Growth and Maturation in the Parthenogenetic Eggs of magna Strauss By Hyman Lumer Department of Biology, Western Reserve University Cleveland,Ohio With 4 Plates (36 Figures) ReceivedOctober 19, 1935 Introduction It has recently been shown (Banta and Brown, '29 a, b) that the sex of offspring developing from parthenogenetic eggs of some is definitely subject to environmental control. These find ings raise the significant question of whether or not chromosomal differences exist between such environmentally determined male and female-producing eggs. Allen and Banta ('29) sought an answer to this question in their investigation on Moina macracopa, but were unable to reach any definite conclusion. It seemed of interest to continue the attack on this problem in another species of Cladocera; hence the present investigation was undertaken. was selected because of its suitability for experimental work, and because it seemed at the time that it might possess certain advantages as cytological material. Due to difficulties which have arisen in the attempt to control the sex of the offspring in this species, it has not been possible thus far to achieve the entire objective of the investigation. However, the growth and maturation of the eggs has been studied, and the diploid chromosome number determined. The present paper deals only with the parthenogenetic eggs, but it is hoped that a similar study may be made shortly on the sexual eggs.

Materials and Methods The used were obtained from a clone derived from Dr. A. M. Banta's stock at Brown University, and were reared in a manure-soil medium (Banta, '21). It was found that the growth stages could best be studied in immature animals, where the situation is not complicated by the presence in the ovary of fully grown eggs. A series for these stages was obtained by isolating a number of broods at the time of release; from these, animals were removed for fixation every twenty-four hours up to end of the first adult instar. Cytologia1937 8. 1 2 H. LUMER Cytologia 8

Considerable difficulty was experienced in timing the early maturation stages. Maturation begins shortly before molting; at

the time the molt occurs, the chromosomes are already in the meta

phase or early anaphase. At room temperature (20-22•Ž), the eggs are extruded about five minutes afterward, and the maturation divi

sion is rapidly completed in the brood chamber. Thus, while the molt offers a means for timing the later stages, serious difficulty

arises in the case of those occuring prior to molting, since no reliable criterion has been found for determining in advance when molting

will occur. The previous brood is released some time before the molt, but the time between release and molting is so variable, even in

animals reared under as nearly identical conditions as possible, that it is worthless as a means of determining the stage of the eggs at

the time the is fixed.

Attempts to time these stages in this manner were finally given up, and the following procedure was adopted instead. A large

number of females from broods released on the same day were reared individually in 50cc. vials. These were placed in racks, each hold ing twenty vials. After each animal released its first brood, the vials

were rearranged in the racks in the order of time of release. The animals were then observed at intervals of 15 minutes, until ap

proximately one-third of those in a given rack had molted, at which time all twenty were fixed. In this way a fairly complete series for the maturation division was obtained. Early cleavage stages were also fixed at this time.

Several efforts were made to induce male production by crowd ing the mothers, but without success. In experiments involving several hundred animals, males were produced in only a few isolated cases, in some of which the controls produced a higher percentage of males than the crowded animals. It would appear from these results that in this species crowding is apparently not in itself an important factor in controlling sex. It has thus been impossible to secure a sufficient percentage of male-producing eggs to permit an investigation of possible cyto logical differences connected with sex determination. Before this can be done, it will be necessary to develop a suitable technique for inducing male production with some degree of consistency.

Of the many fixatives tried, a combination of Ohlmacher's fluid and Allen's B15, as employed by Allen and Banta ('29), gave the best results. The animals were fixed whole in the first for one hour at room temperature, then in the second for one hour at 40•Ž.

Sections were cut 5-7,u in thickness. The presence of numerous large yolk globules occasioned some difficulty in sectioning the later 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 3

stages, particularly those in the brood chamber. Some improve ment was obtained by placing the animals, during the course of dehydration, in a 4 per cent solution of phenol in 80 per cent alcohol

for twenty-four hours (Slifer and King, '33).

Most of the sections were stained with Heidenhain's hema toxylin. They were mordanted twenty-four hours in a 1per cent

solution of iron alum, and stained for an equal length of time in a 2per cent solution of hematoxylin. Some of the sections were

counterstained with eosin, but in most cases no counterstain was used. Feulgen's reaction was employed, generally with fast green as a

counterstain, in an effort to determine the distribution of the chro

matin during the growth stages. Flemming's triple stain was tried on some of the material, but no sample of safranin could be obtained

which would yield satisfactory results.

For examining the maturation stages, a Zeiss 1.5mm., 120•~,

apochromatic oil immersion objective was used, in combination with a 10•~ compensating ocular.

I am deeply indebted to Dr. J. C. Gray for his helpful sugges

tions during the course of the work, and for making the photographs

accompanying this paper, also to Dr. B. G. Anderson for his in valuable aid in the task of securing the maturation stages.

Observations

The oogonia

The ovaries are a pair of elongated organs lying on either side

of the gut, and somewhat ventral to it. Each is enveloped by an

ovarian epithelium consisting of a single layer of squamous cells, and has a short oviduct leading dorsally from its posterior end into the

brood chamber. The oogonia lie in the posterior portion of the

ovary, and move anteriorly as development progresses. The fully

grown eggs at the anterior end must then squeeze back past the developing germ cells to reach the oviduct.

The oogonia are small, loosely-packed cells with vesicular nuclei

and a small amount of granular cytoplasm (fig. 1). Each nucleus

contains from one to three nucleoli and a number of smaller granules.

These bodies take nuclear stains, and give a positive reaction with Feulgen's test, indicating that they are chromatin material. They

are embedded in a flocculent achromatic substance, which stains only faintly with nuclear dyes, and takes counterstains readily.

In immature animals, these cells exhibit numerous mitotic divi

sions. Two such dividing cells are shown in figure 1. The chromo

somes are extremely small and closely massed together, so that it is

1* 4 H. LUMER Cytologia8 difficult to count them even in polar views. However, their number appears to be between six and eight. No mitoses have been observed in the oogonia of mature animals.

The growth stages The more anterior of the oogonia begin to increase in size and to move forward. The onset of the growth period is characterized by a change in the structure of the nucleus, in which there now appears a large, solid, spherical body, staining deeply with nuclear stains (figs. 1, 2, 3). Within it a few small, refractive bodies may occasionally be seen. Although this structure has been called a nucleolus by previous investigators, and will be designated as such here, its nature is not at all clear. As growth progresses, it begins gradually to disintegrate, becoming vacuolated and irregular in out line (fig. 4). This process will be considered in detail later. The chromatin granules which were present in the nuclei of the oogonia are no longer visible. The achromatin has increased in volume and become more prominent, but exhibits no change in its staining properties. Occasionally it presents the appearance of a network of faint, irregular threads. The location of the chromatin in these stages is problematical. In sections to which Feulgen's test has been applied, none of the extranucleolar material stains; only the nucleolus gives a faint, doubtfully positive reaction. The cytoplasm increases in volume, but shows no changes in structure, except for the appearance of several small, ovoid bodies, which lie against or near the nuclear membrane (figs. 2 and 3), or, less frequently, are scattered through the cytoplasm. These bodies take nuclear stains, but give a negative reaction with Feulgen's test. They persist up to about the time of the first appearance of the yolk. Similar structures were observed by Schrader ('25) and by Allen and Banta ('29). As previously described by Claus (1876) and Weismann (1877), the growing germ cells become segregated into groups of four. One cell of each group eventually becomes an egg, while the remaining three become nurse cells. The former is at first morphologically indistinguishable from the latter. Both Claus and Weismann .assert that the potential egg is always the third cell of the group from the posterior end. I have been unable to verify this, however, due to the irregularity of the arrangement of the cells in many of the groups. The prospective egg, nevertheless, first becomes clearly re cognizable by its position. It comes to lie ventro-medial to the nurse cells, and continues to grow after they have reached their maximum size. As it grows, it becomes cup-shaped, with the three nurse cells 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 5 lying in more or less tandem fashion in the hollow of the cup. As a matter of convenience, this cell will be termed the egg from this point on. Numerous yolk globules and vacuoles now make their appearance in its cytoplasm. Staining with Sudan III and osmic acid shows that in the living egg the latter contain fat droplets. These inclusions increase in size and number until in the fully-grown egg, they almost completely fill the cytoplasm. These stages are illustrated in figures 4 to 9. While the deutoplasm is being formed, the egg nucleus under goes a series of changes. The disintegration of the nucleolus, men tioned above, now proceeds rapidly, giving rise to a granular sub stance which has a considerable affinity for nuclear stains (figs. 4 10). As it disintegrates, it becomes irregular in outline, and develops numerous small vacuoles (figs. 4-9). In the majority of cases, these flow together to produce a large central cavity, the nucleolus thus assuming the form of a spherical shell. In sections cut through its center it appears more or less ring-shaped (fig. 10). The central cavity becomes filled with the same granular material which is present outside the nucleolus. This is most probably due to the disintegra tion of the interior nucleolar surface. Such an interpretation is supported by the fact that the wall of the nucleolus becomes pro gressively thinner without any significant decrease in its external diameter. It does not disintegrate completely, but remains at the end of the growth period as a thin-walled shell (fig. 11). In some instances, on the other hand, the vacuoles do not fuse, but persist as discrete cavities. In this situation the nucleolus be comes progressively smaller in diameter, and remains a fairly solid body (fig. 12). In the early stages of this process, the granules are comparatively few in number, and may become lined up in such a way as to present the appearance of rough threads (figs. 4, 5, 6). Occasionally two such structures may lie near and approximately parallel to one an other, giving the impression of paired threads. Such formations, however, appear to be entirely fortuitous. There is no evidence of the consistant appearance of paired threads, such as were described by Kuhn ('08) in Daphnia pulex, nor even of single threads. More over, it is extremely unlikely that this substance is chromatin, inas much as it gives a distinctly negative reaction to Feulgen's test. Sub sequently the granules fill the nucleus almost completely, so that thread-like formations are no longer discernable (figs. 7-10). Shortly before the onset of the maturation division, these granules lose their affinity for nuclear stains to a considerable degree (figs. 11, 12). 6 H. LUMER Cytologia8

Toward the close of the growth period, the nucleus, which thus far has occupied a central position, migrates to the periphery of the egg (figs. 11, 12). Here it is found most frequently on the side of the egg adjacent to the gut, and occasionally at the dorsal or ventral surface, but never on the side adjacent to the nurse cells. The nuclear membrane is still intact, and as far as the chromatin is concerned, the entire period may be considered as purely a resting period.

Fate of the nurse cells; the "degenerate body" The nurse cells reach their maximum size shortly after the first appearance of deutoplasm in the egg, although their nuclei continue to grow for some time afterward. They exhibit no appreciable change in structure, however, until a much later stage, when the process of yolk-formation in the egg has nearly reached completion. At this time a series of degenerative changes sets in. The first of these is a change in the structure of the cytoplasm, which becomes highly alveolar in appearance, and develops several spheroidal bodies with an affinity for nuclear stains (figs. 13, 14). Meanwhile the nucleolus, whose behavior has thus far paralleled that of the egg nucleolus, becomes irregular in shape, and subsequently breaks up into several large fragments. Following this, the staining capacity of the cytpolasm increases somewhat while that of the nuclear contents is largely lost (fig. 15). The cytoplasm then begins to decrease in volume. The nuclear membrane disintegrates, and the entire cell shrinks until only a small remnant, containing a few irregular masses, is left (fig. 16). Such remnants may be observed in the ovary until the eggs are extruded. Their subsequent fate has not been determined. During the later stages of this process, there appears in the ad jacent cytoplasm of the egg a yolk-free, alveolar region, containing numerous minute granules and a few large, irregular masses stain ing deeply with nuclear dyes (figs. 16, 18). This region persists up to the completion of the growth period, when it becomes filled with small yolk globules and disappears. The degenerating nurse cells lie near or in contact with this portion of the egg, but in no case has cytoplasmic fusion between them been observed. The cell membranes remain intact throughout. Moreover, it is certain that the remnants of the nurse cells are not engulfed by the egg, since they can still be seen in the ovary at the time the eggs are being extruded. During these stages there appears at the periphery of the egg a structure which closely resembles the "degenerate body" described 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 7

by Allen and Banta ('29) in Moina macrocopa. It is first visible as an ill-defined mass lying in the alveolar region of the egg cyto plasm (fig. 16). It has an affinity for nuclear stains, but fails to stain with Feulgen's reagent. From its original position it migrates to the periphery, where it gradually assumes the form of a hemi sphere, bounded by a distinct membrane, and containing a finely granular material (figs. 17, 18, 19). This structure persists until the early cleavage stages, when it loses its staining capacity, sinks beneath the surface, and becomes lost to view among the yolk globules.

On the basis of its history, this body appears to be associated with the degeneration of the nurse cells and the appearance of the alveolar region in the egg cytoplasm. Its true significance, however,

is as yet obscure; hence it seems advisable to retain the name "de

generate body" for the present.

The maturation division

The condition of the egg nucleus at the close of the growth period has been described above (Lu 6, line 1). It now undergoes a single

maturation division, without reduction of chromosome number.

During the metaphase or early anaphase of this division molting

occurs, and shortly afterward the eggs are extruded into the brood chamber. The length of time elapsing between the onset of matura

tion and molting is not definitely known, although an estimate made from data collected during the fixation of these stages indicates that at

room temperature (20-22•Ž) it may in some cases be as long as

five hours. The prophase begins soon after the nucleus has reached the

periphery of the egg. The nucleolus, which heretofore has exhibited the physical characteristics of a fairly rigid body, now apparently

becomes plastic. It exhibits a great variety of shapes, and soon separates into a few large fragments. At the same time, the nucleus

assumes a highly irregular contour. The nuclear membrane becomes

faint, and disintegrates at various points, permitting the granular material to escape into the surrounding cytoplasm. These stages

are illustrated in figures 20 and 21. At this point the chromatin comes to view in the form of

granules of varying size and shape, lying in a hyaline region within the nuclear area (fig. 22). Soon after its appearance, this region

exhibits faint striations, suggesting the structure of a spindle (fig. 23). The chromatin granules occasionally seem to be joined together

by fine threads (fig. 24). More frequently, they tend to clump to

gether to produce aggregates of various sizes and shapes. In some cases two or three large masses may be produced, as in figure 21, 8 H. LUMER Cytologia8

but in most instances the aggregates are smaller and more numerous (fig. 25). The significance of this phenomenon is not at all clear, since these formations occur with no apparent regularity. After a short time, they break up again, giving rise directly to a group of small chromosomes. Meanwhile the striations appearing in the hyaline region gradually become more distinct, and finally develop into a short, barrel-shaped, anastral spindle, on which the chromosomes align themselves to form a more or less regular equatorial plate. By this time the nuclear membrane and the granular material have disap peared completely, and the spindle lies, variously oriented, in a large, yolk-free region of granular cytoplasm (figs. 27, 29). The nucleolar fragments may occasionally still be observed in this region, or in the process of migrating out among the yolk globules where they soon disappear from view. A large number of metaphases was obtained in which the chro mosomes could be counted. The sections illustrated in figures 26-29 show definitely the presence of eight of these bodies. In favorable sections, such as the polar view shown in figure 29, it may also be observed that four of them have the form of short rods, while the remaining four are ellipsoidal. This distinction is not always evi dent in equatorial views, in some of which all of the chromosomes appear to be ellipsoidal. However, it is apparent in practically all cases that some are appreciably more elongated than the others (see fig. 27). In some of the eggs the chromosomes divide at this time. Generally they remain in the early anaphase until the eggs are ex truded, although occasionally a somewhat more advanced stage is attained (see fig. 32). More frequently, however, the metaphase persists, the chromosomes dividing only after the eggs have entered the brood chamber. That the interval between the onset of the metaphase and the extrusion of the eggs is of comparatively long duration is indicated by the large proportion of these stages occurr ing in the fixed material. During this period the cytoplasmic region containing the spindle becomes filled with yolk, and the spindle is crowded to the very periphery of the egg (figs. 26, 28). Here its long axis, in the majority of cases, is parallel to the egg surface (fig. 28); less frequently it is perpendicular to it or at an inter mediate angle (fig. 26). The nucleolar fragments are now no longer visible. Several anaphases permitting a detailed study of the chromo somes were found. In the earliest stages, the two daughter groups are indistinguishable in equatorial views, due to their irregular dis 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 9 position on the spindle. Chromosome counts could be most readily made in polar views, in some of which the entire complex can be observed. Two such views are shown in figures 30 and 31; in each of these, sixteen chromosomes are discernable. In figure 31 , some of them have not yet completely separated. The more advanced stages , such as that shown in figure 32, provide more favorable material for observation of the chromosomes. This figure shows two groups of eight chromosomes each. Four of those in each group are rod shaped, and four are ellipsoidal. It may be noted that two of the rod-shaped chromosomes lie in close contiguity. Although this has been observed in other complexes (cf. fig. 29), it is as yet doubtful whether any special significance may be attached to it. During the anaphase, the spindle increases considerably in length; this is also shown in figure 32. It has already been remarked that the maturation division is completed in the brood chamber. The late anaphase and telophase are apparently passed through very rapidly, since it was difficult to secure more than a few eggs in these stages. In almost all the eggs fixed within a short time after they had entered the brood chamber, either the chromosomes were still in early anaphase, or else the divi sion had gone to completion. In the few telophases which were studied, it was evident that no polar body is budded off, the polar body chromosomes being retained in the egg cytoplasm, where they rapidly clump together. In the telophase shown in figure 33, the clumping has progressed to the point where the individual chromo somes of the group are no longer discernable, whereas those of the other group are still quite distinct. The process results in the forma tion at the periphery of the egg of a small, deeply staining mass, which disappears almost immediately. Precisely what becomes of it has not been determined. In the next stage to be found, the egg chromosomes have become transformed into a faintly-staining, vesicular nucleus. This struc ture remains for a short time at the periphery of the egg, then sinks in among the yolk globules, accompanied by a thin layer of yolk free cytoplasm. Owing to its faintness, and to the slight amount of clear cytoplasm surrounding it, I have been unable to trace the path of its migration through the deeply-staining yolk to the center of the egg, where it subsequently reappears prior to the first cleavage. Schrader ('25) reports that whether or not a polar body is budded off depends on the orientation of the spindle with reference to the surface of the egg. It may be that this is the case here also, but that because of the small number of these stages found, no in stance of the formation of a distinct polar body has come to light. 10 H. LUMER Cytologia 8

The early cleavages Cleavage in these eggs is of the superficial type, with the early interkinetic nuclei each surrounded by a large area of yolk-free cytoplasm (figs. 35, 36). At room temperature, the first division occurs about one hour after the eggs have entered the brood chamber. The chromosomes in these stages differ from those of the matura tion division. They have the form of short rods or threads, and are rather uniform in size and shape. There is no change in their number (fig. 34). The cleavage spindles possess rather poorly defined asters, which appear as areas of finely granular cytoplasm at whose periphery a few radiating fibers are visible. The interkinetic nuclei are highly vesicular structures (figs. 35, 36). The appearance of their chromatin suggests the possible ex istence of chromosome vesicles, but it could not be determined whether these actually are present, as Kuhn ('08) finds in Daphnia pulex. As cleavage progresses, the cytoplasmic areas and the chromo osmes become progressively smaller, and migrate to the periphery of the egg. In several chromosome counts made during these stages, the somatic number was always found to be eight.

Discussion The present observations agree in most respects with those of previous investigators. The diploid chromosome number in Daphnia magna is clearly eight. The same number was reported by Kuhn ('08) in both Daphnia pulex and pediculus. The number is much higher, however, in the other forms investigated. Schrader ('25) found twenty-four in the species of Daphnia studied by him, while Allen and Banta ('29) found twenty-two in Moina macrocopa. The nuclear phenomena during the growth period present several points of interest. A particularly striking element in these stages is the large, prominent nucleolus. It differs in its behavior from that of the other Cladocera which have been studied. Kuhn ('08) finds that in Daphnia pulex and Polyph.emus pedieulus this body breaks up and disintegrates completely prior to the appearance of the chromosomes, while Schrader ('25) reports the formation of a spherical shell, which disappears at the close of a period in which the entire nucleus stains intensely with iron hematoxylin. Neither of these investigators describes the peculiar fragmentation observed in the present case (see figs. 20, 21). In Moina macrocopa, accord ing to Allen and Banta ('29), the nucleolar material is present from the start in the form of several discrete bodies, whose fate is similar to that of the nucleolus in Daphnia pulex. 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 11

Nothing is known about the nature and function of the nucleolus. That it is of much importance as a source of nutritive material , as Allen and Banta ('29) assert, seems unlikely in this case . Although its disintegration coincides with the period of most rapid growth of the egg, its granular remains seem to be retained in the nucleus until yolk-formation is practically complete. Both Kuhn ('08) and Allen and Banta ('29) maintain that it is not chromatin. Its reaction to Feulgen's test here is too faint to be interpreted as a positive one; nevertheless, the possibility must be recognized that chromatin may be distributed within it. If Feulgen's test may be accepted as a criterion, it is quite evi dent, on the other hand, that the granular material to which the nucleolus gives rise is not chromatin. The same may be said of the flocculent achromatic matrix. Moreover, the threadlike configurations which appear in the earlier portion of the growth period seem to represent largely a result of the fortuitous distribution of the granules among the achromatin, rather than a consistent nuclear element. An examination of Kuhn's figures indicates that the structures which he interprets as chromatin threads are probably just such granular configurations. On the basis of the foregoing evidence, I am inclined to believe that the chromatin must be either contained in the nucleolus, or, as Allen and Banta ('29) think, in a vesicular state. It is perhaps significant in this connection that the subsequent appearance of the chromatin is correlated with the final fragmentation of the nucleolus. The prophase is markedly atypical. Aside from the extremely irregular clumping of the chromatin, there is no evidence of the formation of a spireme. It is unlikely that this clumping represents the formation of chromosome-aggregates, such as were described by Schrader ('25). It occurs before the chromosomes are discernable as such, and although a large number of these stages was examined, no manner of uniformity could be observed in the process. A study of the corresponding stage in the maturation of the sexual eggs may perhaps yield some information of value in interpreting this phenomenon. The chromatin clumps give rise directly to rod-shaped and ellipsoidal chromosomes. The paired threads and tetrad-like struc tures described by Kuhn in the prophase and metaphase respectively are lacking here. It is to be noted that the individual chromosomes exhibit some differences in structure, which have not been observed in the other forms investigated. The behavior of the nurse cells in the latter part of the growth period, together with the correlated changes in the adjacent cyto 12 H. LUMER Cytologia8

plasm of the egg, indicates that they play some part in supplying the egg with nutritive material. It is evident, however, that this material must be received in dissolved form, and not by the incorporation of the nurse cell cytoplasm. Similar changes in the egg cytoplasm are reported by Kuhn ('08) in Daphnia pulex (see his fig. 10), and by Allen and Banta ('29) in the sexual eggs of Moines macrocopa (their fig. 6). It is interesting to note that such changes are lacking in the parthenogenetic eggs of the latter species, where, according to Allen and Banta, no nurse cells are produced. That the degenerate body is in some way associated with the above phenomena seems likely from its history. A similar structure has been described only in Moina macrocopa, in which it apparently has a different function. Allen and Banta think that it arises from reorganized nucleolar material. "After the complete dissolution of the nuclear membrane, it appears in the region of the germinal vesicle. It is then an ill-defined, darkly-staining homogeneous mass. In this form it moves to its position at the opposite side of the egg..." (p. 134). Kuhn describes no such body in Daphnia pulex. Unfortunately, it has been impossible thus far to investigate the existence of chromosomal sex differences. Daphnia, magna would appear to be particularly suitable for such a study, in view of the comparatively small number of its chromosomes, and of the existence of differences among them in size and shape. On the other hand, little is known about the environmental control of sex in this species. It will be necessary to investigate this phase more thoroughly before any correlation with cytological findings can be sought. The results of the efforts made to induce male production in the present in vestigation indicate that probably environmental factors other than crowding are to be looked for as the significant elements in sex determination. In Moina macrocopa, it has been shown that the sex of the offspring to develop from parthenogenetic eggs is fixed about four hours before the eggs are extruded (Banta and Brown, '29 b) . It would be desirable to determine the corresponding effective period in Daphnia magna. It is hoped that these investigations may be carried out in the near future.

Summary 1. The parthenogenetic egg develops from one of a group of four cells, of which the remaining three become nurse cells. These degenerate, presumably supplying the egg with nutritive material , but are never engulfed by it. 2. Associated with these phenomena is the appearance in the 1937 Growth and maturation in the parthenogenetic eggs of Daphnia magna 13 egg cytoplasm of a body resembling in appearance the "degenerate body" previously described in Moina macrocopa. 3. The location of the chromatin during the growth period of the egg is uncertain. It is probably either in a vesicular state, or contained in the large, prominent nucleolus present in these stages. 4. The nucleolus partially disintegrates, producing a mass of granular material, which, on the basis of Feulgen's test, is not inter preted as chromatin. Neither is it considered to be nutritive material. 5. The chromatin is first visible in the prophase of the single maturation division, in the form of small granules. These exhibit an extremely irregular clumping, the significance of which iss un known. 6. The diploid chromosome number is eight. No reduction occurs in the maturation division, the egg developing with eight chromosomes. 7. It would be desirable to investigate the possible existence of chromosomal sex differences in these eggs. This is not feasible, however, until further information is secured concerning environ mental sex control in this species.

Literature Cited Allen, Ezra, and A. M. Banta 1929. Growth and maturation in the parthenogenetic and sexual eggs of Moina macrocopa. J. Morph. and Physiol., vol. 48, pp. 123-152. Banta, A. M. 1921. A convenient culture medium for daphnids. Science, vol. 53, pp. 557-558. -and L. A. Brown 1929a. Control of sex in Cladocera. I. Crowding the mothers as a means of controlling male production. Physiol. Zool., vol. 2, pp. 80-92. -1929b . Control of sex in Cladocera. III. Localization of the critical period for control of sex. Proc. Nat. Acad. Sci., vol. 15, pp. 71-81. Claus, C. 1876. Zur Kenntniss der Organization and das feineren Bau der Daphniden and verwandten Cladoceren. Zeit. f. wiss. Zool., Bd. 27, S. 362-402. Kiihn, Alfred 1908. Die Entwicklung der Keimzellen in den parthenogenetischen Generationen der Cladoceren Daphnia pulex de Geer and de Geer. Archiv f. Zellforsch., Bd. 1, S. 538-586. Schrader, Franz 1925. The cytology of pseudosexual eggs in a species of Daphnia. Zeit. f. Indukt. Abstammungs- u. Vererbungslehre, Bd. 40, S. 1-27. Slifer, E. H., and L. R. King 1933. Grasshopper eggs and the paraffin method. Science, vol. 78, p. 366. Weismann, August 1877. Beitrage zur Naturgeschichte der Daphnoiden. Theil II. III, and IV. Zeit. f. wiss. Zool., Bd. 28, S. 93-240.

Explanation of Plates

The magnifications given for the individual figures are those of the original pho tomicrographs and drawings. Plates 1 and 2 have been reduced two-fifths from the originals, while Plates 3 and 4 have been reduced one-sixth. Wherever the photo micrographs fail to show adequate detail, they are accompanied by line drawings. The rough outlines of all the drawings were either traced from the photomicrographs or drawn with camera lucida. The details were drawn in from the sections, using a Zeiss 1.5mm. apochromatic objective (N. A. 1.3) and a 10•~ compensating ocular. 14 H. LUMER rytologia 8

Plate 1

Figs. la, lb. Section through posterior end of ovary, showing oogonia and cells in early growth stage. •~1500. Figs. 2 and 3. Cells in later growth stages. •~825. Figs. 4, 5, 6. Egg and nurse cell. The formation of deutoplasm in the egg has just begun. •~645. Fig. 7. Egg and nurse cell, showing later stage of yolk formation. •~600. Fig. 8. Somewhat more advanced stage than that shown in figure 7. •~450. Fig. 9. Yolk formation almost complete. •~430.

Plate 2

Fig. 10. Egg nucleus, showing disintegration of nucleolus, with formation of a large central cavity. •~645. Figs. 11 and 12. Egg nuclei at end of growth period, showing the two types of nu cleolar disintegration. The staining capacity of the granular material has been largely lost. •~770. Fig. 13. Degenerating nurse cell. •~1500. Fig. 14. Degenerating nurse cell, and adjacent germ cells in growth stage. Some what earlier than the stage shown in figure 13. •~770. Fig. 15. Two nurse cells in late stage of degeneration, •~915. Fig. 16. Egg, showing yolk-free, alveolar cytoplasmic region, and adjacent remnant of a nurse cell. The degenerate body may be seen as a black, irregular mass in the alveolar egg cytoplasm.•~350. Figs. 17 and 18. Degenerate body at the periphery of the egg. Figure 18 shows also a portion of the alveolar egg cytoplasm. •~915. Fig. 19. Degenerate body at the periphery of the egg. It has assumed the definite hemispherical shape which it retains until its disappearance during the early cleavages. •~600. Fig. 20. Egg nucleus at beginning of prophase of the maturation division, showing distorted nucleo'.ar fragments. Note the irregularity of its contour. •~1600. Fig. 21. Same stage as figure 20. The chromatin is visible as a few large clumps within the hyaline region. •~1500.

Plate 3

Figs. 22a, 22b, 23a and 23b. Egg nuclei in prophase, showing the chromatin in the form of small granules. Figure 23b shows faint striations, probably the

precursors of the spindle fibers, in the hyaline region. •~1015. Figs.24a and 24b. Prophase. The chromatin granules are joined together by fine threads.•~1015. Figs. 25a and 25b. Prophase, showing chromatin in the form of sevaral clumps.•~ 1015. Figs. 26a, 2,b, 27 and 28. Metaphase, equatorial views. Note the variation in the orientation of the spindle. •~1600.

Plate 4

Fig. 29. Metaphase, polar view. •~1600. Figs. 30a, 30b and 31. Early anaphase, polar views. In figure 31, some of the daughter chromsomes are still joined. •~1600. Figs. 32a and 32b. Advanced anaphase, equatorial view. •~1600. Fig. 33. Telophase, in egg shortly after extrusion, showing clumping of the polar body chromosomes. Fig. 34. Interkinetic nuclei, two cell stage. •~450. Fig. 35. Cleavage spindle in early anaphase. The spindle has been cut obliquely, so that the asters are not visible. •~1500. Fig. 36. Interkinetic nucleus of two-cell stage. •~1015. Cytologia 8, 1937 Plate I

Lumer: Growth and Maturation in the Parthenogenetic Eggs of Daphnia magna Strauss Cytologia 8, 1937 Plate 2

Lumer: Growth and Maturation in the Parthenogenetic Eggs of Daphnia magna Strauss Cytologia 8, 1937 Plate 3

Lumer: Growth and Maturation in the Parthenogenetic Eggs of Daphnia magna Strauss Cytologia 8, 1937 Plate 4

Lumer: Growth and Maturation in the Parthenogenetic Eggs of Daphnia magna Strauss