V. COMPARISONS AND CONCLUSIONS.
The literature relating to the spermatocytes of insects was reviewed at some length in my previous paper upon the history of these cells in the Acrididæ (17). It is not my purpose to go over this same ground again except in so far as increased knowledge makes it necessary. More recent papers by Montgomery, Wilcox and others will, however, be discussed in detail. The policy previously announced, of restricting comparisons to results derived from insects, will again be adhered to. I believe that the main features of the maturation divisions are essentially the same in all insects, and I desire to see this belief either well established or overthrown. If it can be demonstrated that so large a class as the insects are characterized by a common process, it will be a firm basis upon which to conduct further comparative studies into more comprehensive groups. On the contrary, if it is shown that there is no type, even in the class, then it is useless to seek agreements between widely removed species.
(a) Nomenclature.
A necessary basis for any comparative work is a common terminology. Confusion inevitably follows the loose application of names to the structures compared. This is perhaps unavoidable in the early stages of an investigation, but should be overcome as soon as possible. There is surely no reason for continuing uncertainty after terms have received general acceptance. Believing this, I feel called upon to repeat my criticisms of Montgomery’s application of the well-accepted terms “prophase,” “metaphase,” “anaphase,” and “telophase.”
In reply to my previous objection directed against this part of his work, Montgomery acknowledges the validity of the criticism so far as it relates to the metaphase, but denies the application to the other phases, particularly to the anaphase. He alleges in support of his position that the introduction of an unusual condition, the “synapsis,” makes it impossible to correlate strictly the stages of the germ-cells with those of ordinary divisions. Upon this point I must again disagree with him. It is impossible for any known modification of the prophase to change the essential character of the anaphase, so as to make it precede instead of follow the metaphase. This stage marks the movements of the chromosomes from the equatorial plate to the poles, and terminates when they are massed around the centrosomes. How can the “synapsis” in the least affect the duration or character of this process? It is apparent enough, I think, that Montgomery’s subphases of the “anaphase” do not belong to this portion of the mitotic cycle at all, but are really portions of the telophase of the spermatogonia and prophase of the first spermatocyte. Further, it may be noted that, even were these subphases properly included in the anaphase, they would belong to the spermatogonia and not to the spermatocytes.
Montgomery himself seems to be rather uncertain of the position of his “anaphase.” In the first paper, upon Euchistus (12), it was put down as the anaphase of the first spermatocyte; in his later paper (14), upon Peripatus, it is recorded as the anaphase of the spermatogonia. Still more confusing is his use of the “telophases,” for in the article upon Peripatus (14) it is, in the “Contents,” placed as a substage of the spermatogonial anaphase, and in the body of the work, page 307, as the telophase of the spermatocyte! Neither the anaphase nor the telophase can, by any possible construction of their proper meanings, be made to apply to the “growth period” of the germ cycle, as Montgomery insists; they are the last stages of the “division period,” in reality. The prophase of the first spermatocyte is the initial stage in the constructive process marking the growth period.
Montgomery’s translocation of the terms makes the “synapsis” occur in the anaphase. This is manifestly an impossible condition of the chromatin at this time, and his figures show definitely enough that it is a prophase, or, at the earliest, a spermatogonial telophase, that witnesses the contraction of the chromatin. The objection urged in my earlier paper (17) to the use of the term as a designation for the mere contracted condition of the chromatin cannot apply to Montgomery’s latest use of it; for he here recognizes the justice of my contention that it was primarily designed to indicate the fusion of the spermatogonial chromosome to produce the chromosomes of the spermatocyte. He states this clearly in the following words: “Moore (1895) first gave the name ‘synaptic phase’ to that stage in the growth period of Elasmobranchs when the reduction in the number of chromosomes takes place. Accordingly, the criterion of the synapsis stage is, first of all, the combination of univalent chromosomes to form bivalent ones; whether the chromosomes are then densely grouped or not is of secondary importance.”
(b) The Spermatocytes of the Locustidæ and Acrididæ.
The formation of the first spermatocyte chromosome gives us an insight into the later changes undergone by these elements such as cannot be obtained in any other way. The great importance attaching to this part of the spermatogonial process renders it desirable to exhaust every effort in obtaining a knowledge of the actual changes here taking place. This thought has been held constantly in mind during the progress of these investigations, and every point of resemblance or of difference between the various species studied has received careful attention. Despite variations in details, however, I must state that the essential features of the maturation divisions are the same in all species of the Orthoptera examined. It is true that as yet only two families, the Acrididæ and the Locustidæ, have been worked out in a detailed way, but the close agreement between these raises a strong presumption in favor of the general prevalence of the type. The processes of the two families have already been described in detail, but it will perhaps be well to call particular attention to some points worthy of mention.
The general appearance of the material derived from the two families is quite different in sections. Even the hastiest observation will show this. The spermatocytes of the Locustid testis are much smaller, denser and more deeply staining than those of the Acrididæ. The relative quantity of chromatin is greater, so that it is possible by microscopical examination of a section to tell whether it was prepared from Locustid or Acridian material.
The transformation from the telophase of the last spermatogonial division to the prophase of the first spermatocyte is marked by practically the same changes in both families. It is to be observed, however, that the derivation of the spireme from the disintegrating chromosomes of the previous generation is not so clearly indicated in the Locustid cells, and it was for this reason that in the examination of Xiphidium I was not able to determine certainly that the accessory chromosome came over from the spermatogonia into the spermatocytes as a formed element. Upon this point, as upon others, my later material is clearer, and I was able to reconcile the appearances in the two families. In both, unfortunately, it has been found impossible to determine the exact origin of the first spermatocyte chromosomes.
In connection with the transformation of the chromatin from the spermatogonial condition to that of the spermatocyte, we must take notice of that stage which is commonly denominated the “synapsis.” The evidence afforded by the Orthopteran cells is entirely negative regarding this. In properly fixed material there is no distortion of the chromatin in the nucleus at any time. It would, if present, be particularly easy to observe, as was stated in my previous paper, for during the entire winter the spermatocytes exist in the spireme stage, and in a longitudinal section of a follicle all stages may be discerned. On the other hand, in poorly fixed or hastily prepared material the synapsis is present, and always in such a form as to indicate its artificial character. What is here said regarding the synapsis refer to the appearance commonly thus designated, but, as has already been stated, such an application of the term does not meet the spirit of the definition as intended by Moore (20). A fusion of the spermatogonial chromosomes of some sort must certainly occur, but that it is always marked by a unilateral massing of chromosomes, I deny.
During the prophase the chromatin segments in the cells of Orchesticus and other species of the Locustids are heavier, more granular and denser than they are in Hippiscus. It is to be observed, also, that there is a greater variation in the size of the elements. This fact is observable from the earliest appearance of definite segments down through both the spermatocyte mitoses. This disproportion may be such that one chromosome will exceed another in the same cell by twenty or thirty times its volume. We have here, as is pointed out in another place, a strong proof concerning the individuality of the chromosomes, for in some species it is possible to distinguish a particular chromosome in all the spermatocytes. This is strikingly the case in Anabrus, where there is always one chromosome very much larger than any of the others. It exceeds in size even the accessory chromosome, and might be mistaken for it were it not for the difference in form. It is, however, typically a tetrad, and shows the four chromatids, while the accessory chromosome exhibits the usual spermatogonial condition.
As was indicated under the head of “Observations,” the prophase tetrad characteristic of Anasa and Hippiscus is again exemplified in the Locustid cells. So close is the resemblance of the maturation chromosomes of these various insect cells in their early stages, that I now regard it as practically established that they are commonly present in all insect spermatocytes. No more important evidence regarding chromosome structure and behavior can be obtained than that afforded by these elements. Particularly are the ring figures of value in the determination of the sequence of the longitudinal and cross divisions, and upon this point the material from the two families is equally convincing and positive in demonstrating that the first spermatocyte mitosis witnesses a separation along the longitudinal cleft of the spireme thread.
I should like to emphasize the fact that the chromosomes in both the Orthopteran families studied have been carefully traced from their earlier appearance down to the time of their dissolution in the spermatid through such a gradual series of changes that there can be no reasonable doubt of the accuracy of the conclusion set forth in these papers. The Orthopteran material possesses one distinct advantage over the Hemipteran, in that the point of cross-division is always marked by the same sort of a protuberance as is to be distinguished in the early chromatin segments. When the two free ends of the element are brought around to form a closed ring, the last particle of doubt regarding the position of the planes of separation marked out for the two spermatocyte divisions is dispelled.
This diagnostic character seems to be lacking in the chromosomes of the Hemiptera, and Paulmier, in his work on Anasa, depends for his criteria of orientation upon the relative lengths of the chromosome axes. Such a feature would be valueless in Orthopteran cells, because, as has been shown, the chromatids move upon each other in such a way as to exactly reverse the preexisting relation between the axes. How applicable this observation may be to conditions in the Hemipteran cells, I do not know; but, judging from the great resemblance of the elements in the prophase, it would seem most reasonable to expect a similarity of the divisions.
Paulmier (22) advances the suggestion that in the double-V figures we may find a structure that will serve to reconcile the divergent accounts concerning the longitudinal and cross divisions of the tetrads. The only way in which this might be accomplished would be to suppose that each of the interspaces represents a longitudinal cleavage of the thread, the first being at right angles to the second. I have given this suggestion careful consideration, and find no evidence to support it. The double Vs are only of rare occurrence, the common element being a straight rod, in the center of which is a diamond-shaped clear spot representing the two planes of division laid out for the spermatocyte mitoses. If two longitudinal divisions occur, one must precede the other considerably and the resulting halves become mutually repulsive, so that they move apart and lie in one plane with only a slight connection at the point of final separation. Moreover, the second cleavage must begin at the opposite end of the segment and proceed in a reverse direction from the first. Not only this, but the first spermatocyte mitosis divides the elements along what is generally conceded to be the longitudinal split, and this must necessarily succeed the supposititious first longitudinal cleavage by some time. Without going into a consideration of these points, I may say that they suggest such deviation from normal processes that only extensive and accurate observations would make Paulmier’s suggestion worthy of further consideration.
(c) Formation of the Tetrads.
In my former paper I reviewed the results obtained by Montgomery upon the Hemiptera, but further notice of his work will now be necessary, since on almost every important point relating to chromosome structure he has changed his opinion. His late extensive comparative study upon the Hemipteran cells, as well as that upon Peripatus, will at the same time receive consideration.
It appears from Montgomery’s account that at the point where the Orthoptera are least valuable in demonstrating chromosomal relations the Hemiptera and Peripatus are most convincing. I refer here to the derivation of the first spermatocyte chromosomes from the chromatin of the spermatogonia. He claims to have observed the union by pairs of the secondary spermatogonial chromosomes during the anaphase (his synapsis) so clearly as to be positive of this fusion. I hope this may be verified, for it offers a logical explanation of the process of reduction, and is a confirmation of what has previously been assumed true without sufficient basis in observed fact, as was suggested in my paper on Hippiscus. This, if established, would also be a strong support of the theory relating to the constancy of the chromosomes. If this true synapsis is accomplished at this time, however, it must be noted that it occurs during the last phase of the final spermatogonial mitosis, and is not an act of the spermatocyte prophase. But as to the exact location of this point no contention need be made, for it is conceivable that the time of its occurrence might vary considerably without affecting the essential nature of the process.
With regard to such an origin of the first spermatocyte chromosomes, there is an important difference to be noted between the earlier and later work of Montgomery, and one which he fails to mention. In his paper (12) upon Euchistus he states the matter as follows: “But in the post synapsis we do not find seven chromosomes, the definitive number present in the spermatocyte divisions, but a smaller number; hence, in the synapsis the true (i. e., exactly half) reduction of the chromosomes does not take place, but the number is reduced to less than one-half.” This statement is based, he says, upon a most careful and painstaking enumeration of the chromatic segments in a number of nuclei, and is unhesitatingly declared correct.
In his later paper, on the contrary, he is just as positive that the definitive reduction is here accomplished, for he says: “Since then I have been able to demonstrate that this numerical reduction is effected in the synapsis by the union into seven pairs of the fourteen chromosomes, each of the seven bivalent chromosomes (pairs) being composed of two univalent chromosomes joined end to end.” This statement is made without adducing any specific proof, as was formerly done. By what means we are to reconcile these diametrically opposite statements Montgomery does not say. He, however, insists that he has always known that the fusion by pairs takes place. How this was to be brought about under his previous assumption that one of the fourteen spermatogonial chromosomes became removed from participation in the usual processes of the cell to form a “chromatin nucleolus,” he fails to state. Until the confusion is cleared up by corroborative evidence on one side or the other, a most important part of Montgomery’s work must still be regarded as uncertain.
Despite his recognition of the fusion of the chromosomes in the synapsis as the essential feature of this stage, Montgomery is insistent upon the concentration of the chromatin as its distinguishing characteristic. Regarding this he says: “McClung considers the appearance of the synapsis stage as artefacts. It is hardly necessary to reply to this criticism, since in all Metazoa where the spermatogenesis has been carefully examined, with the exception of certain Amphibia, the dense massing of the chromosomes (?) in the synapsis stage has been shown to be a perfectly normal phenomenon.”
Concerning two points in this statement I wish to take exception. First, as was suggested in my previous paper (17), the term synapsis is usually applied to a condition of the prophase in which the apparently unsegmented spireme exists. It must be remembered that most investigators consider that the reduction of the chromosomal number takes place by the segmentation of a spireme into half the usual number of segments. In the second place, I must resent the implication that the work done in this laboratory is not “carefully” conducted. Many “Metazoa” have been examined “carefully,” and in none has the “synapsis” occurred when the material was well fixed and prepared. It has, moreover, been found possible to produce the appearance at will. One case of this kind is sufficient to raise the presumption that it may not be normal even when constantly found in certain preparations. I have not, however, absolutely denied the possibility of such an occurrence, because it is conceivable that from the telophase of the preceding division the massing of the chromosomes may persist during their elongation. My contention is that the appearance is not a constant or necessary condition in “all the Metazoa,” and this I have proven.
In rather striking contrast to the work of Montgomery, in which an effort is made to formulate a typical process for the entire Metazoa from the study of a single order, is that of Wilcox, wherein a general denial of any apparent system in the maturation divisions of animals is based practically upon the study of a single species. As was stated in my former paper, I regard Wilcox entirely in error upon the vital point of his theory of tetrad formation, not by “forced interpretation” of his own views, but by an actual examination of the object upon which he worked. There is no point upon which Orthopteran material affords more indisputable evidence than upon the occurrence of the longitudinal division of the chromatin thread in the early prophase. My statement regarding Wilcox’s position on this subject was in no sense “misdirected criticism,” but an actual statement of fact; it was not an attempt to explain away “abundant and evident cases which cannot be made to fit into the scheme,” but simply the presentation of proof that one case was wrongly interpreted.
Wilcox claims the distinction of being the first and only investigator to doubt the hypothesis that longitudinal and cross divisions of the chromatic thread produce chromosomes of a different character. It is perhaps well that this is so, in view of the reasoning by which such a distinction is secured. Upon his own unconfirmed and disputed statement that there is no longitudinal division of the spireme, Wilcox presumes to disparage the accepted view of practically all cytologists. The constructive thought of the last two decades is summarily disposed of by this author in the following language: “The whole question, therefore, whether a certain division is longitudinal or transverse loses its practical significance, since the theoretical interpretation which has long been placed upon these divisions is shown to be impossible and absurd!” The showing alluded to consists in the statement that the chromosomes consist of an indefinite number of granules, which cannot be expected to arrange themselves in any order, and which, therefore, may be divided in any way without affecting the results.
Laying aside for a moment the question as to the occurrence of a longitudinal division, we may well inquire whether the belief that, “In view of this manner of the formation of the chromosomes (by the aggregation of the chromomeres), it seems absurd to assume that the separation of an individual chromosome by one plane could be quantitative while the separation by another plane was qualitative,” is well founded. At the basis of such an assumption lies the implication that any definite arrangement of chromomeres is impossible; for if any definite order were possible, then the supposed argument against the longitudinal disposition of the chromomeres would be invalid.
The argument of Wilcox is therefore directed against order in general, and not against order in any one particular, as he would have it appear. For it must be admitted that if it is possible for the scattered chromatic granules of the early prophase to arrange themselves at all (and this even Wilcox does not deny), it is equally possible for them to come together in a definite order. That they do this is amply evidenced by the fact that later they appear in definite groups or chromosomes. It is to be noted, moreover, that the later investigations tend to suggest that the apparently unorganized chromatic granules in the first spermatocyte prophase are really bound together and represent merely a diffuse condition of the spermatogonial chromosomes.
Wilcox’s chief error, however, is not to be sought in speculative theories, but rather in his faulty observations. He repeatedly denies the occurrence of any longitudinal split in the chromatic thread of the first spermatocyte prophase. That he is mistaken here I am thoroughly convinced, both from a study of his own object and from investigations upon many other species of the same family. At the present time, also, practically every spermatologist is aligned in support of the view denounced by Wilcox. For a while Wilcox had some backing, but most of those who advocated only cross divisions of the thread have later been able to demonstrate the longitudinal cleavage in better prepared material.
There is general acceptance of the opinion that the chromomeres of the last secondary spermatogonia appear in a linear arrangement to form what is commonly known as the “spireme.” Wilcox declared that while in a very fine condition this thread breaks across into segments, which unite by pairs to form the chromosomes of the first spermatocyte. The great majority of other investigators are unanimous in the opinion that this fine thread, made up of granules, becomes double by the division of each granule individually, thus producing a double thread. Thus it is that the two halves of a longitudinally divided chromosome are made equivalent, not by the sifting apart of preexisting granules, but by the division of these after they are arranged in a linear series. It need hardly be mentioned that the formation of the thread has here a reason for existence which is entirely lacking according to Wilcox’s scheme.
This much space has been devoted to Wilcox’s statements, not because they present any arguments against the generally accepted views of his fellow workers, but because he represents a rapidly lessening minority which is content to work in a very limited field and to resort for the explanation of diverse results to the very convenient theory that great differences may be expected in the normal processes of even closely related forms. One needs only to glance at the work of all insect spermatologists to see how closely the agreement now is upon the important points of the process. This accordance of results Wilcox notes, but interprets in his own way, which may be regarded as not exactly complimentary to the skill and judgment of his colaborers. “It is only necessary,” he says, “to refer to any recent publication on the subject to find examples of this attempt to force the divergent processes in different species to fit the same formula.” This is certainly a very easy and convenient way to dispose of the accumulated observations of the many careful investigators who have come to an agreement upon the important questions under discussion, but I venture to think will hardly satisfy any one except its sponsor.
After handing in this article for publication, I fortunately secured a copy of the paper by R. de Sinéty (37) in which the spermatogenesis of various Orthopteran species is described. I regret that the available time is so short that I shall not be able to bestow upon this contribution to insect spermatogenesis the attention it deserves, but I shall try at least to consider the principal points wherein a difference exists between the results of de Sinéty and of myself.
It is unfortunate that we have here a further complication of the problem concerning the character of the two maturation divisions in insects. At this time it had begun to appear as if there was every possibility of insect spermatologists coming to an agreement with regard to the maturation processes. Indeed, with the exception of Wilcox, who occupies a unique and solitary position in the field, workers upon the subject are committed to a belief in the occurrence of a cross and a longitudinal division of the chromosomes in the spermatocyte mitoses. The sole difference of opinion relates to the sequence of the divisions. We have now to consider in connection with insects the remaining possibility in tetrad formation—that of two longitudinal divisions—which finds an advocate in de Sinéty.
Because of a thorough acquaintance with the forms upon which this author has worked, I do not hesitate to say that he is entirely mistaken with regard to the character of the second spermatocyte division. I am convinced of this because of the fact that in the early period of my work upon Orthopteran spermatogenesis I was inclined to place just such an interpretation upon the phenomena encountered in the spermatocytes of the Acrididæ as does de Sinéty. I soon became convinced, however, that I was proceeding upon a wrong assumption, and abandoned it in favor of the one which more extended observation taught me is correct. I hope to demonstrate here the ground for my plain statement that de Sinéty is in error upon the question of a double longitudinal division of the chromatin thread during the formation of the tetrads in insect spermatocytes.
It is fortunate that our author has properly appreciated the value of the early prophase in the determination of the structure of the first spermatocyte chromosomes, for we are here upon common ground, and need only compare like stages in order to reach our conclusions. As will be recalled, the statement is made elsewhere in this paper that the typical chromosome of the first spermatocyte is an approximately straight rod, split longitudinally, and again cleft in its middle by a second fissure at right angles to the first. Such an element is represented in figures 15a, 17, D and E of my paper upon the Acrididæ, and in figures 7, 9, 11 and 38 of the present one. Although this is extremely common, and, as the photomicrographs show, undeniably present, de Sinéty does not figure it at all. The nearest approach to such a structure is found in figure 123c, where a cross with two nearly equal arms is represented. My interpretation of this figure, based upon a great number of careful observations, is that this represents merely an extension of the shorter arms at the expense of the longer ones. In support of this, I have stated that all intermediate stages between a rod with a mere enlargement at the center and a cross with equal arms could be found. How, according to de Sinéty’s conception of overlying free elements, could these structures be explained?
It is not necessary, however, to have these gradations in order to disprove the theory under discussion. One needs only to carefully examine one of these crosses to be convinced that the two arms lie in one plane where they intersect, and are not superimposed one upon the other as de Sinéty shows in his figure 123. Our author clearly realizes the importance of the cross, as may be judged by the following quotation:
“La croix est de toutes ces figures celle dont la genèse peut le plus facilement donner lieu à des interprétations en sens contraire.—C’est précisément pour cette raison que nous croyons devoir l’étudier spécialement au point de vue critique, persuadé que, cette figure une fois rattachée à une théorie, les autres doivent en suivre le sort.”
It is unfortunate, therefore, that he was not able to trace the formation of the element in its very early stages and through the various modifications which connect it with the typical rod already described.
As the simplest modification of this basic form, we find the one where it is evident that the change consists merely in a flexure of the rod at the weak spot in its center. Such forms are shown in figure 14 of my former paper (17) and in figures 8, 9 and 11 of this one, but are not illustrated by de Sinéty. It occasionally happens that in chromosomes of this character the halves diverge widely at the center, producing the double-Vs of Paulmier, as is represented in figure 14 of my paper upon the Acrididæ (17) and in figure 8 of the present one. These structures are not shown by de Sinéty and would be difficult to explain in agreement with his conception of the tetrad.
I have consistently placed great reliance upon the frequent ring-shaped chromosomes in determining the structure of the first spermatocyte elements, and have no occasion to change my opinion of them since examining the work of de Sinéty. This investigator joins issue with me upon my interpretation of these structures, and states his attitude in the following language:
“McClung fait grand fond, pour appuyer son interprétation, sur une forme spéciale, la forme en anneau, qui pour lui dérive du bâtonnet a′ b′
a′ b′’, supposé placé transversalement sur le fuseau, inséré par son milieu et incurvé en dehors jusqu’ à rapprochement et soudure de ses extrémités.
“Le chromosome en anneau est en effet très fréquent chez les acridiens; mais il nous a été possible d’en reconstituer l’histoire, grâce à des détails qui ne semblent pas s’être rencontrés dans les figures de McClung. On se souvient que nous avons établi les deux points suivants en complet désaccord avec la théorie de l’auteur américain:
“1. Les deux moitiés de l’anneau proviennent de la première division longitudinale.
“2. L’insertion est terminale.”
With equal emphasis, I must deny that the enclosed space in the ring represents any plane of division in the chromatin thread; and that the insertion of the spindle fibers is at any place except at the center of what would be the typical rod-shaped chromosome were the ring straightened out. We encounter in de Sinéty’s interpretation of these rings the very error against which I was careful to caution elsewhere in this paper, i. e., of regarding the points where the fibers are attached as the crossed ends of a simple segment. This mistake de Sinéty has made, and has thereby vitiated all his conclusions concerning the structure of the tetrads. It is not necessary to repeat here the proof which I have brought forward in support of my views. No one, I am sure, will find difficulty in reducing the various forms of chromosomes found in the first spermatocytes to the type of a doubly split rod, in which one plane of division is parallel to the long axis and the other at right angles to it. The explanation offered by de Sinéty requires us to conceive a doubly split rod in which one separating space may vary indefinitely while the other is constant. There is here no common type, but an infinitely variable one, which differs with every modification of the interspace between the first pair of chromatids in each chromosome.
As a constructive basis for the foundation of his theory of a double longitudinal division, de Sinéty uses particularly the chromosomes of Œdipoda (Hippiscus) miniata, represented in figures 129 and 130, concerning which he says:
“Survient le phénomène exceptionnellement important de la seconde division longitudinale; nous regardons comme un point capital dans notre travail d’en mettre l’existence hors de doute et pour cela nous désirons ne faire appel qu’à des images extrêmement claires. Nous considérons comme telles les fig. 129 et 130 rapprochées l’une de l’autre.
“Il est de toute évidence que le chromosome a, fig. 130, n’est que le chromosome de même désignation, fig. 129, dont les deux anses jumelles se sont clivées. De même, le chromosome en forme de boucle, c, fig. 129, dont les deux branches représentent, comme nous l’avons fait remarquer, deux anses jumelles, se retrouve avec un clivage très évident en d, fig. 123. On pourrait faire les mêmes rapprochements entre b, fig. 105, et a, fig. 107; ici, le clivage est moins avancé, mais les granules sont nettement divisés.”
I am obliged to confess that I have never seen in other species of this genus any appearances that would incline me to place an interpretation upon them such as does our author upon these. I would venture to suggest, on the contrary, that the chromosomes represented in figure 129 have not as yet demonstrated any division, but show merely irregular spaces between chromosomes. At even an earlier stage (figs. 5, 37, and 38), I have shown the formation of the tetrads by means of simultaneous cross and longitudinal divisions so clearly that presumed successive divisions, as represented by de Sinéty, cannot be regarded as occurring.
Finally, I would emphasize the fact mentioned in connection with the discussion of the cross-shaped chromosomes, that where the elements of one of these compound chromosomes intersect they lie in one plane, and are not superimposed upon each other, as de Sinéty’s theory demands and as his figures represent. This was shown clearly in Paulmier’s figures as well as in my own, and is even more clearly demonstrated, if possible, in the very long, slender chromosomes of the myriapods, which I have observed in Mr. Blackman’s preparations. This, and the continuity of the chromatin in contiguous arms of the cross, is alone sufficient to disprove de Sinéty’s theory, and, fortunately, is easily demonstrated. This same fault of de Sinéty’s is encountered, in another form, in his discussion of the ring figures. He asserts that the halves of the rings are pulled past each other while they lie in the plane of the spindle axis. Herein my observations fail entirely to agree with his. The rings lie in the plane of the equator, and no elements of the mitotic figure show a lateral displacement of the separating halves equal to the width of the chromosome when viewed in this plane.
(d) The Spermatocyte Divisions.
I approach a discussion of Montgomery’s conclusions regarding the form of the chromosomes in the first spermatocyte, and the sequence of their divisions, with considerable hesitation, because of the difficulty I experience in appreciating his exact position. This is due, not to any lack of positive statements on his part, but to the partial contradictions that result from his frequent changes of opinion. The most important statement in his first paper upon Euchistus reads as follows: “From the resting stage of the first spermatocyte to the formation of the spermatid, there is absolutely no longitudinal division of the chromosomes. I have studied hundreds of nuclei in these stages, and at the first with a hope of finding a trace of such a process, but observation shows that all divisions of the chromatin elements are transverse divisions.”
This would certainly seem to be as strong a stand as one could take upon the subject, but in later papers Montgomery assumes with equal assurance the opposing position, which holds for a longitudinal division. Regarding this he says: “During the synapsis stage the chromosomes become split longitudinally, as was first shown by Paulmier (1898, 1899) for Anasa—a process that I had overlooked (!) in my former paper (1898).” Throughout his later investigations this hypothesis serves as the basis of all his theories, and the careful longitudinal division of the thread is assigned an important role in the maturation process. So far as positive assertions to the contrary are concerned, a general acceptance of the theoretical importance attaching to this act is to be supposed.
Notwithstanding this, I find nowhere in his later writings any statement that he abandons the conception formerly entertained regarding the non-importance of the longitudinal cleavage. This attitude is indicated in the following language: “If it can be proved that the mode of division of a chromosome, i. e., the axis of the line of division, is merely a function of its chromomeres, then it would be of no theoretical value whether the division be longitudinal (equation) or transverse (reduction). But it happens that the postulated difference forms one of the main premises of Weismann’s theoretical superstructure. On account of the differences observed in different objects in regard to the modes of division of the chromosomes, it would appear that the differences have no theoretical value, but that the halving of the mass of chromatin is the process of importance—the standpoint taken by Hertwig.
“In the two reduction divisions the chromosomes may split by two longitudinal divisions, by two transverse divisions, by one longitudinal and one transverse division, or by one division (longitudinal or transverse) preceded or followed by an elimination division. The facts show already that there is no general uniformity in the mode of division of the chromosomes in the reduction mitoses. The long line of observations on different objects show this to be the case, and demonstrates that the expected uniformity does not occur.”
Herein lies the essential conclusion of the work upon Pentatoma, which, so far as a specific retraction is concerned, stands yet. If this be abandoned, then the first work upon the chromatin structure of Pentatoma is practically discredited, for Montgomery has definitely retreated from his positions concerning the absence of the “chromatin nucleolus” in the spermatogonia, the non-occurrence of a longitudinal cleft in the spireme thread, the lack of an equational division of the chromatin in the spermatocyte, the origin of the “chromatin nucleolus,” and the fragmentation of the “chromatin nucleolus.” In addition to these specifically acknowledged errors, we may infer that Montgomery (12) considers himself at fault in his views upon the production of chromosomes from the “three to six chromatin loops” by breaking apart in the prophase, and upon the occurrence of both longitudinal and cross divisions of ordinary chromosomes in the same mitosis. The observations recorded in his last paper (15) upon the production of the spermatocyte chromosomes by the end-to-end union of those in the last spermatogonial division warrant this assumption.
It follows from all this that we may practically disregard Montgomery’s earlier work upon chromosomal structure and take his views as expressed in the later papers (14, 15) as representing his opinions upon the subject. These later theories are largely the result of his investigations upon Peripatus, but they seem to be carried over and applied to the Hemiptera without essential modifications, and we may regard this concept as applicable to the forms studied by him.
I called attention in my previous paper to the fact that, by many investigators, the definitive form of the chromosome is used as the basis for determining the direction and sequence of the chromosome divisions. This fact and the danger attending the practice was partly realized by Montgomery in his work upon Euchistus (12), for he devotes considerable space to a consideration of the prophase segments, but in determining the character of the second spermatocyte division he regards only the formed element. With respect to this he says: “And now a fact may be determined which is of the greatest importance in estimating the morphological value of the second division of the chromosomes. While the latter are still parallel to the axis of the spindle, there may be clearly seen in some cases a transverse constriction on some of the chromosomes, so that they already acquire a dumb-bell shape.” This constriction is not correlated with any similar one on the prophase elements, and is here observed for the first time.
In his paper upon Peripatus, however, he definitely supports the contention that it is only in the prophase of the first spermatocyte that we can learn the construction of the chromosomes, for he says: “The early stages in the prophase are of the greatest importance in determining the exact constitution of the chromosomes of the first maturation division.... Since, then, as has been shown in another section of the present paper, the split of the univalent chromosome of the second spermatocyte is a true longitudinal split, corresponding perfectly in position with the longitudinal split of the early prophase, it follows that the univalent chromosome does not become turned upon its axis to take its place on the equator of the spindle.” Orientation is in both spermatocytes based, accordingly, upon planes determined in the prophase. Upon this point Paulmier and Montgomery, as students of Hemipteran spermatogenesis, are now agreed, and their results correspond with observations made upon Orthopteran cells.
It is upon the sequence of divisions in the spermatocyte that differences now exist between these investigators and myself. In my previous paper I took occasion to elaborate the proof in support of my position regarding the early occurrences of the longitudinal division in the Orthopteran spermatocytes. Montgomery follows Paulmier in ascribing the reduction division to the first spermatocyte, and takes no account of my results upon Hippiscus. The objections that I previously urged against Paulmier’s conclusions apply equally well to Montgomery’s. Until the chromosomes are traced in a more detailed way through the prophase to the metaphase, I shall consider the presumption against the occurrence of the cross-division in the first spermatocyte mitosis. In this I believe that I am justified by the definite proof of my position brought forward in the work upon Hippiscus. Here, it may be recalled, I observed and photographed in the same mitosis all stages of movement by the chromatids along the plane of the longitudinal split. In addition, I was able to locate definitely the position of the future cross-division in the ring figures, so that it is impossible to mistake the character of the first division in them. These two proofs I consider incontrovertible so far as they apply to the Orthopteran families studied.
Paulmier judged the planes of the division by the relative lengths of the chromosome axes, but, as I pointed out, this is not conclusive unless it can be shown that they have not shifted, as it is possible for them to do, during the prophase. The value of the ring figure, which is formed at such an early stage that it would be impossible for the shifting of the axis to occur, is here evident.
Montgomery finds these rings in Peripatus, and realizes the importance of their evidence in determining the planes of division, but places his conclusions upon a much more insecure footing than those founded upon the Orthopteran cells, because of the criterion used in determining which point represents the junction of the paired chromosomes. The diagnostic feature he uses is the linin connection persisting between the “central ends” of the chromosome, which holds them together until the “distal fibers” connect with the centrosomes and cause the rupture of the “central” fiber. Since the whole of his elaborate theory regarding the continuance of the linin spireme is practically a theoretical conception with little basis in observed fact, the value of such proof cannot compare with that furnished by the definitely formed chromosomes themselves in the Orthopteran cells.
In view of all these facts, I think it must still be held an open question as to which is the reduction and which the equation division in the Hemipteran spermatocytes, although it is not to be doubted that the probability of the first spermatocyte being witness of the reduction division is much increased when thus interpreted by two independent observers.
(e) The Accessory Chromosome.
I have already, in another paper (19), taken up a comparative study of the accessory chromosome in different insect spermatocytes, and shall not be obliged, for that reason, to enter into a very lengthy discussion of the subject here. The great interest attaching to this structure, however, compels me to consider the work that has been done since the manuscript of the earlier article was sent in for publication. This review will concern, very largely, the investigations of Montgomery upon a considerable number of Hemipteran species, which are set forth in his paper under the pretentious title “A Study of the Chromosomes in the Germ Cells of Metazoa.”
In his first work upon Euchistus, Montgomery describes a cell element under the name “chromatin nucleolus” which corresponded so closely to my accessory chromosome that I concluded the two structures were identical. These similarities were, the origin from a spermatogonial chromosome, the integrity and constancy of staining power and position during the spermatocyte prophase, and participation in the division act during metakinesis of a spermatocyte.
Among the numerous changes of opinion recorded by Montgomery in his latest work, there are several relating to his “chromatin nucleolus” that materially alter the aspect of the question. Perhaps the most important of these concerns the origin of the element. I was some time in determining that the accessory chromosome is a spermatogonial chromosome which divides in the spermatogonia with the other chromatin elements and comes over into the first spermatocyte as a formed structure. The work of Sutton upon the early history of the element in Brachystola, however, was convincing in this respect and confirmed me in the opinion I had already formed. I therefore gave Montgomery the credit for this discovery, and set it down as strong confirmation of the assumption that we were dealing with similar structures in the two orders of insects.
Upon this point Montgomery now completely reverses himself, and declares that his “chromatin nucleolus” is not a spermatogonial chromosome, but may be noted in the earlier generations as a nucleolar structure, which, however, divides in metakinesis. The most important feature to be noted in this connection is the fact that the structure does not exist as a simple element, but is observed as a number of granules, and that this number varies considerably in different species. These granules fuse during the “synapsis stage,” as do the chromosomes, to produce in the spermatocyte half the number of “chromatin nucleoli” that were present in the spermatogonia. In this respect the “chromatin nucleolus” differs radically from the accessory chromosome, which has the same valence in both cell generations. The indefinite number and insignificant size of Montgomery’s structures are other characters that point to extensive differences between them and the accessory chromosome.
In his work upon Peripatus, Montgomery states that in restudying his preparations of Euchistus he observes a continuous linin spireme which involves the “chromatin nucleolus” as well as the chromosomes. Here, again, there is a difference between the Hemipteran element and the accessory chromosome; for the latter is entirely free from linin connections in the prophase and is usually surrounded by a hyaloplasmic investment.
According to Montgomery, also, his “chromatin nucleolus” usually takes part in both spermatocyte mitoses. In this respect there exists an essential difference between his element and that found in the Orthoptera, for, after extended and most critical studies, I have become convinced that only one division takes place in the spermatocytes. In those cases where Montgomery admits but a single division, it is stated to occur in the first spermatocyte, while in the Orthoptera the accessory chromosome remains undivided here and is halved in the second spermatocyte.
If, therefore, Montgomery’s recent observations are correct, it must follow, I think, that his “chromatin nucleolus” and the accessory chromosome are different structures. I am free to admit, however, that his statements are far from convincing. So much dependence is placed upon the numerical relationships of elements that are admittedly very minute, and so little corroborative proof is given, that I entertain serious doubts as to the accuracy of the observations. In this connection I would suggest a comparison between the figures of the “chromatin nucleolus” in the first paper upon Euchistus (figs. 55–68) (12) and those in the last one (figs. 1–17) (15). The showing here made would alone be sufficient to raise a question as to the nature of the “chromatin nucleolus,” and until further evidence is forthcoming the character of the peculiarly modified chromosomes in the spermatocyte of the Hemiptera must remain in doubt.
Aside from definite retractions that Montgomery has made regarding his earlier views on the character of the “chromatin nucleolus,” there are noticeable different attitudes toward it in his earlier and later works. Thus, in his lecture at Woods Holl (13a), we find the following: “These remarkable ‘nucleolar’ structures which stain like chromatin have been observed by numerous writers, but as yet no satisfactory description has been given of their mode of origin. They have been observed by me in spermatocytes of various insects, in hypodermal and other cells of Carpocopsa, and in follicle cells of the testicles of Plethodon and Mus.” At this early stage of Montgomery’s investigations it is apparent that he views his “chromatin nucleolus” primarily as a nucleolus with chromatic origin and characters, but the fact is equally apparent that he now regards it primarily as a “chromosome” with nucleolar attributes. This is made evident in his recent definition, which reads: “The chromatin nucleoli are morphologically chromosomes, undergoing division in mitosis like the other chromosomes, but differing from them in the rest stage by preserving a definite (usually rounded) form.”
What has here been said regarding the “chromatin nucleolus” applies to those structures in Euchistus and other Hemiptera to which Montgomery has given the name without qualification. According to his definition, however, there is present in the cells of Protenor and other species another form, the “chromosome x.” Not only by inference is this classification operative, but by direct statement we learn that Montgomery regards this element as a member of the class of bodies which he calls “chromatin nucleoli.” In speaking of Protenor chromosomes, he says: “This is the only case in the Hemiptera where one chromosome becomes differentiated into a ‘chromatin nucleolus’ for the first time in the spermatocyte generation.”
The noteworthy thing about this “chromosome x” is the fact that in every essential detail it corresponds to the accessory chromosome of the Orthoptera. It is a spermatogonial chromosome that comes over intact into the spermatocyte; it retains its form and staining power unchanged through the prophase of the spermatocyte; it divides in only one of the spermatocyte mitoses; and is a large and conspicuous element of the cell at all times.
This “chromosome x” agrees just as closely in its description to the accessory chromosome as do the ordinary ones of the two orders, and, if Montgomery’s account is correct, there would seem to be no reason for doubting their identity. In two respects, however, there are differences between these structures. First, it is to be noted that the “chromosome x” divides in the first spermatocyte, while the accessory chromosome undergoes separation in the second spermatocyte. Should Montgomery’s observations prove correct, it would yet indicate no fundamental difference in the character of the element, for the result is the same whether division takes place in the first or second mitosis. In either event, one-half the spermatozoa are provided with the odd chromosome while the remaining half are not.
The second point of difference would seem to be a more serious one. Montgomery states that during the spermatogonial mitosis the “chromosome x” regularly divides as do all the other chromosomes, i. e., longitudinally. In the spermatocyte mitosis, however, the element is broken across, and the longitudinal split, which is apparent in the early stages, disappears and is not utilized in division. We have here the remarkable occurrence of a chromosome entirely unchanged in its structure, but merely differing in its surroundings, which, instead of dividing along the plane marked out for it, as it has done in all preceding mitoses, breaks across after it is a formed element. An occurrence of this kind, so different from the usual method of division, would require strong proof to establish it, and this, in my opinion, Montgomery has not brought forward.
A criticism of the degeneration theory as advocated by Paulmier and Montgomery has already been given (17), so that it would not be necessary to consider it here except in so far as it has been modified since its promulgation. As a rule, Montgomery refers to his “chromatin nucleoli” throughout his late paper (15) as degenerating chromosomes, but in discussing their function specifically he makes important changes in this conception. These are stated as follows: “When we find, accordingly, the mutual apposition of them (true nucleoli) to chromatin nucleoli, it would be permissible to conclude that the chromatin nucleoli are chromosomes which are especially concerned with nucleolar metabolism. And this, I think, would be the correct interpretation. The chromatin nucleoli are in that sense degenerate that they no longer behave like the other chromosomes in the rest stages, but they would be specialized for a metabolic function; and from this point of view they would certainly seem to be much more than degenerate organs.”
It is difficult to comment upon a contradictory statement like this; but, fortunately, it is not necessary to do so, since it carries with it its own refutation. The conception of a chromosome specialized in the direction of increased metabolic activity as being in the process of disappearing from the species can hardly be regarded seriously.
Taking everything into consideration, it may be said that Montgomery’s work upon the Hemiptera has left the subject in a very disturbed condition, and any prospect of a complete agreement between the accessory chromosome of the Orthoptera and the “chromatin nucleolus” of the Hemiptera is made more remote than was previously the case. This, I think, is largely due to the inferior character of the Hemipteran material, which has lead to misconception of phenomena that are clearly marked in Orthopteran cells.
It is gratifying to note that the recent work of de Sinéty (37) practically corroborates the conclusions herein set forth regarding the history of the accessory chromosome. Aside from failure to observe the important spireme condition of this element in the first spermatocyte prophase, de Sinéty describes the same series of processes with scarcely an exception. His summary contains the following account of the accessory chromosome:
“Le ‘chromosome accessoire,’ découvert par McClung chez Xiphidium fasciatum, se retrouve chez les locustiens que nous avons étudiés. Chez Orphania, il se divise dans les spermatogonies en deux masses volumineuses et allongés, que l’on reconnait dans les nucléoles, également volumineux et allongés, des spermatocytes de premier ordre en prophase. A la métaphase de la première cinèse, on le trouve situé excentriquement et plus près de l’un des pôles; il va tout entier a l’une des cellules-filles. Dans celle-ci, il se divise comme un chromosome ordinaire, d’où il suit que sur quatre spermatides formant la descendance d’un spermatocyte, deux se trouvent privilegiees. Par ce partage inégal, non réalise dans Xiphidium fasciatum, d’après McClung, le chromosome spécial d’Orphania rappelle celui des hémiptères.”
A like series of processes is recognized in the Phasmids.
As is elsewhere explained in this paper, the occurrence of two divisions of the accessory chromosome in Xiphidium, which was mentioned as a possible occurrence in my preliminary paper, is shown not to take place. While it is much more difficult to demonstrate the undivided condition of the accessory chromosome in one of the spermatocyte mitoses of Xiphidium than it is in the cells of Orchesticus, Anabrus, Scudderia, and Microcentrum, I am convinced that it does not differ from the other Locustids in this respect.
We may therefore feel assured that our knowledge of the morphological character of the accessory chromosome in the Orthoptera is fairly well established. This gives us a good base from which to conduct further comparative studies into other groups, and it is to be hoped that our knowledge of this element will rapidly increase.
Unfortunately, de Sinéty has chosen to add another name to the already overburdened list of synonyms, and “chromosome spécial” now takes its place in the literature of insect spermatogenesis. The reason for adding this name—
“Il reçu successivement leg noms de ‘accessory chromosome’ (McClung), ‘small chromosome’ (Paulmier), ‘chromatin nucleolus’ (?), ‘chromosome x’ (Montgomery). Nous avons préféré éviter ces appellations, qui semblent toutes supposer une signification qui n’a jamais été définie ou s’appuyer sur des caractères plus ou moins secondaires, pour adopter un nom indifférent, celui de ‘chromosome spécial,’ nous conformant à l’idée de Wilson, pour qui c’est un ‘extra chromosome,’”
would seem to be at least insufficient, since “accessory chromosome” can scarcely be regarded as implying any more primary or secondary function than can “chromosome spécial.”
(f) Individuality of the Chromosomes.
In each of my preceding papers I took the opportunity to point out the fact that, even were the accessory chromosome of no other value, it would certainly be worthy of study for the light it throws upon the question of the individuality of the chromosomes. On this point Montgomery has much to say in his late paper (15). I think it cannot be questioned that we have here indisputable proof that at least one chromosome may be identified through all the cell generations of the testis. While this does not prove that chromosomes are persisting and independent structures, it does evidence the fact that they may be, and greatly strengthens the hypothesis that they are.
In addition to the evidence here offered by the accessory chromosome, there must be noted that derived from a study of spermatocytes in which there is always present one ordinary chromosome that greatly exceeds the others in size. Such a condition is found in the cells of Anabrus. The disproportion in size of the elements is here so striking that it would be impossible to fail in distinguishing the giant chromosome. In each of the spermatocytes of Anabrus there are therefore two chromosomes which are plainly recognizable. It may be observed further that the remaining chromosomes are quite different in size, and it may be possible within reasonable limits of certainty to pick out one or more other chromosomes in each cell. Unless this could be done for each element, however, it would not definitely prove that all the chromosomes are distinct and recognizable structures. The actual recognition of two elements in each cell of the same generation and its ancestors or descendants in other generations goes far, however, to render probable the individuality of each chromosome.
Beyond this point studies upon the Orthopteran cells will not permit me to go; but Montgomery has been fortunate enough to find in Peripatus an object in which he considers it possible to demonstrate the continuity of the chromosomes from one generation to another, and their fusion by pairs in the early history of the spermatocyte to bring about the reduced number. This is, in the main, a logical conclusion to my own work, and I am therefore bound to regard his results as probably correct. While doing this, however, I recognize that the absolute proof he brings forward in support of his hypothesis is very slight. I consider any deductions based upon observations of linin structures as very insecure, and it is upon these that Montgomery principally relies to demonstrate his theory. Further observations upon the behavior of the chromosomes between the spermatogonia and the spermatocytes in objects favorable for study will be awaited with interest. In the meantime it must be conceded that the work upon insect spermatogenesis has at least lent strong support to the theory of the individuality of the chromosomes in general and has definitely shown that there is such a thing in some instances.
(g) Nucleoli.
Considerable importance is attached by some investigators to the nuclear structures, properly called plasmasomes, that occur in the spermatocytes. It is probable that there are marked differences between the cells of various species in regard to the occurrence of these bodies, for in the Orthoptera they either do not appear at all, or, if present, they are minute and inconspicuous. This fact would tend to disprove any theory which would attach a fundamental importance to these structures, such as is conceived for the chromatin. The Orthopteran cells do not allow any observations which would add to our positive knowledge of the nucleoli, and I include this brief statement merely for the negative value it may possess.
(h) Rest Stage.
In his first paper upon Euchistus, Montgomery assigns an important and conspicuous place to the “rest stage” among his numerous subphases preceding the first spermatocyte mitosis. As a result of his later comparative work upon the Hemiptera, however, we learn that in certain families no trace of such a condition of diffusion on the part of the chromatin is observable, from which we conclude that “accordingly such a stage would appear to have no broad significance.” It has already been announced that nothing like a rest stage intervenes between the spermatogonia and spermatocytes of the Orthoptera, and the work of most investigators would tend to indicate that it is the exception rather than the rule. In those cases where such a condition of the nucleus exists, it would seem to be true that nothing more unusual than an excessive diffusion of the spermatogonial chromosomes occurs, and this is of hardly sufficient importance to receive a special designation.
The existence of a rest stage between the first and second spermatocytes is also negatived by the conditions found in the Orthopteran cells. The formation of chromosomes in the prophase of the first spermatocyte that are already prepared for two divisions would a priori render improbable the intervention of a rest stage here; and the actual observed persistence of the chromosomes, as such, through the telophase of the first spermatocyte and through the modified prophase of the second spermatocyte gives actual proof in support of the view that commonly prevails regarding the suppression of the second spermatocyte rest stage.
Observations upon numerous species tend to show that the behavior of the chromatin during the period between the two spermatocyte mitoses varies considerably with the species and even within the species itself. The amount of diffusion would, in some measure, seem to be related to the form of the chromosomes and to vary correspondingly in those individuals where the chromosomes are of diverse forms. Thus, where the elements of the second spermatocyte metaphase appear as short double rods, the amount of diffusion is slight, and the individual chromosomes may be distinguished throughout the telophase of the first spermatocyte; but in those cases where the members of the mitotic figure are much elongated the diffusion is more extensive and the distinction between elements is made difficult or impossible. Since these two conditions may prevail in the same testis, it is probably only a question as to the extent of elongation on the part of each chromosome. In those cases where the elements become very much extended the appearance of the resting condition would be simulated closely, while, on the contrary, chromosomes consisting of spherical or short cylindrical chromatids would never give a suggestion of such a stage. In this we may find, I think, an explanation for those cases in which a rest stage is described as occurring between the spermatocyte generations.