Absorption of Water by the Egg of the Garden Chafer, Phyllopertha Horticola L

A good deal of attention has been paid to the acridiid egg in which water enters through the hydropyle (Slifer, 1938), a specialized area of cuticle at the posterior end of the egg. The water content of the egg is closely correlated with the developmental stages of the embryo. However, the mechanism controlling water content is obscure and little understood. For example, Matthée (1951) shows that eggs of Locustana pardalina cannot absorb water in the absence of oxygen. He concludes that the hydropyle cells secrete water into the egg. Yet Slifer (1938) showed that the hydropyle cells of Melanoplus differentialis often become detached from the hydropyle at the beginning of blastokinesis without affecting the course of water uptake and embryonic development.

Among the lamellicorns, eggs of Anisoplia austríaca (Kerenski, 1930), Melolontha melolontha (Schuch, 1938) and Popillia japónica (Ludwig, 1932) have been studied. Water is absorbed over the whole shell and not through a specialized hydropyle.

Kerenski found that eggs of Anisoplia would swell and develop normally on filter-paper wetted with distilled water, soil filtrate, insect Ringer’s solution, NaCl solution up to 4%, 2% KNO₃ solution or 2% BaCl₂ solution. Schuch notes that anything less than 100% relative humidity retards development, produces small larvae and increases egg mortality steeply. He also found that Melolontha eggs would develop normally if dipped in water (i.e. half submerged), but would die, sometimes swelling up and bursting, if completely submerged. Ludwig gives weight-increase curves for eggs of Popillia at two different temperatures (20 and 25⁰ C.), showing that the rate of water absorption and the rate of development both increased at higher temperatures.

The garden chafer is a lamellicorn with an annual life cycle. In the field, adults are only seen for 4-6 weeks of the year (May-June). The females lay about a dozen eggs, depositing them singly about $112$ in. deep, each in a small cavity in the soil. New-laid eggs are large and white (weight 1 mg., length 1·5 mm., diameter 1 mm., approximately) and their shape is usually that of a prolate spheroid. At least 95 % of eggs laid are fertilized and usually at least 90 % hatch. There is some evidence that the proportion of fertilized eggs decreases with the age of the female (Raw, 1951; Milne & Laughlin, 1956). In the field the embryonic period lasts a month or more, according to the prevailing soil temperatures.

Adults were kept at room temperature, twenty or thirty beetles to a cage, and provided with food and a tray of sifted soil. The females would burrow into the soil to lay and the eggs were sieved out once or twice a day. Thus the term ‘new-laid eggs ‘refers to eggs less than 24 hr. old.

During the incubation period the egg trebles its weight by taking up water from the surroundings. The water content increases from 50 % in the new-laid egg to around 80 % in the swollen egg. The dry weight remains constant, as far as it is possible to tell from the following data : A batch of twenty eggs was divided into two lots of ten after the eggs had been weighed individually. One lot was oven-dried (12 hr. at no⁰ C.) and the eggs weighed again. The other lot was kept at 20° C. for 6‒7 days in a damp atmosphere before drying. The mean dry weights of the two lots (0·574 and 0-569 mg. for new-laid and swollen eggs respectively) did not differ significantly (P= >0·5).

The water content of individual eggs of the same 4ge varies very little. The water content of twenty new-laid eggs ranged from 48·4 to 51·9 % (mean 50·2 %) and of twenty swollen eggs from 79·6 to 83-1 % (mean 81-8%).

At 20° C. the incubation period lasts about 21 days. For the first 2 days the egg remains chalky-white and sausage-shaped. On the second or third day the weight begins to rise and by the seventh day has doubled or trebled. The egg now appears pearly and translucent and is more nearly spherical in shape. From the seventh day to the end of the incubation period the weight continues to rise slowly (Fig. 1).

Twenty eggs were kept at 25° C. and weighed individually each day. Each day also, two or three eggs were taken, frozen into a drop of water by spraying drop and egg with ethyl chloride; the frozen egg was cut in half with a razor blade. The frozen half-eggs were immediately dropped into fixative and finally vacuum-embedded in paraffin wax for sectioning.

The chorion of the new-laid egg (Fig. 2 a) is about 7 μ thick, the surface granules being 3·4 μ in diameter and the shell itself about 3-5 μ thick. Two layers can be seen at this stage, but only one layer of chorion is visible in older eggs. At 2·3 days (Fig. 2,b) the serosa is present and the epembryonic cuticle already about 4 μ thick. At 3·4 days (Fig. 2,c) the epembryonic cuticle is 4·5 μ thick and the serosal nuclei are darker and the cell boundaries less clearly defined. At 4·5 days the rate of water uptake is slowing down (Fig. 2,f) and a second layer of epembryonic cuticle has been laid down (about 4 μ thick). There is also an inner layer (5·10 μ thick) which appears in the sections as loosely packed fibres. The shell is now 9·11 μ thick, not counting the granules or the inner fibrous layer. It remains in this form until the embryo is ready to hatch. The epembryonic cuticle is then resorbed. Sections of vacated shell show only the chorion (Fig. 2 e).

The chorion does not crack or break as the egg swells and so must stretch and become thinner. It measures 3‒3·5 μ in the new-laid egg sections but only 1·5·2 μ in the swollen egg. In the vacated shell it is about 2 μ thick. The chorion is noticeably elastic. Strips of shell from a swollen egg always curl up with the granules on the inner side of the curve. Also, when the epembryonic cuticle is resorbed, the egg loses its shape and the chorion clings to the embryo. This plays an important part in the hatching mechanism, for the chorion is pressed closely against the eggbursting spines on the third thoracic segment of the first instar larva (Rittershaus, 1925). At the least movement of the larva the spines pierce the chorion and the shell cracks and begins to slide off the emerging grub.

Eggs were kept in a damp atmosphere at various constant temperatures. The eggs were weighed individually at intervals to follow the course of water uptake. Absorption begins 1-6 days after laying (after 1 day at 26° C. and after 6 days at 11° C.). A period of rapid uptake follows, occupying about one-third of the total incubation period. The absorption rate then falls to a low level until the egg hatches.

Table 1 shows the length of the incubation period at various constant temperatures between 11 and 26° C. The rate of development (Fig. 3) appears to be proportional to the temperature. The rate of water uptake and the length of the rapid absorption period are also proportional to temperature. All three variables have the same temperature coefficient as far as it is possible to tell from Fig. 4. Fig. 4 shows the mean water contents of three groups of eggs (incubated at 11, 17 and 25° C. respectively) plotted against time expressed as a percentage of the total incubation period.

The rate at which the egg loses water at 0 % relative humidity changes with the age of the egg. To measure the desiccation rate a small torsion balance was made from a galvanometer by fixing a pan to the pointer. Eggs dropped in this pan deflected the pointer against the restoring spring of the instrument. The balance was calibrated and mounted in a square glass jar containing phosphorus pentoxide. The jar was then sealed. Eggs could be dropped into the pan through a tube without opening the jar. Normally the tube was also sealed. When the eggs had been desiccated they were ejected from the pan by discharging a condenser through the galvanometer to flick the pointer against a stop.

The sealed jar was kept at 20° C. and left for 4 days before taking the first reading. Eggs of different known ages were weighed and measured and dropped through the tube. The weight was read immediately and at intervals up to 5 hr. The successive weights were plotted against time, and from these curves the initial rate of water loss was calculated. This figure was divided by the surface area of the egg and the desiccation rate expressed as milligrams of water lost per square centimetre of surface per hour (Table 2).

The surface area of the egg was calculated from the length and breadth before desiccation, on the assumption that the shape was that of a prolate spheroid. It was also assumed that evaporation takes place over the whole surface of the egg. The shell appears to be quite uniform, in thickness and in surface appearance. No account was taken of the granular surface of the egg. The evaporating surface could be effectively higher or lower than the figures used, but any error should be similar for all eggs measured.

Fig. 5 shows the relation between the absorption curve (broken line) and the desiccation rate (continuous line) at 20° C. The desiccation rate curve is only intended to indicate the trend suggested by the data. On the first day after laying, the egg loses water at the rate of about 1 mg./sq.cm./hr. On the second day the rate increases, rising to a maximum of perhaps 20 mg./sq.cm./hr. at about the fifth day. Thereafter the rate falls and by the tenth day has dropped back to about i mg./sq.cm./hr. The desiccation rate starts to increase just before the egg begins to take up water and starts to decrease before the end of the rapid absorption period.

The rate of loss of water gives little information about the rate at which water can pass into the egg. It is well known that the surface membranes of insects show asymmetry, in that water passes across them more rapidly in one direction than the other (Beament, 1954). Furthermore, absorption of water may entail the condensation of water vapour while desiccation requires the evaporation of water from the surface of the egg. However, changes in desiccation rate should reflect changes in permeability to inward flow.

Eggs were immersed in paraffin oil and pricked. A blob of fluid was squeezed out and a small sample drawn into a fine silica capillary. The capillary was sealed and its freezing-point determined as described by Ramsay (1949). Eggs were kept at 22° C. and the freezing-points of samples from eggs of different ages were determined. Eggs up to 4 days old were spun in an air centrifuge for $112$-2 min. This separated out the fat droplets in the yolk which, if included in the sample, made the ice crystals very difficult to see. A centrifuged egg shows four layers. The fatty material forms a cap at one end with a clear region between it and the third layer. At the other end of the egg the densest constituents form a granular fourth layer merging into the third layer. Samples were taken from the third or fourth layers. By the fourth day after laying, at 22° C. the embryo has begun to form and there is an appreciable amount of extra-embryonic fluid. For eggs of this age and older, centrifuging was omitted and the sample drawn from the space above the embryo.

It was found that the freezing-point of a sample dropped rapidly at room temperature (about 20° C.). Thus one sample gave a reading of ‒1·07° C. after 11 min. at room temperature and a reading of ‒ 1·14° C. after a further 30 min. Two samples from another egg were frozen after 7 and 30 min. at room temperature respectively.

Their freezing-points were ‒0·92 and ‒0·99° C. respectively. The drop is almost certainly due to autolysis of the organic constituents of the egg. The freezingpoint (and hence the osmotic pressure) at the time of pricking must remain unknown, but it is evident that sampling must be done quickly and in a reasonably standard time. Samples were frozen within 12 min. of starting the centrifuge (average, 8 min.). Uncentrifuged eggs were sampled and the samples frozen within 3 min. of pricking. This difference in timing, and perhaps the centrifuging itself, affected the readings for centrifuged eggs. Three new-laid eggs which were sampled without centrifuging (i.e. frozen within 3 min. of pricking) gave significantly higher readings than the centrifuged new-laid egg samples (Table 3).

Thus there is an error in all the osmotic pressure values obtained. By standardizing the procedure the error is made, as nearly as possible, the same for all eggs sampled. In general the values given are too high, particularly in the case of eggs under 4 days old.

Eggs will sit on the surface of distilled water, absorbing water and developing normally. A series of solutions of sucrose was made up, giving osmotic pressures from 2·5 to 25 atm., with intervals of 2·5 atm. Fifty-five eggs between 2 and 3 days old at 20° C. (i.e. eggs which had just begun to swell) were divided into eleven groups of five. Ten of the groups were floated on the ten sucrose solutions, and the eggs of the last group were weighed individually and floated on distilled water. All the groups were kept at 20° C. Five days later the eggs were washed in distilled water, rolled on filter-paper to dry off excess water, and weighed individually. Table 4 shows the figures obtained. The ‘change-in-weight’ line was obtained by subtracting the mean new-laid weight of the five eggs in distilled water from the mean weight of each group at the end of the experiment.

The amount of water absorbed by the eggs in 5 days decreases with increasing sucrose concentration. Above 15 atm. little or none is taken up. Above 15 atm. it would seem that the difference between the osmotic pressure of the solution and the force drawing water into the egg is not large enough to stretch the shell.

New-laid and swollen eggs were suspended over distilled water and over saturated solutions of potassium bichromate or potassium nitrate at 25° C. These should have given relative humidities of 100, 98·00 and 92·48% respectively (Solomon, 1951), but local fluctuations of temperature within the containers probably caused a good deal of variation from these figures.

The airtight jars containing solutions and eggs were looked at every 3 days but were not opened. At 92% relative humidity all eggs, swollen and new-laid, collapsed within the first 3 days. At 98 % relative humidity the swollen eggs collapsed eventually but took rather longer to do so. The new-laid eggs managed to take up a little water at this humidity but not enough to allow the embryo to survive. In ‘saturated air’ the new-laid eggs absorbed water normally and all the eggs eventually hatched. Thus the new-laid egg cannot extract water from air that is much below saturation, and even swollen eggs are killed by prolonged exposure to unsaturated air.

The egg is not resistant to desiccation. Prolonged exposure to a relative humidity of 98 % or less kills both swollen and new-laid eggs. This is probably of very little importance in the field. The humidity of small cavities in soil under permanent pasture must rarely, if ever, drop below saturation.

In a saturated atmosphere water will tend to flow into the egg. To regulate absorption, water must be kept out. The process of regulation is highly efficient. The water contents of individual eggs of the same age and at the same temperature show very little variation; further, while rate of embryonic development varies widely with temperature, the water content of the egg remains closely associated with the stages of embryonic development.

The changes in desiccation rates suggest that the shell is relatively impermeable to water before and after the rapid absorption period; that the embryo removes a waterproofing layer, lets in so much water, and then re-erects some ‘water barrier’. This may be partly true but is certainly not the whole explanation.

It seems quite likely that the new-laid egg depends chiefly on a waterproof shell to keep water out; unfertilized eggs usually remain small and unswollen for days until the whole system breaks down and decomposes.

The increase in shell permeability during the rapid absorption period facilitates the entry of water but does not necessarily control it. The rate of uptake during this period will also depend on the elasticity and plasticity of the shell. Yet neither of these physical mechanisms can explain the close relation between rate of uptake and embryonic development over a wide range of temperatures.

Similarly, low shell permeability and high hydrostatic pressure within the egg will tend to prevent entry of water in the latter part of the embryonic period but could not of themselves control the water content accurately.

1. The garden chafer egg absorbs nearly twice its own weight of water in the early stages of the incubation period.

2. The chorion is thin, uniform and elastic, and the surface is covered with granules. Several layers of epembryonic cuticle are added as the embryo develops. These layers are resorbed before hatching takes place.

3. The rate of development, the rate of water uptake and the length of the rapid absorption period are equally affected by temperature.

4. Eggs are most easily desiccated in the rapid absorption period.

5. The yolk of the new-laid egg has an osmotic pressure of about 13 atm. The extra-embryonic fluid of the swollen egg has an osmotic pressure of about 8 atm.

6. Eggs absorb little or no water from sucrose solutions with osmotic pressures of 15 atm. or over.

7. New-laid eggs cannot absorb water from air at 98% relative humidity. Swollen eggs placed in air of this humidity collapse and die.

I would like to thank Dr J. W. L. Beament for his help and advice on the work described above. Also Dr A. Ramsay for the use of apparatus for determining the freezing-point of yolk samples.