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, x₁, x₂, and m₁, m₂) 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.