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STUDIES IN SPERMATOGENESIS

PART II.

(PAGES 33-74. PLATES VIII-XV.)

A COMPARATIVE STUDY OF THE HETEROCHROMOSOMES
IN CERTAIN SPECIES OF COLEOPTERA,
HEMIPTERA AND LEPIDOPTERA, WITH
ESPECIAL REFERENCE TO

SEX DETERMINATION.

BY N. M. STEVENS

[Illustration]

WASHINGTON, D. C.:
Published by the Carnegie Institution of Washington
October, 1906

CARNEGIE INSTITUTION OF WASHINGTON

PUBLICATION NO. 36, PART II.

FROM THE PRESS OF
THE WILKENS-SHEIRY PRINTING CO.
WASHINGTON, D. C.




STUDIES IN SPERMATOGENESIS.--II.

A COMPARATIVE STUDY OF THE HETEROCHROMOSOMES IN CERTAIN SPECIES OF
COLEOPTERA, HEMIPTERA, AND LEPIDOPTERA, WITH ESPECIAL REFERENCE TO SEX
DETERMINATION.

By N. M. STEVENS.




INTRODUCTION.


In Part I of this series of papers, the spermatogenesis of five species
belonging to four different orders of insects was considered. In two
species of Orthoptera an "accessory chromosome" was found; in _Tenebrio
molitor_, one of the Coleoptera, an unequal pair of chromosomes was
described; in the other species no heterochromosomes were discovered.
The apparent bearing of the chromosome conditions in _Tenebrio molitor_
on the problem of sex determination has led to a further investigation
of the germ cells of the Coleoptera. One of the Hemiptera homoptera and
two of the Lepidoptera have also been examined for comparison with the
Coleoptera and the Hemiptera heteroptera.


METHODS.

As a result of previous experience with similar material, only two
general methods of fixing and staining have been employed: (1) Fixation
in Flemming's strong solution or Hermann's platino-aceto-osmic, followed
by either Heidenhain's iron-hæmatoxylin or Hermann's safranin-gentian
staining method (Arch. f. mikr. Anat. 1889). (2) Fixation after Gilson's
mercuro-nitric formula, followed by iron-hæmatoxylin, Delafield's
hæmatoxylin and orange G, Auerbach's combination of methyl green and
acid fuchsin, or thionin.

The iron-hæmatoxylin with either mode of fixation gives by far the most
satisfactory preparations for general study. The other stains were used
mainly for the purpose of distinguishing between heterochromosomes and
plasmosomes in resting stages of the nucleus.




COLEOPTERA.


Trirhabda virgata (Family Chrysomelidæ).

Two species of _Trirhabda_ were found in larval, pupal, and adult stage
on _Solidago sempervirens_, one at Harpswell, Maine, the other at Woods
Hole, Massachusetts. The adult insects of the two species differ
slightly in size and color, the germ cells mainly in the number of
chromosomes, _Trirhabda virgata_ having 28 and _Trirhabda canadense_ 30
in spermatogonia and somatic cells.

In _Trirhabda virgata_, the metaphase of a spermatogonial mitosis (plate
VIII, fig. 3) contains 28 chromosomes, one of which, as in _Tenebrio
molitor_ is very much smaller than any of the others. The maternal
homologue of the small chromosome is, as later stages show, one of the
largest chromosomes. In _Tenebrio_ the unequal pair could not be
distinguished in the growth stages of the spermatocytes. In _Trirhabda_
it has not been detected in the synizesis stage (fig. 4), but in the
later growth stages (figs. 5-7) this pair is conspicuous in preparations
stained by the various methods cited above, while the spireme is pale
and inconspicuous. The size of the heterochromosome pair varies
considerably at different times in the growth period, and in some nuclei
(fig. 7) both chromosomes appear to be attached to a plasmosome. The
ordinary chromosomes assume the form of rings and crosses in the
prophase of the first maturation mitosis (fig. 8), but usually appear in
the spindle as dumb-bells or occasionally as tetrads (fig. 10), or
crosses (fig. 11). The unsymmetrical pair is plainly seen in figures 9
and 11, but is not distinguishable in a polar view of the metaphase
(fig. 13). In the anaphase (figs. 14-16) the larger and the smaller
components of the pair separate as in _Tenebrio_. This is, therefore,
clearly a reducing division as far as this pair is concerned, and
probably for all of the other pairs, though neither the synapsis stage
nor the prophase forms are so clear on this point as in some of the
other species studied. Figures 17 and 18 show metaphases of the two
classes of second spermatocytes, the chromosomes varying somewhat in
form in different preparations and even in different cysts of the same
preparation. An early anaphase of this mitosis is shown in figure 19;
here the small chromosome is already divided. It was impossible to find
good polar views of the daughter plates in the two classes of second
spermatocytes, but it is evident from figure 19 and other similar views
of the second spermatocyte spindle that, as in _Tenebrio_, one-half of
the spermatids will contain one of the derivatives of the small
chromosome, the other half one of the products of its larger homologue.

Sections of male pupæ were examined for equatorial plates of somatic
mitoses. Figure 1 is a specimen of such plates. As might be expected,
this figure resembles quite closely the spermatogonial equatorial plate
(fig. 3) in number, form, and size of chromosomes, the small one being
present in both. Figure 2 is from the follicle of a young egg; here we
find 28 chromosomes, but no small one. The chromosome corresponding to
the larger member of the unequal pair in the male evidently has a
homologue of equal size in the female. The chromosome relations in the
male and female somatic cells are therefore the same as in _Tenebrio
molitor_, and must have been brought about by the development of a male
from an egg fertilized by a spermatozoön containing the small
chromosome, and a female from an egg fertilized by a spermatozoön
containing the larger heterochromosome.


Trirhabda canadense.

In _Trirhabda canadense_ the spermatogonial chromosomes are invariably
smaller than in _T. virgata_, but similar size relations prevail. The
spermatogonial plate (fig. 21) contains 30 chromosomes, 29 large and 1
extremely small. In the growth stages the association of the two
unequally paired chromosomes with a rather large plasmosome is more
evident than in _T. virgata_ (figs. 22-23). In this species the unequal
pair is more often found at a different level from the other chromosomes
in the early metaphase of the first maturation mitosis (fig. 24), but it
later comes into the plate with the other chromosomes (figs. 25-27), and
divides earlier than most of the other bivalents (fig. 27). In a polar
view of this metaphase the largest chromosome often appears double (fig.
28); in a front view it is a tetrad as in _T. virgata_, figure 10.
Figure 29 is the equatorial plate of a metaphase in which the larger
component of the unequal pair has been removed in sectioning. The
daughter plates of a first spermatocyte in anaphase (fig. 30) show the
separation of the components of the heterochromosome pair; and
equatorial plates of the resulting two classes of second spermatocytes
(fig. 31) show the same conditions. Figures 32 and 33 are prophases of
the second division, figure 33 showing the small chromosome ready for
metakinesis. It was impossible here also to get good drawings of
daughter plates of the second spermatocytes to show the content of the
two classes of spermatozoa, but there is no doubt that all of the
chromosomes divide in the second mitosis, giving one class of spermatids
containing the small chromosome, the other class its larger homologue.

No male somatic cells were found in mitosis, but they would, if found,
show the same conditions as in the spermatogonia. One of many good
equatorial plates from egg follicles (fig. 20) shows 30 large
chromosomes, indicating an equal pair in place of the unequal pair of
the male.


Chelymorpha argus (Family Chrysomelidæ).

This species was found in larval and adult stages on _Convolvulus
arvensis_ at Harpswell, Maine, in July and August. It shows the same
conditions as _Trirhabda_ and _Tenebrio_, so far as the unequal pair of
chromosomes is concerned, and is especially favorable for study of
synapsis stages. The number of chromosomes in the spermatogonia (plate
IX, fig. 36) is 22. Here the components of the unequal pair are the
small spherical chromosome and one of the several chromosomes third in
size, forming a comparatively small unsymmetrical bivalent (figs.
47-49). The spermatogonia occupy the outer end of each follicle, and
next to them comes a layer of cysts in which the chromosomes from the
last spermatogonial division are closely massed in the form of short
deeply staining loops at one side of the nuclear space (fig. 37).
Following this synizesis stage comes one in which some of the short
loops have straightened, their free ends extending out into the nuclear
space (figs. 38 and 39). Figure 40 shows the nucleus of a slightly later
stage in which the free ends of two straightened chromosomes are on the
point of uniting. In figures 41 and 42 the point of union of homologous
chromosomes is indicated in some cases by a knob, in others by a sharply
acute angle. In a slightly later stage (fig. 43), when all of the short
loops have straightened and united in pairs, the point of union is no
longer visible, all of the loops being rounded at the bend and of equal
thickness throughout. My attention was first called to this method of
synapsis by the conspicuous difference in number and length of loops in
the synizesis stage compared with the later bouquet stage just before
the spireme is formed. Following the synapsis stage shown in figure 43
comes one in which the loops lose their polarized arrangement and unite
to form a continuous spireme (figs. 44 and 45). In this form, the
heterochromosome pair could not be distinguished until the spireme
stage, and it is, therefore, uncertain whether these chromosomes remain
condensed after the last spermatogonial divisions and are hidden among
the massed and deeply staining loops of the synizesis and synapsis
stages, or whether they pass through the same synaptic phases as the
other chromosomes, condensing and remaining isolated at the beginning of
the spireme stage. An early prophase of the first maturation mitosis
(fig. 46) shows segments of the spireme longitudinally split, and in
some cases transformed into crosses which show a transverse division
also. Most of the equal bivalents have the dumb-bell form in the
spindle (figs. 47-49). One is ring-shaped, the ring being formed by
union of the free ends of the segment so that the spindle fibers are
attached to the middle of each univalent chromosome (fig. 49). This
method of ring formation, like that described by Montgomery ('03) for
the Amphibia, is of very frequent occurrence in the spermatocytes of the
Coleoptera. The dumb-bells are so bent at the ends (fig. 52) that the
spindle fibers, here also, are attached at or near the center of each
univalent component of a bivalent chromosome, and the separated,
univalent chromosomes go to the poles of the spindle in the form of Vs.
As in _Tenebrio_ the heterochromosome pair is late about coming into the
equatorial plate (figs. 47-48), but it does finally take its position
with the others (fig. 49) and separates into its component parts
somewhat earlier than the other bivalents (figs. 52, 53). Figures 50 and
51 show polar views of the metaphase, the smaller element (_x_) being
the unequal pair. The chromosomes in late anaphase are too much crowded
to give clear drawings. As in all the beetles so far studied there is no
rest stage between the two maturation divisions, but the late anaphase
of the first mitosis passes over quickly into the second spindle.
Figures 54 and 55 are typical equatorial plates of the second division,
one showing the small chromosome (_s_), the other its mate more nearly
spherical than the others (_l_). An anaphase including the small
chromosome is shown in figure 56. As in the species previously described
the spermatozoa are evidently dimorphic.

Female somatic equatorial plates from egg follicles are shown in figures
34 and 35; 22 chromosomes are present and no one is without an equal
mate.


Odontota dorsalis (Family Chrysomelidæ).

_Odontota dorsalis_ is a small leaf-beetle found on _Robinia
pseudacacia_. The chromosomes are comparatively few in number, 16 in the
spermatogonia (figs. 58 and 59), and of immense size when one considers
the smallness of the beetle. In some of the spermatogonial cysts many of
the chromosomes are V-shaped as in figure 58, while in others all, with
the exception of the small one, are rod-shaped as in figure 59, which
looks like a hemipteran equatorial plate. The spermatogonial resting
nucleus (fig. 60) contains a large plasmosome (_p_), but no condensed
chromatin. The synizesis and synapsis stages are similar to those in
Chelymorpha (figs. 61 and 62). The spireme stage (figs. 63, 64)
contains, in addition to the pale spireme, a very conspicuous group
consisting of a large plasmosome with a large and a small chromosome
attached to it. In the prophase, before the nuclear membrane has
disappeared, this group is easily distinguished from the other
dumb-bell and ring-shaped bivalents (figs. 65-67). In preparations much
destained (fig. 67) the small chromosome component of the group retains
the stain longer than the larger one. The spindle in prophase (fig. 68)
is much elongated and the 8 chromosomes are often spread out upon it so
as to be easily counted. In the early metaphase the parachute-like
heterochromosome group is always nearer one pole of the spindle (plate
X, figs. 69 and 70). The equatorial plate often shows both the larger
component of the pair and the plasmosome (fig. 71). Figures 72-74 show
the metakinesis of the heterochromosome bivalent. In figure 74 the two
unequal elements are completely separated and the plasmosome has
disappeared. The equatorial plates of the two resulting kinds of second
spermatocytes appear in figures 75 and 76. In the anaphase of the second
division all of the chromosomes are divided quantitatively as may be
seen in figures 77 and 78. A few dividing male somatic cells were found
in the walls of the testis. Figure 57 (plate IX) is an equatorial plate
from one of these. The chromosomes are like those of the spermatogonia
(figs. 58 and 59), 15 large and 1 small. No dividing female somatic
cells were found.

A few drawings of developing spermatids are given to show the
transformations of a peculiar body which seems to be characteristic of
insect spermatids. Figure 79 is a very young spermatid showing only
diffuse chromatin in the nucleus. The nucleus soon enlarges (fig. 80)
and a large dense body (_n_) appears which stains like chromatin with
various staining media. A little later (fig. 81) the chromatin forms a
homogeneous, more or less hemispherical or sometimes crescent-shaped
mass which stains an even gray in iron-hæmatoxylin. In addition the
nucleus contains a body (_n_) smaller than in the preceding stage, but
staining the same. As the nucleus condenses and elongates to form the
sperm head, a light region containing this deeply staining body is seen
on one side (figs. 82, 83). A little later the body is divided into two,
which appear sometimes spherical (fig. 84), sometimes elongated (fig.
85). As the sperm head elongates still more, approaching maturity, these
bodies diminish in size (figs. 86, 87) and ultimately disappear. A cross
section of the sperm head at such a stage as figure 87 shows the
chromatin in crescent shape with material which stains very little
within (fig. 88). The chromatin-like body described above was observed
in _Tenebrio_ in a stage corresponding to figure 81, and it was thought
that the larger body seen in some cases and the smaller one in others
might be the larger and smaller heterochromosomes, but a study of this
element in more favorable material disproves that supposition by showing
that the different sizes are merely different phases in the evolution of
the body. Throughout its history it stains like dense chromatin, and my
only suggestion as to its origin is that it seems, from a study of this
and other species of beetles, to be a derivative of the chromatin of the
spermatid, increasing in size for a time, then decreasing, and finally
breaking up into granules and dissolving in the karyolymph. Whether it
has any function connected with the development of the spermatozoön, or
whether it is merely material rejected from the chromosomes, as in many
cases in oögenesis, one can only surmise.

In one testis a peculiar abnormality was found. In all of the perfect
spermatogonial plates two small chromosomes were present (figs. 89, 90).
Nineteen such plates were counted in five different cysts. All of the
equatorial plates of the first spermatocytes showed 8 chromosomes, as
usual. In a few favorable growth stages (fig. 91) the two small
chromosomes were seen to be combined with the larger heterochromosome
and a plasmosome, and one first spermatocyte spindle was found in which
the same combination could be clearly seen (fig. 92). All of the second
spermatocyte metaphases in which a small chromosome occurred, contained
two small ones, making 9 in all (fig. 93). The others contained 8 large
chromosomes, as usual. The only explanation suggested by the conditions
is that somewhere in its history, the small chromosome had undergone an
extra division, and that ever afterward the two products behaved like
the one small heterochromosome of a normal individual. The chief
interest in this abnormality centers in the fact that the two small
chromosomes of this specimen behave exactly like the usual single one,
emphasizing the individuality of this particular heterochromosome. Both
evidently have the same individual characteristics and affinities as the
one in other cases.


Epilachna borealis (Family Coccinellidæ).

_Epilachna borealis_ was found in abundance on squash vines at Woods
Hole, Massachusetts, in September. The testes, unlike those of most of
the Coleoptera, consist of many free follicles similar to those of the
Orthoptera. The germ glands were rather far advanced, but some good
spermatogonial and spermatocyte cysts were found. In figure 94, a
spermatogonial metaphase, the small chromosome is shown with 17 larger
ones. The heterochromosome pair appears in condensed form in the spireme
stage (fig. 95), and again in the first maturation spindle (figs. 96,
97). The varying forms of the ordinary chromosomes are shown in figure
98. Figures 99 and 100 are equatorial plates of the first mitosis. The
unequal pair is shown by itself in figure 101, and the separation of the
heterochromosomes is seen in figure 102. Equatorial plates of the
second division, one containing the small chromosome (_b_), are shown in
figure 103. A prophase of the same division (fig. 104) proves that the
small chromosome divides quantitatively like the others. It was
interesting to find here and there in this material whole cysts in which
the nuclei were like those described by Paulmier ('99) for _Anasa
tristis_ (plate XIII, fig. 14) as cells which were being transformed to
serve as food for the glowing spermatids (figs. 105, 106). The only
occasional appearance of these cysts seems to me to preclude their being
a special dispensation to furnish the spermatids with nutrition during
their transformation. Their appearance and size make me suspect that
they are giant spermatids due to the failure of one of the
spermatogonial or spermatocyte mitoses. The smaller chromatin body seems
to correspond to that described for the spermatids of _Odontota
dorsalis_.


Euphoria inda (Family Scarabæidæ).

Of _Euphoria inda_ only one male was captured, but the numerous testes
furnished abundant material in desirable stages. The spermatogonial
equatorial plate (fig. 107) contains 20 chromosomes of which the two
smallest (_l_ and _s_) form the unequal pair. The resting spermatogonium
contains a two-lobed plasmosome (fig. 108). The growth stages are
similar to those in _Tenebrio_ in showing no distinct bouquet stage, but
there is a spireme stage in which the heterochromosome pair is clearly
seen (fig. 109). Figure 110 (plate XI) is an early prophase, and figure
111 one in which the unequal pair appears with a tetrad and several
dumb-bell forms. The prophase of the spindle, as in _Odontota_, is much
elongated (fig. 112). In figures 113-116 the small heterochromosome pair
is shown in various positions with reference to the other chromosomes of
the metaphase of the first spermatocyte. Figure 117 shows it more deeply
stained than the others in the equatorial plate. This pair divides in
advance of the others, and the larger and smaller elements are plainly
seen nearer the poles in anaphase than the other univalent chromosomes
(figs. 118-120). Daughter plates of the first spermatocyte are shown in
figure 121, and equatorial plates of the second spermatocyte in figure
123. Figure 122 shows the telophase of the first division with the
spindle for the second division forming. In figures 124 and 125 we have
daughter plates of the two classes of second spermatocytes, showing the
content of the two equal classes of dimorphic spermatozoa, as this was
shown in _Tenebrio_. Figures 126 and 127 are anaphases showing the
division of the heterochromosomes (_l_ and _s_). Figures 128-130 are
early stages in the development of the spermatid showing the chromatin
nucleolus (_n_) in various phases.


Blepharida rhois (Family Chrysomelidæ).

The testes were rather too far advanced when this material was collected
and no dividing spermatogonia were present. The growth stages (figs.
131, 132) show a faintly staining spireme and a heterochromosome group
similar to that of _Odontota_, a large and a small chromosome attached
to a large plasmosome. The spireme appears to go directly over by
condensation and segmentation into the dumb-bell-shaped figures seen in
the first maturation spindle (figs. 133, 134), though cross-shaped
bivalents occasionally occur (fig. 135). The heterochromosome pair,
slightly separated by plasmosome material, is usually found at the
periphery of the plate (figs. 133-136). Figure 137 is an exceptional
anaphase in which the heterochromosome elements are not mingled with the
polar masses of chromatin. Figures 138 _a_ and _b_ are equatorial plates
of the second mitosis, and figures 139 and 140 are pairs of daughter
plates from second spermatocytes showing again the dimorphism of the
spermatozoa as to their chromatin content. As in several of the forms
studied, material was collected for examination of the somatic cells,
but no favorable cases of mitosis were to be found.


Silpha americana (Family Silphidæ).

Only one male of this species was secured, but the large testes gave all
stages in abundance. The chromosomes, however, were very small and too
numerous, 40 in the spermatogonia (fig. 141). The small chromosome is,
nevertheless clearly distinguished in many of these plates (_s_). The
resting spermatogonium contains one very large plasmosome and often one
or two smaller ones (fig. 142, _p_). The unequal pair is seen in the
growth stages (figs. 143, 144), and may frequently be seen outside of
the equatorial plate of the first spermatocyte spindle (fig. 146). In
favorable sections it may also be found in the plate among the other
bivalents (fig. 147). Figure 145 is a prophase showing the bivalent
chromosomes still connected by linin fibers. An equatorial plate of the
first division is shown in figure 148, and a pair of corresponding
plates of the second spermatocyte in figure 149. The small
heterochromosome divides in the second spindle in advance of the others
as seen in figure 150. Therefore, although this form is not especially
favorable for detailed study on account of the large number of small
chromosomes, the conditions are evidently the same as in the other
species described--an unsymmetrical heterochromosome bivalent in the
first spermatocyte, giving rise by the second maturation division to
equal numbers of dimorphic spermatozoa, one class receiving the large
heterochromosome, the other class the small one.


Doryphora decemlineata (Family Chrysomelidæ).

_Doryphora decemlineata_ has been the most difficult one of the
collection to work out satisfactorily. The chromosomes in the
spermatogonial plates were in most cases much tangled, and the behavior
of the heterochromosome pair was such as to suggest an "accessory
chromosome" rather than an unequal pair. Abundant material for the study
of somatic cells was at hand, but nothing favorable could be found in
the sections.

Two spermatogonial plates, containing 36 chromosomes, are shown in
figures 151 and 152 (plate XII). The small heterochromosome (_s_) is
slightly elongated. The synizesis and synapsis stages are especially
clear. The chromosomes, after the last spermatogonial mitosis go over
immediately into a synizesis stage consisting of a polarized group of
short loops, which later straighten and unite in pairs (figs. 153 and
154). From these loops are formed the spireme (figs. 155-158), which
splits and segments, producing various cross, dumb-bell, and ring forms
(figs. 159-163). As in most of the other species of Coleoptera, the
unequal pair is not distinguishable until the spireme stage. Figure 162
is an unusual prophase in which all of the equal pairs show a
longitudinal split as well as a transverse constriction, and the larger
heterochromosome (_l_) is also split. Figure 163 shows a somewhat later
and more common prophase in which the unequal pair, one ring, crosses,
and dumb-bells may be seen. This figure, as well as figures 164-168,
show the unequal pair in various relations to the other chromosomes.
This pair in _Doryphora_ consists of a large V-shaped chromosome with a
small spherical one attached to it in different positions. When the
small one is behind the V, the group has the appearance of an
orthopteran "accessory."

Figures 169-171 show the separation of the two elements outside of the
equatorial plate, while in figure 168 the unequal pair is in line with
the other chromosomes. In figure 172, an anaphase, the unequal elements
are barely separated, while the metakinesis of the other pairs is much
further advanced.

Figures 173 and 174 are equatorial plates of the first division, one
showing only the larger element of the heterochromosome pair (fig. 174,
_x_), the other both elements (fig. 173, _l_ and _s_). In the late
anaphase (fig. 175) the larger heterochromosome is often seen outside of
the polar mass, reminding one again of the "accessory" in the
Orthoptera. Occasionally it is found in some other isolated position
(fig. 176). Equatorial plates of the second division show the same
conditions as in the other species; some contain the larger
heterochromosome, others the smaller one (fig. 177, _a_ and _b_). It was
impossible to draw anaphases of the second division from a polar view
and the lateral view showed nothing unusual, merely the longitudinal
division of all of the chromosomes.

The spermatids show some interesting variations from the other species
which have been examined. In figures 178 and 179 we have telophases of
the second spermatocyte, showing centrosome and archoplasm (fig. 178)
and certain masses of deeply staining material in the cytoplasm (fig.
179, _a_{1}_). Figures 180 and 181 are young spermatids showing the
archoplasm from the second spindle (_a_{2}_) and a smaller, more deeply
staining mass (_a_{1}_), derived from the irregular masses of the
earlier stage (fig. 179, _a_{1}_). In figures 182 and 183, the axial
fiber has appeared and the larger mass of archoplasm (_a_{2}_) is being
transformed into a sheath. The other body remains unchanged. During the
following stages this smaller archoplasmic body (_a_{1}_) lies in close
contact with the axial fiber and sheath (_a_{2}_), and gradually
decreases in size (figs. 184-186) until it disappears in a slightly
later stage. The acrosome seems to develop directly out of the
cytoplasm. The enigmatical body (_a_{1}_), which is probably archoplasm
from the first maturation spindle, as it is not found in the cytoplasm
of the first spermatocyte, may serve as nutriment for the developing
axial fiber. The sperm head has a peculiar triangular form, staining
more deeply on two sides.


Miscellaneous Coleoptera.

Considerable material from the spruce borers was collected at Harpswell,
Maine, but the species were not identified. Although these insects were
in the pupa stage, most of the testes were too old. There were no
dividing spermatogonia and few spermatocyte mitoses. Most of the
spermatocytes contained 10 chromosomes, one of which was plainly an
unequal pair. In a few testes the number was 11, indicating that pupæ of
two species had been collected. Figure 187 shows the metaphase of first
spermatocyte mitosis with the unequal pair in metakinesis. Figures 188
and 189 are first spermatocyte equatorial plates of the two species,
containing 10 and 11 chromosomes respectively. Figure 190 is a first
spermatocyte spindle in anaphase, showing the unequal pair behind the
other chromosomes. Figure 191 is an equatorial plate from a second
spermatocyte, showing the small chromosome. In figure 192 are shown
several of the bivalent chromosomes, including the unsymmetrical pair,
from nuclear prophases of the first division, all from the same cyst.

_Adalia bipunctata_ (family Coccinellidæ), the common lady beetle, has a
very conspicuous pair of unequal heterochromosomes, as may be seen in
figures 193-197 (plate XIII). This would seem to be a favorable form for
determining the chromosome conditions in somatic cells, but no clear
equatorial plates were found in either larvæ or pupæ.

In _Cicindela primeriana_ (family Cicindelidæ) there are 18 chromosomes
in the spermatogonium (fig. 198), one being small. The heterochromosome
group is blended into a vacuolated sphere in growth stages (figs. 199,
200). In the metaphase of the first division it is trilobed, or
tripartite (fig. 201), and in metakinesis, a small spherical chromosome
separates from a much larger V-shaped one (fig. 202). Equatorial plates
of first and second spermatocytes are shown in figures 203 and 204.
Whole cysts of giant first spermatocytes were found both in growth
stages (fig. 205) and prophases (fig. 206). Here the heterochromosome
group is plainly double (fig. 205), and the conditions observed must
have been due to the failure of a spermatogonial mitosis to complete
itself.

Several of the Carabidæ have been studied, and the material, though not
especially favorable, is interesting in that some members of the family
have an unequal pair of heterochromosomes, others an odd one. _Chlænius
æstivus_ (figs. 207-212), _Chlænius pennsylvanicus_ (figs. 213-215), and
_Galerita bicolor_ (fig. 216) have the unequal pair, while _Anomoglossus
emarginatus_ (figs. 217-223) has an odd heterochromosome (_x_), which
behaves exactly like the larger heterochromosome in other carabs.

In the Elateridæ and Lampyridæ we also have examples of the second type
with the odd chromosome. Two Elaters, species not determined (figs.
224-229 and 230-235), have each 19 chromosomes in the spermatogonia
(figs. 224 and 230), and in the first spermatocyte division an odd
chromosome (_x_) which is in each case the smallest. In the first of
these Elaters, the female somatic number was determined to be 20 (fig.
229). In the second Elater the pairs of second spermatocytes, containing
9 and 10 chromosomes respectively in the two cells, were in nearly every
case connected as shown in figure 235, one pair of chromosomes not
having separated completely in the first mitosis. Of _Ellychnia
corrusca_ (family Lampyridæ) only the spermatogonial equatorial plate,
containing 19 chromosomes (_x_, the odd one) is given, as no material in
maturation has yet been obtained, and a comparative study of the germ
cells of the Elateridæ and Lampyridæ will be made as soon as suitable
material can be secured.

In addition to the species of Coleoptera described here, two others,
_Coptocycla aurichalcea_ and _Coptocycla guttata_ have been studied by
one of my students and the results published elsewhere (Nowlin, '06). In
both an even number of chromosomes (22, 18) was found in the
spermatogonia, one being very small and forming with a larger one an
unequal pair which remained condensed during the growth stage and
separated into its larger and smaller components in the first
spermatocyte mitosis. The result of maturation, as in the other species
here described and in _Tenebrio molitor_, is dimorphism of the
spermatozoa. The method of synapsis in Coptocycla is like that described
for _Chelymorpha argus_.




HEMIPTERA HOMOPTERA.


Aphrophora quadrangularis.

The abundance of Aphrophora at Harpswell, Maine, in June and July, 1905,
suggested that it might be well to examine at least one more of the
Hemiptera homoptera for comparison with the many species of Hemiptera
heteroptera which have been recently reexamined by Wilson ('05, '05,
'06).

The larvæ only were collected, as they gave all the desired stages for a
study of the spermatogenesis, and also oögonia and synizesis and
synapsis stages of the oöcytes. In the first collections the testes were
dissected out, but the many free follicles break apart so easily that
the later material was prepared by cutting out the abdominal segments
which contained the reproductive organs, and fixing those without
dissection. The same methods of fixation and staining were employed as
for the Coleoptera. Hermann's safranin-gentian method was especially
effective with this material.

In _Aphrophora_ the follicles of each testis are free, forming a dense
cluster, each follicle being connected with the vas deferens by a short
duct. The very young follicles are spherical, the older ones ovoid in
form. The primary spermatogonia (plate XIV, fig. 237)--very clear cells
with a lobed nucleus which stains slightly--occupy the tip of the
follicle. Next to these comes a layer of cysts of secondary
spermatogonia which are conspicuous for their deeper staining quality
(fig. 238). There appears to be no plasmosome in either class of
spermatogonia. Figure 239 is the equatorial plate of a secondary
spermatogonium. There are 23 chromosomes, two of which are conspicuously
larger than the others and evidently form a pair. The odd one is one of
the three next in size.

Next to the secondary spermatogonia are cysts of young spermatocytes,
whose nuclei show a continuous spireme and an elongated deeply staining
chromatin rod which is the odd chromosome (fig. 240). This is often more
elongated than in the figure and more or less wormlike in appearance. A
pair of smaller chromatin masses may sometimes be detected at this
stage, and are readily found a little later (fig. 241) when the nucleus
has enlarged and the spireme has become looser and stains less deeply.
Here the odd chromosome is more condensed, or shortened, and split.
There is no synizesis and no polarized or bouquet stage, but the nuclei
of all of the spermatocytes contain a continuous spireme throughout the
growth stage. Synapsis must occur at the close of the last
spermatogonial mitosis before the spireme is formed. Figures 242 and 243
show a slightly later growth stage. The form and connection of the
"_m_-chromosome" pair (Wilson, '05_{b}) comes out clearly here. Figure
244, from a safranin-gentian preparation, shows both the odd chromosome
and the _m_-chromosomes. Some time before the first mitosis, the spireme
splits and the pairs of granules embedded in linin are wonderfully
distinct, both in iron-hæmatoxylin and safranin-gentian preparations
(fig. 245). The _m_-chromosomes have here formed a precocious tetrad
(_m_). Figure 246 is a similar stage from a safranin-gentian
preparation. Figures 247 and 248 show the condensation of chromatin
granules to form tetrads of various sizes, still embedded in the linin
spireme. As these tetrads come into the spindle without losing their
elongated form, it is evident that each one consists of two
longitudinally split chromosomes united end to end in synapsis and
separated in the first maturation mitosis, which is therefore
reductional. The odd chromosome and the _m_-chromosomes show no
longitudinal split in these figures, but they may appear as in figure
249. Occasionally one of the tetrads takes the form of a cross (fig.
249). In this figure the split "accessory" (_x_) lies against the
nuclear membrane and the archoplasmic material for the spindle is seen
along one side of the nucleus. It is certain here that the spindle
fibers come from extranuclear material, not from nuclear substance, as
Paulmier ('99) describes for _Anasa tristis_.

Figures 250 and 251 show the first maturation mitosis as it usually
appears in sections from mercuro-nitric material stained with
iron-hæmatoxylin. The odd chromosome is always more or less eccentric
and is attached by a spindle fiber to one pole. In Hermann material,
considerably destained, the tetrads and the odd chromosome appear as in
figures 252, 253, and 254, the tetrads being in position for a
transverse division. The odd chromosome is always so placed that its
longitudinal split is at right angles to the axis of the spindle, as
though it were to divide in this mitosis. It does not do so, however,
but goes to one daughter cell, always lagging behind, as is shown in
figures 255 and 256. Figures 257, _a_ and _b_, are polar plates of the
first mitosis with 11 and 12 chromosomes, respectively, and figures 258,
_a_, _b_, and _c_, show the polar plates (_a_ and _c_) each containing
11 chromosomes, and the odd chromosome at a different level (_b_). The
latter is a view of the anaphase which one often gets at three foci in
one section. Figures 259, _a_ and _b_, are equatorial plates of the
second mitosis with 11 and 12 chromosomes respectively. Figure 260 shows
a side view of the second spindle in metaphase, and figure 261 in
anaphase. Figures 262 and 263 are daughter plates from two spindles
showing the chromosome content of the two equal classes of spermatozoa,
one class containing 11 ordinary chromosomes, the other 11 ordinary
chromosomes plus the odd heterochromosome, for the odd chromosome
divides with the others in the second spindle as in Orthoptera (McClung
and Sutton).

In figures 264 and 265 (plate XV) are seen the telophase of the two
kinds of second spermatocytes, one (fig. 265) showing the divided odd
chromosome, which continues to stain more deeply after the others have
become diffuse. All of the spermatids (figs. 266-268) contain, in the
early stages of development, a body (_n_) which stains like chromatin,
but increases in size from a small granule in the telophase (figs. 264,
265) to the large dense body (_n_) seen in figure 267. This is probably
homologous with the chromatin nucleolus described for the spermatids of
the Coleoptera. In addition to this, in one-half of the spermatid nuclei
there is a condensed mass of chromatin which is evidently the derivative
of the odd chromosome of the spermatogonia and spermatocytes (figs. 267
and 268, _x_). In common with the spermatids of other Hemiptera these
show two masses of archoplasm, the larger of which forms the sheath
(_s_) of the axial fiber of the tail, and the smaller the acrosome
(_a_). The axial fiber grows out directly from the centrosome, on either
side of which there is a dense band forming the lateral boundary of the
middle piece. It will be seen that the odd chromosome of Aphrophora is
in its behavior precisely like the typical Orthopteran "accessory" of
McClung, and similar to the odd chromosome of the Coleoptera.

In various parts of the young male larvæ dividing cells were found and
the number 23 determined (fig. 269). Turning now to the female larvæ to
determine the somatic number, the oögonia proved to be more favorable
for counting. Twenty-four chromosomes were present in equatorial plates
of oögonial mitoses (fig. 270), thus confirming Wilson's results for the
_Anasa_ group of the Hemiptera heteroptera.

In examining sections of female larvæ stained with
safranin-gentian-violet, I was surprised to see a very marked polarized
or bouquet stage and to find among the loops something resembling the
odd chromosome of the growing spermatocytes. It was difficult to get a
clear view of this body as it lay within the loops. In one section of a
slightly earlier stage before synapsis, there were found two pairs of
chromosomes (fig. 271, _x_{1}_, _x_{2}_, and _m_{1}_, _m_{2}_) which
were stained with safranin in contrast with the violet spireme. These
two pairs I interpret as being (1) the homologues of the pair of
_m_-chromosomes, which remain condensed during the growth stage of the
spermatocytes, and (2) a pair of heterochromosomes corresponding to the
odd chromosome of the male. Various combinations of these
heterochromosomes are shown in figures 272-277. Figures 278 and 279 were
taken from mercuro-nitric material stained with iron-hæmatoxylin. In
section 278 the "bouquet" was cut through, showing the bivalent
corresponding to the larger pair in figure 271, and in figure 279 this
element is seen behind the paler loops. The history of these two pairs
of heterochromosomes, which have not, so far as I know, been found
before in oöcytes, should be followed up in older ovaries, and related
species should be examined for similar phenomena.




LEPIDOPTERA.


Cacoecia and Euvanessa.

I had no intention of making an extended study of the spermatogenesis of
the Lepidoptera, but was interested to see if anything corresponding to
the heterochromosomes of other orders could be found. The material
studied was the testes of the larvæ of _Cacoecia cerasivorana_ and
_Euvanessa antiopa_. The number of chromosomes is large, but the
equatorial plates are diagrammatically clear. In both species 30
chromosomes are found in both first and second spermatocytes. In both,
one chromosome is larger (figs. 290 and 293, _x_). In the growth stage
(figs. 283, 284) there is a two-lobed body (or sometimes two separate
spherical bodies) which seems to correspond in size to the larger pair
of chromosomes in the first spermatocyte. In iron-hæmatoxylin
preparations this pair is often obscured by parts of the spireme which
are tangled around it. In safranin-gentian preparations it stains, not
like a plasmosome, but red like the heterochromosomes, while the spireme
is violet. The staining reaction at least suggests that this equal pair
of chromosomes, which may be traced through the synizesis stage (fig.
280), synapsis stage (figs. 281, 282), growth stages (figs. 283, 284),
and prophases (figs. 285-287), into the first spermatocyte spindle
(figs. 288, 290), and on to the second spermatocyte (figs. 289, 291,
292), is an equal pair of heterochromosomes comparable to the equal pair
of "idiochromosomes" found by Wilson in _Nezara_ ('05). As the various
stages are practically the same in _Euvanessa antiopa_, but somewhat
clearer in _Cacoecia_, only one figure is given for _Euvanessa_--the
equatorial plate of the first spermatocyte (fig. 293).




SUMMARY OF RESULTS.


(1) An unequal pair of heterochromosomes has been found by the author in
19 species of Coleoptera belonging to 8 families:

FAMILY.                 SPECIES.

I. Buprestidæ        Two spruce-borers, species not determined.

                     { 1. _Chlænius æstivus._
II. Carabidæ         { 2. _Chlænius pennsylvanicus._
                     { 3. _Galerita bicolor._

                     { 1. _Blepharida rhois._
                     { 2. _Chelymorpha argus._
                     { 3. _Coptocycla aurichalcea._
III. Chrysomelidæ    { 4. _Coptocycla guttata._
                     { 5. _Doryphora decemlineata._
                     { 6. _Odontota dorsalis._
                     { 7. _Trirhabda virgata._
                     { 8. _Trirhabda canadense._

IV. Cicindelidæ      _Cicindela primeriana._

V. Coccinellidæ      { _Adalia bipunctata._
                     { _Epilachna borealis._

VI. Scarabæidæ       _Euphoria inda._

VII. Silphidæ        _Silpha americana._

VIII. Tenebrionidæ   _Tenebrio molitor._

(2) An odd chromosome, which behaves during the growth stage of the
first spermatocytes like the "accessory" of the Orthoptera, has been
found in 4 species of Coleoptera,[A] belonging to 3 families:

FAMILY.             SPECIES.

I. Carabidæ       _Anomoglossus emarginatus._

II. Elateridæ     Two Elaters; species not determined.

III. Lampyridæ    _Ellychnia corrusca._

(3) In most of the species of Coleoptera examined, the unequal pair or
the odd chromosome remains condensed during the growth period of the
first spermatocyte, like the "accessory" of the Orthoptera and the
various heterochromosomes of the Hemiptera.

(4) Several of these species of Coleoptera have a synizesis stage in
which the spermatogonial number of short loops is massed at one side of
the nucleus. This is followed by a synapsis stage in which the loops
straighten and unite in pairs, forming longer loops which soon spread
out in the nuclear space, and, with the exception of the
heterochromosomes, unite to form a continuous spireme.

(5) In several of the species of Coleoptera and in Aphrophora, it has
been shown that a body staining like chromatin develops in the
spermatids, increasing in size for a time, then breaking up into
granules and disappearing. This body evidently has no relation to the
heterochromosomes, as it is the same for all of the spermatids. Its
staining qualities suggest that it may be material derived from the
chromosomes. It is finally dissolved in the karyolymph.

(6) In iron-hæmatoxylin preparations the heterochromosomes of the
Coleoptera vary greatly in their staining properties during mitosis. In
some species they stain exactly like the ordinary chromosomes, in others
the larger one of the unequal pair holds the stain more tenaciously than
the others and also than its smaller mate, and this is true in several
cases where the heterochromosome is smaller than the other chromosomes,
which destain more readily. The odd chromosome of the Elaters stains
less deeply than the others in the first spermatocyte. In the growth
stage they stain more deeply, as a rule, than the spireme, with
iron-hæmatoxylin or thionin, stain red with safranin-gentian and green
with Auerbach's methyl green-fuchsin combination.

(7) _Aphrophora quadrangularis_ agrees with the _Anasa_ group of
Hemiptera heteroptera in having a pair of _m_-chromosomes and an odd
chromosome in the spermatocytes, but differs from many of that group in
that the odd chromosome divides in the second mitosis instead of the
first. It also differs from other known forms in exhibiting
heterochromosomes in certain stages of the oöcytes.

(8) The two species of Lepidoptera examined have an equal pair of
heterochromosomes.

FOOTNOTES:

[A] AUG. 20, 1906.--Since this paper was prepared, 19 other species of
Coleoptera have been studied. Of these, 17 have an unequal pair of
heterochromosomes in the spermatocytes. Six belong to the Chrysomelidæ,
making 14 of that family that have been examined. Representatives of 4
new families--Melandryidæ, Lamiinæ, Meloidæ, Cerambycinæ have been
studied. In only two species--1 Elater and 1 Lampyrid--has the odd
chromosome been found in place of the unequal pair. No species of
Coleoptera has yet been examined in which one or the other of these two
types of heterochromosomes does not occur in the spermatocytes. Of the
42 species of Coleoptera whose germ cells have been studied, 85.7 per
cent are characterized by the presence of an unequal pair of
heterochromosomes in the male germ cells, 14.3 per cent by the presence
of an odd chromosome.




COMPARISON OF RESULTS IN DIFFERENT SPECIES OF COLEOPTERA.


In number of chromosomes there is great variation, the smallest number
(16) having been found in _Odontota dorsalis_, and the largest (40) in
_Silpha americana_. The difference in size is also very marked, as may
be seen by comparing the spermatogonial plates in figures 3 and 58 with
those shown in figures 94 and 141.

No other species of the Tenebrionidæ has yet been secured, and all of
the other beetles examined differ in a marked degree from _Tenebrio
molitor_ in the growth stages of the spermatocytes. While in _Tenebrio_
the chromatin stains very dark throughout the growth stage, and the
unequal pair can not be distinguished until the prophase of division
('05, plate VI, figs. 171-180), in most of the others there are very
distinct synizesis and synapsis stages, following the last
spermatogonial mitosis, then a spireme stage in which the condensed
unequal pair of heterochromosomes or the odd chromosome is conspicuous
in contrast with the pale spireme, whether the preparation is stained
with iron-hæmatoxylin, gentian, or thionin. In _Tenebrio molitor_, the
unequal pair behaved in every respect like the other bivalent
chromosomes. In the other forms, though it behaves during the two
maturation divisions like the symmetrical bivalents, it remains
condensed during the growth period like the "accessory" of the
Orthoptera, the odd chromosome, "_m_-chromosomes," and "idiochromosomes"
of the Hemiptera. In several cases the heterochromosomes of the
Coleoptera are associated with a plasmosome (figs. 22, 23, 63, 132, 158,
217), as is often true in other orders. This peculiar pair of unequal
heterochromosomes varies considerably in size during the growth stage in
some of the species studied, but changes very little in form, differing
in this respect from the "accessory" in some of the Orthoptera (McClung,
'02) and from the large idiochromosome in some of the Hemiptera (Wilson,
'05).

The odd chromosome, so far as it has been studied, behaves precisely
like the larger member of the unequal pair without its smaller mate
(figs. 219, 220, 226, 233). In the growth stage it remains condensed and
either spherical or sometimes flattened against the nuclear membrane
(figs. 217, 225, 231). In the first maturation mitosis it is attached to
one pole of the spindle, does not divide, but goes to one of the two
second spermatocytes (figs. 233, 235). In the second spermatocyte it
divides with the other chromosomes, giving two equal classes of
spermatids differing by the presence or absence of this odd chromosome.

All of the evidence at hand leads to the conclusion that in the
Coleoptera, the univalent elements of all the pairs, equal and unequal,
separate in the first spermatocyte mitosis and divide quantitatively in
the second. In this respect the behavior of the chromosomes in this
order appears to be much more uniform than in the Orthoptera and
Hemiptera.




COMPARISON OF THE COLEOPTERA WITH THE HEMIPTERA AND LEPIDOPTERA.


As has been seen above, the conditions in the Coleoptera, so far as the
heterochromosomes are concerned, correspond very closely in final
results with those in the Hemiptera heteroptera and the Orthoptera. In
minor details these chromosomes are less peculiar in the Coleoptera than
in either of the other orders. Even condensation during the growth stage
is not universal, and synapsis of the heterochromosomes apparently
occurs simultaneously with that of the ordinary chromosomes, instead of
being delayed, as in many of the Hemiptera heteroptera.

_Aphrophora_ (Hemiptera homoptera) agrees with the _Anasa_ group of the
Hemiptera heteroptera in having a pair of condensed _m_-chromosomes, in
the growth stage, but this pair is already united in synapsis when first
seen. It differs from _Anasa_, but agrees with _Banasa_ and _Archimerus_
in exhibiting a typical odd chromosome which goes to one pole without
division in the first spermatocyte, and divides with the other
chromosomes in the second spermatocyte. The odd chromosome in this
species of Hemiptera, therefore, behaves like that in the Coleoptera and
Orthoptera. The most interesting points in the results of this study of
the germ cells of _Aphrophora_ is the discovery of two pairs of
condensed chromosomes in certain phases of the growth stages of the
oöcytes. This has not been shown to be the case in any other species of
Hemiptera, so far as I can ascertain. It is now evident that in the
Heteroptera homoptera there are at least two distinct classes as to
behavior of chromosomes. In one class we have the Aphids (Stevens, '05
and '06) and Phylloxera (Morgan, '06) in which no heterochromosomes have
been found, while in the other class are such forms as Aphrophora with
both a pair of _m_-chromosomes and a typical odd heterochromosome.

The two species of Lepidoptera examined indicate that here we may have
conditions comparable to those in _Nezara_--an equal pair of
heterochromosomes whose only apparent peculiarity is their condensed
form during the growth stage. Doubtless the results of other
investigators will soon throw more light on the heterochromosomes of
this order.




GENERAL DISCUSSION.


It will be seen from the foregoing that the results obtained in the
study of the germ cells of _Tenebrio molitor_ have been confirmed in
full for several species of Coleoptera, and in part for 19[B] different
species belonging to 8[B] families. It has also been shown that a
different type of Coleopteran spermatogenesis exists in at least 3
families, where an odd chromosome like that in the Orthoptera occurs in
place of the unequal pair. In all of these insects the spermatozoa are
distinctly dimorphic, forming two equal classes, one of which either
contains one smaller chromosome or lacks one chromosome.

The most difficult part of the work has been the determination of the
somatic number of chromosomes in the male and female. In some cases
suitable material has been lacking; in others, though material was
abundant, no metaphases could be found in which the chromosomes were
sufficiently separated to be counted with certainty. In three species
(in addition to _Tenebrio molitor_) where the unequal pair is present,
the female somatic cells have been shown to contain the same number of
chromosomes as the spermatogonia, but an equal pair in place of the
unequal pair of the male. In two new cases the male somatic number and
size have been shown to be the same as in the spermatogonia. In one of
the Elateridæ, where the spermatogonial number is 19, the female somatic
number is 20, and in _Aphrophora_ the numbers in male and female cells
are respectively 23 and 24. No exception has been found to the rule
established by previous work on the Coleoptera (Stevens, '05) and on the
Hemiptera (Wilson, '05 and '06), that (1) in cases where an unequal pair
is present in the male germ cells, it is also present in the male
somatic cells, but is replaced in the female by an equal pair, each
component being equal in volume to the larger member of the unequal
pair, and (2) in cases where an odd chromosome occurs in the male, a
pair of equal size are found in the female. It is therefore evident that
an egg fertilized by a spermatozoön (1) containing the small member of
an unequal pair or (2) lacking one chromosome, must develop into a male,
while an egg fertilized by a spermatozoön containing the larger element
of an unequal pair of heterochromosomes or the odd chromosome must
produce a female.

Whether these heterochromosomes are to be regarded as sex chromosomes in
the sense that they both represent sex characters and determine sex, one
can not decide without further evidence.

Comparison of the two types in Coleoptera, especially where, as in the
Carabidæ, both occur in one family, has suggested to me that here it is
possible that the small chromosome represents not a degenerate female
sex chromosome, as suggested by Wilson, but some character or characters
which are correlated with the sex character in some species and not in
others. Assuming this to be the case, a pair of small chromosomes might
be subtracted from the unequal pair, leaving an odd chromosome. The two
types would then be reduced to one. It may be possible to determine the
validity of this suggestion for particular cases by observation or
experiment.

Since the first of this series of papers was published, there have
appeared three important papers by Prof. E. B. Wilson, bearing on the
problem of sex determination in insects. These papers are based on a
study of many species of the Hemiptera heteroptera. These insects fall
into two classes--one in which a pair of "idiochromosomes," usually of
different size, remain separate and divide quantitatively in the first
spermatocyte, conjugate and then separate in the second maturation
mitosis; and another class in which an odd chromosome--the
"heterotropic" chromosome--divides in one of the maturation mitoses, but
not in the other. Wilson regards the odd chromosome as the equivalent of
the larger of the "idiochromosomes," its smaller mate having
disappeared. In the somatic cells of the former class he finds in the
male the unequal pair, in the female an equal pair, the smaller
chromosome being replaced by an equivalent of the larger
"idiochromosome." In the latter class the male somatic cells contain the
odd number, the female somatic cells and oögonia an even number, the
homologue of the odd chromosome of the male being present and giving to
the female one more chromosome than are found in the male.

In his latest paper Wilson ('06) makes a variety of suggestions as to
sex determination. He shows that if the "idiochromosomes" and the
heterotropic chromosome be regarded as sex chromosomes in the double
sense that they both bear sex characters and determine sex, the
following scheme accounts for the observed facts in all cases where an
unequal pair or an odd heterochromosome have been found:


            Sperm.                               Egg.
   {Large [Male] "idiochromosome"}
I. {or                           } + Large [Female] sex chromosome = a [Female]
   {Odd chromosome.              }

II. {Small [Female] "idiochromosome"}
    {or                             } + Large [Male] sex chromosome = a [Male]
    {No sex chromosome              }

Here we know that such a combination of gametes must occur to give the
observed results, but we are not certain that we have a right to
attribute the sex characters to these particular chromosomes or in fact
to any chromosomes. It seems, however, a reasonable assumption in
accordance with the observed conditions. The scheme also assumes either
selective fertilization or, what amounts to the same thing, infertility
of gametic unions where like sex chromosomes are present. It also
assumes that the large female sex chromosome is dominant in the presence
of the male sex chromosome, and that the male sex chromosome is dominant
in the presence of the small female sex chromosome. Or, it might rather
be said that these are not really assumptions, but inferences as to what
must be true if the heterochromosomes are sex chromosomes. This theory
of sex determination brings the facts observed in regard to the
heterochromosomes under Castle's modification of Mendel's Law of
Heredity ('99).

The question of dominance is a difficult one, especially in
parthenogenetic eggs and eggs which are distinctly male or female before
fertilization. It may be possible that the sex character of the egg
after maturation is always dominant in the fertilized egg, as appears to
be the case in these insects (see scheme). Conditions external to the
chromosomes may determine in certain cases, such as Dinophilus, which
sex character shall dominate in the growing oöcyte, and maturation occur
accordingly. It is evident that this reasoning would lead to the
conclusion that sex is or may be determined in the egg before
fertilization, and that selective fertilization, or infertility of
gametic unions containing like sex characters, has to do, not with
actual sex determination, but with suitable distribution of the sex
characters to future generations. If both sex characters are present in
parthenogenetic eggs, as appears to be the case in aphids and
phylloxera, dominance of one or the other must be determined by
conditions external to the chromosomes, for we have both sexes at
different points in the same line of descent without either reduction or
fertilization.

Wilson suggests as alternatives to the chromosome sex determinant theory
according to Mendel's Law, (1) that the heterochromosomes may merely
transmit sex characters, sex being determined by protoplasmic conditions
external to the chromosomes; (2) That the heterochromosomes may be
sex-determining factors only by virtue of difference in activity or
amount of chromatin, the female sex chromosome in the male being less
active. The first of these alternatives is an attempt to cover such
cases as _Dinophilus_, _Hydatina_, and _Phylloxera_ with large female
and small male eggs. Here Morgan's ('06) suggestion as to degenerate
males seems much to the point. The male sex character, having become
dominant in certain eggs at an early stage, may, from that time on,
determine the kind of development. As to the second alternative, I see
no reason for supposing that the small heterochromosome of a pair is in
any different condition, as to activity, from the large one. The
condensed condition may not mean inactivity, but some special form of
activity. And, moreover, it has been shown that in certain stages of the
development of the oöcyte of one form, _Aphrophora quadrangularis_,
there are pairs of condensed chromosomes corresponding to those of the
spermatocyte, so that there would hardly seem to be any basis for
Wilson's attempt to associate the difference in development of male and
female germ cells with activity or inactivity of chromosomes, as
indicated by condensed or diffuse condition of the chromatin.

On the whole, the first theory, which brings the sex determination
question under Mendel's Law in a modified form, seems most in accordance
with the facts, and makes one hopeful that in the near future it may be
possible to formulate a general theory of sex determination.

This work has been done in connection with a study of the problem of sex
determination, but, whatever may be the final decision on that question,
it brings together a mass of evidence in favor of the belief in both
morphological and physiological individuality of the chromosomes, as
advocated by Boveri, Sutton, and Montgomery. It also gives the strongest
kind of evidence that maternal and paternal homologues unite in synapsis
and separate in maturation, leaving the ripe germ cells pure with regard
to each pair of characters.

BRYN MAWR COLLEGE, _June 7, 1906_.

FOOTNOTES:

[B] AUG. 20, 1906.--36 species belonging to 12 families. See note, p.
49.




BIBLIOGRAPHY.


BOVERI, TH.

     '02. Ueber mehrpolige Mitosen als Mittel zur Analyse des
     Zellkerns. Verh. d. phys.-med. Ges. Würzburg, N. F., vol.
     35.


CASTLE, W. E.

     '03. The heredity of sex. Bull. Mus. Comp. Zoöl. Harvard
     College, vol. 40, no. 4.


MCCLUNG, C. E.

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

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

     '01. Notes on the accessory chromosome. Anat. Anz., vol. 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.

     '05. The chromosome complex of orthopteran spermatocytes.
     Biol. Bull., vol. 9, no. 5.


MONTGOMERY, THOS. H., JR.

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

     '03. The heterotypic maturation mitosis in Amphibia and its
     general significance. Biol. Bull., vol. 4, no. 5.

     '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.

     '05. The spermatogenesis of _Syrbula_ and _Lycosæ_ and
     general considerations upon chromosome reduction and
     heterochromosomes. Proc. Acad. Nat. Sci. Phila., 1905.


MORGAN, T. H.

     '06. The male and female eggs of Phylloxerans of the
     Hickories. Biol. Bull., vol. 10, no. 5.


NOWLIN, W. N.

     '06. A study of the spermatogenesis of _Coptocycla
     aurichalcea_ and _Coptocycla guttata_. Journ. of Exp. Zoöl.,
     vol. 3, no. 3.


PAULMIER, F. C.

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


DE SINÉTY.

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


STEVENS, N. M.

     '05. A study of the germ cells of _Aphis rosæ_ and _Aphis
     oenotheræ_. Journ. of Exp. Zoöl., vol. 2, no. 3.

     '05_a_. Studies in spermatogenesis, with especial reference
     to the "accessory chromosome." Carnegie Inst. of Wash., pub.
     no. 36.

     '06. Studies on the germ cell of Aphids. Ibid., pub. no. 51.


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.


WILSON, E. B.

     '05. Studies on chromosomes. I. The behavior of the
     idiochromosomes in Hemiptera. Journ. Exp. Zoöl., vol. 2, no.
     3.

     '05_a_. The chromosomes in relation to the determination of
     sex in insects. Science, vol. 22, no. 564.

     '05_b_. Studies on chromosomes. II. The paired
     microchromosomes, idiochromosomes, and heterotropic
     chromosomes in Hemiptera. Journ. Exp. Zoöl., vol. 2, no. 4.

     '06. Studies on chromosomes. III. The sexual differences of
     the chromosome-groups in Hemiptera, with some considerations
     of the determination and inheritance of sex. Ibid., vol. 3,
     no. 1.




DESCRIPTION OF PLATES


[The figures were all drawn with Zeiss oil-immersion 2 mm., oc. 12, and
have been reduced one-third, giving a magnification of 1,000 diameters.]


PLATE VIII.


_Trirhabda virgata_ (_Family Chrysomelidæ_).

FIG. 1. Equatorial plate from somatic tissues of a male pupa, 27 large
chromosomes, 1 small one.

2. Equatorial plate from an egg follicle, 28 large chromosomes.

3. Equatorial plate of spermatogonium, 27 large chromosomes, 1 small
one.

4. First spermatocyte, synizesis stage.

5. First spermatocyte, early spireme stage, showing unequal pair of
chromosomes.

6-7. First spermatocyte, later growth stages.

8. First spermatocyte, prophase.

9-12. First spermatocyte, metaphase.

13. First spermatocyte, equatorial plate.

14-15. First spermatocyte, anaphase, showing separation of the elements
of the unequal pair (_l_ and _s_).

16. First spermatocyte, daughter plates.

17. Second spermatocytes, equatorial plates.

18. Second spermatocytes, equatorial plates showing V-shaped
chromosomes.

19. Second spermatocyte, early anaphase, the small chromosome in
metakinesis.


_Trirhabda canadense._

20. Equatorial plate from egg follicle, 30 large chromosomes.

21. Equatorial plate of spermatogonium, 29 large chromosomes, 1 small
one.

22. First spermatocyte, growth stage showing the heterochromosome group.

23. Heterochromosome group. _p_ = plasmosome, _l_ = large
heterochromosome, _s_ = small heterochromosome.

24-27. First spermatocyte, metaphase.

28. First spermatocyte, equatorial plate.

29. First spermatocyte, equatorial plate, small member of the unequal
pair only present.

30. First spermatocyte, daughter plates.

31. Second spermatocytes, equatorial plates.

32-33. Second spermatocytes, prophase.

[Illustration: STEVENS PLATE VIII

N. M. S. del.

COLEOPTERA]


PLATE IX.


_Chelymortha argus_ (_Family Chrysomelidæ_).

FIGS. 34-35. Equatorial plates from egg follicles, 11 equal pairs, no
small chromosome.

36. Equatorial plate of spermatogonium, 21 large chromosomes, 1 small
one.

37. First spermatocyte, synizesis stage.

38-40. First spermatocyte, synapsis stage.

41-43. First spermatocyte, bouquet stage after synapsis.

44-45. First spermatocyte, spireme stage showing the unequal pair of
heterochromosomes.

46. First spermatocyte, prophase.

47-49. First spermatocyte, metaphase.

50-51. First spermatocyte, equatorial plates, _x_ the heterochromosome
pair.

52. First spermatocyte, showing metakinesis of the unequal pair.

53. First spermatocyte, anaphase.

54-55. Second spermatocyte, equatorial plates.

56. Second spermatocyte, anaphase.


_Odontota dorsalis_ (_Family Chrysomelidæ_).

57. Equatorial plate of male somatic cell from walls of the testis, 15
large chromosomes, 1 small one.

58-59. Equatorial plates of spermatogonia, 15 large chromosomes, 1 small
one.

60. Resting nucleus of spermatogonium, showing plasmosome (_p_).

61. First spermatocyte, synizesis stage.

62. First spermatocyte, synapsis stage.

63-64. First spermatocyte, spireme stage, showing the larger and smaller
heterochromosome associated with a plasmosome.

65-68. First spermatocyte, prophases.

[Illustration: STEVENS PLATE IX.

N. M. S. del.

COLEOPTERA]


PLATE X.


_Odontota dorsalis._

FIGS. 69-70. First spermatocyte, metaphase.

71. First spermatocyte, equatorial plate.

72. First spermatocyte, metaphase, showing metakinesis of the
heterochromosomes.

73-74. First spermatocyte, anaphase.

75-76. Second spermatocyte, equatorial plates.

77. Second spermatocyte, showing metakinesis of the small chromosome
(_s_).

78. Second spermatocyte, prophase, showing chromosomes longitudinally
split.

79-80. Young spermatids, _n_ the chromatin nucleolus.

81-87. A series of stages in the development of the sperm head, showing
the various phases in the history of the chromatin nucleolus (_n_).

88. Cross-sections of nearly mature sperm heads.

89-90. Equatorial plates of spermatogonia of abnormal individual, 15
large chromosomes, 2 small ones.

91. First spermatocyte from same testis, spireme stage, showing 2 small
chromosomes associated with 1 large one and a plasmosome.

92. First spermatocyte from the same testis, metaphase showing a similar
heterochromosome group.

93. Second spermatocyte from same testis, equatorial plate, showing 2
small chromosomes.


_Epilachna borealis_ (_Family Coccinellidæ_).

94. Equatorial plate of spermatogonium, 17 large chromosomes and 1 small
one.

95. First spermatocyte, spireme stage, showing the unequal pair.

96-97. First spermatocyte, late prophases.

98. First spermatocyte, metaphase, showing chromosomes of different
forms.

99-100. First spermatocyte, equatorial plate.

101. Unequal heterochromosome pair from a metaphase.

102. First spermatocyte, anaphase; ordinary chromosomes stippled to show
more clearly the metakinesis of the unequal pair.

103. Second spermatocyte, equatorial plates.

104. Second spermatocyte, prophase.

105-106. Abnormal giant spermatids, probably in process of degeneration.


_Euphoria inda_ (_Family Scarabæidæ_).

107. Equatorial plate of spermatogonium, 20 chromosomes. The 2 smallest
are the unequal pair of heterochromosomes (_l_ and _s_).

108. Resting spermatogonium, showing plasmosome (_p_).

109. First spermatocyte, spireme stage.

[Illustration: STEVENS PLATE X.

N. M. S. del.

COLEOPTERA]


PLATE XI.


_Euphoria inda._

FIGS. 110-111. First spermatocyte, prophases.

112-113. First spermatocyte, late prophase.

114-116. First spermatocyte, metaphase.

117. First spermatocyte, equatorial plate, _x_ the unequal pair.

118-120. First spermatocyte, anaphase.

121. First spermatocyte, daughter plates.

122. Second spermatocyte, prophase.

123. Second spermatocyte, equatorial plates.

124-125. Second spermatocyte, daughter plates of the two classes.

126-127. Second spermatocyte, anaphase.

128-130. Spermatids, _n_ the chromatin nucleolus.


_Blepharida rhois_ (_Family Chrysomelidæ_).

131-132. First spermatocyte, spireme stages, showing the
heterochromosome group.

133-135. First spermatocyte, beginning of metakinesis.

136. First spermatocyte, equatorial plate, _x_ the unequal pair.

137. First spermatocyte, late anaphase, showing the heterochromosomes
_l_ and _s_.

138. Second spermatocyte, equatorial plates.

139-140. Second spermatocyte, daughter plates of the two classes.


_Silpha americana_ (_Family Silphidæ_).

141. Equatorial plate of spermatogonium, 40 chromosomes--39 large, 1
small.

142. Resting nucleus of spermatogonium, showing 2 plasmosomes (_p_).

143-144. First spermatocyte, spireme stage.

145. First spermatocyte, prophase.

146-147. First spermatocyte, metaphase.

148. First spermatocyte, equatorial plate.

149. Second spermatocyte, equatorial plates.

150. Second spermatocyte, showing metakinesis of the small chromosome.

[Illustration: STEVENS PLATE XI.

N. M. S. del.

COLEOPTERA]


PLATE XII.


_Doryphora decemlineata_ (_Family Chrysomelidæ_).

FIGS. 151-152. Equatorial plates of spermatogonia, 36 chromosomes--35
large, 1 small.

153. First spermatocyte, synizesis stage.

154. First spermatocyte, synapsis stage.

155-158. First spermatocyte, spireme stages.

159. First spermatocyte, spireme segmented and split.

160-163. First spermatocyte, prophases.

164-171. First spermatocyte, metaphase.

172. First spermatocyte, anaphase.

173-174. First spermatocyte, equatorial plates.

175-176. First spermatocyte, late anaphase.

177. Second spermatocyte, equatorial plates.

178-179. Second spermatocyte, telophase, _a_{1}_, archoplasmic material.

180-186. Spermatids in different stages; _a_{1}_, archoplasmic material
from first spermatocyte spindle, _a_{2}_ archoplasmic material from
second maturation spindle.


_Spruce-borers_ (_Family Buprestidæ_).

187. First spermatocyte, metaphase.

188-189. First spermatocyte, equatorial plates of two species, 10 and 11
chromosomes.

190. First spermatocyte, anaphase.

191. Second spermatocyte, equatorial plates containing 9 large
chromosomes and 1 small one.

192. Chromosomes from prophase of the first spermatocyte, all from the
same cyst.

[Illustration: STEVENS PLATE XII.

N. M. S. del

COLEOPTERA]


PLATE XIII.


_Adalia bipunctata_ (_Family Coccinellidæ_).

FIG. 193. Equatorial plate of spermatogonium, 20 chromosomes--19 large,
1 small.

194. First spermatocyte, spireme stage, _x_ the heterochromosome group.

195. First spermatocyte, metaphase.

196. First spermatocyte, equatorial plate.

197. Second spermatocyte, equatorial plates.


_Cicindela primeriana_ (_Family Cicindelidæ_).

198. Equatorial plate of spermatogonium, 20 chromosomes--19 large, 1
small.

199. First spermatocyte, spireme stage, _x_ the heterochromosome group.

200. First spermatocyte, prophase.

201. First spermatocyte, metaphase, _x_ the unequal pair in tripartite
form.

202. First spermatocyte, showing metakinesis of the heterochromosomes
(_l_ and _s_).

203. First spermatocyte, equatorial plate.

204. Second spermatocyte, equatorial plates.

205. Giant spermatocyte, spireme stage, heterochromosome group double
the usual size.

206. Giant spermatocyte, prophase.


_Chlænius æstivus_ (_Family Carabidæ_).

207. First spermatocyte, spireme stage, showing the unequal pair
associated with a large plasmosome.

208. First spermatocyte, metaphase.

209-210. First spermatocyte, beginning of metakinesis.

211. First spermatocyte, equatorial plate, 17 chromosomes.

212. First spermatocyte, anaphase, showing elongated centrosome and
diverging univalent chromosomes.


_Chlænius pennsylvanicus._

213. First spermatocyte, spireme stage.

214. First spermatocyte, equatorial plate, _x_ the unequal bivalent.

215. First spermatocyte, late prophase.


_Galerita bicolor_ (_Family Carabidæ_).

216. Equatorial plate of spermatogonium, 30 chromosomes--29 large, 1
small.


_Anomoglossus emarginatus_ (_Family Carabidæ_).

217. First spermatocyte, growth stage, _x_ the odd chromosome.

218. First spermatocyte, prophase.

219-220. First spermatocytes, metaphase, _x_ the odd chromosome.

221. First spermatocyte, equatorial plate.

222. First spermatocyte, daughter plates containing 18 and 19
chromosomes, respectively.

223. Second spermatocytes, equatorial plates.


_Elater I_ (_Family Elateridæ, species not determined_).

224. Equatorial plate of spermatogonium, 19 chromosomes, _x_ the odd
one.

225. First spermatocyte, spireme stage, _x_ the odd chromosome.

226. First spermatocyte, metaphase.

227. First spermatocyte, prophase.

228. First spermatocyte, equatorial plate.

229. Equatorial plate from egg follicle, 20 chromosomes, _x_{1}_ and
_x_{2}_ the pair corresponding to _x_ in the spermatogonium.


_Elater II_ (_Species not determined_).

230. Equatorial plate of spermatogonium, 19 chromosomes, _x_ the odd
one.

231. First spermatocyte, spireme stage.

232. First spermatocyte, prophase.

233. First spermatocyte, beginning of metakinesis.

234. First spermatocyte, equatorial plate, _x_ the odd chromosome.

235. A pair of second spermatocytes in metaphase, two chromosomes
connected, _x_ the odd chromosome.


_Ellychnia corrusca_ (_Family Lampyridæ_).

236. Equatorial plate of spermatogonium, 19 chromosomes.

[Illustration: STEVENS PLATE XIII.

N. M. S. del.

COLEOPTERA]


PLATE XIV.


_Aphrophora quadrangularis_ (_Hemiptera homoptera_).

FIG. 237. Resting primary spermatogonium with lobed nucleus.

238. Resting secondary spermatogonium, with nucleus staining much more
deeply.

239. Equatorial plate of secondary spermatogonium, 23 chromosomes.

240. First spermatocytes, very early growth stage, _x_ the odd
chromosome.

241-243. First spermatocyte, later spireme stages, showing the odd
chromosome (_x_) and a pair of _m_-chromosomes (_m_).

244. Similar stage from a safranin-gentian preparation.

245. First spermatocyte, split-spireme stage, _x_ the odd chromosome,
_m_ the _m_-chromosome tetrad.

246. Similar stage from a safranin-gentian preparation.

247-248. First spermatocyte, condensation of chromatin granules to form
tetrads in the linin spireme.

249. Later tetrad stage.

250-251. First spermatocytes, metaphase from mercuro-nitric material.

252-254. Similar stages from Hermann material, showing longitudinal
split in both the bivalents, and the odd chromosome (_x_).

255. First spermatocyte, anaphase.

256. First spermatocyte, telophase.

257. First spermatocyte, daughter plates containing 11 and 12
chromosomes, respectively.

258. First spermatocyte, _a_ and _c_ daughter plates, each containing 11
chromosomes, _x_ the odd chromosome at a different level (_b_).

259. Second spermatocyte, equatorial plates of the two classes.

260. Second spermatocyte, metaphase.

261. Second spermatocyte, anaphase.

262-263. Second spermatocyte, daughter plates of the two classes.

[Illustration: STEVENS PLATE XIV.

N. M. S. del.

HEMIPTERA HOMOPTERA]


PLATE XV.


_Aphrophora quadrangularis._

FIGS. 264-265. Second spermatocyte, telophase, showing chromatin
nucleolus (_n_) and the products of division of the odd chromosome
(_x_).

266. A spermatid containing the chromatin nucleolus (_n_).

267-268. Spermatids containing both the chromatin nucleolus (_n_) and
the odd chromosome (_x_), _a_ the acrosome.

269. Equatorial plate from a somatic cell of a male larva, 23
chromosomes.

270. Equatorial plate of an oögonium, 24 chromosomes.

271. Resting nucleus of a young oöcyte before synapsis, showing two
pairs of condensed chromosomes, corresponding in size to the
_m_-chromosomes and the odd chromosome of the spermatocytes.

272-277. Sections of nuclei of oöcytes, showing one or more of these
heterochromosomes, from safranin-gentian preparations.

278-279. Bouquet stage from iron-hæmatoxylin preparations, showing the
heterochromosome bivalent (_x_).


_Cacoecia cerasivorana_ (_Lepidoptera_).

280. First spermatocyte, synizesis stage, showing 2 condensed
chromosomes (_x_{1}_ and _x_{2}_).

281-282. First spermatocyte, synapsis stage.

283-284. First spermatocyte, growth stages.

285. First spermatocyte, prophase.

286-287. First spermatocyte, later prophases, showing the
heterochromosome pair (_x_).

288. First spermatocyte, metaphase.

289. Second spermatocyte, metaphase.

290. First spermatocyte, equatorial plate.

291-292. Second spermatocyte, equatorial plates.


_Euvanessa antiopa_ (_Lepidoptera_).

293. First spermatocyte, equatorial plate.

[Illustration: STEVENS PLATE XV.

N. M. S. del.

HEMIPTERA AND LEPIDOPTERA]






End of Project Gutenberg's Studies in Spermatogenesis, by Nettie Maria Stevens