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STUDIES IN SPERMATOGENESIS

WITH ESPECIAL REFERENCE TO THE

"ACCESSORY CHROMOSOME"

BY N. M. STEVENS.

[Illustration]

WASHINGTON, D. C.:
Published by the Carnegie Institution of Washington
September, 1905.

CARNEGIE INSTITUTION OF WASHINGTON

PUBLICATION No. 36

FROM THE PRESS OF
THE HENRY E. WILKENS PRINTING CO.
WASHINGTON, D. C.




STUDIES IN SPERMATOGENESIS WITH ESPECIAL REFERENCE TO THE "ACCESSORY
CHROMOSOME."

By N. M. STEVENS.


In connection with the problem of sex determination it has seemed
necessary to investigate further the so-called "accessory chromosome,"
which, according to McClung ('02), may be a sex determinant. This view
has been supported by Sutton ('02) in his work on _Brachystola magna_,
but rejected by Miss Wallace ('05) for the spider.

The forms selected for study have been taken from several groups of
insects, and are all species whose spermatogenesis has not been
previously worked out. They are (1) a California termite, _Termopsis
angusticollis_; (2) a California sand-cricket, _Stenopelmatus_; (3) the
croton-bug, _Blattella germanica_; (4) the common meal-worm, _Tenebrio
molitor_; and (5) one of the aphids, _Aphis oenotheræ_.

A brief account of a chromatin element resembling the accessory
chromosome in _Sagitta_ has been added for comparison. The
spermatogenesis of each form will be described in detail, and a general
discussion of the results and their relation to the accessory chromosome
and sex determination will follow. The spermatogenesis of the aphid has
been included in another paper, but a summary of results and a few
figures will be given here for reference in the general discussion.


METHODS.

The testes were fixed in various fluids--Flemming's strong solution,
Hermann's platino-aceto-osmic, Gilson's mercuro-nitric, Lenhossek's
alcoholic sublimate acetic, and corrosive acetic. Flemming's and
Hermann's fluids followed by safranin gave good results in most cases.
The mercuro-nitric solution and Lenhossek's fluid gave excellent
fixation and were preferable to the osmic mixtures when it was desirable
to stain the same material with iron-hæmatoxylin, and also with various
anilin stains.

Heidenhain's iron-hæmatoxylin, either alone or with orange G or
erythrosin, was used more than any other one stain. With osmic fixation
safranin gave better results in some cases, because of the abundance of
spindle fibers and sphere substance which were stained by hæmatoxylin.
The safranin-gentian combination used by Miss Wallace and others in the
study of the accessory chromosome did not prove to be especially helpful
with these forms. Thionin was found to be a very useful stain for
distinguishing between the accessory chromosome and an ordinary
nucleolus. Licht-grün was often used in combination with safranin.




RESULTS OF INVESTIGATIONS.


Termopsis angusticollis.

In the termite it was not found to be practicable to dissect out the
testes. The tip of the abdomen was therefore fixed and sectioned, young
males whose wings were just apparent being used. The cells are all
small, and could not be studied to advantage with less than 1500
magnification (Zeiss oil immersion 2 mm., oc. 12).

In the spermatogonium there is a very large nucleolus (plate I, fig. 1),
which in the iron-hæmatoxylin preparations is very conspicuous, but does
not stain like chromatin with thionin or other anilin stains, nor does
it behave like an accessory chromosome during the maturation mitoses.
Before each spermatogonial division it divides as in figures 2 and 3,
and the same is true for each maturation mitosis. Figure 4 shows the 52
chromosomes of a spermatogonial division in metaphase. Figures 5 and 6
are young spermatocytes, showing the division of the nucleolus. Figures
8, 9, and 10 show a stage immediately following that shown in figure 6
and evidently persisting for some time. The spireme thread is very fine,
stains deeply, and is wound into a dense ball, often concealing one
(fig. 10) or both nucleoli (fig. 8). Figure 11 shows the next stage; the
bivalent chromosomes are so disposed as to give the familiar "bouquet
stage," with the loops directed away from the centrosome and sphere
(_c_). Figures 12, 13, and 14 show the later development of the same
stage, the chromatin loops becoming thicker by the concentration of the
smaller granules to form the larger ones seen in figure 14. The loops
now straighten out and extend in various directions across the nuclear
space (figs. 15, 16, 17). In fig. 18_a_ a longitudinal split is seen in
several chromosomes. Figures 18_b_, 19, 20, and 21 show various stages
in the contraction of these split bivalent chromosomes to form
diamond-shaped tetrads, each side of which is a univalent daughter
chromosome. The tetrads come into the spindle in this form (figs. 22,
23), and change to the form shown in figure 24 during the metaphase
(figs. 22, 26, 28). Figures 25 and 27 show the 26 bivalent chromosomes,
or tetrads, in early and late metaphase, respectively, and figures 29,
30, and 31 in anaphase. This is certainly a reduction division, for the
tetrads are always somewhat elongated and come into the spindle with
their longer axes parallel with the axis of the spindle. The aberrant
bodies in these figures are probably remains of the nucleoli; they are
found only in iron-hæmatoxylin preparations. Figures 31 and 32 show
exceptional cases where the cell has divided. Usually the two daughter
nuclei are formed in an undivided cell. The resting-stage between the
two divisions is only partial. The nucleolus appears and divides into
two (figs. 33-36), and the chromosomes change into the dyad form (fig.
36), in which they come into the second maturation spindle (figs. 37,
38). The equatorial plate again shows 26 chromosomes (fig. 39). The
formation of the spermatozoa is peculiar in that the original
spermatocyte cell-body, as a rule, does not divide; but the four nuclei
resulting from the two maturation divisions develop into sperm-heads in
one cell. All have a nucleolus (fig. 41), and in a slightly later stage
(fig. 42) the elongated nuclei have a distinct centrosome and sphere at
the posterior end. Later stages are shown in figures 43, 44, and 45.

The points of greatest interest in the spermatogenesis of _Termopsis
angusticollis_ are, (1) the fact that no accessory chromosome is
present; (2) that the method of tetrad formation and reduction are
clear, despite the fact that the cells and the chromatin elements are
quite small; and (3) the failure of the cell-bodies to divide and the
consequent development of four spermatozoa in one cell.


Stenopelmatus.

The spermatogonium of _Stenopelmatus_ contains from one to three large
nucleoli, which stain much less with thionin than does the spireme
(plate II, figs. 46, 47, 48). As the distinct chromosomes come into view
in the prophase of mitosis, two are seen to be nearly twice as long as
the others, but of equal length (figs. 48, 49, 50.) There are 46
chromosomes in the equatorial plate of a spermatogonial spindle (fig.
50). Besides the nucleolus (_n_), there appears in the young
spermatocyte a conspicuous element (_x_) which stains deeply with all
chromatin stains (fig. 51). It is closely applied to the nuclear
membrane and is connected with an end of the spireme (figs. 51-54). At
first it is quite small, and it gradually increases in size during the
spireme stage. There is no "bouquet stage" in this form. Figure 55 shows
the spireme segmented and split longitudinally. The segments have begun
to open out at the center to give the cross which is the typical tetrad
form in _Stenopelmatus_. Figures 56, 58, 59, and 60 show various stages
in the contraction of the split segments to form crosses and
diamond-shaped rings. The tetrads usually remain connected by delicate
linin threads, as shown in figures 57 and 60, also in figures 62 and 63,
the latter taken from the metaphase of the first maturation spindle. If
these linin connections persist, as they appear to do, from the
segmentation of the spireme to metakinesis, the first division of the
contracted tetrads must be longitudinal, corresponding to the split in
the segments of figures 55, 57, 58, etc. The chromosomes in the
metaphase usually appear as dumbbells (fig. 66) or elongated crosses
(fig. 67), but occasionally one can be found which still shows its
tetrad nature (fig. 64), so clearly indicated in the quadrivalent
crosses of figure 59. In the anaphase the chromosomes are often split as
in figure 68, and occasionally the two components can be seen as plainly
as in figure 65. Figure 61 shows the various shapes assumed by the
element _x_ during the tetrad-stage of the chromosomes. This element _x_
almost invariably appears in a vesicle near one pole of the spindle
(figs. 67, 68); in exceptional cases it is found nearer the equatorial
plate, as in figure 66, or even in the same plane with the ordinary
chromosomes, but always somewhat isolated from them. In position and
form this element resembles the accessory chromosomes described by
Baumgartner ('04) for _Gryllus domesticus_; in its mode of origin it
seems to differ from the other accessory chromosomes yet described.

Figures 69 and 70 show the 23 bivalent chromosomes in metaphase; in
figure 69 the element _x_ is shown partly behind the large chromosome
and at a different level. In figures 66 and 67 the one exceptionally
large chromosome doubtless represents the two larger ones of the
spermatogonia. In the anaphase the element _x_ is sometimes as
conspicuous as in figure 71; in other cases it is concealed either
behind or within the polar mass of chromatin. In this form there is a
distinct resting stage between the two maturation mitoses (figs. 72-75).
The element _x_ is conspicuous in one-half of the cells (figs. 72, 73);
it may be included in the nucleus as in figure 72, or it may be partly
or wholly outside, as in figures 74, 75, and 76. In the latter case, but
not in the former, it is surrounded by its own membrane. As the
chromatin begins to condense for the second mitosis, disintegration of
the element _x_ becomes apparent. This is most easily made out in cases
where the element is isolated, as in figures 75 and 76; but there seems
to be little doubt that it disappears before the metaphase of the second
maturation mitosis. It is not possible to count the chromosomes in this
stage, they are so crowded together, but it is not probable that such a
conspicuous chromatin element as that seen in the first division could
escape detection, even if it were in the equatorial plate among the
chromosomes. No aberrant element is ever seen in these spindles; and,
moreover, all of the spindles and all of the spermatids appear to be
exactly alike at the same stage. The chromosomes are double in the
prophase (fig. 77) and always appear double in the equatorial plate
(fig. 78), the paired elements corresponding to those of figure 65.

In figure 80, plate III, a pair of spermatids is shown with nuclear
membrane formed and the spindle fibers twisted in a characteristic
manner. Figure 81 is a slightly later stage with the spindle-remains
massed against the nuclear membrane. Curiously enough there appears in
the nucleus of every spermatid a body similar to the element _x_ of the
spermatocytes of the first order (figs. 82-86). This body is often
applied to the nuclear membrane and connected with the spireme (figs.
84-86). It decreases in size and finally disappears (figs. 88-91). The
spindle-remains divides (fig. 83), and a small part of it (_a_) goes to
form the acrosome at the apex of the head (figs. 85-92). The larger part
is probably utilized in the formation of the tail, for it gradually
disappears as the tail develops.

The centrosome which, although small, is conspicuous in each mitosis, is
seen in figure 83 between the two parts of the spindle-remains, applied
to the outside of the nuclear membrane. In figures 85, 86, and 87 the
relation of the tail (or its axial fiber) to the centrosome is shown. In
figures 87 and 88, instead of the small spherical centrosome of figures
83 to 86, we have a much elongated body, at first (fig. 87) applied for
its whole length to the nuclear membrane, but later lying along one side
of a middle piece (_m_), as shown in figure 89, and in a later stage in
figures 90 to 92. The mature spermatozoön with its forked anterior end
appears in figure 93.

The points of especial interest in the spermatogenesis of
_Stenopelmatus_ are the development of the aberrant chromatin element
_x_ during the growth stage of the spermatocyte of the first order, its
distribution to one-half of the spermatocytes of the first order, its
disappearance during the rest stage between the two maturation
divisions, and the development of a similar, though smaller, element in
all of the spermatocytes.


Blattella germanica.

Unlike the spermatogonia of _Stenopelmatus_, those of _Blattella_ have
both a faintly-staining nucleolus and a deeply-staining chromatin
element (_x_), and moreover the two are always closely associated (figs.
95, 96). The number of chromatin elements in the equatorial plate of
late spermatogonial mitoses is 23 (fig. 97). Later events indicate that
one of the 23 is the element _x_, but it is impossible to distinguish it
here. Figure 98 is a very early stage of the spermatocyte of the first
order, showing the element _x_ as a U-shaped body. The centrosome was
also conspicuous in all of the cells of this group. The spireme here, as
also in figure 99, is fine and closely interwound. In figure 99 and
again in figure 100 the element _x_ is joined to the spireme as it is
throughout the spireme stage. In the "bouquet" or "polarized" stage the
combined nucleolus and element _x_ are always at one side of the group
of loops and down very close to the base of the figure (figs. 101, 103).
In figure 102 most of the loops are cut across. The stage shown in
figures 104 and 105 is a later one than that just described. Here we
have again a continuous spireme connected with the element _x_, making
it seem improbable that the bivalent chromosomes are really separated in
the bouquet stage. Figure 106 gives some of the variations in form of
the combined nucleolus and element _x_. The last of the five figures was
taken from a giant cell containing at least twice the usual amount of
chromatin. In one giant cell four unusually large combinations of this
kind were found, and a corresponding amount of chromatin in the spireme.
In figure 107 one sees the spireme divided into segments still joined by
linin bridges. In figure 108 similar segments may be seen, one of them
showing a longitudinal split. The element _x_ is present, but the
nucleolus has disappeared. In many cases the split, if it appears at
all, closes quickly and the chromosome bends in U-shape, as in figure
109, plate IV. This figure also shows two centrosomes (_c_). In other
cases the split persists as in figure 110 and leads to the formation of
crosses of a tetrad character (figs. 111, 112, 113), as in
_Stenopelmatus_ and many other insects. Figures 114 to 117 show later
stages of the U-shaped chromosomes. Perfect rings are rare. All sorts of
variations are seen, broad and narrow U-shapes, rings split at one point
or the opposite points, a U split at the bottom (fig. 114), pairs of
parallel rods (fig. 115), and occasionally rods constricted in the
middle and showing a longitudinal split in each half, as in figure 116.
Figure 117 shows different views of the split rings. Apparently all of
these forms straighten out so that the two components of the bivalent
chromosome stand end to end as dumbbells or compressed crosses in the
metaphase of the first maturation spindle (figs. 123-125). The element
_x_ remains concentrated and more or less spherical in form. Figures
118-122 are equatorial plates, with _x_ absent in figure 120, in the
same plane as the 11 other chromosomes in figure 119, far to one side in
figure 118, and near one pole of the forming spindle in figure 122. It
is also shown in various positions with regard to the spindle in figures
123 to 126 and 128 to 132. In figure 125 it is apparently double, and
again in figure 129. In figure 130 one lagging chromosome shows the dyad
nature of the products of the division of the tetrad. In this form
there can be no doubt that reduction occurs in the first spermatocyte
division. The element _x_ is very often concealed by the polar
aggregation of chromatin, but it is sometimes as conspicuous as in
figures 131 and 132. The spermatocytes of the second order go into a
complete resting stage before they are completely separated, and one of
a pair shows the element _x_, while it is lacking in the other (fig.
133). At the close of the resting stage the chromosomes appear as 11
pairs of rods of considerable length, which gradually shorten and
thicken and usually bend at the center, forming U's or V's (figs.
134-138). In one stage these double U's look much like tetrads (fig.
138). The rods straighten again as they shorten still more (fig. 139),
become more closely approximated, and finally form dumbbells, as in
figure 141.

The element _x_ is, of course, present in only one-half of these nuclei.
In the equatorial plate, figure 142, it is absent; in figure 143 it is
present, but can not be distinguished from the other chromosomes, while
in figure 144 it is rendered conspicuous by its spherical form and
isolated position. In only a few cases has it been possible to
distinguish _x_ in the spindle. Figures 146 and 147 show two of these
cases where this element is clearly double and of different form from
the other chromosomes. It is probable that it divides and so goes into
one-half of all of the spermatids, as in McClung's typical cases of the
accessory chromosome. Figure 145 shows the usual appearance of the other
chromosomes in metaphase. The two spermatids of a pair are always alike
so far as any evidence of the presence of the element _x_ is concerned
(fig. 148). Figure 149 is an exceptional case, where one chromatin
element (possibly _x_) has evidently divided late and been left out in
the cytoplasm; a smaller chromatin granule is also present in the
cytoplasm of each spermatid. All of the spermatids, as in
_Stenopelmatus_, develop a deeply-staining body, which, however, in this
case is usually centrally located and often appears double (figs.
150-152).

The spindle-remains (_Spindelreste_) forms a very conspicuous body at
one side of the nucleus in the spermatids, and occasionally a mass of
chromatin, probably due to imperfect mitosis, is found near the
spindle-substance (fig. 150). The mass of spindle-substance at first
appears structureless, but soon assumes the condition shown in figures
150 to 152. In one individual many of the spermatids had two balls of
spindle-material (fig. 152), and the resulting later stages were
double-tailed (fig. 153). Figure 156 shows how the spindle-substance
goes into the tail and gradually disappears as the tail lengthens.

The centrosome is evidently applied to the nuclear membrane, as in
_Stenopelmatus_, and the middle-piece is developed in connection with
it, as in figures 156-157, 154-155, 158-160. The element _x_ of the
spermatids gradually disappears (figs. 150, 159). An acrosome develops
at the anterior end, the head condenses and lengthens, and we have the
ripe spermatozoön (fig. 161). The tail is very long and is shown only in
part.

Of the forms studied, _Blattella_ alone has many degenerate spermatozoa.
Some follicles have none, others a number varying perhaps from
one-fourth to three-fourths of the whole number. No evidence of
degeneracy was detected among the young spermatids up to the stage shown
in figures 154-155, where a few like figure 162 were found. Most of the
degenerate forms occur among the nearly ripe spermatozoa or in the
sperm-ducts. Such are shown in figures 163 to 168. The chromatin is
strangely broken up into irregular clumps, and probably no two of these
degenerate sperm-heads can be found which are alike. The tails are
always imperfect. The distribution and varying numbers of these
degenerate spermatozoa make it impossible to interpret their condition
as due to the absence of the accessory chromosome, as Miss Wallace does
in the spider. The only probable explanation, it seems to me, is
imperfect mitosis. Cases where more or less chromatin is left behind in
the cytoplasm, especially in the first spermatocyte mitosis, are very
common, and such cases as those shown in figures 149 and 150 are not
rare. The giant cells, so far as I have been able to trace them, do not
develop into spermatozoa.

The most important points are:

(1) The presence of the element _x_ in the spermatogonia, closely
associated with the nucleolus.

(2) The uneven number of chromatin elements in the metaphase of
spermatogonial divisions.

(3) The connection of the element _x_ with the spireme up to the stage
where the spireme segments to form the bivalent chromosomes.

(4) The varied character of the tetrads, showing the first spermatocyte
division to be a reducing division in the sense that it separates whole
chromosomes.

(5) The fact that the element _x_ fails to divide in the first
maturation division, does divide in the second, but can not be traced
beyond the equatorial plate of the latter mitosis.

(6) The similarity of all the normal spermatids, though one-half of them
must contain the element _x_, the other half not.

(7) The varying and often large number of degenerate spermatozoa.

An attempt was made to determine the somatic number of chromosomes. The
dividing cells of the follicles of young eggs seemed to afford the most
favorable material, but even here there was so much overlapping of the
ends of the chromosomes that it was impossible to be absolutely certain
of the number. In the two most favorable cases 23 were counted (fig.
94). This differs from McClung's count for similar cases among the
Orthoptera, and Sutton's for _Brachystola magna_. The eggs have so far
resisted all efforts to learn what part the odd chromosome may play in
fertilization.


Tenebrio molitor.

In the metaphase of all spermatogonial mitoses where it was possible to
count accurately, 20 chromosomes were found, 19 large ones of
approximately equal size, and 1 small spherical one (figs. 169, 170).
There is nothing in the resting nucleus of the spermatogonia which would
suggest either a nucleolus or an accessory chromosome. The chromatin
stains well during the whole growth period of the spermatocytes, but it
is impossible to separate the period into so definite stages as in most
other forms.

In the youngest spermatocytes one finds occasionally a cyst containing
cells with nuclei like those of figures 171 and 172, indicating that a
brief "synapsis" or condensation stage occurs at the close of the last
spermatogonial mitosis. During the greater part of the period the
chromatin forms a heavy, irregular, and often segmented spireme (figs.
173, 174). Shortly before the first maturation division, such split
segments as appear in figure 175 are sometimes found; some of these
simulate tetrads with slender connecting bands between the paired
elements. Again, one finds a few cases like figure 176, where the
spireme is segmented into bivalent chromosomes, each component showing a
longitudinal split. This figure also shows the small chromosome.
Usually, however, the irregular and much tangled spireme (figs. 173,
174) condenses into a heavy segmented band variously disposed in the
nucleus (fig. 177). This band soon separates into the bivalent
chromosomes shown in figures 178 and 179, giving 9 symmetrical pairs and
1 unsymmetrical one (fig. 179 _s_) composed of the small chromosome and
a much larger mate. In the prophase of the spindle, in rare cases, some
of the chromosomes are longitudinally split and transversely
constricted, forming tetrads (fig. 180), but more often they appear as
in figure 181. The unequal pair appears in each figure at _s_. In the
metaphase (fig. 182) it is the last to come into the equatorial plate,
possibly because of its lack of symmetry. The smaller component of this
pair is always directed toward the equator of the spindle. Figure 183
shows a small tangential section of a spindle in metaphase, containing
the unequal pair and one equal pair. In figure 184 a polar view of a
metaphase is shown, the unequal pair, which was somewhat below the
others, being indicated by stippling. Figures 184 _a_ and 185 show that
the unequal components of the unsymmetrical pair, as well as the equal
components of the symmetrical pairs, are separated in metakinesis,
making this clearly a reduction division. Two polar plates are shown in
figures 186 and 187, one containing 10 equal elements, the other 9 equal
ones and 1 small one. The telophase is shown in figure 188. There is no
resting stage, but the new spindle is formed from the remains of the old
one, and the spindle-shaped mass of chromatin seen in figure 188 either
passes into the center of the new spindle or becomes enveloped by it.
The double chromosomes separate as in figures 189 and 190. Figure 190
shows the small dyad, and figure 189 an aberrant one which may be its
mate. The spindle in both divisions is peculiar in having outside of the
spindle proper a dense mass of fibers which, in osmic material, stain
deeply with iron hæmatoxylin. These fibers are shown in all the figures
from 174 to 196. Figures 191 and 192 are equatorial plates of the two
kinds of spermatocytes of the second order, figure 191 showing the small
chromosome. An early anaphase appears in figures 193 and 194, which show
both the small and larger chromosomes in metakinesis. Figure 195 is a
later anaphase containing the divided small chromosome. In figure 196
are shown the two polar plates of a spindle corresponding to that of
figure 195, and in figure 197 the polar plates of a spindle in which 10
equal chromosomes have been divided. In _Tenebrio molitor_ the
spermatids are therefore certainly of two distinct kinds, so far as the
chromatin content is concerned.

In most of the young spermatids, after the nuclear membrane has formed,
there appears an isolated chromatin element, which corresponds fairly
well to the large or to the small component of the unsymmetrical pair,
separated in the first mitosis and divided in the second. The clear
portion of the nucleus containing this isolated element is at first
turned toward the spindle-remains (fig. 198), but before the tail
appears either the whole nucleus or its contents have rotated 180° (fig.
199). Various stages in the development of the spermatid are seen in
figures 200 to 203. The clear region and the isolated element finally
disappear (fig. 202 _b_), and the chromatin breaks up into coarser and
then into finer granules within the sperm-head. In the later stages the
centrosome is clearly seen at the base of the head (fig. 203).

In order to determine, if possible, the value of the unsymmetrical pair
of chromatin elements, very young ovaries and ovaries with egg-tubes
were sectioned and the chromosomes counted in the dividing cells of the
egg-follicle (Female somatic cells), and in dividing oögonia. In
both cases 20 large chromosomes were found. Figure 207 is the equatorial
plate from a female somatic cell of a young egg-follicle. Figure 208 _a_
and _b_ shows two sections of an oögonium in the prophase of mitosis. In
order to determine the number and character of the chromosomes in the
male somatic cells, several male pupæ were sectioned. As in the
spermatogonia, 19 large chromosomes and 1 small one were found. Figure
204 shows the equatorial plate of a dividing male somatic cell, and
figures 205 to 206 are daughter plates from a similar cell. (Three large
chromosomes of the plate shown in figure 206 are in another section.)

From these facts it appears that the egg-pronucleus must in all cases
contain 10 large chromosomes, while the spermatozoön in fertilization
brings into the egg either 10 large ones or 9 large ones and 1 small
one. Since the somatic cells of the female contain 20 large chromosomes,
while those of the male contain 19 large ones and 1 small one, this
seems to be a clear case of sex-determination, not by an accessory
chromosome, but by a definite difference in the character of the
elements of one pair of chromosomes of the spermatocytes of the first
order, the spermatozoa which contain the small chromosome determining
the male sex, while those that contain 10 chromosomes of equal size
determine the female sex. This result suggests that there may be in many
cases some intrinsic difference affecting sex, in the character of the
chromatin of one-half of the spermatozoa, though it may not usually be
indicated by such an external difference in form or size of the
chromosomes as in _Tenebrio_. It is important that related forms should
be studied in order to ascertain whether the same chromatic conditions
prevail in other species of this genus or possibly in the Coleoptera in
general.[A]

[Footnote A: Prof. E. B. Wilson has recently found a similar dimorphism
in the spermatozoa of _Lygæus_ and other of the _Hemiptera
heteroptera_.]


Aphis oenotherae.

The spermatogenesis of _Aphis_ has been fully described in another paper
and will merely be briefly summarized here for the purpose of comparison
with other forms.


The spermatogonia contain a large nucleolus, which gradually disappears
in the prophases of mitosis (plate VII, figs. 209-211). The youngest
spermatocytes closely resemble the spermatogonia (fig. 212). There is no
bouquet stage and no such marked spireme stage as in many other
insects. The true synapsis occurs, as shown in figure 213, by pairing of
like chromosomes side by side. This conjugation of like chromosomes is
followed by a stage in which they are massed together at one side of the
nucleus (fig. 214). In these latter stages the nucleolus has entirely
faded out and nothing suggesting an accessory chromosome is present.
Figures 215 and 216 are equatorial plates of the first spermatocyte
mitosis. There are 5 chromosomes of different sizes and shapes, and
figure 216 shows each one double. The first division of the chromosomes,
though apparently longitudinal, is evidently a separation of the
elements paired in a preceding stage, and is therefore a reducing
division.

The anaphase of the same mitosis is shown in figures 217 and 218; it is
peculiar in that one chromosome always divides more slowly than the
others, the two elements hanging together at one end. In figure 219 are
sister spermatocytes of the second order, the "lagging" chromosomes
still connected. The second maturation division is seen in metaphase in
figure 220 and in anaphase in figure 221. Figure 222 shows a young
spermatid, the five chromosomes still preserving their characteristic
form. Figure 223 is the equatorial plate of the first maturation
division of the winter egg, showing the same form and size relations of
the chromosomes as in the spermatocyte divisions. Figures 224 and 225
are equatorial plates of a polar spindle (fig. 224) and of a
segmentation spindle (fig. 225) of the parthenogenetic egg, where 10
chromosomes are present, 2 of each of the sizes found in the sexual germ
cells.

So far as an accessory chromosome or any other visible evidence of a sex
determinant are concerned, the results are entirely negative. The
conditions shown do, however, support Mendel's conception of the "purity
of the germ-cells," and also afford evidence in favor of Boveri's theory
of the individuality of the chromosomes.


Sagitta bipunctata.

In connection with these insect forms it is of interest to find in the
spermatogenesis of _Sagitta_ a body which stains like chromatin and
behaves somewhat like the accessory chromosome. It is found in all
resting stages of the spermatogonia, closely applied to the nuclear
membrane (fig. 226). It divides before each spermatogonial mitosis (fig.
227) and, though not often discernible in the spindle, appears in the
next generation. Figure 228 is the last spermatogonial mitosis, and
figure 229 shows the element _x_, and the chromosomes paired at one pole
of the spindle. During the various phases of the growth stage (figs.
230-232) the element _x_ is again applied to the nuclear membrane.

In the prophase of the first maturation division this element divides
(figs. 233-234), and in metakinesis the two elements are found in
various positions with regard to the spindle (figs. 235-237), often as
conspicuous as in these figures, but sometimes concealed among the
chromosomes. Before the spindle for the second division forms, this
element divides again and one of the products goes into each spermatid
(figs. 238-241).

As _Sagitta_ is hermaphrodite, there would appear to be no question of
sex determination by any special chromatic element. The size of the
element _x_, its evident chromatic nature, its division before each
mitosis, and its presence in mitosis and in the spermatids, with the
same staining qualities as in the previous rest stages, certainly
indicate some important function, either in the whole process of
spermatogenesis or in the formation of the sperm-head, of which it
finally becomes a part. In _Sagitta_ this element certainly can not be
regarded as a specialized spermatogonial chromosome, or as chromatin
rejected from the spireme. No such element is present in the ovogenesis
of _Sagitta_ (Stevens, '03), nor has any been detected in connection
with fertilization. It is certain that none is present in the first
segmentation spindle of the egg.




GENERAL DISCUSSION.


THE "ACCESSORY CHROMOSOME."

The literature bearing on the "accessory chromosome" of McClung, the
"small chromosomes" of Paulmier, and the "chromatin nucleoli" of
Montgomery has been fully discussed by McClung in the paper entitled,
"The accessory chromosome--sex determinant?" ('02), and will therefore
be considered here only in its relation to the several forms studied.
The present status of the question has been well summarized more
recently by Montgomery under the heading "Heterochromosomes" in the
paper, "Some observations and considerations upon the maturation
phenomena of the germ cells."

Three theories as to the function of the "heterochromosomes" have been
advanced: (1) That of McClung that they are sex-determinants, since in
the forms which he has examined these chromatin bodies occur in only
one-half of the spermatozoa, and the sex-character is the only character
which divides the individuals of a species into two approximately equal
groups. (2) That of Paulmier and Montgomery that they are degenerating
chromatin. Montgomery regards them as "chromosomes that are in the
process of disappearance in the evolution of a higher to a lower
chromosome number." (3) That of Miss Wallace, who suggests that in the
spider only the one out of each four spermatids which contains the
accessory chromosome is capable of developing into a functional
spermatozoön, while the other three degenerate, as do the polar bodies
given off by the egg. McClung is inclined to believe that the accessory
chromosome is an element common to all of the male reproductive cells of
Arthropods, and probably to vertebrate spermatocytes as well ('02).

Of the insects considered in this paper _Aphis_ and _Termopsis_ have no
"accessory chromosome" or "heterochromosome" of any kind. The fact that
no males develop from the fertilized eggs of _Aphis_ might be offered as
a reason for its absence, but such an argument would not apply to
_Termopsis_. The sex-character may indeed be represented in the
chromatin of some one of the pairs of paternal and maternal chromosomes
of the spermatocytes, but there is no evident peculiarity by which
one-half of the spermatozoa can be said to be different from the other
half. As to McClung's statement ('02) "that if there is a cross-division
of the chromosomes in the maturation mitosis, there must be two kinds of
spermatozoa, regardless of the presence of the accessory chromosome," it
appears to me that in a case like the aphid, where the paired elements
of the five bivalent chromosomes are separated in the first maturation
mitosis, there may be as many as seventeen kinds of spermatozoa instead
of two. If, however, we suppose that the sex characters are segregated
in the first maturation mitosis, there would, of course, be two equal
classes of spermatozoa with reference to that character.

In _Stenopelmatus_ the element _x_ in certain stages closely resembles
the "accessory chromosome" of McClung, and especially that described by
Baumgartner for _Gryllus domesticus_, but its origin and fate are
different. It first appears attached to an end of the spireme in the
growth stage of the young spermatocytes, where it is much smaller than
in later growth stages. It gradually increases in size, is a conspicuous
element in the first maturation spindle, goes into one of each pair of
spermatocytes of the second order, and there degenerates during the rest
stage between the two maturation mitoses. The whole history of this
element suggests that it may be rejected chromatin analogous to that
observed in the ovogenesis of many forms. In _Sagitta_, for example, a
considerable quantity of chromatin granules is given off by the
chromosomes and cast out into the cytoplasm near the close of ovogenesis
(Stevens, '03). Rückert ('92) has described a similar casting out of
chromatin material by the chromosomes of the oöcytes of _Pristiurus_.

The spermatogenesis of _Stenopelmatus_, therefore, differs from that of
the other Orthoptera which have been described in having (1) a larger
number of chromosomes (46), (2) an even number in the spermatogonia, (3)
an accessory chromatin structure in the spermatocytes of the first
order, which disappears before the second maturation division.

In _Blattella_ we have a typical "accessory chromosome," according to
McClung. It appears (1) in all resting spermatogonia closely associated
with a nucleolus, (2) in the spermatogonial mitoses as an odd chromatin
element, making 23 in all, (3) in the growth stage of the spermatocytes
connected with an end of the spireme and also with the nucleolus. It
becomes separated from the other chromatin in the tetrad-stage, remains
nucleolus-like in form, and later appears in the first maturation
division either among the chromosomes or in a more or less aberrant
position. It passes into one of each pair of spermatocytes of the second
order, persists during the rest stage, appears in the second mitosis as
a dyad and then divides, going into one-half of the spermatids. The
spermatids, however, as in _Stenopelmatus_, all have the same
appearance: each has in the center--not against the nuclear membrane--a
small element that stains like chromatin. Occasionally a mass of
chromatin is found outside the nucleus, but this is not constant enough
to support the contention of Moore and Robinson ('05) that the
"nucleolus" of the related form, _Periplaneta americana_, is fragmented
and cast out into the cytoplasm. The spermatids all appear to develop
equally well for some time, but as they approach maturity a varying
proportion of them become degenerate. This can not, however, be due to
absence of the accessory chromosome, as Miss Wallace supposes, in the
spider; for in some follicles no degenerate spermatozoa are found, and
in others more than half may be degenerate. All attempts to study
fertilization stages of the egg have so far failed, and the chromosomes
in the female somatic cells have not proved favorable for counting.
Twenty-three have been counted in several cases, but there was always
some chance of error. If 23 is the somatic number in both sexes, it must
be maintained by union of sex-cells containing 11 and 12 chromosomes,
respectively, the same unequal number occurring in the maturated eggs as
in the sperm. Under such conditions it is difficult to see how the odd
chromatin element of the spermatozoa can determine sex.

The brief description of the chromatin element _x_ in _Sagitta_,
introduced here because it behaves like the accessory chromosome in many
particulars, serves as an example of the occurrence of such an element
in the spermatogenesis of a hermaphrodite form, where it can not
possibly be conceived of as a sex determinant. In _Sagitta_ it is known
to be confined to the male germ-cells. No such element occurs in the
ovogenesis, in the sperm nucleus in the egg, or in the first
segmentation spindle. Its function must, therefore, be confined to the
process of spermatogenesis.

From the standpoint of sex determination, we have in _Tenebrio molitor_
the most interesting of the forms considered in this paper. In both
somatic and germ cells of the two sexes there is a difference not in the
number of chromatin elements, but in the size of one, which is very
small in the male and of the same size as the other 19 in the female.
The egg nuclei of the female must be alike so far as number and size of
chromosomes are concerned, while it is absolutely certain that the
spermatids are of two equal classes as to chromatin content of the
nucleus--one-half of them have 9 large chromosomes and 1 small one,
while the other half have 10 large ones. Since the male somatic cells
have 19 large and 1 small chromosome, while the female somatic cells
have 20 large ones, it seems certain that an egg fertilized by a
spermatozoön which contains the small chromosome must produce a male,
while one fertilized by a spermatozoön containing 10 chromosomes of
equal size must produce a female. The small chromosome itself may not be
a sex determinant, but the conditions in _Tenebrio_ indicate that sex
may in some cases be determined by a difference in the amount or quality
of the chromatin in different spermatozoa. This is much the most
suggestive part of the work, and it will be followed up by the study of
related forms.

There appears to be so little uniformity as to the presence of the
heterochromosomes, even in insects, and in their behavior when present,
that further discussion of their probable function must be deferred
until the spermatogenesis of many more forms has been carefully worked
out.

BRYN MAWR COLLEGE, _May 15, 1905_.




BIBLIOGRAPHY.


BAUMGARTNER, W. J.

     '04. Some new evidences for the individuality of the
     chromosomes. Biol. Bull., vol. 8, no. 1.


MCCLUNG, C. E.

     '99. A peculiar nuclear element in the male reproductive
     cells of insects. Zool. Bull., vol. 2.

     '00. The spermatocyte divisions of the Acrididæ. Kans. Univ.
     Quart., vol. 9, no. 1.

     '01. Notes on the accessory chromosomes. Anat. Anz., bd. 20,
     nos. 8 and 9.

     '02. The accessory chromosome--Sex determinant? Biol. Bull.,
     vol. 3, nos. 1 and 2.

     '02_a_. The spermatocyte divisions of the Locustidæ. Kans.
     Univ. Quart., vol. 1, no. 8.


MONTGOMERY, THOS. H., JR.

     '01. A study of the chromosomes of the germ-cells of
     Metazoa. Trans. Amer. Phil. Soc., vol. 20.

     '01_a_. Further studies on the chromosomes of the _Hemiptera
     heteroptera_. Proc. Acad. Nat. Sci. Phila. 1901.

     '04. Some observations and considerations upon the
     maturation phenomena of the germ-cells. Biol. Bull., vol. 6,
     no. 3.


MOORE, J. E. S., and ROBINSON, L. E.

     '05. On the behavior of the nucleolus in the spermatogenesis
     of _Periplaneta americana_. Quart. Jour. of Mikr. Sci., n.
     s., no. 192 (vol. 48, part 4).


PAULMIER, F. C.

     '93. Chromatin reduction in the Hemiptera. Anat. Anz., vol.
     14.

     '99. The spermatogenesis of _Anasa tristis_. Journ. of
     Morph., vol. 15.


RÜCKERT, J.

     '92. Zur Entwickelungsgeschichte des Ovarialeies bei
     Selachiern. Anat. Anz., vol. 7, no. 4 and 5.


DE SINÉTY, R.

     '01. Recherches sur la biologie et l'anatomie des phasms. La
     Cellule, vol. 19.


STEVENS, N. M.

     '03. On the ovogenesis and spermatogenesis of _Sagitta
     bipunctata_. Zool. Jahrb., vol. 18.


SUTTON, W. S.

     '02. On the morphology of the chromosome group in
     _Brachystola magna_. Biol. Bull., vol. 4, no. 1.

     '03. The Chromosomes in heredity. Biol. Bull., vol. 4, no.
     5.


WALLACE, L. B.

     '00. The accessory chromosome in the spider. Anat. Anz.,
     vol. 18.

     '05. The spermatogenesis of the spider. Biol. Bull., vol. 8,
     no. 3.


WILCOX, E. V.

     '95. Spermatogenesis of _Caloptenus femur-rubrum_ and
     _Cicada tibicen_. Bull. Mus. Comp. Zool. Harvard Univ., vol.
     27.

     '96. Further studies on the spermatogenesis of _Caloptenus
     femur-rubrum_. Bull. Mus. Comp. Zool. Harvard Univ., vol.
     29.

     '97. Chromatic tetrads. Anat. Anz., vol. 14.

     '01. Longitudinal and transverse division of chromosomes.
     Anat. Anz., vol. 19, no. 13.




DESCRIPTION OF PLATES.


[The figures of plates I-VI were all drawn with Zeiss oil-immersion 2
mm., oc. 12, and have been reduced one-third; those of plate VII with
oc. 8, not reduced.]


PLATE I.


_Termopsis angusticollis._

FIGS. 1-3. Resting nuclei of spermatogonia, showing division of
nucleolus.

4. Equatorial plate of spermatogonial mitosis, 52 chromosomes.

5-6. Young spermatocytes, showing division of nucleolus.

7. First maturation spindle, and two nuclei (6 and 8) in same cyst.

8-10. Skein-stage--so-called synapsis-stage.

11-14. Bouquet-stage, showing two nucleoli, centrosome (_c_) in fig. 11,
and loops made up of fine, then coarser granules.

15-17. Stage following preceding; loops straightened out and extending
in various directions through nucleus.

18. _a_, Chromosomes much shortened and longitudinally split; _b_,
chromosomes contracted to form diamond-shaped figures.

19. Stage between 18_a_ and 18_b_.

20. Stage between 19 and 18_b_.

21. Stage similar to 18_a_, one chromosome in double diamond form.

22. First maturation spindle in metaphase, chromosomes in single and
double diamond shapes.

23. Chromosome in single diamond or tetrad form, as they usually come
into the spindle.

24. Double diamond-form assumed before metakinesis.

25. The 26 chromosomes of an early metaphase.

26. First maturation spindle in metakinesis.

27. Equatorial plate of first maturation spindle in metakinesis.

28. Another spindle, showing three granules which are probably remains
of nucleoli.

29. Anaphase of first maturation mitosis, one centrosome divided.

30. Late anaphase.

31-32. Telophase, exceptional cases of division of the cell.

33-36. Partial rest stage between first and second maturation divisions,
two nucleoli present. Chromosomes in fig. 36 in form of double diamonds
ready for metakinesis.

37-38. Second maturation spindle in metaphase.

39. Equatorial plate of second maturation spindle, 26 chromosomes.

40. Same in anaphase.

41. Four spermatid nuclei in one cell, each nucleus containing one
nucleolus.

42. A later stage, showing elongation of nuclei, centrosome and sphere
at posterior end.

43-45. Later stages in the development of the spermatozoa, nucleolus
grows gradually smaller.

[Illustration: STEVENS. PLATE I.

N. M. S. del.

TERMOPSIS ANGUSTICOLLIS.]


PLATE II.


_Stenopelmatus._

FIGS. 46-47. Nuclei of spermatogonia, showing 2 and 3 nucleoli (_n_).

48-49. Prophase of spermatogonial mitosis, showing two exceptionally
large chromosomes of equal length.

50. Equatorial plate of spermatogonial mitosis, 46 chromosomes.

51-54. Spermatocytes in spireme stage, nucleus containing a nucleolus
(_n_), and a chromatin element (_x_), which is attached to one end of
spireme and gradually increases in size during growth stage of
spermatocytes.

55. Spireme longitudinally split and showing the beginning of cross
formation.

56. Spireme segmented, tetrads forming.

57. One split segment and a part of another connected by bands of linin.

58. More open cross and diamond forms; element _x_ conspicuous.

59-60. More contracted cross and diamond-shaped tetrads; linin bands
shown in 60, where element _x_ is also present.

61. Different forms assumed by element _x_ during tetrad stage (figs.
56-60).

62-63. Diamond-shaped and contracted cross-shaped tetrads from metaphase
of first maturation mitosis, showing linin connections.

64. Diamond-shaped tetrad with spindle-fibers attached; _a-a_, probably
halves of one univalent chromosome; _b-b_, halves of the other.

65. Dyad from anaphase of first maturation mitosis.

66-67. Metaphase of first maturation spindle, showing element _x_ in
different positions.

68. Late anaphase of same.

69-70. Equatorial plate of first maturation spindle, 23 chromosomes and
element _x_ below, in fig. 69.

71. Chromatin massed at poles of spindle; element _x_ isolated at one
pole.

72-73. Two resting spermatocytes of the second order, one containing
element _x_, the other not.

74-76. Successive stages of breaking down of element _x_.

77. Prophase of second division; dyads evident, but no sign of _x_ in
this or following stages.

78. Second spermatocyte division--metakinesis.

79. Same; late anaphase.

[Illustration: STEVENS. PLATE II.

N. M. S. del.

STENOPELMATUS.]


PLATE III.


_Stenopelmatus._

FIG. 80. Telophase of second maturation mitosis.

81. Young spermatid, showing spindle-remains at _s_.

82. Spermatid showing a conspicuous chromatin element in nucleus, and
spindle-remains (_s_) elongated.

83. Spermatid, showing centrosome (_c_) and divided spindle-remains (_s_
and _a_).

84. Older spermatid, showing centrosome (_c_), axial fiber of tail, and
spindle-remains (_s_).

85. Spermatid, showing acrosome material (_a_) migrating to side of
nucleus opposite centrosome.

86. Slightly older spermatid.

87. Later stage of spermatid, showing condensed chromatin, elongated
centrosome (_c_), acrosome material (_a_), and spindle-remains (_s_).

88-89. Older spermatids, showing formation of acrosome (_a_) and middle
piece (_m_).

90-92. More advanced stages.

93. Mature spermatozoön.


_Blattella germanica._

FIG. 94. Somatic cell from egg follicle, 23 chromosomes.

95. Spermatogonium, showing chromatin element (_x_) associated with a
nucleolus (_n_).

96. Same, prophase of mitosis.

97. Equatorial plate of spermatogonial mitosis, 23 chromatin elements.

98. Young spermatocyte, showing centrosome (_c_) and U-shaped element
(_x_).

99. Young spermatocyte, element _x_ attached to one end of a long, fine
spireme.

100. Coarser spireme stage.

101-103. Bouquet stage.

104-105. Later spireme stage.

106. Various forms assumed by the combined nucleolus and element _x_;
last figure from a giant cell.

107. Segmenting spireme.

108. Similar stage to fig. 107, one chromosome longitudinally split;
element _x_ present.

[Illustration: STEVENS. PLATE III.

N. M. S. del.]


PLATE IV.


_Blattella germanica._

FIG. 109. Similar stage to figs. 107 and 108; chromosomes U-shaped and
not longitudinally split; two centrosomes present (_c_).

110. Longitudinally split chromosomes.

111-113. Various stages in formation of cross-shaped tetrads.

114-117. Bent rods, U-shapes, split rings, pairs of rods, and rod-shaped
tetrads (116), which are equivalent to the crosses of figs. 112-113.

118-122. Metaphase of first maturation division, showing the element _x_
in various positions.

123-127. First maturation spindle in metaphase.

128. Same in anaphase.

129-132. Late anaphase, showing element _x_ double in 129, and a lagging
tetrad in 130.

133. Telophase, with the element _x_ in one daughter cell.

134-136. Prophase of second maturation mitosis, showing dyads and
element _x_.

[Illustration: STEVENS. PLATE IV.

N. M. S. del.

BLATTELLA GERMANICA.]


PLATE V.


_Blattella germanica._

FIGS. 137-141. Dyads contracting for second maturation mitosis.

142. Equatorial plate of second maturation spindle, containing 11
chromosomes.

143-144. Same, with 11 chromosomes and the element _x_.

145-147. Sections of second maturation spindles; element _x_ dividing in
146 and 147.

148. Telophase of second mitosis.

149. Telophase of second mitosis, showing masses of chromatin left
behind in cytoplasm.

150. Spermatid with extranuclear chromatin (_a_).

151. Similar stage; different view of spindle-remains (_s_) and of
chromatin element (_x_{2}_).

152-153. Spermatid with divided spindle-substance and the corresponding
double-tailed form.

154-155. Stages between 156 and 158.

156-157. Older spermatids than 151, showing spindle-remains (_s_) and
centrosome (_c_).

158-160. Later stages in development of sperm-head.

161. Ripe spermatozoön.

162-168. Degenerate spermatids and spermatozoa.

[Illustration: STEVENS. PLATE V.

N. M. S. del.

BLATTELLA GERMANICA.]


PLATE VI.


_Tenebrio molitor._

FIGS. 169-170. Equatorial plates of spermatogonial mitosis, showing 19
large and 1 small chromosome.

171-175. Condensation stage, bouquet stage, spireme stage, and rather
rare tetrad stage of young spermatocyte.

176. Bivalent chromosomes, with longitudinal split; small chromosome
shown at _s_.

177. Bivalent chromosomes condensed into a close spireme.

178-179. Bivalent chromosomes separating for mitosis. The unsymmetrical
pair shown in fig. 179.

180. Prophase of first maturation mitosis, showing the unsymmetrical
pair and the tetrad nature of the symmetrical pairs.

181. Prophase of same mitosis, showing symmetrical and unsymmetrical
pairs, as in figs. 178 and 179.

182. Metaphase, unsymmetrical pair out of the equatorial plane.

183. Tangential section of a spindle in metaphase, showing the
unsymmetrical pair and one symmetrical pair.

184. Equatorial plate of same mitosis, 10 chromosomes.

184_a_. Early anaphase, showing separation of the elements of the
unsymmetrical pair.

185. Later anaphase.

186. Polar plate, showing 9 large and 1 small chromosome.

187. Polar plate, showing 10 large chromosomes.

188. Condensation stage between the two maturation divisions.

189-190. Prophase of second maturation division, fig. 189 showing 10
equal dyads, and fig. 190, showing 9 equal and 1 small dyad.

191. Equatorial plate, showing 1 small chromosome and 9 large ones.

192. Equatorial plate, showing 10 large chromosomes.

193-194. Tangential sections of spindle in metakinesis.

195. Anaphase of same mitosis.

196. Polar plates of a spindle, showing in each 1 small chromosome and 9
large ones.

197. Polar plates of another spindle, 10 large chromosomes in each.

198. Young spermatid, showing isolated small chromosome.

199. Young spermatid, showing isolated large chromosome and rotation of
nuclear contents.

200-202_a_, _b._ Older spermatids.

203. Sperm-heads, showing centrosome and granular chromatin.

204. Equatorial plate from dividing somatic cell of male pupa, showing
19 large and 1 small chromosome.

205-206. Daughter plates of a similar spindle, showing small chromosome
in each; three of the large chromosomes missing in 206.

207. Equatorial plate of a dividing cell of follicle of a young egg,
showing 20 large chromosomes.

208. Prophase of mitosis in a young oögonium, showing 20 large
chromosomes in two sections, _a_ and _b_.

[Illustration: STEVENS. PLATE VI.

N. M. S. del.

TENEBRIO MOLITOR.]


PLATE VII.


_Aphis oenotherae._

FIG. 209. Spermatogonium.

210-211. Spermatogonia in prophase of mitosis.

212. Young spermatocyte of first order.

213. Spermatocytes of first order; conjugation of the chromosomes.

214. Condensation of chromatin--spermatocytes of first order immediately
before mitosis.

215. Equatorial plate of first maturation division.

216. Same, side view, showing chromosomes double.

217-218. Anaphase of same mitosis.

219. Daughter spermatocytes of second order.

220. Equatorial plate of second maturation mitosis.

221. Anaphase of same.

222. Young spermatid.

223. Equatorial plate of first polar spindle of winter egg.

224. Equatorial plate of polar spindle of parthenogenetic egg.

225. Equatorial plate of segmentation spindle of parthenogenetic egg.


_Sagitta bipunctata._

FIG. 226. Resting spermatogonia.

227. Prophase of spermatogonial mitosis.

228. Last spermatogonial mitosis, metakinesis.

229. Anaphase of same, showing synapsis of chromosomes at pole of
spindle, and element _x_.

230. Resting spermatocyte of first order.

231. Bouquet stage.

232. Later growth stage.

233. Prophase of first maturation mitosis, some of the chromosomes split
longitudinally.

234. Later stage, chromosomes condensing and element _x_ dividing.

235-237. First maturation mitosis.

238. Division of element _x_ between the two maturation divisions.

239. Second maturation mitosis.

240. Anaphase of same, showing the element _x_ more deeply stained than
the chromosomes.

241. Young spermatids; element _x_ still conspicuous.

[Illustration: STEVENS. PLATE VII.

N. M. S. del

APHIS OENOTHERA--SAGITTA BIPUNCTATA.]