Growth and Maturation in the Parthenogenetic Eggs of Daphnia

Growth and Maturation in the Parthenogenetic Eggs of Daphnia 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 Cladocera 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. Daphnia magna 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 animals 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 animal 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.

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