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Further information concerning the envelopessurrounding dipteran eggs

By R. C. KING

(From Northwestern University, Evanston, Illinois, U.S.A.)

With z plates (figs, i and 2)

SummaryA centripetal migration of follicle cells, which results in a separation of egg and nursechambers, occurs in higher dipterans like Drosophila melanogaster but not in lowerdipterans like Anopheles maculipennis. In Anopheles and Drosophila, while the vitellinemembrane can be secreted in the absence of the oocyte, the follicle cells must bepresent. This suggests that the follicle cell is the governing agent in the synthesis ofthe vitelline membrane. The envelopes of Drosophila embryos differ from thosesurrounding ovarian eggs in that a membrane about 0-04 fi thick lies directly outsidethe vitelline membrane. This thin layer is thought to represent a waterproofing waxwhich forms once the egg leaves the ovariole.

IntroductionT H E mature oocyte of most dipterous insects is surrounded by an innervitelline membrane and an outer chorion. King and Koch (1963) demon-strated for Drosophila melanogaster that the vitelline membrane is an inter-cellular structure which is always bordered on the outer surface by the plasmamembranes of follicle cells. The inner surface is usually bordered by theoocyte plasmalemma; but under abnormal circumstances a plasma membranesupplied by another type of cell can serve the same function. The fact thatvitelline membrane can be secreted in the absence of an oocyte but onlywhen follicle cells are present suggested that the follicle cell plays the majorrole in the secretion of precursor material of the vitelline membrane. Datadealing with mosquito oogenesis will be presented in this paper which sup-port this contention. King and Koch also presented information which sug-gested that a waterproofing wax is laid down between the vitelline membraneand the chorion once the egg leaves the ovariole. Further data bearing on thiswaterproofing layer are presented here.

Material and methodsThe slides of sectioned ovaries from Anopheles maculipennis examined in

this study were the same that Dr. A. J. Nicholson used for his classical paper(1921) on the oogenesis of this mosquito.

Newly laid embryos of wild type Drosophila melanogaster were fixed for15 min in 1% buffered OsO4 at 500 C. The dehydration and infiltrationprocedures of Staubli (1963) were followed to produce plastic embedments.Thin sections cut from these blocks with an ultrotome were photographedwith a Siemens elmiskop I.

[Quart. J. micr. Sci., Vol. 105, pt. 2, pp. 209-11, 1964.]

21 o King—Envelopes of dipteran eggs

Results and conclusionsDifferences between Anopheles and Drosopkila in the behaviour of ovarian

follicle cells

In the Lepidoptera, the Hymenoptera, the adephagous Coleoptera, and inthe higher Diptera, the nurse cells are gradually separated from the oocyteby a wedge-shaped layer of follicle cells which advances centripetally fromthe lateral follicular envelope (see fig. 2 of King and Koch, 1963). Thevitelline membrane is extended inwards by these cells, and eventually theoocyte is completely surrounded by this envelope. The centripetal migrationof follicle cells does not occur in mosquitoes (see figs. 1 to 5 of Nicholson,1921). Instead, as the growth rate of the oocyte exceeds that of the nursecells, the ooplasm extends laterally between the follicular envelope and thecluster of 7 nurse cells. Eventually this group of nurse cells is completelyenveloped by ooplasm save for a narrow isthmus which connects it to theexterior (fig. 1, D). At a later stage (fig. 1, E) the nurse cell nuclei are foundoutside the oocyte, and presumably these have been forced out through theisthmus by the pressure exerted by the growing ooplasm. The vitellinemembrane (or the 'inner wall' as Nicholson called it) forms by the fusion ofsmall droplets which accumulate between the follicular epithelium and theoocyte. This membrane is clearly visible in all the photomicrographs offig. 1. A region of particular interest is shown in fig. 1, A and at higher magni-fications in fig. 1, B and c. It is clear that in this region precursor dropletsfor the future vitelline membrane have formed at an interface between folliclecells and nurse cells. Thus as in Drosophila there is evidence pointing to thefollicle cell as the controlling agent in the secretion of the vitelline membrane.

It should be recalled that Nicholson came to the same conclusion and foran equally good reason. He noticed at a later stage in oogenesis that an isolateddeposit of material with staining properties similar to vitelline membranewas formed between certain specialized follicle cells and the nurse cells. Thisdeposit he called the 'stopper', since it was destined to form a plug below themicropylar perforation of the vitelline membrane (see figs. 39 and 41 ofNicholson, 1921). The stopper also appears in fig. 1, D and E.

From a comparison of the morphology and development of the egg shellsin the 2 species, it is clear that the endochorion of Drosophila corresponds tothe inner portion of the outer wall of the Anopheles egg. There is no struc-ture in the Drosophila egg shell that can be homologized with the floats

FIG. I (plate). Light photomicrographs of sections of egg chambers of Anopheles maculi-pennis. A, B, D, and E, phase contrast; c, bright field. A, D, and E, show a series of advancingstages in the development of the mosquito egg chamber. B and c are higher magnificationsof A. Note how the cluster of nurse cells become enveloped by ooplasm (A VS D). At a laterstage (E) the degenerating nurse cell nuclei lie outside the oocyte. In figs, B and c arrows pointto precursor droplets for the future vitelline membrane which have formed between thefollicle cells and the nurse cells. The nurse cell nuclei contain banded polytene chromosomesand a large nucleolus. / , follicular epithelium; o, ooplasm containing yolk spheres; n, nursecell cluster; s, stopper; v, vitelline membrane.

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FIG. 2

R. C. KING

King—Envelopes of dipteran eggs 211

of Anopheles. However, the cells forming the floats resemble the cells whichproduce the chorionic appendages in Drosophila in that both omit vitellinemembrane production and proceed directly with the secretion of endochorion.

The envelopes of Drosophila embryos

The shell surrounding the young embryo is similar to that of the ovarianegg (contrast fig. 2 of the present paper with fig. 8 of King and Koch, 1963).There is an exochorion of variable thickness (generally between 0-5 and 1 /x).The outer endochorion is 0-2 JU. thick, and the air-filled endochorionic spaceaverages about 1 (i in width, although it varies considerably. An inner endo-chorion about 0-04 fM thick lies above a slightly thicker membrane which isthought to be the remains of the fused plasma membranes of the dead folliclecells. Below this lies a structure seen in the embryo, but not in the ovarianegg; a membrane which has a thickness of about 0-04 /n. This layer pre-sumably represents the waterproofing wax. The vitelline membrane is 0-3 to0-4 {i thick. In places where the vitelline membrane is pulled apart from theendochorion the layer of wax follows the convolutions of the vitelline mem-brane and is generally separated from it by a space about 0-05 JU, wide. Theembryo also differs from the ovarian egg in that the cellular covering of theexochorion is missing from most of its surface. The waxy layer appears in arecently published electron micrograph of Mahowald (1962, his fig. 1).

This research was performed during my tenure of a National ScienceFoundation Senior Postdoctoral Fellowship at the Division of Entomology,Commonwealth Scientific and Industrial Research Organization, Canberra,Australia while on leave from Northwestern University. I am grateful to theDivision Chief, Dr. Douglas Waterhouse, for his generous hospitality, toProfessor Edgar Mercer for making available the electron microscope facilitiesof the John Curtin School of Medical Research, and to Dr. A. J. Nicholsonfor letting me examine his Anopheles slides and for his useful comments uponthe finished manuscript. Excellent technical assistance was performed byMr. Surinder Aggarwal.

ReferencesKing, R. C. and Koch, E. A., 1963. Quart. J. micr. Sci., 104, 297.Mahowald, A. P., 1962. J. exp. Zool., 151, 201.Nicholson, A. J., 1921. Quart. J. micr. Sci., 65, 395.Staubli, W., 1963. J. Cell Biol., 16, 197.

FIG. 2 (plate). An electron micrograph of a segment of the envelopes surrounding an earlyembryo of Drosophila melanogaster. end^, outer endochorion; enda, inner endochorion;endp, endochorionic pillar; ends, endochorionic space; ex, exochorion; o, ooplasm; v, vitellinemembrane; wl, waxy layer.