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Mitosis and meiosis in Rhodnius Prolixus: The fine structure of the spindle and diffuse kinetochore
© 1967 by Academic Press Inc.
J. ULTRASTRUCTURE RESEARCH 18, 489-501 (1967)
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Mitosis and Meiosis in Rhodnius Prolixus: The Fine Structure of the Spindle and Diffuse Kinetochore 1 ROBERT C. BUCK
Department of Anatomy, University of Western Ontario, London, Canada Received July 18, 1966 The chromosomes of mitotic cells of the epidermis of Rhodnius prolixus possess an extensive layer of material interpreted as the diffuse kinetochore. Only a few fibers insert into it. Some bundles of continuous spindle fibers pass through the plane of the metaphase plate in gaps between the chromosomes. Other bundles perforate the chromosomes. Meiotic cells do not show the diffuse kinetochore. The dyads, however, are perforated by continuous spindle fibers. The fibers are not in bundles, but each passes separately through the chromosomes. The chromosomes of hemipteran insects are characterized by the absence of a localized point of attachment of the spindle (4). The spindle fibers appear to extend from the whole polar surface of the chromosome, so that during anaphase the chromatids separate parallel to each other, rather than with trailing arms, as in the conventional anaphase. At no time do the chromosomes show any differentially stained region that could be interpreted as a localized kinetochore. Although there are great variations in spindle morphology among members of the Hemiptera, the intensive study of these insects particularly by Schrader (15), Hughes-Schrader (3), Ris (11), and HughesSchrader and Ris (5) has led to the conclusion that the diffuse kinetechore is invariably present. Certain other organisms also have a diffuse kinetochore, in particular, the plant Luzula (1) and probably also the Brazilian scorpion (see discussion in 5). Accounts of the fine structure of the spindle in hemipteran insects appear to be confined to a study (19) in which the meiotic chromosomes of the milkweed bug were observed by a new spreading technique. The bug Rhodnius offers favorable material for the electron microscopic study of mitosis because of the ease with which dividing epidermal cells can be found by means of the method described by Wigglesworth (18). Meiotic cells are readily observed in testis. 1 Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.
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R.C. BUCK MATERIALS AND METHODS
To induce mitosis in epidermal cells, insects of fourth or fifth instar were placed on the ears of rabbits until they bad taken a full meal of blood. They were then maintained at 26°C in a humid atmosphere for 5 or 6 days. The cuticle and adherent layer of epidermal cells was then dissected off under the surface of fixative. It was left in fixative, which was 4 % glutaraldehyde in 0.1 M p h o s p h a t e buffer (pH 7.0), for 7 hours at 4°C. After it was thoruughly washed in 5.4% buffered sucrose solution, the tissue was placed in 2 % osmium tetroxide for 1 hour, dehydrated in alcohol and acetone, and embedded in Vestopal W. Small squares of the tissue were positioned at the ends of the block so that sections could be cut parallel to the surface. F o r the study of meiosis the testis of adults fed a few weeks previously was dissected under the surface of fixative, then processed as above. After lead staining (10) the sections were observed in an R C A E M U 3 D electron microscope operated at 65 kV. OBSERVATIONS
Mitosis in epidermal cells Several specimens showed division figures, a n d over 80 cells in all stages of mitosis have been studied. A n a d v a n t a g e of studying cells in the single layer of Rhodnius epidermis is t h a t sections always pass t h r o u g h the cell parallel to the long axis of the spindle. The interp r e t a t i o n of the i m a g e is t h e r e b y greatly simplified. Fig: 1 shows a typical m e t a p h a s e configuration. The spindle takes u p a large p a r t of the cell, the m i t o c h o n d r i a a n d m e m b r a n e s of the e n d o p l a s m i c r e t i c u l u m being c r o w d e d to the p e r i p h e r a l region. T h e p r i n c i p a l c o m p o n e n t s of the spindle zone are the spindle fibers (or tubules) a n d the m a n y r i b o s o m e s t h a t lie between them. Scattered small dense granules of u n k n o w n c h a r a c t e r are also found. There is a t e n d e n c y for spindle tubules to be a r r a n g e d in bundles, p a r t i c u l a r l y as they a p p r o a c h the region of the m e t a p h a s e plate. The bundles can be seen in Fig. 1, b u t they are m u c h m o r e obvious in thicker sections because their density is c o n s i d e r a b l y lower t h a n that of the mass of r i b o s o m e s t h r o u g h which they pass. The bundles of fibers pass c o m p l e t e l y t h r o u g h the p l a n e of the m e t a p h a s e plate. Here their course takes one of two forms, namely, between the c h r o m o s o m e s (arrows A) or t h r o u g h the substance of a c h r o m o -
FIG. 1. Metaphase in epidermal cell. The chromosomes lie in the plane of the equator. They appear rather short in this picture because they have been sectioned transversely. A central light area is seen between each pair of chromatids. Diffuse kinetochores do not show to advantage in this plane of sectioning, but dense material representing the kinetochores lies on the polar surface of two chromosomes. Spindle fibers are mainly in bundles, four of which are marked by arrows. Arrows A indicate bundles of continuous fibers that pass through the plane of the metaphase plate in gaps between chromosomes. Arrows B indicate bundles of fibers that perforate the chromosomes. The fibers converge toward the poles. A centriole at one of the poles has been sectioned, x 15,200.
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some (arrows B). Where a bundle passes through a chromosome it creates a light streak with straight and parallel margins (Figs. 1 and 2), in contrast to the gaps between chromosomes, which have irregular margins. If this were not the case, it might be difficult to distinguish a chromosome perforated by a bundle from two separate chromosomes. The spindle tubules converge to the centrosphere, where they come into close association with the centrioles. The details of this association have not been observed, but there seems to be no unusual feature of this part of the mitotic spindle of Rhodnius. The metaphase chromosomes of the epidermal cells of Rhodnius are strikingly different from those of nonhemipteran species: (a) They show consistently such obvious and frequent perforations by spindle tubules bundles, as just mentioned. (b) The chromosomes exhibit a light central area (Figs. 1 and 3). This appearance represents the characteristic disjoined form of the chromatids in species with diffuse kinetochores. Light cores are, of course, not seen in the anaphase chromosomes (Fig. 2). (c) An extensive, but incomplete, band of rather dense material lies close to the surface of the chromosomes (Fig. 3). This layer is similar in density to the substance of the kinetochore described by numerous authors, and similar in its relation to the surface of the chromosome, from which it is separated about 100-300 A. It possesses a finer granularity than that of the chromosome. Only occasionally are fibers of the spindle seen passing into this material. They are hard to identify because they enter it singly, rather than in the bundles characteristically found in species with localized kinetochores. I have not been able to determine whether the diffuse kinetochore material is in the form of a tube around the metaphase chromosome, or whether it coats only the polar surfaces. In anaphase it seems to be confined to the polar surface. Meiosis in Rhodnius testis
The metaphase configuration of the spindle in a primary spermatocyte is shown in Fig. 4. The spindle region of this cell is characterized by great numbers of membranes of the endoplasmic reticulum that radiate from the poles toward the chromosomes (Figs. 4 and 5). The chromosomes are partially enclosed by similar double membranes (Figs. 4 and 6). FIG. 2. Anaphase in epidermal cell. The light core seen in metaphase chromosomes is not present at anaphase. A perforating bundle of spindle fibers is marked by the arrow, x 16,500. Fx~. 3. A chromosome at metaphase in an epidermal cell. The chromosome has been sectioned along its long axis, and a part at the middle lies almost out of the plane of the section. The disjunction of the chromatids is represented by the light central core. The polar surfaces show finely granular dense material along most of the length, interpreted as the diffuse kinetochore. There is a distinct paucity of spindle fibers inserting into the kinetochores, although they are preserved in this specimen. × 20,300.
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Spindle tubules in bundles are not obvious. Careful examination at high magnification, however, reveals a large number of spindle tubules that lie separate from each other (Fig. 6). Some of them pass through the metaphase plate in gaps between the chromosomes (Fig. 6). Others lie between the chromosome and the incomplete membrane that surrounds it (Fig. 7). Many spindle tubules pass singly through the substance of the chromosome. When the metaphase plate is sectioned at right angles to the polar axis the profiles of spindle tubules embedded in chromosome substance can be seen (Fig. 7). It is only possible to see them because each tubule is surrounded by a light halo about 4 0 0 / ~ in diameter. The tubules enter the chromosomes in gaps between pieces of membrane (Fig. 6). The extensive layer of dense material that was found applied to the chromosomes in the epidermal cells is missing in the spermatocytes. There are, however, occasional dense points at the polar surface of the chromosomes which may represent kinetochores. Another striking difference from the epidermal mitotic cells is in the shape of the metaphase and anaphase chromosomes. The bivalents are positioned, not in the transverse axis, but in the longitudinal axis of the cell (Figs. 1 and 6). Their separation at anaphase involves the production of a thin, tapering connection between them that probably represents the terminalization of chiasmata. Similar connections have been observed in certain aphids (11). An incidental finding in a Rhodnius spermatocyte seems worth reporting in view of recent work on other species in which a new organelle has been described (17). The organelle is shown in Fig. 8. In association with a well condensed prometaphase chromosome is a banded structure consisting of 4 rows of dense granules embedded in some almost homogeneous material of lower density. In the original print there is a fine dense line mid-way between the rows of granules. According to Wettstein and Sotelo (17) these structures, which they have called "composite bodies," are related to the synaptinemal complexes, and they are found only in primary spermatocytes. DISCUSSION The kinetochore in conventional mitosis is the region of the chromosome at which the chromatids are held together at metaphase. Also, when the anaphase chroFIo. 4. Metaphase in primary spermatocyte. The cell is cut parallel to the spindle axis, as centrioles at both poles are sectioned. The spindle contains many radiating membranes, but spindle fibers are not visible at this magnification. The chromosomes are surrounded by incomplete double membranes. The very dense, coarsely granular material at the equator is thought to be derived from the nucleolus. x 16,400.
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mosomes move toward the poles they do so with the kinetochores leading, and the chromosome arms trailing behind. The kinetochore is recognized in stained preparations as a spot that is Feulgen negative (primary constriction). The observation that in hemipteran insects the chromosomes remain parallel to each other as they separate, and also that no kinetochore is morphologically recognizable, led Schrader (15) to postulate the existence of a diffuse kinetochore extending along the length of the chromosome. The observations on this type of kinetochore were extended by HughesSchrader (3) working particularly with coccids and by Brown (1) on the plant Luzula. Experimental evidence for the existence of a diffuse spindle attachment in Steatococcus was obtained by Hughes-Schrader and Ris (5), who observed that fragmented chromosomes produced by irradiation behaved as intact chromosomes in subsequent divisons. This was interpreted to indicate that their activity was mediated through a part of the kinetochore that remained with the fragments. By contrast, fragments of orthodox chromosomes are not mitotically active. In the epidermal cells of Rhodnius a structure of the character postulated by these workers has, in fact, been observed in the present study. The diffuse kinetochore appears to be composed of dense material comparable to that of the localized kinetochore except that it extends over most of the length of the chromosome. The question of its kinetic function cannot be settled by electron microscopy. However, it is somewhat disturbing to find so few spindle fibers inserting into the diffuse kinetochore. Of course, if the number of fibers converging to a localized kinetochore, only 10 or less in the sea urchin (2), were to be spread over the relatively huge area of the diffuse type, their concentration would certainly be very low. On the other hand, the fact is that large expanses of the diffuse kinetochore do not show any chromosomal fiber insertions. This finding appears to be difficult to reconcile with the suggestion that the density of the conventional kinetochore may be due to dense material closely associated with the individual tubules as they insert into it (6, 8). Moreover, if the whole kinetochore has a function related to chromosome movement it becomes difficult to visualize such a kinetic action operating through the considerable distance between the fibers. Why is there so much dense material per spindle fiber? Is this the substance of the postulated chromosome pellicle (16)? One might speculate on the possible stiffening effect provided by such a layer on chromosomes showing parallel disjunction, but this does not answer the question of whether
FIG. 5. Polar region at early anaphase in spermatocyte. Between the membranes of the spindle are many tubules that appear to radiate from the immediate vicinity of the centrioles. Part of a chromosome is seen at the right margin, x 34,000. FiG. 6. Disjunction in spermatocyte. The dyads develop tapering processes as they move apart. At several points spindle tubules are seen entering the substance of the chromosome. × 27,500.
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or not the diffuse kinetochore has the active role in chromosome movement that has been ascribed to the localized kinetochore (reviewed by Mazia, 9). It should be emphasized that although the coccid division figure studied in detail by Hughes-Schrader (3) is an example of the diffuse kinetochore type, there are peculiarities of the mitotic apparatus in these species that are not found throughout hemipteran insects. The coccid mitotic apparatus is very simple. According to Hughes-Schrader (4), "Mitosis is possible with no new structural differentiations in the cell other than chromosomal fibers and interzonal connectives. Asters and centrioles may be absent, and in none of the variant coccid figures are any continuous spindle fibers demonstrable." The term "interzonal fibers" describes a part of the spindle extending only between disjoining chromosomes. Hughes-Schrader believes that elongation of a stem body of interzonal origin is part of the mechanism of anaphasic movement of the chromosomes in coccids. The mitotic apparatus of Rhodnius obviously differs from that of the coccids in that orthodox continuous fibers are demonstrable. However, the presence of the diffuse kinetochore in Rhodnius tempts one to try to interpret the coccid figure in the light of the present findings. In a study of meiosis in the cockroach Krishan and Buck (7) stated that "... it is possible that the fibers identified at metaphase as continuous ones, become the interzonal fibers of anaphase . . . . If such a case is proved by further observation, the distinction between the interzonal and continuous fibers will tend to diminish." It is now apparent in Rhodnius that spindle fibers passing between disjoining chromosomes do extend through into the polar spindle, and thus constitute a special part of the continuous fiber system. Perhaps they should be called perforating fibers. They are found in both mitotic and meiotic cells, although in the former t h e fibers are in bundles, and in the latter they are individual units. A similar finding has been demonstrated in other species. 1 In a paper on the ameba Pelornyxa Roth and Daniels (13) have illustrated the uninterrupted passage of spindle fibers through the chromosomes, although a recent report from the same laboratory (14) stated that chromosomes of the corn borer spermatocyte were not traversed by 1 Since this paper was submitted for publication O. Behnke and A. Forer have independently described spindle tubules that penetrate the telophase nucleus of the crane fly spermatocyte in the same manner as those in Rhodnius spermatocytes [O. Behnke and A. Forer, Science 153, 1536 (1966).
FIG. 7. Metaphase in spermatocyte. The plane of the section is at right angles to the polar axis. Several spindle tubules perforating the chromosome are shown with short arrows. Each is surrounded by a light halo. Some of the spindle tubules lying between the chromosome and the incomplete membrane around it are indicated by long arrows, x 100,000 FIG. 8. Prometaphase in spermatocyte. A "composite body" is shown in association with a chromosome. x 73,000.
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spindle fibers. Robbins and Gonatas (12) observed that in HeLa cells continuous spindle tubules were sometimes seen traversing a chromosome. They suggested that this feature might result in anomalous chromosome movement at anaphase. These observations, and those on Rhodnius, cannot be ascribed to the artifact mentioned by Harris (2) of the apparent fusion of bundles of chromosomal and continuous fibers. Returning to the descriptions of the coccid figure, it is obvious that light microscopic examination will not differentiate between interzonals and this type of continuous fiber. In view of the present findings in Rhodnius, it seems reasonable to suggest that the stem body in coccids may be composed of continuous tubules that have passed through the chromosomes. It is regrettable that the fine structure of the coccid spindle has not yet been studied, for as electron microscopic observations accumulate, the existence of interzonal fibers, in the original meaning of the term, becomes questionable. The observation that some of the continuous fibers pierce the chromosomes has a bearing on the understanding of anaphasic movements. Theories of karyokinesis would have to incorporate the idea that chromatids or homologues would remain aligned on the long axis of the spindle by these perforating fibers. The relationship between the perforating fibers and the chromosomes might take any of three forms: (a) Only a passive positioning, like a bead on a thread; (b) a firm attachment between the chromosome and the fibers; (c) some more active process might be involved, perhaps comparable to the activity ascribed to the kinetochore. On the evidence yet available there seems to be no basis for choosing among these possibilities. In view of the perforation of chromosomes by the fibers in Rhodnius epidermal cells, it is not difficult to understand how they might assist in maintaining the characteristic parallel disjunction. Obviously, no chromosome arms could form if the whole chromosome were held in position by continuous fibers. I am grateful to Dr. W. F. Baldwin, Chalk River Nuclear Laboratories, for his encouragement and for supplying me with the insects used in this study. Mr. William Daniels and Mr. Charles Jarvis provided technical assistance. REFERENCES 1. BROWN, S. W., Univ. California Publ. Botany 27, 231 (1954). 2. HARRIS, P., J. Cell Biol. 14, 475 (1962). 3. HUGH~S-SCHRADER,S., J. Morphol. 70, 261 (1942). 4. - - - - Advan. Genet. 2, 127 (1948). 5. HUGHES-SCHRADER,S. and RIs, H., J. Exptl. Zool. 87, 429 (1941). 6. KgISHAN, A. and BUCK, R. C., J. Cell Biol. 24, 433 (1965). 7. - - - - Y. Ultrastruct. Res. 13, 444 (1965). 8. LUYKX, P., Exptl. Cell Res. 39, 643 (1965).
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9. MAZIA, D., in BRACHET, J. and MIRSKY, A. E. (Eds.), The Cell, Vol. III. Academic Press, New York, 1961. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). RIs, H., Y. Exptl. Zool. 90, 267 (1942). ROBBINS,E. and GONATAS,N. K., J. Cell Biol. 21, 429 (1964). ROTH, L. E. and DANIELS, E. W., J. Cell Biol. 12, 57 (1962). ROTH, L. E., WILSON, H. J. and CHAKRABORTY,J., .[. Ultrastruct. Res. 14, 460 (1966). SCHRADER, F., Cytologia 6, 422 (1935). -Mitosis, 2nd ed. Columbia Univ. Press, New York, 1953. WETTSTEIN, R. and SOTELO, J. R., Chromosoma 17, 246 (1965). WIG~LESWORTH,V. B., The Control of Growth and Form: A Study of the Epidermal Cell in an Insect. Cornell Univ. Press, Ithaca, New York, 1959. 19. WOLFE, S. L. and JOHN, B., Chromosoma 17, 85 (1965). 10. 11. 12. 13. 14. 15. 16. 17. 18.
J. ULTRASTRUCTURE RESEARCH 18, 489-501 (1967)
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Mitosis and Meiosis in Rhodnius Prolixus: The Fine Structure of the Spindle and Diffuse Kinetochore 1 ROBERT C. BUCK
Department of Anatomy, University of Western Ontario, London, Canada Received July 18, 1966 The chromosomes of mitotic cells of the epidermis of Rhodnius prolixus possess an extensive layer of material interpreted as the diffuse kinetochore. Only a few fibers insert into it. Some bundles of continuous spindle fibers pass through the plane of the metaphase plate in gaps between the chromosomes. Other bundles perforate the chromosomes. Meiotic cells do not show the diffuse kinetochore. The dyads, however, are perforated by continuous spindle fibers. The fibers are not in bundles, but each passes separately through the chromosomes. The chromosomes of hemipteran insects are characterized by the absence of a localized point of attachment of the spindle (4). The spindle fibers appear to extend from the whole polar surface of the chromosome, so that during anaphase the chromatids separate parallel to each other, rather than with trailing arms, as in the conventional anaphase. At no time do the chromosomes show any differentially stained region that could be interpreted as a localized kinetochore. Although there are great variations in spindle morphology among members of the Hemiptera, the intensive study of these insects particularly by Schrader (15), Hughes-Schrader (3), Ris (11), and HughesSchrader and Ris (5) has led to the conclusion that the diffuse kinetechore is invariably present. Certain other organisms also have a diffuse kinetochore, in particular, the plant Luzula (1) and probably also the Brazilian scorpion (see discussion in 5). Accounts of the fine structure of the spindle in hemipteran insects appear to be confined to a study (19) in which the meiotic chromosomes of the milkweed bug were observed by a new spreading technique. The bug Rhodnius offers favorable material for the electron microscopic study of mitosis because of the ease with which dividing epidermal cells can be found by means of the method described by Wigglesworth (18). Meiotic cells are readily observed in testis. 1 Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.
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To induce mitosis in epidermal cells, insects of fourth or fifth instar were placed on the ears of rabbits until they bad taken a full meal of blood. They were then maintained at 26°C in a humid atmosphere for 5 or 6 days. The cuticle and adherent layer of epidermal cells was then dissected off under the surface of fixative. It was left in fixative, which was 4 % glutaraldehyde in 0.1 M p h o s p h a t e buffer (pH 7.0), for 7 hours at 4°C. After it was thoruughly washed in 5.4% buffered sucrose solution, the tissue was placed in 2 % osmium tetroxide for 1 hour, dehydrated in alcohol and acetone, and embedded in Vestopal W. Small squares of the tissue were positioned at the ends of the block so that sections could be cut parallel to the surface. F o r the study of meiosis the testis of adults fed a few weeks previously was dissected under the surface of fixative, then processed as above. After lead staining (10) the sections were observed in an R C A E M U 3 D electron microscope operated at 65 kV. OBSERVATIONS
Mitosis in epidermal cells Several specimens showed division figures, a n d over 80 cells in all stages of mitosis have been studied. A n a d v a n t a g e of studying cells in the single layer of Rhodnius epidermis is t h a t sections always pass t h r o u g h the cell parallel to the long axis of the spindle. The interp r e t a t i o n of the i m a g e is t h e r e b y greatly simplified. Fig: 1 shows a typical m e t a p h a s e configuration. The spindle takes u p a large p a r t of the cell, the m i t o c h o n d r i a a n d m e m b r a n e s of the e n d o p l a s m i c r e t i c u l u m being c r o w d e d to the p e r i p h e r a l region. T h e p r i n c i p a l c o m p o n e n t s of the spindle zone are the spindle fibers (or tubules) a n d the m a n y r i b o s o m e s t h a t lie between them. Scattered small dense granules of u n k n o w n c h a r a c t e r are also found. There is a t e n d e n c y for spindle tubules to be a r r a n g e d in bundles, p a r t i c u l a r l y as they a p p r o a c h the region of the m e t a p h a s e plate. The bundles can be seen in Fig. 1, b u t they are m u c h m o r e obvious in thicker sections because their density is c o n s i d e r a b l y lower t h a n that of the mass of r i b o s o m e s t h r o u g h which they pass. The bundles of fibers pass c o m p l e t e l y t h r o u g h the p l a n e of the m e t a p h a s e plate. Here their course takes one of two forms, namely, between the c h r o m o s o m e s (arrows A) or t h r o u g h the substance of a c h r o m o -
FIG. 1. Metaphase in epidermal cell. The chromosomes lie in the plane of the equator. They appear rather short in this picture because they have been sectioned transversely. A central light area is seen between each pair of chromatids. Diffuse kinetochores do not show to advantage in this plane of sectioning, but dense material representing the kinetochores lies on the polar surface of two chromosomes. Spindle fibers are mainly in bundles, four of which are marked by arrows. Arrows A indicate bundles of continuous fibers that pass through the plane of the metaphase plate in gaps between chromosomes. Arrows B indicate bundles of fibers that perforate the chromosomes. The fibers converge toward the poles. A centriole at one of the poles has been sectioned, x 15,200.
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some (arrows B). Where a bundle passes through a chromosome it creates a light streak with straight and parallel margins (Figs. 1 and 2), in contrast to the gaps between chromosomes, which have irregular margins. If this were not the case, it might be difficult to distinguish a chromosome perforated by a bundle from two separate chromosomes. The spindle tubules converge to the centrosphere, where they come into close association with the centrioles. The details of this association have not been observed, but there seems to be no unusual feature of this part of the mitotic spindle of Rhodnius. The metaphase chromosomes of the epidermal cells of Rhodnius are strikingly different from those of nonhemipteran species: (a) They show consistently such obvious and frequent perforations by spindle tubules bundles, as just mentioned. (b) The chromosomes exhibit a light central area (Figs. 1 and 3). This appearance represents the characteristic disjoined form of the chromatids in species with diffuse kinetochores. Light cores are, of course, not seen in the anaphase chromosomes (Fig. 2). (c) An extensive, but incomplete, band of rather dense material lies close to the surface of the chromosomes (Fig. 3). This layer is similar in density to the substance of the kinetochore described by numerous authors, and similar in its relation to the surface of the chromosome, from which it is separated about 100-300 A. It possesses a finer granularity than that of the chromosome. Only occasionally are fibers of the spindle seen passing into this material. They are hard to identify because they enter it singly, rather than in the bundles characteristically found in species with localized kinetochores. I have not been able to determine whether the diffuse kinetochore material is in the form of a tube around the metaphase chromosome, or whether it coats only the polar surfaces. In anaphase it seems to be confined to the polar surface. Meiosis in Rhodnius testis
The metaphase configuration of the spindle in a primary spermatocyte is shown in Fig. 4. The spindle region of this cell is characterized by great numbers of membranes of the endoplasmic reticulum that radiate from the poles toward the chromosomes (Figs. 4 and 5). The chromosomes are partially enclosed by similar double membranes (Figs. 4 and 6). FIG. 2. Anaphase in epidermal cell. The light core seen in metaphase chromosomes is not present at anaphase. A perforating bundle of spindle fibers is marked by the arrow, x 16,500. Fx~. 3. A chromosome at metaphase in an epidermal cell. The chromosome has been sectioned along its long axis, and a part at the middle lies almost out of the plane of the section. The disjunction of the chromatids is represented by the light central core. The polar surfaces show finely granular dense material along most of the length, interpreted as the diffuse kinetochore. There is a distinct paucity of spindle fibers inserting into the kinetochores, although they are preserved in this specimen. × 20,300.
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Spindle tubules in bundles are not obvious. Careful examination at high magnification, however, reveals a large number of spindle tubules that lie separate from each other (Fig. 6). Some of them pass through the metaphase plate in gaps between the chromosomes (Fig. 6). Others lie between the chromosome and the incomplete membrane that surrounds it (Fig. 7). Many spindle tubules pass singly through the substance of the chromosome. When the metaphase plate is sectioned at right angles to the polar axis the profiles of spindle tubules embedded in chromosome substance can be seen (Fig. 7). It is only possible to see them because each tubule is surrounded by a light halo about 4 0 0 / ~ in diameter. The tubules enter the chromosomes in gaps between pieces of membrane (Fig. 6). The extensive layer of dense material that was found applied to the chromosomes in the epidermal cells is missing in the spermatocytes. There are, however, occasional dense points at the polar surface of the chromosomes which may represent kinetochores. Another striking difference from the epidermal mitotic cells is in the shape of the metaphase and anaphase chromosomes. The bivalents are positioned, not in the transverse axis, but in the longitudinal axis of the cell (Figs. 1 and 6). Their separation at anaphase involves the production of a thin, tapering connection between them that probably represents the terminalization of chiasmata. Similar connections have been observed in certain aphids (11). An incidental finding in a Rhodnius spermatocyte seems worth reporting in view of recent work on other species in which a new organelle has been described (17). The organelle is shown in Fig. 8. In association with a well condensed prometaphase chromosome is a banded structure consisting of 4 rows of dense granules embedded in some almost homogeneous material of lower density. In the original print there is a fine dense line mid-way between the rows of granules. According to Wettstein and Sotelo (17) these structures, which they have called "composite bodies," are related to the synaptinemal complexes, and they are found only in primary spermatocytes. DISCUSSION The kinetochore in conventional mitosis is the region of the chromosome at which the chromatids are held together at metaphase. Also, when the anaphase chroFIo. 4. Metaphase in primary spermatocyte. The cell is cut parallel to the spindle axis, as centrioles at both poles are sectioned. The spindle contains many radiating membranes, but spindle fibers are not visible at this magnification. The chromosomes are surrounded by incomplete double membranes. The very dense, coarsely granular material at the equator is thought to be derived from the nucleolus. x 16,400.
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mosomes move toward the poles they do so with the kinetochores leading, and the chromosome arms trailing behind. The kinetochore is recognized in stained preparations as a spot that is Feulgen negative (primary constriction). The observation that in hemipteran insects the chromosomes remain parallel to each other as they separate, and also that no kinetochore is morphologically recognizable, led Schrader (15) to postulate the existence of a diffuse kinetochore extending along the length of the chromosome. The observations on this type of kinetochore were extended by HughesSchrader (3) working particularly with coccids and by Brown (1) on the plant Luzula. Experimental evidence for the existence of a diffuse spindle attachment in Steatococcus was obtained by Hughes-Schrader and Ris (5), who observed that fragmented chromosomes produced by irradiation behaved as intact chromosomes in subsequent divisons. This was interpreted to indicate that their activity was mediated through a part of the kinetochore that remained with the fragments. By contrast, fragments of orthodox chromosomes are not mitotically active. In the epidermal cells of Rhodnius a structure of the character postulated by these workers has, in fact, been observed in the present study. The diffuse kinetochore appears to be composed of dense material comparable to that of the localized kinetochore except that it extends over most of the length of the chromosome. The question of its kinetic function cannot be settled by electron microscopy. However, it is somewhat disturbing to find so few spindle fibers inserting into the diffuse kinetochore. Of course, if the number of fibers converging to a localized kinetochore, only 10 or less in the sea urchin (2), were to be spread over the relatively huge area of the diffuse type, their concentration would certainly be very low. On the other hand, the fact is that large expanses of the diffuse kinetochore do not show any chromosomal fiber insertions. This finding appears to be difficult to reconcile with the suggestion that the density of the conventional kinetochore may be due to dense material closely associated with the individual tubules as they insert into it (6, 8). Moreover, if the whole kinetochore has a function related to chromosome movement it becomes difficult to visualize such a kinetic action operating through the considerable distance between the fibers. Why is there so much dense material per spindle fiber? Is this the substance of the postulated chromosome pellicle (16)? One might speculate on the possible stiffening effect provided by such a layer on chromosomes showing parallel disjunction, but this does not answer the question of whether
FIG. 5. Polar region at early anaphase in spermatocyte. Between the membranes of the spindle are many tubules that appear to radiate from the immediate vicinity of the centrioles. Part of a chromosome is seen at the right margin, x 34,000. FiG. 6. Disjunction in spermatocyte. The dyads develop tapering processes as they move apart. At several points spindle tubules are seen entering the substance of the chromosome. × 27,500.
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or not the diffuse kinetochore has the active role in chromosome movement that has been ascribed to the localized kinetochore (reviewed by Mazia, 9). It should be emphasized that although the coccid division figure studied in detail by Hughes-Schrader (3) is an example of the diffuse kinetochore type, there are peculiarities of the mitotic apparatus in these species that are not found throughout hemipteran insects. The coccid mitotic apparatus is very simple. According to Hughes-Schrader (4), "Mitosis is possible with no new structural differentiations in the cell other than chromosomal fibers and interzonal connectives. Asters and centrioles may be absent, and in none of the variant coccid figures are any continuous spindle fibers demonstrable." The term "interzonal fibers" describes a part of the spindle extending only between disjoining chromosomes. Hughes-Schrader believes that elongation of a stem body of interzonal origin is part of the mechanism of anaphasic movement of the chromosomes in coccids. The mitotic apparatus of Rhodnius obviously differs from that of the coccids in that orthodox continuous fibers are demonstrable. However, the presence of the diffuse kinetochore in Rhodnius tempts one to try to interpret the coccid figure in the light of the present findings. In a study of meiosis in the cockroach Krishan and Buck (7) stated that "... it is possible that the fibers identified at metaphase as continuous ones, become the interzonal fibers of anaphase . . . . If such a case is proved by further observation, the distinction between the interzonal and continuous fibers will tend to diminish." It is now apparent in Rhodnius that spindle fibers passing between disjoining chromosomes do extend through into the polar spindle, and thus constitute a special part of the continuous fiber system. Perhaps they should be called perforating fibers. They are found in both mitotic and meiotic cells, although in the former t h e fibers are in bundles, and in the latter they are individual units. A similar finding has been demonstrated in other species. 1 In a paper on the ameba Pelornyxa Roth and Daniels (13) have illustrated the uninterrupted passage of spindle fibers through the chromosomes, although a recent report from the same laboratory (14) stated that chromosomes of the corn borer spermatocyte were not traversed by 1 Since this paper was submitted for publication O. Behnke and A. Forer have independently described spindle tubules that penetrate the telophase nucleus of the crane fly spermatocyte in the same manner as those in Rhodnius spermatocytes [O. Behnke and A. Forer, Science 153, 1536 (1966).
FIG. 7. Metaphase in spermatocyte. The plane of the section is at right angles to the polar axis. Several spindle tubules perforating the chromosome are shown with short arrows. Each is surrounded by a light halo. Some of the spindle tubules lying between the chromosome and the incomplete membrane around it are indicated by long arrows, x 100,000 FIG. 8. Prometaphase in spermatocyte. A "composite body" is shown in association with a chromosome. x 73,000.
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spindle fibers. Robbins and Gonatas (12) observed that in HeLa cells continuous spindle tubules were sometimes seen traversing a chromosome. They suggested that this feature might result in anomalous chromosome movement at anaphase. These observations, and those on Rhodnius, cannot be ascribed to the artifact mentioned by Harris (2) of the apparent fusion of bundles of chromosomal and continuous fibers. Returning to the descriptions of the coccid figure, it is obvious that light microscopic examination will not differentiate between interzonals and this type of continuous fiber. In view of the present findings in Rhodnius, it seems reasonable to suggest that the stem body in coccids may be composed of continuous tubules that have passed through the chromosomes. It is regrettable that the fine structure of the coccid spindle has not yet been studied, for as electron microscopic observations accumulate, the existence of interzonal fibers, in the original meaning of the term, becomes questionable. The observation that some of the continuous fibers pierce the chromosomes has a bearing on the understanding of anaphasic movements. Theories of karyokinesis would have to incorporate the idea that chromatids or homologues would remain aligned on the long axis of the spindle by these perforating fibers. The relationship between the perforating fibers and the chromosomes might take any of three forms: (a) Only a passive positioning, like a bead on a thread; (b) a firm attachment between the chromosome and the fibers; (c) some more active process might be involved, perhaps comparable to the activity ascribed to the kinetochore. On the evidence yet available there seems to be no basis for choosing among these possibilities. In view of the perforation of chromosomes by the fibers in Rhodnius epidermal cells, it is not difficult to understand how they might assist in maintaining the characteristic parallel disjunction. Obviously, no chromosome arms could form if the whole chromosome were held in position by continuous fibers. I am grateful to Dr. W. F. Baldwin, Chalk River Nuclear Laboratories, for his encouragement and for supplying me with the insects used in this study. Mr. William Daniels and Mr. Charles Jarvis provided technical assistance. REFERENCES 1. BROWN, S. W., Univ. California Publ. Botany 27, 231 (1954). 2. HARRIS, P., J. Cell Biol. 14, 475 (1962). 3. HUGH~S-SCHRADER,S., J. Morphol. 70, 261 (1942). 4. - - - - Advan. Genet. 2, 127 (1948). 5. HUGHES-SCHRADER,S. and RIs, H., J. Exptl. Zool. 87, 429 (1941). 6. KgISHAN, A. and BUCK, R. C., J. Cell Biol. 24, 433 (1965). 7. - - - - Y. Ultrastruct. Res. 13, 444 (1965). 8. LUYKX, P., Exptl. Cell Res. 39, 643 (1965).
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9. MAZIA, D., in BRACHET, J. and MIRSKY, A. E. (Eds.), The Cell, Vol. III. Academic Press, New York, 1961. REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). RIs, H., Y. Exptl. Zool. 90, 267 (1942). ROBBINS,E. and GONATAS,N. K., J. Cell Biol. 21, 429 (1964). ROTH, L. E. and DANIELS, E. W., J. Cell Biol. 12, 57 (1962). ROTH, L. E., WILSON, H. J. and CHAKRABORTY,J., .[. Ultrastruct. Res. 14, 460 (1966). SCHRADER, F., Cytologia 6, 422 (1935). -Mitosis, 2nd ed. Columbia Univ. Press, New York, 1953. WETTSTEIN, R. and SOTELO, J. R., Chromosoma 17, 246 (1965). WIG~LESWORTH,V. B., The Control of Growth and Form: A Study of the Epidermal Cell in an Insect. Cornell Univ. Press, Ithaca, New York, 1959. 19. WOLFE, S. L. and JOHN, B., Chromosoma 17, 85 (1965). 10. 11. 12. 13. 14. 15. 16. 17. 18.