The Origin and fate of the Membranes surrounding the Grasshopper Egg; together with some Experiments on the Source of the Hatching Enzyme

After the morphological studies to be described below had been completed a tantalizing problem remained. What was responsible for the rapid dissolution of the tougher portions of the egg coverings immediately before hatching occurred? In 1924 Hoffmann, Dampf, and Varela had noted this phenomenon in the eggs of Schistocerca paranensis, and had ascribed it to the action of fluids which came from the embryo’s mouth. Their idea, however, was backed by no proof of any sort. The second part of the present paper, therefore, deals with experiments designed to settle this point.

For this purpose Melanoplus differentialis (Thomas) eggs secured immediately after they had been laid were fixed at once or else incubated at 25° C. until a desired pre-diapause age had been reached. For post-diapause stages eggs were selected which had been exposed to low temperatures so that their development might be completed when returned once more to 25° C. At least three, and usually more, eggs were fixed and sectioned for each of the thirty-eight days during which active development occurs, so that well over a hundred eggs were examined. Ovarian eggs (more properly, oocytes) were secured by killing several dozen females and fixing the ovaries entire. Bouin’s solution was found excellent as a fixative, and sectioning was carried out with the phenol/water-soaking method described earlier (Slifer and King, 1933). Heidenhain’s iron-haematoxylin, Mallory’s connective tissue stain, Ehrlich’s haematoxylin with triosin, borax carmine with picro-nigrosin, and the Feulgen method were used for staining. The first two listed were found most satisfactory.

Besides the eggs of Melanoplus differentialis a few freshly laid eggs of Chortophaga viridifasciata (De Beer), Romaica microptera (Beauvois), and Locusta migratoria migratorioides (Rch. & Frm.)¹ were examined. In the case of this last species eggs in which development had been three-fourths completed were also sectioned and studied.

The acridian ovary has no nurse-cells, and a series of oogonia and oocytes in various stages of development may be found, one behind the other, in the same ovariole. In the upper end of the ovariole shortly after growth has begun (fig. 1, Pl. 18) the oocytes are surrounded by an extremely thin epithelial wall (ep.) in which mitotic figures are common. Outside of this is found a layer (olm.) of connective tissue (Tunica propria, Korschelt, 1887; outer limiting membrane or membrane propria, Nelsen, 1934 a). Beyond this is a delicate network (am.) of much flattened cells (Peritoneal-hfille, Korschelt, 1887; accessory membrane, Nelsen, 1934 a). Duringyolk deposition (fig. 2, Pl. 18) the ovariole epithelium thickens and tiny particles of yolk may be seen along its inner surface in addition to the large, heavily stained masses (yo.) already present in the interior of the young egg. The cells of the epithelium as they enlarge become rounded, and draw apart leaving between them spaces which fill with droplets of material resembling yolk.

Shortly before the egg is laid the vitelline membrane makes its appearance as a delicate but distinct structure (vm., fig. 3, Pl. 18). At this stage, or a bit earlier, the ovariole cells, which are now enormous in size, begin to secrete the inner layers of the chorion (ch.). When the outermost of the several layers of the chorion has been completed the egg passes into the oviduct and there awaits laying. The germinal vesicle has, in the meantime, broken down and the chromosomes of the first maturation metaphase lie at the periphery about 500 μ from the posterior end of the egg (Slifer and King, 1934).

As is commonly the case in insects, maturation is arrested at this point, and is not completed until after the egg has been deposited. A cross-section through the surface of an egg which has just been laid is shown in fig. 4, Pl. 19. The outermost layer of the chorion, it should be noted, has applied to it a thin coating (tc.) which during the next few days shrivels and disappears. Ayers, in 1884, described a similar mucous covering on the eggs of Oecanthus. The vitelline membrane is distinct at this time, but is so fragile that it will fragment and be difficult to find unless the egg is handled with great care in preparation for sectioning. The unusually large eggs of Romalea micropt era are especially valuable for demonstrating the vitelline membrane, for in this species it is quite a substantial structure.

In fact the present investigator first found the vitelline membrane in Romalea eggs, then looked for and found a more delicate but similar covering in the eggs of Melanoplus differentialis, Chortophaga viridifasciata, and Locusta migratoria migratorioides ¹ Shortly after development begins in Melanoplus differentialis the vitelline membrane can no longer be found, and whether it disintegrates or is incorporated into the serosa was not determined. This point could, undoubtedly, be settled more readily with Romalea eggs.

The vitelline membrane in the grasshopper egg was correctly described and pictured by Korschelt (figs. 16, 17, 1887) for Gomphocerus dorsatus. Meissner (1855) and Leuckart (1855) had, earlier, studied the egg coverings of a large number of insects, and had come to the general conclusion that the vitelline membrane is thin, delicate, and essentially structureless. The membrane which Korschelt described for Gomphocerus was of this type.

Recent investigators have failed to find such a membrane, and have assumed that it was lacking or else have given the name to some entirely different structure. Riley, Packard, and Thomas (1877) described the egg-shell of Melanoplus spretus as consisting of two layers. The outer they called the chorion but the inner they left nameless. However, they described this membrane as smooth, yellow, thick, translucent, tough, and present near hatching, so it is easily recognized as the chitinous cuticle or Blastodermhaut, which is an embryonic serosal product formed after development has bégun. Uvarov (1928) in his book ‘Grasshoppers and Locusts’ follows Riley and his co-workers, but assumes that the chitinous cuticle is the vitelline membrane. McNabb (1928) states that the surface of the egg of Chortophaga viridifasciata consists of a chorion below which lies an inner protoplasmic layer. Nelsen (1931) labels the inner layers of the chorion ‘vitelline membrane’ in Melanoplus differentialis, but in a later paper (1934 b) calls this, now correctly, the chorion. Slifer and King (1934) failed to find the vitelline membrane in Melanoplus differentialis and Chortophoga viridifasciata eggs in their studies on maturation and early development. Husain and Roonwal (1933) label what is apparently the inner layer of the chorion ‘vitelline membrane’ in eggs of Schistocerca gregaria which have just been laid. Roonwal, in fig. 2a of his 1936 paper, repeats this error with newly laid eggs of Locusta migratoria migratorioides and in fig. 2b confuses the situation still further by calling the chitinous cuticle of an older egg the ‘vitelline membrane’. In view of its delicacy it is not surprising that the vitelline membrane has eluded so many investigators. The really surprising fact is that earlier workers were able to find and to describe it so precisely.

The formation of the blastoderm and its differentiation into serosa and germ-band occur during the next few days (Slifer, 1932b; Slifer and King, 1934). By the sixth day at 25° C. the serosa, which now completely encloses the yolk and the embryo, has begun to secrete a new membrane at its outer surface—the chitinous cuticle or Blastodermhaut. Campbell, in 1929, showed that this membrane contains chitin. The extreme outer layer of this covering (yc., fig. 5, Pl. 19), however, is non-chitinous and this portion (which is laid down during the sixth and seventh days at 25° C.) has been called the yellow cuticle by Jahn (1935 a, 1935 b, 1936) who has made a careful study of its chemical and physical properties. Although the yellow cuticle is extremely thin—less than a micron in thickness—Jahn has found that it possesses a high degree of ionic impermeability. He suggests that it may be related to the cuticulin of the insect exoskeleton described by Wigglesworth (1933, 1934). Upon close examination the outer surface of this layer is seen to be covered with minute ridges and tubercles. As soon as this layer has been completed—this is usually accomplished by the seventh day—the serosa begins to deposit a second layer.¹ This layer is white, very tough, and has a fibrous appearance (wc., fig. 6, Pl. 19). Jahn (1935 b) has shown this to be either chitin or a compound closely similar to it in its properties, thus confirming Campbell’s observations. Microscopically it appears to be composed of innumerable fine threads tangled closely together. The formation of this layer occupies approximately a week, and when it is completed it is usually thicker than is the chorion. The condition of the egg coverings on the twenty-first day is shown in fig. 7, Pl. 20. The serosa (se.) has pulled away from the finished cuticle (which has a rather sharply delimited inner edge) and a liquid¹ fills the space between the two. The egg after the formation of the white cuticle is very efficiently protected. The yellow layer confers a high degree of impermeability; while the white layer is responsible for a greatly increased toughness and resistance to mechanical injury.

Following blastokinesis the embryo grows rapidly in length, and within a few days engulfs the remaining yolk and the serosa. An exoskeleton is then secreted over the entire body surface with the exception of the pleuropodia. This, the embryonic cuticle, is moulted immediately after the insect leaves the egg. The true nature of this covering was correctly pointed out by Carothers (1923). Earlier workers had called this membrane the amnion, which is an altogether different structure.

The white cuticle because of its toughness would be a barrier of formidable proportions to the young grasshopper when the time for hatching arrives.² So, in preparation for this event, the embryo produces an enzyme which, during the days immediately preceding emergence, dissolves the white fibrous layer. Three days before hatching the white cuticle has already decreased considerably in thickness (fig. 8, Pl. 20), and on the day of hatching nothing, or only a trace of it, remains (fig. 9, Pl. 20). The yellow cuticle is not affected by the enzyme and persists unchanged to the end. The chorion is usually worn and cracked by this time and shows a tendency to laminate.¹ As the young grasshopper hatches, then, it leaves behind only the chorion and the yellow cuticle. A few moments later it sheds its embryonic exoskeleton.

In 1924 Hoffmann, Dampf, and Varela had stated that the fluids which soften the grasshopper egg-shell in preparation for hatching originated from the embryo’s mouth. Since they had not tested their hypothesis an interesting field lay open to experimentation.

These experiments were designed to isolate the digestive fluids in one end or the other of the egg by applying a ligature before the enzyme made its appearance. For this purpose eggs which were scheduled to hatch seven or eight days later were selected. A cotton thread was tied firmly around each, and all were then placed in an 18 per cent, sodium chloride solution and allowed to remain there for about twenty-four hours.² At the end of this time the eggs had lost much of their original turgidity and by tightening the ligature each egg could now be constricted much farther than had been possible at first. The eggs were then washed in distilled water and placed on damp filter paper in covered dishes to incubate at 25° C. When the time for hatching arrived the eggs were fixed in Bouin’s solution at 55° C., sectioned, and stained with Mallory’s connective tissue stain. Between two and three hundred eggs were ligated and examined after sectioning, but only nineteen of these gave entirely satisfactory results.³ In most cases either the ligature was too tight and the embryo died or the ligature was too loose and the enzyme leaked through. The embryo’s movements are pronounced during the last week of development and this keeps the egg contents agitated so that the tendency for leakage past the ligature is increased. It is also possible that some secretion of the enzyme had already taken place in many of the eggs at the time when they were ligated.

The ligatures were applied at all levels from a point near the extreme anterior end to one near the extreme posterior end of the animal. It soon became apparent that the enzyme was being produced near the middle of the egg, and with continued experimentation it was finally traced to the region containing the pleuropodia (appendages of the first abdominal segment). Eggs which had been ligated below the pleuropodia and had successfully.attained the hatching stage gave results of the type shown diagrammatically in Text-fig. 1 C. The white cuticle below the ligature was intact while above the ligature it had entirely disappeared. Eggs, on the other hand, which had been ligated above the pleuropodia appeared as in Text-fig. 1 D. The results here were so decisive that little doubt remained that the pleuropodia were the source of the hatching enzyme. However, to make the case doubly convincing, it was felt that the effects of removing the pleuropodia should be determined. Experiments of this sort are described below.

Eggs which were due to hatch seven or eight days later were used in these experiments also. The chorion was first removed from each egg so that the embryo could be seen through the transparent chitinous cuticle. This was easily accomplished by alternately wetting and drying the eggs (Slifer, 1932 a). The pleuropodia were then visible behind the upper ends of the metathoracic femora. Each egg during the operation (which was carried out under a binocular microscope) was immersed in 70 per cent, alcohol.¹ The egg was placed ventral side up and a fine, sharp needle inserted through the cuticle above each pleuropodium. Both needles were introduced simultaneously and withdrawn simultaneously so that the pressure inside the egg might serve to force both pleuropodia out. If the operation was successful both pleuropodia were instantly extruded from the egg and, a moment later, were coagulated and whitened by the alcohol. This allowed the operator to be certain that an entire trilobed pleuropodium had been pushed out on each side. The hardened pleuropodia were easily brushed off, and the egg was transferred to a dish containing filter paper dampened with distilled water. The process was then repeated with the next egg. If the least doubt was felt concerning the complete removal of each pleuropodium that egg was discarded at once. During the incubation period which followed the operation the eggs were examined daily. If a leak was found at the site of a wound a drop of 70 per cent, alcohol touched to the spot quickly dried and sealed the injury. Seventy-five eggs were successfully operated in this manner and seventy-two of these developed to the hatching stage¹ with no signs of injury or abnormality in so far as the embryos were concerned. The remaining three failed to complete their development.

Twenty-four of the eggs treated as described above were fixed as soon as they had reached the hatching stage.² These were sectioned and stained, and in every case the white cuticle, although sometimes reduced in amount,³ was heavy and conspicuous. Forty-six of the eggs, when hatching was due, were opened as carefully as possible with needles and the embryos extracted. This was a very difficult performance because of the toughness of the cuticle and the delicacy of the young grasshopper. In fifteen cases where the embryos were removed with little or no injury each shed its embryonic exoskeleton in an entirely normal manner. Eight of these animals were raised to maturity. Two of the operated eggs were allowed to develop for several days past the time when they were ready to emerge. The embryos in these eggs worked vigorously to free themselves from the chitinous cuticle, but without success. One was finally removed artificially with needles but died soon afterwards. The cuticle of this egg was tough and heavy. The other egg was fixed and sectioned. Microscopic examination showed that here, too, the greater part of the white cuticle was still present.

It could be argued that immersion in alcohol or the shock of the operation might have influenced the disappearance of the white cuticle. Thirty-three control experiments were run, therefore, in which a portion of the prothoracic or mesothoracic leg was removed instead of the pleuropodia. In other respects the operations were conducted in exactly the same manner as those in which the pleuropodia had been removed. Twenty-five eggs of this sort hatched and the grasshoppers which emerged from them behaved quite normally. Thirteen of these individuals reached maturity. The cuticula left behind by these animals upon hatching were carefully examined. Each was extremely delicate indicating that all, or most, of the white cuticular material had been digested away. Six eggs of this sort were fixed when hatching was due. In four of these the white cuticle had entirely disappeared, but in two a considerable amount was still present. Two eggs out of the thirty-three failed to hatch.

The function of the pleuropodia has long been a puzzle to students of insect embryology. According to Wheeler (1890) they were first described by Rathke in 1844 in Gryllotalpa. Since that time they have been found and reported in many other insect embryos. In 1890 Wheeler devoted an interesting paper to the pleuropodia and suggested that they might represent odoriferous organs. Somewhat earlier Ayers (1884) had stated that these appendages in Oecanthus functioned as gills. According to the literature some insect embryos possess pleuropodia and others lack them; in some these structures are evaginated from the body-wall and in others they are embedded in it. They are glandular, in some cases at least are not covered by the embryonic exoskeleton, and are sloughed off or disappear at hatching (Wheeler, 1890; Hirschler, 1928).

In Melanoplus differentialis the pleuropodia are trilobed stalked bodies about millimetre in diameter, and are made up of a single layer of very large cells. Preliminary studies have shown the presence of a considerable number of granules and vacuoles in the cytoplasm during the latter part of embryonic development. A more detailed investigation of their structure is in progress.

In this species of grasshopper it may be concluded, then, that the pleuropodia produce the enzyme which destroys the white portion of the chitinous cuticle. Perhaps in other insects these structures serve the same purpose; perhaps they do not. It would be interesting to extend these experiments to other orders of insects where the embryos are also provided with pleuropodia.

1. The eggs of Melanoplus differentialis, Chortophaga viridifasciata, Romalea microptera, and Locusta migratoria migratorioides possess a delicate vitelline membrane. This was seen by earlier workers in other species of grasshoppers but has been overlooked by recent investigators.

2. In Melanoplus differentialis eggs the yellow cuticle is deposited by the serosa during the sixth and seventh days at 25° C. and the greater part of the white cuticle during the following week.

3. During the last few days of incubation the white cuticle is rapidly digested by a hatching enzyme.

4. The hatching enzyme, in Melanoplus differentialis, is produced by the pleuropodia.

All figures are of cross-sections through the surface of eggs or of oocytes of Melanoplus differentialis and all are reproduced at a magnification of 735.

am,, accessory membrane; ch., chorion; cy., cytoplasm; ep., ovariole epithelium; olm., outer limiting membrane; se., serosa; sp., space filled with liquid; tc., temporary coating; vm., vitelline membrane; wc., white cuticle; yc., yellow cuticle; yo., yolk.

Fig. 1.—Young oocyte in ovariole.

Fig. 2.—Oocyte during deposition of yolk. Note increase in size of cells of epithelium.

Fig. 3.—Oocyte during secretion of chorion. The vitelline membrane is now distinct.

Fig. 4.—Egg which has just been laid. Note temporary coating outside chorion.

Fig. 5.—Egg which has been incubated at 25° C. for eight days. The serosa has completed the secretion of the yellow cuticle. Note remnants of temporary coating still attached to chorion.

Fig. 6.—Egg which has been incubated at 25° C. for eleven days. The serosa is in the process of secreting the white cuticle.

Fig. 7.—Egg which has been incubated at 25° C. for twenty-one days. Note liquid-filled space between white cuticle and serosa.

Fig. 8.—Egg which is due to hatch three days later (at 25° C.). The thickness of the white cuticle is now much reduced.

Fig. 9.—Egg which is ready to hatch. Nothing remains but the chorion and the yellow cuticle.