The Formation and Structure of a special Waterabsorbing Area in the Membranes covering the Grasshopper Egg

As indirect evidence for such a view we may take those cases reviewed by Needham (pp. 904-5, 1931), Uvarov (pp. 70-3, 1931), and Buxton (pp. 306-13, 1932) where, with few exceptions, high humidities have been found to favour the development of the eggs of various species of insects. Indirect evidence of another sort consists of observations on the increase in size of the eggs as they develop. It is probable that Reaumur (pp. 127-9, 1740) was the first to record this phenomenon. He described the eggs of a saw-fly as doubling in volume during their development, and wondered, whether the shell of the egg might act as a ‘placenta’ to draw nourishment from the willow leaf to which it is fastened. He allowed leaves with eggs attached to dry, and states that the eggs soon shrivelled. The stems of other leaves with eggs on them were placed in water, and the eggs on these remained turgid and hatched from four to seven days later. Réaumur (1926)¹ noticed, also, that the eggs of ants increase in size as they develop. In the two centuries following Reaumur other workers have recorded such a size increase in the eggs of insects belonging to many different orders. Blunck (1914), Needham (1931), Johnson (1934), and Roonwal (1936) list numerous observations of this type….

—and now the evidence becomes direct—Bodine published curves demonstrating that the increase in weight of the egg of the grasshopper, Melanoplus differentialis, as it develops, is due to an actual increase in water content. This was the first detailed, quantitative, and extensive study of the subject. Somewhat earlier Hoffman, Dampf, and Varela (1925) had reported a few experiments in which they showed that the eggs of another grasshopper, Schistocerca paranensis, which had been shrunken by exposure to dry air were able to absorb water and to regain their original turgidity when returned to a moist atmosphere. In 1930 Kerenski proved that the eggs of a beetle, Anisoplia austriaca, are able to increase in weight when moistened with nothing but water. Johnson (1934, 1937) has shown that the increase in size of the eggs of Notostira erratica is due to an absorption of water. Quite recently Roonwal (1936) has found, as had Bodine, that the water content of the grasshopper egg rises as it develops. Roonwal worked with the eggs of Locusta migratoria migratorioides.

Jahn (1935 a) has shown that the chitinous cuticle of the egg of Melanoplus differentialis is impermeable to such materials as K₄Fe(CN)₆ and FeCl₃. This cuticle, a secretion product of the serosa, is laid down during the fifth and sixth days of development at 25° C. and remains intact until shortly before the animal hatches (Slifer, 1937). The delicate, yellow, outer portion of the cuticle is highly resistant to wetting, and closely resembles the epicuticular portion of the body-wall of an adult insect; while the tough, white, inner portion contains chitin and has properties similar to those of the exocuticle and endocuticle of the adult’s exoskeleton. In fact there is reason to believe that the chitinous cuticle represents the first embryonic exoskeleton. During the latter half of the incubation period, and after the embryo has undergone blastokinesis, a second embryonic exoskeleton is secreted. This is shed immediately after the insect leaves the egg. Since the outer yellow layer of the first embryonic cuticle is so resistant to wetting, and since it is present during all but the first few days of embryonic fife, how, then, is water able to enter the grasshopper egg ? The present paper deals with this problem.

A number of years ago while studying the fatty acid content of the eggs of Melanoplus differentialis (Thomas) the present author noticed that the portion of the chitinous cuticle remaining after the eggs had been boiled in a strong solution of KOH showed at the posterior end a small, circular area which was so excessively thin as to be almost transparent. In contrast to this, the cuticle covering all other parts of the egg was, after such treatment, still tough, rather heavy, opaque, and of a whitish colour. The idea immediately presented itself that this circular area might be specialized for the exchange of materials between the egg and its environment. Attempts to prove this hypothesis were made by coating the posterior end of the egg with such materials as paraffin and asphalt varnish. These experiments were unsuccessful—for reasons not apparent at the time but now quite clear—and the work was eventually abandoned.

After Jahn (1935 a, 19355) had published his observations on the properties of the membranes which surround the grasshopper egg the presence of this thin area assumed a greater interest. It now seemed even more probable than before that this region might serve for the exchange of gases and liquids between the egg and its environment.

For microscopical studies a large number of Melanoplus differentialis eggs of various known ages were fixed in Bouin’s or in Carnoy-Lebrun’s solution. The eggs were sectioned longitudinally, at 7·5 microns, with the aid of the phenol water method described in an earlier paper (Slifer and King, 1933). Heidenhain’s iron haematoxylin, Mallory’s connective tissue method, and the Feulgen technique were used for staining.

A longitudinal section through the posterior tip of a 3-day old egg is shown in fig. 1, Pl. 30. It will be noticed that except at the extreme posterior end the chorion is thrown into depressions. These are the imprints of the ovariole epithelial cells which secreted the chorion. A fragment of one of the micropyles, which penetrate the chorion, is visible well back from the tip of the egg in the upper left corner of the drawing. The outermost layer of the chorion in the region posterior to the micropyles, is not clear and transparent, as it is elsewhere, but stains deeply and has a granular appearance. That this region differs from the rest of the surface of the egg can be demonstrated by placing whole eggs for a short time in Carnoy-Lebrun, then washing and staining in Mallory’s connective tissue stain. The results are striking. The surface of the greater part of each egg, except in places where it has been injured, retains its original yellow colour, but the chorion covering the posterior tip is reddened with the fuchsin, indicating that the outer less permeable layer of the chorion is lacking here. In addition, the contents of the micropyles stain brilliantly and, if the egg has been laid recently, each depression left by an ovariole epithelial cell will be filled with a blue-stained secretion, the so-called temporary coating of a previous paper (Slifer, 1937).

The cells of that part of the serosa which lies directly beneath the region of the chorion which contains no epithelial imprints are still thin and flattened in the 3-day-old egg (fig. 1, Pl. 30), but two days later they are found to be greatly enlarged, while the serosal cells elsewhere remain flattened. During the fifth and sixth days at 25° C. the serosa secretes the yellow cuticle over its outer surface (Slifer, 1937), and at the same time the enlarged serosal cells at the posterior tip of the egg secrete a membrane several times thicker than the rest of the yellow cuticle and distinctly different from it in structure (h, figs. 4, 5, and 6, Pl. 30). When examined with the aid of an oil-immersion lens this material, instead of appearing homogeneous, shows close-set and delicate striations running at right angles to the surface (fig. 4, Pl. 30). Following the formation of this striate layer the enlarged serosal cells begin the secretion of a second layer which is continuous with the white cuticle which covers the remainder of the egg and which is secreted by the ordinary serosal cells (Slifer, 1937). It never becomes as thick as the white cuticle found elsewhere but has the same structure, and stains in the same way.

Throughout the rest of this article this special area in the yellow layer of the chitinous cuticle will be referred to as the hydropyle (Gr. water-gate) and the enlarged serosal cells which produce it will be called the hydropyle cells¹. The introduction of these terms at this point is somewhat premature, but it will prevent much use of long descriptive phrases, and their suitability will become apparent towards the close of the paper.

By the time that the embryo is ready to undergo blastokinesis—which occurs on approximately the twenty-second day of active development at 25° 0.—a considerable quantity of liquid has accumulated in the space between the white cuticle and the typical, flattened serosal cells found near the posterior end of the egg (fig. 6, Pl. 30). The hydropyle cells are still firmly attached to that portion of the cuticle which they secreted. From this time on the history of the hydropyle cells can be followed conveniently by making observations on the living egg. This is best accomplished by placing an egg, from which the chorion has been removed, in a drop of water on a cover-glass to which several blobs of paraffin have previously been attached. A smaller cover-glass is then laid over the egg and the whole is inverted over the well of a depression slide. With such a preparation it is possible to study the entire egg with a lowpower objective and the half (or more) near the observer with a 4 mm. objective. Nuclei and cytoplasmic granules, vacuoles, and filaments stand out with surprising clearness in the hydropyle cells at the posterior end of an egg mounted in this way. If it is desired to make a series of observations extending over a long period the egg, when not being studied, may be removed to an incubator and returned to the slide for examination at selected intervals.

In order to learn the fate of the hydropyle cells eighty-one eggs were chosen which were at the stage shown in Text-fig. 1 A. These were studied with the help of the method just described. During the initial stages of blastokinesis the strand of serosal cells by which the embryo and yolk are attached to the hydropyle cells becomes more and more slender (Text-fig. 1 B). Waves of contraction, meanwhile, sweep along the lateral body-walls of the embryo from the posterior towards the anterior end and eventually the attaching strand breaks. In seventy of the eighty-one eggs examined the break occurred in the thinner part of the strand, the condition shown in Text-fig. 1 c resulting. Here the hydropyle cells remain firmly attached to the inside of the tip of the egg. The embryos in such eggs completed their development and hatched, each leaving the hydropyle cells behind inside the discarded yellow cuticle. In the remaining eleven eggs the hydropyle cells were pulled loose entirely from the end of the egg, and for some time could be seen as a conspicuous lump on the serosa (Text-fig. 1 D). Later they disappeared and were, presumably, taken into the mid-gut along with the ordinary serosal cells and the yolk. Eggs in which this had occurred also hatched in an entirely normal manner.

From these results it may be concluded that the special function of the hydropyle cells is to secrete the hydropyle and that portion of the white cuticle which lies directly beneath it. After this has been accomplished they are no longer needed, and whether they remain attached to the posterior tip of the egg or are pulled away from it and digested makes little difference to the developing embryo. In eggs where the cells remain attached to the tip they may still be found, in sectioned material to retain a more or less normal appearance close to the time of hatching.

It has been stated in the preceding section that the thinness and unusual structure of the chitinous cuticle at the posterior tip of eggs which had been boiled in KOH, had aroused the suspicion that this particular area might be more permeable than other parts of the surface. With this in mind a number of reagents were selected which were apt to have some visible effect upon the surface or, should they succeed in entering, upon the contents of the egg. In all of the experiments which follow eggs were used from which the chorion had been removed. Observations were all made under a binocular dissecting microscope.

The effect of strong solutions of this reagent are especially conspicuous if eggs are used in which the embryos have just completed blastokinesis. The tip of the abdomen lies close to the posterior end of such an egg and a long, double row of cells containing white urate crystals is visible on either side of the abdomen. The alkali enters rapidly, and within a few minutes the extreme tip of the embryo’s abdomen begins to show the first signs of disintegration. As the KOH diffuses inwards the urate crystals nearest the posterior tip of the egg suddenly disappear. When they have gone, those just anterior to them dissolve. This continues in a regular succession from the posterior end forwards. The phenomenon is so striking and so orderly that only one explanation is possible. The alkali is entering through the posterior end and is diffusing anteriorly.

For experiments of this type eggs were used which were about to begin blastokinesis. If such eggs are placed in CarnoyLebrun the cells at the posterior end quickly coagulate and this coagulation—which is plainly visible—extends in a regular manner away from the posterior end. Eggs placed in Bouin’s solution behave in the same way, but the fixative enters more slowly. Eggs exposed to 0·5 per cent, osmie acid are particularly interesting. The hydropyle turns brown almost instantly, then, very rapidly, becomes an intense black. Since the rest of the cuticle retains its original yellow colour the area blackened by the osmic acid stands out in sharp contrast.

The yellow cuticle of an egg placed in a 0·05 per cent, solution of KMnO₄ soon turns brown while the hydropyle remains uncoloured. Eggs exposed to this solution for hours are unharmed and hatch at the usual time.

Eggs about to undergo blastokinesis which are treated with 0·3 per cent. AgN0₃ and then placed in the sunlight retain their original colouring except at the hydropyle. Irregular brown patches appear slowly on this specialized area of the cuticle, and finally the whole circular area becomes black. The hydropyle cells also turned black in eggs which were exposed to AgNOg for an hour. After being washed these eggs were transferred to an incubator. No ill effects of the treatment could be observed. The eggs developed and hatched as usual. In some the blackened hydropyle cells were left attached to the posterior tip of the egg at blastokinesis and in others they were pulled away from it and, later, breaking loose from the serosa, were seen floating around in the fluid which bathes the embryo. The blackened cells from an egg of the former type were examined in a yellow cuticle from which an embryo had just hatched. They were extremely hard and brittle.

Eggs containing 17-day-old embryos were allowed to stand in concentrated HC1 at room temperature for two weeks. At the close of this period the eggs had become coal-black but were still entire. The eggs were washed in distilled water and then transferred to 0·3 per cent. AgNO₃. At once a circular column of milky precipitate gushed from the posterior end of each. After a short time white spots appeared at other places on the surface of the eggs. These evidently marked the sites of small wounds. The forcibly ejected column appeared only at the posterior end. Eggs allowed to remain in the AgNO₃ solution eventually burst. This was clearly due to an inward passage of water more rapid than the outward diffusion of HC1. The hydropyle was examined microscopically in eggs which were removed to water from the acid and opened before bursting had occurred. No visible pores could be found in the hydropyle.

When placed in concentrated HNPO₃ a white precipitate appears almost instantly at the posterior end of the egg and spreads rapidly away from it. A moment or two later the egg undergoes a miniature explosion, and a portion of the contents are thrown to a considerable distance through the ruptured hydropyle.

A number of eggs from which the chorion had been removed were exposed for a short time to Carnoy-Lebrun. Later these were washed and some treated with triosin and others with fast green. In both cases the hydropyle became brilliantly coloured while the rest of the cuticle was only faintly tinged by the stain.

From the experiments just described it is apparent that the hydropyle differs markedly from other parts of the cuticle in its reaction to various non-biological materials. It remained to be seen whether such physiological materials as O₂, CO₂, and H₂O enter and leave with more ease at this special area than at other locations on the egg surface. The simplest way to solve such a problem would be to cover the hydropyle with some impermeable material, and to compare the subsequent history of such eggs with the history of other eggs similarly treated but with the impermeable material applied to some other spot on the egg. A number of impermeable or relatively impermeable materials were tried, but only one gave satisfactory results. This was a commercial product sold under the name of O.K. Liquid Solder. This solder dries very quickly, sticks to the cuticle with great tenacity, has no toxic effects, and proved to be very impermeable to water. In all of the experiments described below the chorion was first removed from the eggs. If this were not done the sponge-like chorion (which often has minute cracks in it by this time) would very readily conduct water up under the solder and so defeat the purpose of the experiment. Moreover, in eggs from which the chorion has been removed it is possible to observe the condition of the embryo as often as desired through the transparent chitinous cuticle. Such eggs are much more delicate than ordinary eggs, and extreme care must be taken in handling them. If the slightest wound is made in the cuticle a portion of the liquid bathing the embryo oozes out and hardens into a small red spot. Eggs of this sort, unless the injury has been too great, develop normally; but the presence of these spots would be a possible source of error in experiments of the type to be described below. Consequently all eggs which developed red spots were discarded at once.

One series of 20-day-old and one series of 22-day-old Melanoplus differentialis eggs in which the diapause had been broken by exposure to low temperature were prepared as described above. The tips of the posterior ends of some, and the tips of the anterior ends of others, were then dipped in solder. The amount of oxygen consumed by each lot was then determined in Warburg respirometers.¹ The results are shown in Table I. The figures are remarkably close and leave no doubt that the yellow cuticle as a whole is readily permeable to both 0₂ and CO₂.²

Bodine’s (1929) curves demonstrating the increase in the water-content of the eggs of Melanoplus differentialis as they develop show a gradual but steady rise from the time when the eggs are laid to the onset of diapause at the close of the third week of development at 25° C. The curves then flatten out and remain so for a long period. At the end of diapause the water-content again begins to rise, and does so with increasing velocity until the eggs hatch. The curves secured by the present writer for eggs of the same species agree in their general trend with those of Bodine, but—owing perhaps to the closer intervals at which determinations were made—two rather interesting differences have been found. As may be seen in Text-fig. 2 (which is based on data secured when the eggs were analysed for fatty acids, but which have not been published previously) the average wet weight of the eggs remains almost constant during the first week at 25° C. instead of increasing rapidly from the first day as Bodine’s curves would indicate. This is a point of considerable interest, for it is at the close of the first week that the serosa completes the secretion of the yellow cuticle. The acquisition of the power to absorb water in significant quantities is almost exactly coincident with the formation of this membrane.

In one other respect the curves secured by the present author differ from those given earlier. Instead of rising with increasing rapidity between the time when diapause is broken and hatching, as do Bodine’s, those of the present writer show a rapid rise just before, during, and after blastokinesis. This is succeeded by a period of slower water uptake during the later stages of incubation. The curve, some days before hatching, shows a tendency to flatten out parallel to the base line. It is characteristically concave rather than convex to the abscissa during this period (Text-fig. 3). As the egg increases in size due to the uptake of water the pressure inside the egg naturally increases. The membranes are capable of considerable stretching, but after a time the resistance to further stretching tends to balance the forces leading to the further imbibition of water and the two approach an equilibrium. The great turgidity possessed by the egg during its later development is well known to any one who has attempted to operate on eggs of this sort. A good portion of the egg contents spurt out at a slight prick of a needle.

For the experiments designed to test the effect of an impermeable coating applied to the hydropyle, eggs were chosen which had been kept at 15° C. for seventy-one days after they were laid. Such eggs contain embryos at a stage comparable to that reached after seventeen days at 25° C. (Slifer, 1932). These, when placed at 25° C., begin to develop more rapidly, and the majority hatch about twenty days later. The occurrence of a diapause is thus avoided. Seventeen-day-old eggs are particularly suitable for this purpose for the water-content is still low and the white cuticle is so well developed that the chorion can be removed without much difficulty. In the first few experiments of this type the eggs were weighed daily, but the eggs are delicate and the handling and drying preliminary to weighing caused the appearance of many small scars on the cuticle. It was necessary to discard so many eggs because of this that a new set of experiments was begun in which the weighings were made at longer intervals. The results of this experiment are shown in Text-fig. 4. Curve A shows the increase in weight of normal, untreated control eggs; curve B shows the weight of eggs from the same lot, the anterior ends of which had been dipped in solder, and curve C shows the weight of eggs the posterior ends of which had been similarly coated. Only one conclusion is possible. An impermeable covering applied to the posterior end almost completely prevents the uptake of water, while a similar coating on the opposite end has no such effect. Curve C does show a slight rise. This may be attributed either to some slight permeability of the yellow cuticle, or of the solder, to water, or to minute, unnoticed injuries of the yellow cuticle. Water, it may be concluded, enters (or is lost) in significant quantities through the hydropyle and not elsewhere.

In addition to the weight measurements made on the eggs used in the experiments described in the preceding section, observations were made on the course of development. Two useful and conspicuous landmarks in the latter part of the embryo’s history are (1) the occurrence of blastokinesis and (2) hatching. In Text-fig. 5 the percentages of the total number of eggs which had completed blastokinesis are given for the same eggs whose weights are shown in Text-fig. 4. The number of embryos which succeeded in completing blastokinesis is very much lower in the eggs which had the posterior ends covered than it is in either of the other two groups. By the twenty-first day, at 25° C., 73·5 per cent, of the control eggs, 60 per cent, of the eggs with the anterior end covered, but only 2-8 per cent, of the eggs with the posterior end covered had hatched. Development, then, stops at once or progresses at a greatly retarded rate in eggs when the entrance of water is prevented. It is conceivable, of course, that the solder placed in such close proximity to the head of the embryo might have some toxic effect. This objection was met by removing the solder from twenty-seven of those eggs which had had the posterior end covered for four weeks, and which had showed no signs of developing during that period. This was easily accomplished by using acetone as a solvent for the solder. One week later 60 per cent, of these eggs had completed blastokinesis.

One point remained to be settled. Would the application of solder to the posterior ends of eggs after the greater part of the water had already been taken up have any retarding effect on development ? This question was answered by placing solder on the posterior tips of thirty eggs which were scheduled to hatch about a week later. A like number had the anterior ends covered. The results were clear-cut. All hatched at the same time as the controls. The solder, then, does no harm. It is only the lack of water which stops or slows down development.

A search through the literature on embryology in an attempt to learn whether or not the special area in the chitinous cuticle, here called the hydropyle, is present in other insect eggs has produced little in the way of results. It has, apparently, been the practice of nearly all those who have worked with insect eggs to remove the secreted membranes while preparing them for study. Structures such as the curious ‘dorsal organ’, of unknown function, described for certain apterygote insects, as well as for the eggs of a number of arthropods other than insects,¹ arouse the suspicion that this may correspond to the mass of cells which secretes the hydropyle in the eggs of Melanoplus differentialis. For example, the ‘precephalic organ’ described for the egg ofAnuridamaritimaby Imms (1906) and the ‘indusium’ found in eggs of the Paratenodera sinensis by Hagan (1917) both bear a strong resemblance to the hydropyle cells of the grasshopper egg. But the illustrations accompanying the descriptions are not sufficiently detailed to tell whether or not such a special area exists. Moreover, little is known concerning the water metabolism of these forms.

More data, pertinent to the problem under discussion, are available for the eggs ofNotostira erratica than exist for those of most other forms. The eggs of this hemipteran absorb water, as Johnson (1934,1937) has demonstrated, and a comparison of Johnson’s figures of sectioned eggs (especially of fig. G, pl. II, 1934) with fig. 6, pl. 30 of the present paper is illuminating. The cells which Johnson describes as ‘columnar epithelium of cells lining the yolk-plug wall’ closely resemble the hydropyle cells of the grasshopper egg. The two-layered ‘yolk-plug membrane’ corresponds, apparently, to the chitinous cuticle of the present paper but it is not possible to tell, from Johnson’s figure, whether the membrane is modified above the columnar epitheliun to form a hydropyle. It would be interesting to test this point experimentally with the Notostira egg. Finally, Weber’s (1931) description of the uptake of water by the eggs of a coccid, Trialeurodes vaporariorum, from the tobacco leaves to which they are attached by a small, thin-walled stalk should not be overlooked. A short time after the eggs are laid a brown membrane is formed below the chorion. This extends into the stalk and is thinner in that region than it is elsewhere. Weber states that when leaves with eggs attached to them are allowed to wither the eggs on them soon collapse and die—which recalls Reaumur’s experiments, long ago, with the saw-fly eggs. After the inner brown membrane has formed the eggs are less sensitive to changes in the turgidity of the leaf than they are when newly laid. It seems probable that conditions in the coccid egg bear a rather close resemblance to those in the egg of the grasshopper.

1. Water enters (or leaves) the egg of the grasshopper, Melanoplus differentialis, after the sixth day of development at 25° C., through a small, circular, specialized area in the yellow cuticle located at the posterior end of the egg. This area has been named the hydropyle.

2. The hydropyle is secreted by a group of enlarged and modified serosal cells. These are called the hydropyle cells.

3. The outer layer of the greater part of the chorion consists of a clear material which is not readily permeable to dyes. At the posterior tip of the egg this impermeable layer is lacking.

4. The grasshopper egg first begins to absorb water in considerable quantities immediately after the yellow cuticle is formed.

5. Towards the close of embryonic development the rate of water uptake falls off markedly.

6. The chitincus cuticle as a whole is readily permeable to O₂ and CO₂.

7. If water is prevented from entering the egg by covering the hydropyle with a water-impermeable material development is stopped, retarded, or unaffected depending upon the watercontent of the egg at the time when the impermeable coating is applied.

EXPLANATION OF PLATE 30

Fig. 1.—Longitudinal section through the posterior tip of a Mel ano plus differentialis egg which has been kept at 25° C. for the three days since it was laid. The serosa (s.) and germ-band or embryo (e.) lie directly beneath the chorion (ch.) and at the surface of the yolk (y.). A fragment of a micropyle is visible in the chorion in the upper left-hand corner of the drawing, × 160.

Fig. 2.—Diagrammatic, lateral view of an egg, from which the chorion has been removed, to show the size of the hydropyle at the posterior end of the egg. The concave side ofthe egg represents the ventral surface. ×10.

Fig. 3.—Diagrammatic view of an egg, from which the chorion has been removed, as seen from the posterior end. × 10.

Fig. 4.—A section through the hydropyle at right angles to the surface of an egg fixed twelve days before hatching was due. Note the striated appearance of the modified yellow cuticle as seen under high magnification. × 1,678.

Fig. 5.—A section through a part of the hydropyle and a few of the hydropyle cells cut at right angles to the surface of an egg fixed twelve days before hatching was due. × 680.

Fig. 6.—Longitudinal section through the tip of an egg fixed shortly before blastokinesis (Text-fig. 1 A represents an egg at this same stage). The serosa (s.) has withdrawn from the white cuticle (ivc.) leaving a liquid-filled space between the two. A few particles of yolk are enclosed within the serosa, m., fragment of micropyle; ch., chorion; yc., yellow cuticle; h., hydropyle; he., hydropyle cells. ×160.