fleece is being published separately (Davies, 1948). Further, a study of the egg-shell has been made, on the lines of the work of Beament (1946 a, b), in order to determine the nature of the waterproofing mechanism of the L. sericata egg.

The shell of the ovarian egg about 2 days before laying was studied, when, as far as could be seen in sections, it had reached its final size and shape. At this stage the follicle cells had undergone almost complete necrosis and could no longer add material to the shell. The chorion had the same external morphological features, such as the hexagonal imprints of the follicle cells and the longitudinal hatching pleats (see Sikes & Wigglesworth, 1931) as are found in the chorion of the laid egg. Eggs were removed from flies, fixed in alcoholic Bouin, and paraffin sections made. Evidence on the nature of the shell components at this stage was compared with that on the shell of laid eggs, which were not fixed but embedded in paraffin after preliminary dehydration and clearing.

The chorion of ovarian eggs with completely developed shells seemed identical in microscopic appearance and staining properties with that of laid eggs. About 5·0 µ thick over the main part of the shell, it was thickened to about 10·0 µ in the region of the hatching pleats and at the edges of the circular area surrounding the external micropyle (Weismann, 1863). Staining and other chemical tests showed that the chorion was composed of two main layers with a row of dark bodies forming parts of the boundary between them (Fig. 1). The outer layer, about 3·0 µ thick, was readily stained, whilst the inner layer, less readily stained, was 2·0 µ thick over most of the shell. Where the chorion was thickened, e.g. at the edges of the circular micropylar area, most of the increase in thickness was due to an expansion of this inner layer, the outer layer remaining the same thickness over all parts of the shell.

The columnar structure of the chorion of L. sericata eggs (Fig. 1) agreed with the description given for the eggs of other Tachinids by Pantel (1913).

The outer chorion layer was at all stages weakly stained by acid fuchsin, pale brown by Ehrlich’s haematoxylin, pale blue after Mallory’s triple staining, and pale green after light green, whilst the inner chorion layer was not affected by these stains. Both layers, however, stained deeply in basic fuchsin, gave a positive xanthoproteic test, and negative results with both, ninhydrin and warm Millon’s reagent. It was evident therefore that both chorion layers contained protein, but differences in their staining properties indicated that they were not of identical composition.

The chorion as a whole was resistant to cold concentrated nitric and hydrochloric acids, but dissolved rapidly in saturated caustic potash solution at 150 ° C., and more slowly in warm concentrated nitric acid.

When sections of shells were immersed overnight at room temperature in a saturated solution of p-benzoquinone, the outer chorion layer and the row of dark bodies (Fig. 1) became tanned to a deep brown colour over the whole of the eggshell, whilst the inner layer remained colourless. This result was obtained both with ovarian eggs and with laid eggs. It was concluded therefore that the protein of the inner layer was already tanned—or, alternatively, so modified that no tanning by p-benzoquinone could occur—and that the outer chorion layer was largely composed of protein normally susceptible to tanning agents.

The two-layered nature of the chorion, with one layer untanned and the other possibly tanned may explain some of its peculiar mechanical properties. A strip of chorion removed from an egg and mounted so that one end is attached and the other free, underwent rapid curling movements when subjected to humidity changes. When a wet needle was held near it, the chorion curled downwards. When a warm needle was held near, it curled in the opposite direction, instantly returning to its former condition when the warm needle was removed. These observations could be explained if the untanned protein of the outer chorion layer could undergo volume changes dependent on variations in air humidity, whilst the inner layer, being tanned, would be more rigid. The mechanical properties of the chorion not only affect the hatching process, but may account for various features of the rates of water loss from eggs (see p. 83).

This membrane (hereinafter referred to as the c.v. membrane) was about 3·0 µ thick over the general surface of the egg, but thickened to about 10·0 µ in the region of the micropyle (see Pantel, 1913). It stained heavily in Ehrlich’s haematoxylin, pale blue after Mallory’s triple staining, green after light green, and gave a positive xanthoproteic test. It was tanned a dark brown colour in p-benzoquinone at room temperature overnight and dissolved rapidly in saturated caustic potash solution at 150 ° C. It resisted attack by concentrated nitric acid in the cold for several minutes, while on heating it dissolved more rapidly and gave off oily droplets. In sections of fixed ovarian eggs, treated with 1 % ammoniacal silver nitrate, the c.v. membrane gave the characteristic polyphenol reaction (Lison, 1936), except in the thickened micropylar region. At the edges of the non-stained micropylar region there appeared a row of very fine, jet black granules embedded in the membrane as though emerging from the thin part, but not continuous across the thick part (Fig. 2). This layer of silver-reducing granules may have been continued along the thin region of the c.v. membrane, but were invisible as such even under a 1/12 objective; possibly owing to the excessively fine dimensions of the membrane itself. In the laid L. sericata egg, the c.v. membrane showed no silver-reducing properties.

Eggs removed from the ovary some 2 days before they were due to be laid had no resistance to desiccation, although the shell as seen in sections had reached its final dimensions and the follicle cells had undergone necrosis. Such eggs collapsed immediately in saturated sodium chloride solution at 19 ° C., and swelled and burst in distilled water, indicating that the shell was freely permeable to water in both directions. Fragments of chorion and c.v. membrane from these ovarian eggs, when placed in cold concentrated nitric acid, either alone or saturated with potassium chlorate, showed no change within the first few minutes, but rapidly dissolved in both these media on heating. No oily droplets were formed during their disintegration. The shell of the ovarian egg does not therefore seem to contain any appreciable lipoid material.

The shell had the opaque white appearance of the typical Muscid egg. The chorion, considering its thin nature (5·0 µ. thick) was very strong. The water permeability of the shell was now much lower. Most eggs could complete development whilst immersed in saturated sodium chloride solution or concentrated picric acid at 19 ° C. On placing pieces of chorion or c.v. membrane in cold concentrated nitric acid or cold Schulze’s medium, oily droplets were slowly given off. On heating, droplets were rapidly produced, and these could be stained by Sudan III. This result is in marked contrast to that obtained with the same membranes from ovarian eggs. It appears therefore that both chorion and c.v. membrane of laid eggs contain appreciable lipoid material, whilst in ovarian eggs they contain little or no such substances, although at the latter stage the follicle cells have completed their activity and already undergone necrosis. No layers in the shell of laid eggs were selectively stained by either Sudan III or Sudan Black B. Treatment of the chorion and c.v. membrane in chloroform or ether for 12 hr. at 30 ° C. did not visibly affect the bulk of their lipoid content. After this treatment they gave Sudan staining droplets in warm nitric acid in quantities comparable to that given off by untreated shells. It appears, therefore, that lipoid contained in the chorion and c.v. membrane of the laid egg is bound and cannot be removed by fat solvents. The relatively large quantities of oily droplets given off by the chorion in nitric acid suggests that both its layers contain lipoid incorporated into the protein structure. Moreover, this lipoid would appear to be a product of the oocyte, because it does not appear in the shell until the follicle cells have completely degenerated. Since most of the lipoid cannot be removed by fat solvents it probably forms a lipo-protein with the protein of the shell already secreted by the follicle cells. With the incorporation of this lipoid into the chorion and c.v. membrane, there occurs a marked increase in the rigidity and mechanical strength of the eggshell. It is to be expected that lipidization of the protein of the shell would increase its rigidity. On the other hand, this process alone would not be expected to reduce markedly its water permeability. As will be shown later the appearance of bound lipoid in the shell is not itself responsible for waterproofing the egg.

The effects of fat solvents, such as ether, chloroform and carbon tetrachloride on the water permeability of intact laid eggs was gauged by comparing their behaviour in saturated sodium chloride solution at 19 ° C., before and after treatment in a particular fat solvent. It was found that immersion in chloroform at 30 ° C. for 12 hr. produced a radical increase in the permeability of the shell, and made it freely permeable to water in both directions. This indicated that chloroform soluble material responsible for waterproofing the egg-shell was removed or disorganized during the treatment.

When batches of eggs, each batch laid by one fly, were immersed within 15 min. of laying in saturated saline at 19° C., it was found that a small proportion (usually about 10 %) of the eggs in each batch showed signs of water loss by shrinkage within 15 min. The following observations suggested that these non-waterproofed eggs were those that were laid last by each fly. Eggs were dissected from flies in process of ovipositing, and their behaviour in saturated saline at 19 ° C. compared with that of eggs already laid a few minutes earlier by the same flies. About half the eggs, due to be laid within the next few minutes from various positions in the oviducts and vagina, showed signs of water loss after 15 min. under the above conditions—the rest were unaffected. Only 5 % of the eggs already laid by the same flies were of this non-waterproof type. The process of waterproofing the shell would thus appear to occur shortly before laying, and to be unfinished in some eggs when they are laid.

The following observations show that the waterproofing of the egg was not due to the bound lipoid which appeared in the shell shortly before laying. Eggs of the non-waterproofed type, macroscopically identical with laid eggs, were removed from the oviducts of a fly in process of ovipositing. Fragments of chorion and c.v. membrane from such eggs were placed in warm concentrated nitric acid, and droplets stained by Sudan III were given off. This showed that although the shell protein had been lipidized at this stage, the eggs were still permeable to water in both directions. The waterproofing of the egg must therefore be due to some other change in the shell occurring shortly before laying.

Occasionally laboratory culture flies laid complete batches of non-waterproof eggs with slightly transparent chorions. Such eggs shrivelled up at all humidities below saturation and did not complete development. Gough (1946) records that the wheat bulb fly (Leptohylemyia coarctata Fall) laid a few eggs of this type when the egg batches in question contained more eggs than the average number of ovarioles in that species. In L. sericata, eggs which shrivelled up were not observed to be more frequent in large batches than in small ones.

Batches of eggs, weighing 20 − 30 mg. freshly laid by a laboratory culture of flies, were teased apart on fragments of silver foil so that they formed a layer one egg thick and examined under a binocular microscope to ensure that damaged eggs were not used in experiments. After storage in dry air at room temperature for 30 min. to remove all water from the outside of the shells they were re-examined. The rates of water loss from such batches when exposed to dry air at various temperatures were then measured. The foil with eggs attached was suspended in a corked flask fitted with a thermometer and containing phosphorus pentoxide as desiccant, the eggs being a standard distance above the drying agent. This apparatus was similar to that employed by Wigglesworth (1945). The flask was placed in a thermostatically controlled oven. Weighings were made by a torsion balance (50 mg./0·05 mg.). Owing to the large surface area, water loss decreased rapidly with time at high temperatures owing to depletion of water in the eggs. Short exposures of only 15 min. at each high temperature were therefore used, longer exposures of $34$ or 1 hr. being employed at lower temperatures. Surface areas were estimated by making camera lucida drawings of eggs squashed flat under a cover-slip. In this way the water loss was expressed as mg./sq.cm. surface/hr., and this provided data comparable to that obtained by Wigglesworth (1945) on transpiration through the insect cuticle and by Beament (1946 a) on that through the shell of the Rhodnius egg.

The temperature/water-loss curve obtained in this way is shown in Fig. 3. It will be seen that the rates of water loss at temperatures of 20 − 35 °C were low and increased very slowly with rise in temperature within this range. But at about 38 ° C. the water permeability of the shell increased abruptly. It will be obvious from Fig. 3 that the rapid water loss at the higher temperatures necessitated the use of separate egg batches for each new temperature. This curve (Fig. 3) is very similar to that obtained by Beament (1946 a) in comparable experiments on the Rhodnius egg, which he showed was waterproofed by a wax layer. The curve is also similar to those obtained by Wigglesworth (1945) in experiments on the water loss from adult, pupal and larval insects of various orders, and by Beament (1945) for the permeability of membranes covered with films of waxes extracted from insect cuticles. It appears therefore that the Lucilia sericata egg is waterproofed by a lipoid layer rather similar to that of the Rhodnius egg and to the waterproofing layer of insect cuticle. This lipoid waterproofing layer has a ‘critical temperature’ in the region of 38 ° C.

Other features in the effects of temperature on the permeability of the Lucilia sericata egg-shell are similar to those for insect cuticle. The rate of water loss in dry air at 30 ° C. was found to be of the order of 0·5 mg./sq.cm./hr. (Fig. 3). Batches exposed to 50 ° C. and then placed in dry air at 30 ° C. lost water at slightly over 1·0 mg./sq.cm./hr. Exposure to 40 ° C. for 30 min. had little or no effect on the subsequent water loss rate in dry air at 30 ° C., whilst 10 min. at 50 ° C. produced the increase noted above. High temperatures appear to cause a permanent increase in the permeability of the shell. This was found to be the case with insect cuticle in similar experiments on Rhodnius nymphs by Wigglesworth (1945). Exposure to 50 ° C. greatly increased the permeability of the Lucilia sericata egg-shell in both directions. Batches of eggs desiccated at 50 ° C. until they were heavily dimpled regained their water slowly in saturated air at 37 ° C., and more rapidly in saturated air at 50 ° C., so that the dimples in the shells disappeared. For example, a batch of eggs weighing 20-15 mg. was desiccated in dry air at 50 ° C. for 40 min. Its weight was then 9-8 mg. Placed in saturated air at 37 ° C. it regained 1·65 mg. in 3·5 hr.—a rate of approximately 0·47 mg./hr. It was then placed in saturated air at 50 ° C. and regained 2·75 mg. in 1·3 hr.—a rate of approximately 2·1 mg./hr. In addition these eggs eventually regained water to such an extent that they weighed about 5 % more than their original weight, prior to initial desiccation, so that they appeared slightly fatter than normal laid eggs. Evans (1934) showed that eggs, partly desiccated at temperatures below the critical temperature of 38 ° C. found in the present work, did not regain water in saturated air.

The temperature/water loss curve (Fig. 3) was found to be similar in live and in dead eggs killed with ammonia fumes 24 hr. before use. Thus the waterproofing of the egg is not due to an active physiological mechanism.

The I.C.I. detergent C 09993 was used (see Wigglesworth, 1945). Applied as a watery paste to the outer surface of the intact c.v. membrane after the chorion had been stripped off, it caused a marked increase in the rate of water loss through this membrane in dry air at 19 ° C. Eggs so treated collapsed completely in 12 min. Untreated controls with chorions removed, collapsed under similar conditions in 1 hr. This suggests that an emulsifiable material was present on the outside of the c.v. membrane and was responsible for reducing its permeability to water. Application of the detergent to the outer surface of the chorion of the intact egg also affected the rate of collapse of eggs in dry air (Table 1).

These results (Table 1) suggest that the detergent penetrated the chorionic micropyle and, spreading on to the c.v. membrane, affected its water permeability. Eggs minus the chorion, when placed in strong saline showed slight signs of water loss within 30 min; after washing in ether for 5 min. they collapsed completely in 1 − 2 min. in strong saline. On return to distilled water they regained their original shape within 10 min. and occasionally took up water to such an extent that they burst. Freshly laid eggs minus the chorion required only 5 min. washing with ether to become as permeable as described above. Similar eggs, killed in ammonia fumes and stored for 2 days required immersion for 30 min. to produce the same effect. These results again suggest the existence of a lipoid layer on the outer surface of the c.v. membrane.

The effects of abrasion on the permeability of the shell was gauged by comparing the dimpling rate of eggs drawn several times through a layer of alumina dust on a glass slide, with those of untreated controls. It was found that abrasion of the outside of the chorion produced no appreciable increase in the rate of water loss in dry air at room temperature. Eggs with chorions removed, drawn through alumina so that the outside of the c.v. membrane was exposed to abrasion, lost water very rapidly. Such eggs became heavily dimpled within 3 min. and completely collapsed in 10 min. by which time untreated control eggs with chorions removed had not yet lost their original fat sausage-shape. When the c.v. membrane of eggs with chorions removed was dusted, but abrasion of the membrane avoided by keeping them stationary and not moving them through the dust, no marked increase in water loss occurred. This suggests that the waterproofing layer was not a mobile oil which could be absorbed by alumina dust.

All these experiments strongly suggest that the waterproofing layer is situated between the c.v. membrane and the closely fitting chorion. When the chorion is removed damage to the waterproofing layer would be expected, so that the egg minus the chorion would become very susceptible to desiccation. Possibly some of the waterproofing lipoid is partially removed as a coating on the inside of the chorion. The statement of Evans (1934) that the chorion is responsible for the resistance of the L. sericata egg to desiccation is therefore misleading.

Using the rate of dimpling of the shell in dry air as a measure of water loss, occlusion of the external micropyle and/or the longitudinal hatching strip of the chorion with a layer of cellulose paint or paraffin did not affect the rate of water loss from eggs at room temperatures. Eggs so treated completed development at appropriate humidities. It seems that, in effect, water loss occurred over the whole surface of the shell, and not through a restricted area such as the micropyle or hatching strip.

Eggs were obtained from a laboratory culture of flies kept at 22 − 25 ° C. By careful observation, after meat was placed in the cage, it was possible to determine the time of laying to within 5-10 min. Experiments were made at 30, 34, 37, 38 and 39 ° C. in electric incubators giving fluctuations of no more than ±0·5 ° C. from these temperatures. Relative humidities of from o to 95 % were maintained in desiccators by means of sulphuric acid, solutions, made up at 25 ° C. according to the data of Wilson (1921). The specific gravity of the acid was checked at intervals by weighing 10 c.c. samples, and the humidity within the desiccators checked by paper hygrometers or cobalt chloride papers (Solomon, 1945). Eggs, either separated or as complete batches, were incubated within the desiccators, on 3 × 1 in. slides, which could be transferred from one desiccator to another during the incubation period. This involved little disturbance in humidity conditions. In this way, the effect of short periods at very low R.H.’S could be measured. Where time of hatching (or variations in time of hatching within a group of eggs) had to be determined, desiccators fitted with flat plate glass lids were used to facilitate observation of hatching. In all experiments a set of controls was allowed to develop and hatch at 100 % R.H.

To provide data comparable to that obtained by Larsen (1943) on the eggs of dung-breeding Diptera, experiments were also carried out in vessels similar to those used by her. These were solid watch-glasses sealed by a glass plate, on the underside of which the eggs in question were attached (see Larsen 1943, fig. 2). In the present experiments humidity in these vessels was controlled by sulphuric acid/water mixtures placed in the watch-glasses, so that the eggs lay within 1 cm. of the surface of the desiccant.

Eggs laid by flies from 1 to 4 weeks old were used. No significant differences were found in their humidity relations which could be attributed to differences in age of flies.

The minimum humidity for development at 37 ° C. was 50 % R.H. (23·53 mm. sat. def.). The proportion of eggs reaching the pre-hatching stage at this humidity varied considerably between different batches laid by separate flies, as shown in the results of fourteen experiments given below:

Some of these results were obtained by incubating simultaneously in the same humidity vessel eggs laid by different flies. Even in such circumstances the proportion of eggs completing development showed differences between batches although they had been treated identically. There must, therefore, have been considerable variation in the resistance to desiccation of eggs from different flies, as well as variation within a single batch laid by the same female.

The results of experiments in solid watch-glasses as used by Larsen (1943) were substantially similar to those in which desiccators were used as humidity chambers.

To confirm the above results, eggs were obtained from wild flies in the field by attracting them to oviposit on sheep, using the technique of Hobson (1937). Results identical with those described above were obtained with such eggs. Thus the low minimum humidity figure of 23·5 mm. sat. def. found in the present work could not be due to using laboratory flies accidentally selected for desiccation-resistant eggs. In the circumstances it is, therefore, impossible to account for the much lower figure of approximately 17 mm. sat. def. obtained by Evans (1934).

In saturated air at 37 ° C. development was found to take 1·6 − 7·8 hr., a figure agreeing closely with that given by Wardle (1930). At 50 % R.H. at the same temperature, development was found to take 11·75−14·0 hr., thus showing considerable retardation in the rate of development. Retardation of development in L. sericata eggs, caused by low humidities, was also found by Evans (1934) and in the eggs of dung-breeding Diptera by Larsen (1943).

At 34 ° C. the minimum humidity for development was found to be about 40 % R.H. (23·84 mm. sat. def.), when 70 % of the eggs completed development, and at 30 ° C., 25 % R.H. (23·86 mm.), when 68 % of the eggs completed development. The remainder of the eggs died at an early stage in development. The percentages given above again showed great variation from one experiment to another.

Evidence has been given in this paper that the waterproofing layer of the L. sericata egg has a ‘critical temperature’ in the region of 38 ° C. The humidities required by eggs to complete development at 38 and 39 ° C. were therefore investigated. The results of one experiment are shown in Table 2. It will be seen that at 38 ° C. the minimum humidity for development of eggs was about 75 % R.H., and at 39 ° C. about 80 % R.H. Since, at these temperatures and at 95 % R.H., 90 and 75 % of the eggs respectively completed development it is concluded that the high temperatures alone were causing some mortality apart from the effects of humidity. (At 38 and 39 ° C., fewer eggs completed development in 100 than at 95 % R.H. This was presumably due to condensation on the outsides of the eggs interfering with respiratory exchange. A similar phenomenon is mentioned by Larsen (1943).) At 38° C. development was retarded by high temperature alone, but this itself does not account for the much higher humidity required for development, since at 80 % R.H. 38° C. the product of saturation deficiency x × developmental time (hr.) for L. sericata eggs is about 136, whilst at 50 % R.H. 37° C. it is about 282. Since at 95 % R.H. quite high proportions of eggs completed development at 38 and 39° C., this sharp rise of the minimum humidity must be due in part to the harmful effects of the high temperature on the lipoid layer, leading to increased water-penneability of the shell.

In view of the fact that the waterproofing layer of the egg has a ‘critical temperature’ in the region of 38 ° C., the maximum temperatures found in their natural environment—the sheep’s fleece—is of interest. Burtt (1945) found that the temperature as measured by a thermocouple on the face and ears of a sheep, was 35 − 37 ° C. In the present work temperature readings were taken by inserting a clinical thermometer into the fleeces of two sheep under summer field conditions. Sixteen readings taken against the skin at the midback averaged 38·6 ° C. when the sheep were in the shade. When their fleeces were warmed by direct sunlight the skin temperature rose to 39·8 ° C. (average of three readings). Temperatures at 3 cm. off the skin, within the thickness of their fleeces varied greatly, frequently being below 30 ° C. when the sun was clouded, but rising above 40 ° C. in direct sunlight. These observations indicate that L. sericata eggs laid very close to the skin of the sheep may have to develop at temperatures approaching the ‘critical temperature ‘of their waterproofing layer.

Davies & Hobson (1935) found that at 37 ° C. L. sericata eggs required 90–100 % R.H. for rapid hatching. In the present experiments at these high humidities variation in hatching in a batch was about 10 min. With progressively lower humidities, from 90 to 60 % R.H. hatching was found to be less complete, and much slower. The variation in hatching times of the various eggs in a batch was about 2 hr. at 60 % R.H. 37 ° C., and the final percentage of eggs which hatched varied from 40 to 90. The remainder of the eggs at these humidities all contained fully developed larvae imprisoned within the shell. Experiments supported the conclusion of Davies & Hobson (1935), that hatching was completely prevented at 50 % R.H. 37 ° C: At this humidity, however, some hatching occurred in large batches of eggs, incubated as laid by the flies. Forty-three complete unseparated batches were incubated at this humidity. Some hatching occurred in thirteen of them whilst still in air at 50 % R.H. 37 ° C., but never more than 20 % of the total eggs in each batch hatched. This hatching at 50 % R.H. was presumably due to some of the eggs being protected, by close packing, against the effects of the low humidity. In one large mass of approx. 1200 eggs laid by six flies, about 200 eggs batched in air of 50 % R.H. 37 ° C. It was observed that the eggs which hatched at this low humidity were usually those on the edges of the underside of the egg batch. Eggs in the middle of the batch contained fully developed larvae, no doubt unable to break out of their egg-shells because the latter were cemented together into a solid mass by the dried covering of accessory gland secretion coating the eggs. All larvae which emerged at 50 − 70 % R.H. 37 ° C. were weak and shrivelled looking—the ‘chiton-larvae’ of Larsen (1943)—and died soon after hatching.

Davies & Hobson (1935) showed that eggs incubated in saturated air to within 20 min. of hatching, did not hatch if they were transferred to air of 50 % R.H. 37 ° C.—the fully developed larvae being imprisoned within the hardened chorion. Further experiments on this point have shown that larvae could withstand such imprisonment for about 3 hr. Up to that time, the bulk of the larvae hatched when eggs were transferred back to saturated air. Larvae imprisoned for periods longer than 3 hr. showed increasing mortalities—a 100 % mortality being reached after approximately 4 hr. imprisonment.

Immediately after laying, eggs were incubated at 37 ° C. at humidities of 0, 30, 40 and 50 % R.H. for varying periods. They were then quickly transferred to 100 % R.H. 37 ° C. and allowed to complete development. Mortalities produced by exposures of various lengths to these low humidities are shown in Table 3.

It will be seen that at 37 ° C., comparatively long exposures to air of 40 and 30 % R.H. are required to produce a 50 % mortality. Since the R.H. of the air at the base of the sheep’s fleece in summer rarely falls below 30 % R.H. (Davies & Hobson (1935), Macleod (1940)), eggs laid in a dry fleece would therefore be expected to survive if the humidity rose rapidly during the incubation period. Consecutive readings of fleece humidity at standard points in the fleece (Davies, 1948) show that such rises in fleece humidity do occur under natural conditions.

It was found that eggs incubated immediately after laying in saturated air at 37 ° C. were particularly susceptible to desiccation if subsequently incubated at lower humidities. About 30 min. in saturated air was sufficient to cause this effect. Table 4 shows the results of one experiment in which eggs from one batch were incubated in saturated air at 17, 30 and 37 ° C. for 90 min. and then placed for the rest of their developmental period in 55 % R.H. 37 ° C. Twenty eggs in each group were used. It will be seen that this ‘conditioning’ effect was most marked at 37 ° C. and much less so when the period in saturated air was at 30 and 17 ° C. It is well known that the chorion of the Muscid egg hardens in dry air, and softens in humid air. It would appear that L. sericata eggs are laid with the chorion in the ‘hard’ condition, and that incubation in saturated air is needed to soften them. The conditioning of eggs by placing them in saturated air for a time may possibly be explained by this softening of the chorion. The ‘hard’ chorion may allow less rapid water loss than when it has been softened. Water loss through the cuticle of the wireworm was found by Wigglesworth (1945) to diminish rapidly at very low humidities, and he suggested that the lowered permeability resulted from the drying of the cuticle. In the present study it was noticed that eggs conditioned in saturated air at 37 ° C. for 90 min. became dimpled in a shorter time than unconditioned eggs, which had been kept on the bench at about 65 % R.H. for the same time, when both groups were placed in dry air. This was presumably due to the softened chorion of the ‘conditioned’ eggs being less resistant to dimpling than the harder chorion of the ‘unconditioned’ eggs. Dimpling of the shell would be expected to cause deformation of the lipoid waterproofing layer, especially at the edges of the dimple, leading to increased water loss and thus even quicker dimpling. The shells of the ‘unconditioned’ eggs would resist initial dimpling, and thus would lose water more slowly so that more of the eggs would complete development. Since softening of the chorion occurred readily in saturated air at 17 ° and 30 ° C. as at 37 ° C., the less marked ‘conditioning’ effect at the lower temperatures cannot be explained at present.

In view of this ‘conditioning’ effect, experiments of the following type were carried out. A single large egg batch was divided in two, and each half weighed. One half (A) was ‘conditioned’ for 90 min. at 100 % R.H. 37 ° C., reweighed, then placed in 0% R.H. 37 ° C. for 3 hr. The other half (B) was incubated on laying at 0% R.H. for 3 hr. at the same temperature. In one such experiment, batch A in 3 hr. at 0 % R.H. 37 ° C., lost 38 % of its original weight whilst batch B lost only 27 % under the same conditions. This is in agreement with the higher mortalities recorded for ‘conditioned ‘eggs.

The effects of fat solvents, a detergent, an abrasive dust and high temperatures, on the permeability of the L. sericata egg-shell leave little doubt that it is waterproofed by a lipoid layer laid down by the oocyte and situated between the chorion and the c.v. membrane. Its waterproofing mechanism thus appears to be similar in essentials to that described by Beament (1946 a) for the egg of the hemipteron Rhodnius prolixus, and by Wigglesworth (1945) and Beament (1945) for the waterproofing mechanism of insect cuticle. It is interesting to note that an oily layer has been found by Christophers (1945) in the Culex pipiens egg, lying between its exo- and endochorion.

The present work showed that bound lipoid material appeared both in the chorion and the c.v. membrane of the Lucilia sericata egg shortly before laying, and that this lipoid alone did not waterproof the shell. The main function of this bound lipoid would appear to be to strengthen the shell prior to laying down the waterproofing layer. A thin waterproofing layer of orientated molecules would not be effective if laid down on a shell that was soft and easily deformed. Beament (1946 a) showed that the waterproofing layer of-the Rhodnius egg is not laid down until the shell is fairly rigid after the follicle cells have completed the secretion of the endochorion.

Humidity experiments described in this paper showed that Lucilia sericata eggs could withstand considerable desiccation at 37 ° C., with some eggs completing development at 50 % R.H. (23·5 mm. sat. def.). The work of Larsen (1943) showed that eggs of dung-breeding Diptera required much higher humidities for development. It has been shown by previous work (Macleod, 1940; and by Davies, 1947) that the natural environment of the L. sericata egg is normally much drier than that of the eggs of dung-breeding flies. The work of Beament (1946) showed that water loss through the waterproofed Rhodnius egg-shell in dry air at 30 ° C. occurred at about 0·1 mg./sq.cm./hr. (Beament, 1946a, fig. 2). Under the same conditions water loss through the Lucilia sericata egg-shell was found to be in the region of 0·5 mg./sq.cm./hr. Even after allowing for error in calculating the surface area of L. sericata egg batches in this work, it seems that the shell of the latter is more permeable to water than is that of Rhodnius. This greater permeability, coupled with much smaller size and larger surface/volume ratio, account for the more humid conditions required by Lucilia sericata eggs for development.

The results of experiments on the humidity relations of the L. sericata egg in relation to blowfly strike will be discussed in a later paper.

The greater part of this work was carried out at the Zoology Laboratory, Cambridge. My thanks are due to Dr V. B. Wigglesworth, F.R.S., for the facilities he was able to provide and the interest he took in it, to Mr J. B. Cragg for much advice and encouragement, and to Dr J. W. L. Beament for useful discussion. The work was financed by a grant from the Agricultural Research Council.