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STUDIES IN SPERMATOGENESIS

WITH ESPECIAL REFERENCE TO THE

"ACCESSORY CHROMOSOME”

BY N.M; STEVENS.

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

CARNEGIE INSTITUTION OF WASHINGTON

PUBLICATION No. 36

2, LU} O Ly

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

STUDIES IN SPERMATOGENESIS WITH ESPECIAL REF-
ERENCE 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 chromo-
some,’’ which, according to McClung (’02), may be a sex determinant.
This view has been supported by Sutton (’o2) in his work on Brachy-
stola 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, Zermopsis
angusticollis,; (2) a California sand-cricket, Stenopelmatus; (3) the
croton-bug, Blattella germanica,; (4) the common meal-worm, 7ene-
brio molitor ; and (5) one of the aphids, Aphzs enothere.

A brief account of a chromatin element resembling the accessory
chromosome in Sagztfa has been added for comparison. Thespermato-
genesis 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 excel-
lent fixation and were preferable to the osmic mixtures when it was
desirable to stain the same material with iron-hematoxylin, and also
with various anilin stains.

Heidenhain’s iron-hematoxylin, either alone or with orange G
ot erythrosin, was used more than any other one stain. With osmic
fixation safranin gave better results in some cases, because of the

3

4 STUDIES IN SPERMATOGENESIS.

abundance of spindle fibers and sphere substance which were stained by
hematoxylin. ‘The safranin-gentian combination used by Miss Wal-
lace and others in the study of the accessory chromosome did not prove
to be especially helpful with these forms. ‘Thionin was found to bea
very useful stain for distinguishing between the accessory chromosome
and an ordinary nucleolus. Licht-grtin 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. I2).

In the spermatogonium there is a very large nucleolus (plate 1,
fig. 1), which in the iron-hematoxylin preparations is very conspic-
uous, 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 imme-
diately 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 familar ‘‘ 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. 18a a longitudinal split is seen in several chromo-
somes. Figures 186, 19, 20, and 21 show various stages in the con-
traction 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

TERMOPSIS ANGUSTICOLLIS. 5

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-hematoxylin preparations. Figures 31 and 32 show excep-
tional 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
inonecell. 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 Zevmopszs
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 Szenopelmatus contains from one to three
large nucleoli, which stain much less with thionin than does the
spireme (plate 11, 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 (), there appears in the young sperm-
atocyte a conspicuous element (x) which stains deeply with all chro-
matin 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 Stexofelmatus, Figures 56,58, 59, and 60 show various
stages in the contraction of the split segments to form crosses and

6 STUDIES IN SPERMATOGENESIS.

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 chromo-
some and at a different level. In figures 66 and 67 the one excep-
tionally 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 dis-
tinct 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 byits 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 ;

STENOPELMATUS. 7

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 111, 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 centro-
some 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 (7), as shown in figure 89,
and in a later stage in figures 90 to 92. The mature spermatozoon
with its forked anterior end appears in figure 93.

The points of especial interest in the spermatogenesis of Stenopel-
matus 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 Stexopelmatus, 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 98isa very early stage of the spermatocyte of the first
order, showing the element + as a U-shaped body. The centrosome

8 STUDIES IN SPERMATOGENESIS.

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. Inthe ‘‘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.
Io1, 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 rv. 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. III, 112, 113), as in Stexopelmatus 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 matura-
tion 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 chromo-
somes 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

BLATTELLA GERMANICA. 9g

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 spermato-
cytes 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 dis-
tinguish 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 appear-
ance 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 7) 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 sper-
matids, as in Stexopelmatus, develop a deeply-staining body, which,
however, in this case is usually centrally located and often appears
double (figs. 150-152).

The spindle-remains (Spzzdelreste) 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.

IO STUDIES IN SPERMATOGENESIS.

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 spermatozoon (fig.161). The tail is very long and is shown only
in part.

Of the forms studied, Blattella alone has many degenerate sperm-
atozoa. 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 num-
bers 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 chro-
matin is left behind in the cytoplasm, especially in the first spermato-
cyte 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 + 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 spermato-
cyte 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.

BLATTELLA GERMANICA. 1g

An attempt was made to determine the somatic number of chromo-
somes. 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 pos-
sible 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 sperma-
tocytes, 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 con-
taining 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 chro-
mosome. Usually, however, the irregular and much tangled spireme
(figs. 173, 174) condenses into a heavy segmented band variously dis-
posed in the nucleus (fig. 177). This band soon separates into the
bivalent chromosomes shown in figures 178 and 179, giving 9 sym-
metrical 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 ats. In the metaphase (fig. 182) it is the last to come into the
equatorial plate, possibly because of its lack of symmetry. Thesmaller
component of this pair is always directed toward the equator of the

12 STUDIES IN SPERMATOGENESIS.

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 chromo-
somes 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 hematoxylin. 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 chro-
mosome. 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 corre-
sponds fairly well to the large or to the small component of the unsym-
metrical 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 4), 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-

TENEBRIO MOLITOR. 13

tubes were sectioned and the chromosomes counted in the dividing
cells of the egg-follicle (9 somatic cells), and in dividing oogonia.
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 6 shows two sections of an oogonium in the prophase
of mitosis. In order to determine the number and character of the
chromosomes in the male somatic cells, several male pupze were sec-
tioned. 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 spermatozoon in fertili-
zation brings into the egg either 10 large ones or 9 large ones and I
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 7enedrvio. It is important that related
forms should be studied in order to ascertain whether the same chro-
matic conditions prevail in other species of this genus or possibly in
the Coleoptera in general.*

Aphis oenotherae,

The spermatogensis of 4phzs 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 vit, 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

* Prof. E. B. Wilson has recently found a similar dimorphism in the spermatozoa of
Iyge@us and other of the Hemiptera heteroptera.

I4 STUDIES IN SPERMATOGENESIS.

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 acces-
sory 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 ’’ chro-
mosomes 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 Sagztta 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 mem-

SAGITTA BIPUNCTATA. 15

brane. 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 spermato-
genesis or in the formation of the sperm-head, of which it finally
becomes a part. In Sagztia 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 segmenta-
tion 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 sum-
marized more recently by Montgomery under the heading ‘‘ Hetero-
chromosomes ’’ 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-deter-
minants, since in the forms which he has examined these chromatin
bodies occur in only one-half of the spermatozoa, and the sex-char-
acter 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 disappear-

16 STUDIES IN SPERMATOGENESIS.

ance 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 chro-
mosome is capable of developing into a functional spermatozoon, 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 Zermopsis. The sex-character may indeed be repre-
sented 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 thatina
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 G7yllus 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). Riickert (’92) has described a
similar casting out of chromatin material by the chromosomes of the
oocytes of Pristiurus.

GENERAL DISCUSSION. L7

The spermatogenesis of S/enopelmatus, 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 sper-
matogonia, (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,’’ accord-
ing 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 sperma-
tocytes 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. Occa-
sionally 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 degen-
erate. 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 Sagzt/a, intro-
duced 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

18 STUDIES IN SPERMATOGENESIS.

not possibly be conceived of as a sex determinant. In Sag7téfa 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 Zenebrio moli-
tor 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 1o 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 sperm-
atozoon which contains the small chromosome must produce a male,
while one fertilized by a spermatozoon containing 10 chromosomes of
equal size must produce a female. The small chromosome itself may
not be a sex determinant, but the conditions in 7enebrvio 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 pres-
ent, 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. I9

BIBLIOGRAPHY.

BAUMGARTNER, W. J.
'04. Some new evidences for the individuality of the chromosomes. Biol. Bull.,
vol. 8, no. I
McCuune. C. E.
‘99. A peculiar nuclear element in the male reproductive cells of insects. Zool.
Bull., vol. 2.
‘00. The spermatocyte divisions of the Acrididz. Kans. Univ. Quart., vol. 9, no. 1.
‘01. Notes on the accessory chromosomes. Anat. Anz., bd. 20, nos. 8 and g.
'02. The accessory chromosome—Sex-determinant? Biol. Bull., vol. 3, nos. 1 and 2.
02a. The spermatocyte divisions of the Locustidz. Kans. Univ. Quart., vol. 1,
no. 8.
MontTGomery, Tuos. H., Jr.
‘O01. A study of the chromosomes of the germ-cells of Metazoa. Trans. Amer.
Phil. Soc., vol. 20.
‘Ola. Further studies on the chromosomes of the Hemiptera heteroptera. Proc.
Acad. Nat. Sci. Phila. rgor.
04. Some observations and considerations upon the maturation phenomena of the
germ-cells. Biol, Bull., vol. 6, no. 3,
Moore, J. E. S., and Rosinson, L. E.
‘05, On the behavior of the nucleolus in the spermatogenesis of Perzplaneta
americana, Quart. Jour. of Mikr. Sci., n. s., no. 192 (vol. 48, part 4).
PAuULMIER, F. C.
'93. Chromatin reduction in the Hemiptera. Anat. Anz., vol. 14.
99. The spermatogenesis of Avasa trzstis. Journ. of Morph., vol. 15.
RUCKERT, J.
92. Zur. Entwickelungsgeschichte des Ovarialeies bei Selachiern, Anat, Anz.,
vol. 7, no. 4 and 5.
DE SINETY, 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 Sagztta bipfunctata. 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 Calopftenus femur-rubrum and Cicada tibicen. Bull.
Mus. Comp. Zool. Harvard Univ., vol. 27.
96. Further studies on the spermatogenesis of Calopftenus 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,
FAG), 03}.

20

DESCRIPTION OF PLATES.

[The figures of plates 1-vr were all drawn with Zeiss oil-immersion 2 mm.,
oc. 12, and have been reduced one-third; those of plate vir with oc. 8, not reduced. ]

PATE U

Termopsis angusticollis.

. Resting nuclei of spermatogonia, showing division of nucleolus.

. Equatorial plate of spermatogonial mitosis, 52 chromosomes.

. Young spermatocytes, showing division of nucleolus.

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

. Skein-stage—so-called synapsis-stage.

. Bouquet-stage, showing two nucleoli, centrosome (c) in fig. 11, and

loops made up of fine, then coarser granules.

. Stage following preceding; loops straightened out and extending in

various directions through nucleus.

. a, Chromosomes much shortened and longitudinally split; b, chromo-

somes contracted to form diamond-shaped figures.

. Stage between 18a and 18d.

. Stage between 19 and 18).

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

. First maturation spindle in metaphase, chromosomes in single and

double diamond shapes.

. Chromosome in single diamond or tetrad form, as they usually come

into the spindle.

. Double diamond-form assumed before metakinesis.

. The 26 chromosomes of an early metaphase.

. First maturation spindle in metakinesis.

. Equatorial plate of first maturation spindle in metakinesis.

. Another spindle, showing three granules which are probably remains

of nucleoli.

. Anaphase of first maturation mitosis, one centrosome divided.

. Late anaphase.

. Telophase, exceptional cases of division of the cell.

. Partial rest stage between first and second maturation divisions, two

nucleoli present. Chromosomes in fig. 36 in form of double diamonds
ready for metakinesis.

. Second maturation spindle in metaphase.

. Equatorial plate of second maturation spindle, 26 chromosomes.

. Same in anaphase.

. Four spermatid nuclei in one cell, each nucleus containing one nucleolus.
. A later stage, showing elongation of nuclei, centrosome and sphere at

posterior end.

. Later stages in the development of the spermatozoa, nucleolus grows

gradually smaller.

PLATE:

STEVENS.

mpenest / are ae \
: \

Be CO

é, NO oi: Cc
qo |
45° \3
2 4
41 4 43 o¢
N. M. 8. del.

TERMOPSIS ANGUSTICOLLIS.

TE yee
ok mA da iy Fiuil

Fics. 46-47
48-49

50
51-54

55.

56.

57.
58.
59-60.

61.
62-63.

64.

- 65.
66-67.

68.
69-70.

71
72-73

74-76.
77

78
79

DESCRIPTION OF PLATES.

Pirate IT.
Steno pelmatus.

. Nuclei of spermatogonia, showing 2 and 3 nucleoli (n).

. Prophase of spermatogonial mitosis, showing two exceptionally large

chromosomes of equal length.

. Equatorial plate of spermatogonial mitosis, 46 chromosomes.

. Spermatocytes in spireme stage, nucleus containing a nucleolus (),
and a chromatin element (1), which is attached to one end of
spireme and gradually increases in size during growth stage of
spermatocytes.

Spireme longitudinally split and showing the beginning of cross for-
mation.

Spireme segmented, tetrads forming.

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

More open cross and diamond forms; element x conspicuous.

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

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

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

Diamond-shaped tetrad with spindle-fibers attached; a-a, probably
halves of one univalent chromosome; b-b, halves of the other.

Dyad from anaphase of first maturation mitosis.

Metaphase of first maturation spindle, showing element «+ in different
positions.

Late anaphase of same.

Equatorial plate of first maturation spindle, 23 chromosomes and ele-
ment + below, in fig. 60.

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

. Two resting spermatocytes of the second order, one containing ele-

ment +, the other not.

Successive stages of breaking down of element ~*.

. Prophase of second division; dyads evident, but no sign of x in this

or following stages.

. Second spermatocyte division—metakinesis. ~

. Same; late anaphase.

STEVENS. : PLATE Il.

N. M. 8. del.

STENOPELMATUS.

LA
A

ie Ke ; we
FLAT TARY NON

24

DESCRIPTION OF PLATES.

PEATE ble

Steno pelmatus.

Fic. 80. Telophase of second maturation mitosis.

. Young spermatid, showing spindle-remains at s.

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

. Spermatid, showing centrosome (c) and divided spindle-remains
(s and a).

. Older spermatid, showing centrosome (c), axial fiber of tail, and spindle-
remains (s).

. Spermatid, showing acrosome material (a) migrating to side of nucleus

opposite centrosome.

. Slightly older spermatid.

. Later stage of spermatid, showing condensed chromatin, elongated cen-
trosome (c), acrosome material (a), and spindle-remains (s).

. Older spermatids, showing formation of acrosome(a)and middle piece(m).
. More advanced stages.

93. Mature spermatozoéon.

Blattella germanica.

. Somatic cell from egg follicle, 23 chromosomes.

. Spermatogonium, showing chromatin element (4) associated with a
nucleolus (7).

. Same, prophase of mitosis. .

. Equatorial plate of spermatogonial mitosis, 23 chromatin elements.

. Young spermatocyte, showing centrosome (c) and U-shaped element (4).

. Young spermatocyte, element + attached to one end of a long, fine spi-
reme,.

100. Coarser spireme stage.

IOI-103.
104-105.

Bouquet stage.
Later spireme stage.

106. Various forms assumed by the combined nucleolus and element 7; last

figure from a giant cell.

107. Segmenting spireme.
108. Similar stage to fig. 107, one chromosome longitudinally split; element +

present.

STEVENS.

106

STENOPELMATUS—BLATTELLA GERMANICA.

f Ad, te v
(thin

na ae

26

Fic. 100.

II0.
ITI-I13.
II4-117.

118-122.
123-127.
128.

129-132.

133.
134-136.

DESCRIPTION OF PLATES.

Pyate IV.
Blattella germanica.

Similar stage to figs. 107 and 108; chromosomes U-shaped and not longi-
tudinally split; two centrosomes present (c).

Longitudinally split chromosomes.

Various stages in formation of cross-shaped tetrads,

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

Metaphase of first maturation division, showing the element + in various
positions.

First maturation spindle in metaphase.

Same in anaphase.

Late anaphase, showing element x double in 129, and a lagging tetrad
in 130.

Telophase, with the element x in one daughter cell.

Prophase of second maturation mitosis, showing dyads and element x.

STEVENS. PLATE IV.

BLATTELLA GERMANICA.

28

Fics. 137-141.
142.

143-144.
145-147.

148.
140.

150.
I5I.

152-153.

154-155.
156-157.

158-160.
161.
162-168.

DESCRIPTION OF PLATES.

PLATE V.
Blattella germanica.

Dyads contracting for second maturation mitosis.

Equatorial plate of second maturation spindle, containing 11 chro-
mosomes.

Same, with 11 chromosomes and the element +.

Sections of second maturation spindles; element x dividing in 146
and 147.

Telophase of second mitosis.

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

Spermatid with extranuclear chromatin (a).

Similar stage; different view of spindle-remains (s) and of chro-
matin element (#2).

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

Stages between 156 and 158.

Older spermatids than 151, showing spindle-remains (s) and cen-
trosome (c).

Later stages in development of sperm-head.

Ripe spermatozoon.

Degenerate spermatids and spermatozoa.

PEATE V:

BLATTELLA GERMANICA.

30

DESCRIPTION OF PLATES.

PLATE VI.

Tenebrio molitor.

Fics. 169-70. Equatorial plates of spermatogonial mitosis, showing 19 large and

171-175.
176.

WF
178-170.

180.

181.

182.
183.

184.
184a.

185.
186.
187.
188.
189-190.

IOI.
192.
193-104.
195.
190.

197.

108.

199.
200-2024, b.
203.

204.
205-206.

207.

208.

I small chromosome.

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

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

Bivalent chromosomes condensed into a close spireme.

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

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

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

Metaphase, unsymmetrical pair out of the equatorial plane.

Tangential section of a spindle in metaphase, showing the unsym-
metrical pair and one symmetrical pair.

Equatorial plate of same mitosis, 10 chromosomes.

Early anaphase, showing separation of the elements of the unsym-
metrical pair.

Later anaphase.

Polar plate, showing 9 large and 1 small chromosome.

Polar plate, showing 10 large chromosomes.

Condensation stage between the two maturation divisions.

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

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

Equatorial plate, showing Io large chromosomes.

Tangential sections of spindle in metakinesis.

Anaphase of same mitosis.

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

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

Young spermatid, showing isolated small chromosome.

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

Older spermatids.

Sperm-heads, showing centrosome and granular chromatin.

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

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

Equatorial plate of a dividing cell of follicle of a young egg, show-
ing 20 large chromosomes.

Prophase of mitosis in a young odgonium, showing 20 large chro-
mosomes in two sections, a and b.

STEVENS.

PLATE VI.
se ~ ioe pe
: r | og ie F
“s er stan! way) (
Val As1* WV
ps oh AHF 172
7169

183
180 oe oa
e2 e :
“23 ae ie of? eee
® eo *6 oP > o,2
“ery © 0,0 o¢ e
: foe of
- ; ; 186 787 :
ee = 188
eo. e =
® eo ee Ain
‘sy me S3 iu iN ‘
= OO. ode 3>
, 3 e
y a Hue #4 08
194
= 195

207 si
Hy re ee ~
eve 2 (@n \So\
pak SS » ° ) ei
pea ~* vs / 208 b

207, 208 a

TENEBRIO MOLITOR.

Cr
tS)

Fic. 209.
210-211.
212:

213.

214.

215.
216.
217-218.
2109.
220.
221.
222.
22a)
224.
225.

Fic. 226.
227.
228.
220.

230.
231.
232.
233.

234.
235-237.
238.
239.
240.

247.

DESCRIPTION OF PLATES.

Piate VIL.
Aphis oenotherae.

Spermatogonium.

Spermatogonia in prophase of mitosis.

Young spermatocyte of first order.

Spermatocytes of first order; conjugation of the chromosomes.

Condensation of chromatin—spermatocytes of first order immedi-
ately before mitosis.

Equatorial plate of first maturation division.

Same, side view, showing chromosomes double.

Anaphase of same mitosis.

Daughter spermatocytes of second order.

Equatorial plate of second maturation mitosis.

Anaphase of same.

Young spermatid.

Equatorial plate of first polar spindle of winter egg.

Equatorial plate of polar spindle of parthenogenetic egg.

Equatorial plate of segmentation spindle of parthenogenetic egg.

Sagitta bipunctata.

Resting spermatogonia.

Prophase of spermatogonial mitosis.

Last spermatogonial mitosis, metakinesis.

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

Resting spermatocyte of first order.

Bouquet stage.

Later growth stage.

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

Later stage, chromosomes condensing and element x dividing.

First maturation mitosis.

Division of element x between the two maturation divisions.

Second maturation mitosis.

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

Young spermatids; element x still conspicuous.

STEVENS.

PLATE Vil.

@
co
@°
24 215 216 217
a
ae”
222 223
219 220 22I dice!
218
=> a)
= -. @, F, { it
a= 3% Se, )
Qs." : 637 ye {
225
ae, 228 ie

227

232

AHoen& Co Baltimore

APHIS CENOTHERA—SAGITTA BIPUNCTATA.

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2 sn

ote

(i
7
GS

rity