Chapter 2

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.