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.