Observations on the Absorption of Water, Diapause and Embryogenesis in the Eggs of the Cricket Teleogryllus Commodus (Walker)^*

The eggs of Teleogryllus commodus absorb water during their development, provided they have access to free water. In this they resemble the eggs of other crickets (McFarlane, Ghouri & Lennard, 1959).

The period during which water is absorbed by the egg depends on the temperature of incubation (Browning, 1953). Thus at 30° C. water absorption is completed in about 12 hr., whilst at 21° C. absorption continues for 6 days. This suggests that the absorption of water is related to the stage of development of the embryo, a conclusion also reached by McFarlane et al. from similar observations.

Brookes (1952) has described the morphological development of the embryo of T. commodus, recognizing eighteen stages designated A–R. Her nomenclature has been used here in referring to embryonic stages.

A proportion of the eggs enter diapause during their development if they are incubated at a temperature of about 27° C. from time of laying. However, if they are stored for about 1 month at about 12° C. before being brought to a higher temperature, development proceeds without diapause (Browning, 1952; Hogan, 1960). The diapause is thus similar to many other cases of diapause in that a period of low temperature is necessary before embryonic development can proceed.

Hogan (1960) showed that diapause occurs in the eggs of T. commodus at a particular stage in the development of the embryo (stage F-G of Brookes). He concluded that water uptake began at the stage at which the embryo entered diapause and was concluded during diapause. His observations also suggest that water absorption always occurs at this stage.

The observations reported here were made to test the hypothesis that water absorption and embryonic development are related.

A dish of moist sand was placed in a large culture of T. commodus, left for 2 hr. and then removed. The eggs laid in it were sieved out under water and were then incubated in Petri dishes with a layer of moist filter paper. The eggs were placed on numbered squares so that they could be recognized individually. The Petri dishes were kept in desiccators over distilled water, and these were stored in incubators maintained at the required temperature, constant to within ± 0·2° C. The eggs were weighed individually on a torsion balance reading to 0·01 mg.

The stage of development of the embryos was determined by the method of Hogan (1959), in which fixed and cleared embryos can be observed without staining.

Two groups of eggs were stored for 21 days at 13° C. and then incubated at 30° C. One group of twenty eggs was weighed each day. A sample was taken from the second, much larger, group and the stage of development of the embryos was determined.

On the second day most of the eggs had begun to absorb water and on the third day all but three had increased in weight to more than 0·9 mg. from an original mean weight of 0·55 mg. (two of these three later died without absorbing water). Table 1 shows that all developing eggs contained embryos that had reached at least stage F at the time water was being absorbed on the second day (cf. Browning, 1953).

Two groups of eggs were taken and incubated immediately at 30° C. One lot of twenty was weighed each day and a sample was taken daily from the other to observe the embryos.

Of the twenty that were weighed all but four had increased in weight from a mean of 0·5 mg. to greater than 0·9 mg. on the fifth day. In the other group all embryos that could be seen were in stages F–H on the fifth day. In 14 days nine of the twenty eggs had hatched and the remainder appeared healthy. At this stage, in a sample of thirteen eggs that were cleared, five were in an advanced stage of development near hatching, while eight had embryos developed to stage H or less. Most of the eggs that had not reached an advanced stage of development within about 14 days were in stages F–H. There was always a small proportion that had developed beyond stage H. Diapause in these eggs is not as absolute a phenomenon as it is in some other species and some eggs hatch after extended incubation periods (Browning, 1952).

As eggs enter diapause at stages F–H (cf. Hogan, 1960) it seems that whether or not diapause supervenes water is absorbed by the healthy egg at that stage also.

Eggs that had spent 25 days at 13° C. after laying and that weighed between 0·5 and 0·6 mg. were divided into eight groups, four to be weighed and four to be observed for embryonic development. Two groups were placed on filter paper soaked in distilled water, two on paper soaked in 0-5 M sucrose solution, two in 0·3 M sucrose and two in 0·25 M sucrose. Table 2 shows that most eggs in water increased in weight during the first 4 days, whereas in 0·5 M sucrose no egg absorbed water. Furthermore, some of the embryos in the eggs in water developed to stage I during this time, whereas none of those in 0·5 M sucrose progressed beyond stage F. In the other two solutions the results were intermediate between those obtained with 0·5 M sucrose and those obtained with water. The osmotic pressure of 0·5 M sucrose is about 12 atmospheres. Laughlin (1957) found that the minimum pressure required to stop water entering the egg of Phyllopertha was about 15 atmospheres, and Matthée (1951) reported about 14 atmospheres for the egg of Locus tana.

From these experiments it seems clear that eggs in which the embryo had not reached stage F had not begun to absorb water (weight > 0·75 mg.) and eggs in which the embryo had progressed beyond stage F had all commenced to absorb water. And it is clear from Table 2 that not only did the high concentration of sucrose prevent water absorption by the eggs but it also inhibited development beyond stage F.

To try to determine whether embryogenesis and water absorption are causally related attempts were made to interfere with morphogenesis by using metabolic inhibitors. At concentrations lower than 2 × 10⁻² M, potassium cyanide was found to have no influence on development, including hatching (the larvae died as they hatched), nor on the absorption of water. In these experiments the eggs were placed in sealed dishes on filter paper moistened with the solution. The solution, buffered to either pH 6 or pH 8, was renewed each day. Similar results were obtained with sodium azide. Eggs were not observed to absorb water while in dilute formalin, but they developed normally after having been immersed in 0·1% formalin for up to 4 days.

A group of eggs was placed for 1 day at 30° C. in a dilute solution of hydrocyanic acid (10⁻³ N, buffered to pH 3·2, the pH of 10⁻³ N-HCI which was used as the control). They were then removed and half of them were fixed. Of these none had developed beyond stage A-B. But the other half absorbed water and developed normally when incubated in distilled water, but they were about a day behind the controls. Similarly, after 5 days in HCN all the embryos were in stage A–B but three out of ten absorbed water and developed to hatching when removed to water. They hatched 5 or more days later than the controls.

Eggs were incubated for 1 day in a vessel in which the air was replaced by nitrogen. They were then removed and their development was compared with that of a control group. All the eggs developed normally but the mean duration of development for the control group was 15·6 days and that of the eggs from nitrogen was 16·1 days; a difference significant at P = 0·05.

These observations showed that morphogenesis could be inhibited either with HCN or with nitrogen. In the following experiment nitrogen was used because of its convenience.

Two groups of twenty-four eggs were taken from a large number of eggs that had been stored for about a month at 13° C. and then incubated for 2 days at 30° C. One group was chosen from eggs whose weight was less than 0·75 mg., that is, eggs that had not begun to absorb water. The other group was selected from eggs weighing between 0·75 and 1·00 mg., that is, eggs that had begun, but had not yet completed, absorption of water. Half of each group was incubated in air and the other half in an atmosphere of nitrogen. After 2 days the eggs were again weighed. The mean weight of the eggs in air had increased significantly, whilst the weight of the eggs in nitrogen remained unchanged (Table 3) (cf. Matthée, 1951). The nitrogen was then replaced by air and the eggs were returned to the incubator and weighed 1 day later and again 3 days later. Table 3 shows that most of the eggs from nitrogen increased in weight during the second incubation period; they had not been damaged by the period of anoxia.

Banks (1950) showed that the eggs of Corixa punctata absorbed water 3 days after laying when they were incubated at 20° C., whereas water absorption did not begin until about the sixth day at 14° C. Similar observations have been made on the eggs of T. commodus (Browning, 1953), Phyllopertha horticola (Laughlin, 1957) and Acheta domesticas (McFarlane et al. 1959). In all these cases the rate at which the water is taken in is also greater at the higher temperatures. These observations suggest that the absorption of water is dependent upon the embryo reaching a particular stage of development, and that the continued entrance of water into the egg is dependent on the progress of embryonic development. In T. commodus water begins to be absorbed when the embryo is just about to leave the surface of the serosa and sink into the yolk, i.e. between stages F and G. Blocking the development of the embryo with nitrogen, either just before the absorption of water was about to begin or after the egg had begun to absorb water, completely stopped the flow of water into the egg. This makes it seem likely that the process by which water flows into the egg is under the continued control of either the embryo or the serosa.

The most likely mechanisms by which water is drawn into the egg are osmosis or some form of ‘active transport’. Browning & Forrest (1960) showed that the shell of the egg of T. commodus is permeable to water at all times. Since the contents of the egg must have some osmotic pressure, it follows that, during the period when the eggs are not increasing in weight, water must either be continually removed or a hydrostatic pressure must oppose the entry of water. At least three conditions could cause water to enter the egg: (1) The internal osmotic pressure may increase. When the embryo reaches stage F an increase in the concentration of soluble substances in the egg may occur and so increase the osmotic pressure. If this were a gradual process, and dependent on the growth of the embryo, the inflow of water would be inhibited by developmental inhibitors. (2) The hydrostatic pressure within the egg may decrease. The shell of the egg increases considerably in area during the absorption of water and, if this is a growth process and under the control of the embryo or the serosa, it would lead to an inflow of water. Again this would be expected to be inhibited by any inhibitor of growth. (3) Finally if water is being continually pumped out of the egg by some ‘active process’, water would flow in if the pump ceased to function. Nitrogen would be expected to inhibit a ‘pump’, but in these experiments nitrogen inhibited the flow of water. The observations reported here do not allow of a decision between these alternatives, but they do make the concept of an ‘active transport ‘of water seem unlikely. It is significant that Beament, in his recent review (1964) of active transport of water in insects, makes no mention of insect eggs.

The inter-relationship between embryonic development and the water content of the egg is further illustrated in the experiments using solutions of sugar. An external osmotic pressure of about 12 atmospheres was sufficient to prevent water from entering the egg and embryogenesis stopped. The continued development of the embryo thus seems to be dependent upon a particular water content in the egg Hogan (1960), in a study of the embryonic stage at which diapause occurs in T. commodus, concluded that water uptake occurred after the egg had entered diapause. But the observations reported here make it clear that the embryonic stage at which the egg becomes competent to absorb water must occur just before the egg enters diapause, for otherwise the egg would not absorb water until development was resumed, since little morphological development occurs during diapause. Thus the stage at which the egg begins to absorb water and the stage at which diapause supervenes are very close, but the former occurs first.

In the egg of the grasshopper Melanoplus differentialis diapause occurs just before the stage at which water is absorbed, and the first manifestation of post-diapause development is the absorption of water. This led Slifer (1946) to suggest that diapause was due to the presence of an impermeable wax layer in the hydropyle which prevented the entry of water. The elimination of diapause was thought to be due to the gradual breakdown of the waterproof layers.

Many cases of diapause in the eggs of orthopteroid insects are now known and it is clear that the embryonic stages at which morphogenesis ceases and the egg enters diapause and the stages at which water is absorbed are not at all closely related. In Austroicetes cruciata diapause occurs when the embryo is still a plaque of cells at the posterior end of the egg firmly attached to the serosa. Morphological development continues slowly during diapause under certain conditions and some water is absorbed during this time (Andrewartha, 1943). But when diapause is complete and rapid morphogenesis is resumed a sharp increase in the rate of water absorption occurs (Birch & Andrewartha, 1942). In M. differentialis water is absorbed immediately after diapause. In T. commodus the two stages are very close together but water absorption occurs first. In Locustana pardalina most of the water is absorbed during the third to fifth days at 35° C., whereas diapause does not begin until the eighth day (Matthée, 1951). In Melanoplus bivittatus water absorption occurs before the embryo has completed revolution round the posterior pole of the egg, whereas diapause occurs when morphogenesis is nearly complete (Salt, 1949). It is best then to regard water absorption and diapause as quite separate phenomena except that both are intimately related to the morphogenesis of the embryo.