The Segregation of the Germ-Cells in Tricho- Gramma Evaneseens

IN TRICHOGHAMMA EVANUSOENS. 161

The Segregation of the Germ-cells in Tricho- gramma evaneseens.

By .1. Bronte Gnienby, It. V., IJ.Sc, Senior Demy, Magdalen College, Oxford.

With Plate 13 and 1 Text-figure.

CONTENTS. FAOE 1. Introductory ...... 161 2. Previous "Work . . . . .162 3. Technique and Some Remarks on the Sizes of the Eggs . 164 4. Certain Stages in the Later Ovarian Oocyte . . 164 5. The Germ-cell Determinant . . . .165 6. The Segmentation of the Egg . . . .167 7. The Peculiar Form of the Blastoderm Nuclei and its Signifi- cance ...... 169 8. The Central Nuclei which Degenerate . . . 170 9. The Cytoplasm . . . . .171 10. Discussion ...... 171 11. Bibliography ...... 173 12. Explanation of Plate ..... 173

INTRODUCTORY.

IN a previous paper (4) I described a number of stages in the development of Trichogramma evanescens, but at that time I was unable to follow out the segregation of the germ-cells. Since then I have collected more material, and in this paper I am able to describe the missing stages. As far as I am aware, this is the only work that has been VOL. 63, I'AItT 2.—NKW SICKJES. 11 162 J. BRONTE GATENBY. done from the minute cytological aspect on the segregation of the germ-cells, and the segmentation of the egg in a monem- bryonic egg parasite. The presence of a germ-cell determinant at the posterior pole of the egg adds to the interest of a study of this form.

PREVIOUS WORK. Silvestri (1) has examined a form, Oophthora sein- blidis, which is another species of the genus Trichogramma, Oophthora being a synonym for the latter. Oophthora parasitises the eggs of Mamestra brassicte. The young ovarian egg contains a normally reticulate nucleus and this condenses to form a solid mass of chromatin surrounded by zona. (For this process see Text-fig. 1.) This solid nucleus may, according to Silvestri, regain its reticulate open structure ((1) p. 74, PI. 1, fig. 6), but the polar body is drawn dividing without a clear aster or centrosomes almost as I have drawn it in my previous paper. After the extrusion of the polar body or bodies, the egg nucleus looses its solid form and becomes normally reticulate, as does the sperm nucleus if the egg has been fertilised. The two fuse, and Silvestri distinctly draws a normal zygote nucleus. The zygote nucleus now divides with a fairly normal spindle, apparently with centrosomes ((1), p. 74, PL 1, figs. 18 and 14), but without asters around the centrosomes. Subsequently the segmentation nuclei divide many times by mitosis and produce a large number of normally reticulate nuclei. These migrate to the surface of the egg, leaving a few in the centre. After migration the somatic nuclei con- dense to form a solid mass of chromatin surrounded by a lighter sphere. The segregation of the germ-cells is brought about by several of the segmentation nuclei wandering down to the region of the germ-cell determinant. In another later paper (3) Silvestri has examined several other monem- bryonic parasites whose development is similar to that of Oophthora. In all there is a distinct germ-cell determinant. GERM-CELLS IN TRIOHOGRAMMA J'lVANESOENS. 163

TEXT-FIG. 1.

A., B., C, and D. are consecutive stages in the formation of the solid oocyte nucleus from a normal reticulate nucleus (A.). The chromosomes appear (B.), and a spindle is formed (F.), which is not typical; gradually the chromosomes, instead of dividing and passing to the poles of the spindle, conglomerate and entirely fuse to form the solid nucleus in D. (Original, from an Aphid parasite, probably a Chalcid.) E. Extrusion of first polar body in Ageniaspia (Encrytus) showing the non- typical " spindle." (Martin (5).) F. Anaphase of second polar body in Trichogramma showing the non-typical " spindle." (4). Smaller capitals used as reference letters have the following significations : N. Nucleus ; GCD. germ-cell determinant; PB1. and PB'. first and second polar bodies; CH. Chorion. 164 ,T. BRONTE GATJ3NBY.

TECHNIQUE AND SOME REMARKS ON THE SIZES OF THE EGGS. On PL 13 the eggs in figs. 1, 2, 3, 4, 6, and 18 are all drawn to the same scale. It will be noticed that their sizes differ often quite considerably. In PI. 13, figs. 9, 10, 11,12, 13, 14, and 19 are drawn from the widest part of the egg. Here, also, wide differences in size occur. If PI. 13, figs. 2 and 3, and figs. 9 and 19 be compared it will be seen that the amount of chromatin differs considerably in bulk in the several eggs. Moreover, the germ-cell determinants in PI. 13, figs: 1, 2, and 7 are of a different kind from those drawn in the other figures. When I sent a number of Trichogrammas for identifica- tion to the Rev. J. Waterston he pointed out that there might possibly be more than one species, but that this could not be ascertained without closer examination. Never- theless, it should be remembered that all the eggs in the ovary of the insect are graded in size, the lowermost eggs being largest, while those higher up the ovarian tube would be somewhat smaller. In connection with the amount of chromatin in the egg, an explanation might be found in the number of polar bodies extruded (whether one or two) or in whether the egg was fertilised or not. Technique was the same as that previously used, only some material was fixed in Carnoy, while other was treated for differing times in Gilson-Petrunkewitsch (from three to twenty-four hours).

CERTAIN STAGES IN THE LATER OVARIAN OOCYTE. In a very large number of forms (1, 3, 4, 5, 6, 7) it has been found that the egg nucleus while in the ovary, just before deposition, undergoes a most remarkable process. This is shown in Text-fig. 1. At first the oocyte nucleus is normally reticulate, but soon chromosomes appear in it (Text-fig. 1, A. and £.). The nuclear membrane now breaks down, and an imperfect "spindle" becomes established; in some cases the spindle seems to have fibres and a doubtful GEK.\l-CEf;LS IN TKIOHOGKAMMA EVANESCKNS. 165

aster with centrosomes (Martin (5)), in other cases (Hegner (6) and the forms I have examined) there are apparently either spindle fibres without a, cenfcrosome at each pole, or only a clear space in the egg. In this region or on the imperfect spindle the chromosomes lie, and at this stage, instead of splitting and their halves pulling apart as in normal mitosis, the chromosomes gradually close up together and fuse to forma blackly-staining mass (iron hsematoxylin). The " spindle" now disappears (Text-fig. 1, C. and D.) and the nucleus appears to be quite solid. When the first polar body is in process of formation, in most cases no distinct centro- some or aster appears. The chromosomes may appear at this stage (Martin (5)), but each individual chromosome divides by itself ; in other cases, no chromosomes could be found (4) and the polar bodies seem to be extruded by an amitotic figure. Text-fig. 1, E., is a copy of Martin's figure of Encyrtus showing the remarkable "spindle" of the first polar body.

THK GERM-CELL DETERMINANT. (a) In the Unseginented Egg.—In the previous paper I described the germ-cell determinant in the uusegmented egg. It was pointed out that the germ-cell determinant arose as a cloud of granules, which seemed to make their appearance spontaneously in the cytoplasm,.and that in the egg before the formation of the polar bodies, the determinant stained densely black with iron hsematoxylin. I am able to add a few more facts to my original descrip- tion ; in some of the eggs I have examined, the determinant instead of being solid was found to be formed of a number of separate regularly-shaped granules (PI. 13, figs. 1, 2, and 7). In these cases it was of the same size and appearance as the solid determinant, the individual granules staining very darkly. (b) During the Formation of the Germ- cells.—After the segmentation nuclei are from fifteen to twenty-five in number, they have begun to pass to the poles of the Qgg. 166 J. BRONTi GATENBY. Several which go to form the future germ-cell nuclei lie near the determinant. This is seen in progress in PL 13, figs. 4, 8, and 15, and so far the nuclei themselves are unaltered. In PL 13, fig. 16, the nuclei (G.N.) in close proximity to the germ-cell determinant have begun to alter and to differ from the other nuclei (B.N.) which form the blastoderm. Tt is just at and after this stage that the germ-cell determinant begins to alter. In PL 13, fig. 6, an interesting stage is drawn. The blastoderm nuclei are very chromatic, while at the pole of the egg is seen the germ-cell determinant. The latter had become basin-shaped, and in the hollow of the basin lie about half a dozen paler nuclei; the edges of the hollow cup-like determinant have become frayed and pieces are breaking off {F.G.C.D.). In PL 13, fig. 17, the same egg is drawn at a higher power after the microscope had been focussed down a little. The determinant is seen in optical section and in its hollow lie the pale germ-cells. The fragments of the determinant (F.G.G.D.) are here plainly seen, while the difference between blastoderm nuclei (B.N.) and germ-cell nuclei is marked. In PL 13, fig. 5, another egg is drawn nt a slightly earlier stage. The determinant has become a little irreglar in shape, its spherical contour becoming altered to give place to the basin shape. Very often the posterior end of the egg becomes constricted as in PL 13, figs. 5, 16, and 18, and marks the place where the germ cytoplasm joins the blastoderm cytoplasm. It is very difficult to say how this constriction is brought about, but ib is probably caused by a contraction of the cytoplasm in the form of a bandarouud the posterior end of the egg. In other forms (Bncarsia, Silvestri (3)) the constriction and consequent protrusion of the germ-cells is marked. After the stage drawn in PL 13, fig. 17, the determinant fragments more, and gradually becomes absorbed; but before this happens each germ nucleus has formed around it a cell wall containing part of the cytoplasm from the end of the egg. GKHM-CELLS IN TIUOHOGHAMMA EVANESOENS. 167

The loss of chromaticity of the germ-cell nuclei is synchi'onous with the dissolution of the germ-cell determinant. In some eggs it is not a little difficult to say how many nuclei are forming germ-cells at the posterior pole of the egg; and, moreover, at such a stage as that in PI. 13, fig. 16, it becomes somewhat difficult to distinguish between blastoderm and future germ-cell nuclei. The germ-cell determinant appears to be of a plastic nature, for were it not so the occurrences which tiike place as depicted in PL 13, fig. 17, would never happen. Moreover, in many cases the germ-cell determinant in the unsegtnented egg, or during the time the egg is segmenting, often shows curious lumps on its surface, as in PI. 13, fig. 3 or 16 at XX. In a few cases one can find large stray pieces of germ-cell determinant at a stage before the latter begins to fragment (PI. 13, fig. 4, G.X.), and in a very large number of examples one finds small granules scattered in proximity to the germ-cell determinant (PL 13, figs. 1, 3, and 15, G). These granules occur here and there throughout the egg (PL 13, figs. 9, 12, and 13). The exact nature of such bodies is unknown to me. In my previous paper I found them near the second polar body "spindle" ((4), PL 13, fig. 11 A). I feel sure that these grains are not centrosomic in nature, but those found in the segmenting egg may be broken off pieces of chromatin. In PL 13, fig. 19, is drawn a transverse section through the middle of an egg; the latter seemed about to give off a polar body, but the granules (G.) were not, I consider, the degenerated polar bodies. In PL 13, fig. 9, such granules were present in large numbers.

THE SEGMENTATION OP THE EGG. In my previous paper (4) I described the egg just after deposition and the extrusion of the first polar body. In PL 13, figs. 9 and 19, are transverse sections of two eggs ; in both the nucleus is not of the usual reticulate kind, but is formed of an irregularly-shaped, darkly-staining chromatin lump. In PL 13, fig. 19, the nucleus has gone to the surface 168 J. BBONTfi GATENBY. of the egg and seemed to be elongating preparatory to the formation of a polar body. In PI. 13, fig. 9, the nucleus was distinctly constricted in the middle as if it were dividing by amitosis; in my figure of the second polar body in my previous paper (4) the "spindle" is a dumb-bell-shaped chromatin mass, quite unlike any stage in mitoais. In PI. 13, fig. 1, the egg contains two densely staining, apparently solid nuclei; at P. is an elongate body, which is the remains of either one or two polar bodies; in PI. ]3, fig. 13, another egg is drawn in transverse section ; the nuclei are chromatin lumps, and at X. the nucleus is constricting' as if in amitosis. In PI. 13, fig. 2, all the nuclei are apparently in division, and all are solid dumb-bell-shaped figures. Another egg is drawn in PI. 13, fig. 3; at X one nucleus is distinctly constricting; this is more plainly shown at a higher power in PI. 13, fig. 8. I have examined a large number of eggs; stages in division are rather difficult to find, but in no one case did I find any stage resembling karyokinesis; without exception every nucleus in early segmentation stages is solid, blackly staining, and no amount of extraction reveals a reticulate structure. When the nuclei are from twenty to fiftj' in number they gradually disperse as division goes on, till they wander towards the poles of the egg (PL 13, figs. 2, 3, and 8). The germ nuclei are then segregated as previously explained; those which happen to lie near the germ cell determinant eventually forming germ-cells. As soon as segmentation has gone on to such a stage that nuclei occupy the egg in the manner drawn in PI. 13, figs. 5 and 10, segmentation stops; no further nuclear divisions take place till at a later stage. When the egg has reached the stage when the nuclear division temporarily ceases and the end of segmentation arrives, it contains some 120 to 150 nuclei. The number seems very viiriable. Just at or before this final period differences become apparent in the nuclei. In PI. 13, fig. 15, nearly all the nuclei are solid, but at B.N. one nucleus has become frayed at its edges, and a nuclear membrane becomes GERM-CELLS IN TKJCHOGKAMMA EVANESCKNS. 169

demonstrable; the same and later stages of this process am seen in PI. 13, rigs. 5, 16, and 17. By this process all the nuclei eventually become spherical or ovoid structures containing coarse chromatin lumps. Now it is constantly found that the chromatin lumps in the nuclei are specially oriented after a while. The main mass of chromatin is seen to lie outwards, while inwards is left quite often a single grain, or two grains, of chromatin, as shown in PI. 13, fig. 14. The segmentation nuclei lie throughout the whole region of the egg, as shown in PI. 13, fig. 10, or in an earlier stage in PL 13, fig. 3. When the clianges in the solid nuclei take place, which cause the formation of the roughly reticulate nucleus to appear, many, if not all, the nuclei lying in the central middle region of the egg seem to undergo this process, but soon afterwards they become dark again, as shown in PI. 13, fig*. 4, 10, 11, 12, and 13. In PI. 13, fig. 10, this is seen in progress; there are thirteen nuclei in the field, four are markedly blackly staining; one of these lies at the periphery of the egg. In tlie centre are two black nuclei, and one healthy one ; in PI. 13, fig. ] 1, all the central nuclei had shrunken and become black (staining), and one at the upper peripheral region had also changed in this way. In PI. 13, fig. 4, of the twenty-two central nuclei, three were of the rough reticulate form, while all the others were solid; this figure shows that many centrally disposed nuclei do become roughly reticulate before they degenerate. In JJ1. ]3, figs. 5, 6, 12, 14, and 18, subsequent stnges are drawn. The fate of these discarded chromatin lumps has already been followed out (4).

THE PECULIAR FORM OF THE BLASTODKRM NUCLEI AND ITS SIGNIFICANCE. As far as I am aware, no other case is known in which such a peculiarly oriented somatic nucleus is found. In PI. 13, figs. 10, 11,14, 17, and 18, the usual form of somatic nucleus 170 J. BRONTE GATKNBY. is seen; there is nearly always an inner grain and an outer larger lump to the nucleus (see (4) previous paper). In several cases the outer lump was present (PI. 13, figs. 4 and 12), but no inner lump could be found. I am quite at a loss to account for the remarkable form of the nuclei in this Hymenopteron. The only suggestion I can make is that the inner granules represent that chromatin, Avliich is unnecessary and will be extruded, and the reason for the special orientation of this little granule inwards is that the inner region of the egg is at this period the dumping-ground of waste matter. As I pointed out before (4) this inner matter is later extruded. It should also be remembered that the egg at this period is growing without vitellophags or yolk which are found in other insect eggs.

THE CENTRAL NUCLEI WHICH DEGENERATE (PI. 13, figs. 4, 5, 6, 10, 11, 12, 14, and 18). In an ordinary insect's egg, after the zygote nucleus has divided many times in the interior of the egg, a large number of these nuclei migrate to the surface to form the blastoderm. Certain others are left inside the egg, and these form the yolk cells or vitellophags, whose function is to make ready for absorption the stores of yolk and fat which fill the egg. These nuclei, with their island of surrounding cytoplasm, live long after the formation of the mesenteron, and in the majority of cases become very large in the process of attacking the yolk stores. There seems to be little doubt that the centrally placed nuclei which degenerate in the case of Trichogramma are the representatives of the vitellophags of other holometabolous insects. The reason they degenerate seems to be that they have no yolk to utilise for the food of the embryo, since the latter gains its nutriment in another manner. I consider the subsequent extrusion of a part of the centre of the egg containing these nuclei has no other significance than that of a method of removing from the egg waste matter (previous paper (4), p. 162). GERM-CELLS IN TRICHOGFAMMA EVANESCENS. 171

THE CYTOPLASM. The eggs of all the figures, with the exception of PI. 13, figs. 9, 18, were fixed for four hours in Gilson-Petrnnkewitsch ; those of PI. 13, figs. 9 and 18, were fixed overnight in the same fixative; in the latter drawings the vacuoles were more markedly reticulate, while in the other figures the vacuoles were smaller, evener, and the entire cytoplasm less " stringy " ; this is the usual effect, which is due to the length of exposure to the solvent properties of the acetic-alcohol of the fixative. When the egg reaches the stage drawn in PI. 13, figs. 10 and 12, each nucleus is often seen to be surrounded by a halo of cytoplasm. This seems preparatory to the establishment of a cell-wall boundary between each nucleus (see PI. 13, fig. 18). I could not find the boundary line till just before the stage drawn in this figure, and, moreover, this line did not entirely surround the nucleus, only passing inwards and ending abruptly. This does not apply to the germ nuclei, which are surrounded by a complete cell-wall. It seems that in later stages this cell-wall around the outer peripheral edge of each blastoderm nucleus is obliterated when the germ-layers are being formed. In certain other forms Silvestri (2) describes a like occurrence.

DISCUSSION.

Germ-cell Determinant.—It is not proposed to discuss the determinant at any length in this paper, but it can be pointed out that this structure cannot consist altogether, if at all, of mitochondrial matter. This seems to be shown by the fact that Carnoy and Petrunkewitsch and Flemming- acetic apparently do not dissolve away any part of the determinant. The latter appears to react to fixatives and stains in much the same way as Amphibian yolk discs, but at present I take the view that the germ-cell determinant of Trichogramma consists in part or whole of cytoplasmic material. 172 j. BRONT£ GATENBY. Though it is difficult to be sure, I think that the function of the determinant is nutrimental. The germ-cells early become segregated from the somatic region of the egg, and while the somatic nuclei are, so to speak, undergoing phenomenal changes, losing chromatin, becoming altered from time to time or occasionally degenerating, the germ nuclei remain resting. It is tempting to consider that the dense germ-cell determinant is, during this time and later on, acting as a reserve store, while many changes take place in the somatic nuclei. The latter must needs procure their nutriment from the Donacia (host) egg: and during this they are also becoming- disposed to form the germ-layers. The function of the determinant seems to be that of preventing the germ-cell nuclei from being exposed to the uncertain conditions existing- elsewhere in the segmenting egg and embryo. It is also noteworthy that the germ nuclei at first differ not at all from the other segmentation nuclei, and certain nuclei become germ-cell nuclei only because of tlieir accidental position in the egg, towards close of segmentation, in the region of the posterior pole of the egg. With regard to the important question of the segmentation of the egg, it seems clear that typical mitotic spindles do not exist at first. There seems to be a great many interesting facts with regard to the nuclear behaviour during early stages in many of the parasitic Hymenoptera, and I feel sure that further research on this subject would repay the expenditure of time and patience needed to study these interesting forms. It may be possible that the stages I have found which look so much like amitosis are really anaphases during which the chromosomes have fused, but it is now an established fact that in certain forms no centrosomeor normal spindle exists at special periods of segmentation (1, 4, 5), and I consider that neither centrosomes nor mitotic spindles of any sort will be found in Trichogramnia in early stages. (ifiRM-UELLS IN TltlCHUGltAMMA KVANESOENS. 173

BlBLIOGKAl>HY. 1. Silvestri, F,—" Contribuzioni alia eouoscenza biologica degli Irnen- otteri parasitici i-4," 'Boll. Scuol. sup. Agrio. Portici,' T. 1 and 3. 2. " Prime fasi di svillupo del Copidosoma buyssoni (Mayr), Imenottero Calcidide," ' Anat. Anz.,' Bd. 47. 8. " Struttura dell'ovo e prime fasi di sviluppo di alcuni Imen- otteri parassiti," 'Boll. Scuol. sup. Agrie. Portici,' vol. x, 15. 4 Gatenby, J. Bronte.—"The Embryonic Development of Tricho- gramma evalnescens,"" ' Quart. Journ. Micr. Sci.,' vol. 62, pt. 2. 5. Martin, F.—" Zur Entwicklungsgescbichte des polyembryonalen Chalcidiers Ageniaspis (Encyrtus) fuacicollis, Dalai.," 'Zeit. wiss. Zool.,' Bd. 110. 6. Hegner, R.—' The Germ-cell Cycle in Animals,' Macmillan, 1914. 7. " Studies on Germ-cells IT," ' Journ. Morph.,' xxvi, 15.

EXPLANATION OF PLATE 13, Illustrating Mr. J. Bronte Gatenby's paper on "The Segrega- tion of the Germ-cells in Tricliogramma evanescens."

LETTERING. X. dividing nucleus (?). B.N. Blastoderm nucleus. B.N. Degener- ated nucleus. F.G.C.D. Fragments of germ-cell determinant. 0. Granule of unknown nature. O.C. Germ-cells. G.C.D. Germ-cell determinant. G.N. Germ-cell nucleus, N. Nucleus. Figs. 1, 2, 3, 4, 6, and 18, drawn at magnification of 1383. Figs. 5, 7, 8, 9, 10, 11-17, and 19 drawn at magnification of 2100. Fixed either in Carnoy or Gilson-Petrunkewitsch, stained in iron hamatoxylin.

Fig. 1.—Segmentation stage, two nuclei, at P.—either one or two polar bodies came into focus at a different level. Germ-cell determinant granular. Fig. 2.—Later stage, about ten nuclei, apparently dividing by an amitotic figure. Germ-cell determinant as in Fig. 1. Fig. 3.—Later stage, nuclei " nesting " except at X., where one seems to be dividing. Germ-cell determinant solid. Fig. 4.—End of segmentation, inner nuclei necrotic. Staining blackly. 174 J. BRONTfe GATENBY.

Tig. 5.—End of segmentation, showing segregation of germ-cell nuclei near determinant. At B.N. are the inner degenerated nuclei. Kg. 6.—Blastoderm stage nearing the time when the germ-cells become completely isolated. Fig. 7.—Posterior pole of egg showing granular germ-cell determinant. Fig. 8.—Lower part of posterior pole of egg showing dumb-bell shaped " spindles " X., at Y. future germ nucleus, cut in half, another part in the next section. Figs. 9-14.—Transverse sections of mid-region of egg. Fig. 9, egg just laid, nucleus apparently beginning to constrict preparatory to formation of first polar body. Figs. 10, 11, 12, and 14, stages in segmentation showing peculiar nuclei. Fig. 13, early segmentation stage showing irregular nuclei. Figs. 15, 16, and 17.—Three stages in the segregation of the germ- cells. Fig. 17 drawn from same egg as that in Fig. 6, only at a lower focal level. Fig. 18.—Blastoderm after segiegation of germ-cells. Fig. 19.—Unsegmental egg, nucleus near surface preparatory to extruding polar bodies.