In many respects Opalina ranarum, Purk. and Vai., is one of the most interesting of the ciliated Protozoa. Although its adult appearance has been familiar to all zoologists for a long time, it is only in the course of the last year that its interesting life-cycle has become fully known. We owe this knowledge to Neresheimer, who has, however, only given us a preliminary account of his important researches. His full paper will be awaited with much interest.
It will be unnecessary for me to give a detailed description of this very common species. But I would remind the reader that it is a large, multinucleate, holotrichous form found in the large intestine of our common frog and toad (Rana temporaria, L., and Bufo vulgaris, L.). Thenucleimay be regarded as consisting of meganucleus and micronucleus fused to form a synkaryon. For an account of the life-history the reader is referred to accounts already published, but more especially to the memoirs of Zeller and Neresheimer. It will be necessary for me to give a brief account of the earlier part of the life-cycle, however; that is to say, of the part prior to encystment, which takes place in the adult host.
Neresheimer has accurately, though briefly, described this phase, and I have been able to confirm fully his observations.
In the spring, at the frog’s breeding season, Opalina encysts, and is cast out into the water in the excreta. The changes which take place before encystation are as follows :— The adult animal divides in an oblique manner, giving rise to two daughter individuals. Each of these then divides into two, and these again divide until small Opalinæ containing several nuclei are formed. During these divisions important changes occur in the nuclear apparatus. The nuclei are seen to become less distinct, this being due to the fact that the chromatin is cast out into the cytoplasm in the form of small strands and particles, or chromidia. Sometimes I have observed these united to form a network through the creature. Finally, the original nuclei vanish, and we are left with only distributed chromatic material. The chromidia soon become aggregated at certain centres, and thus synthesise new nuclei, which are from two to ten in number. These nuclei are seen to be composed of large chromatin granules, arranged irregularly. They change, however, with the approaching encystment of the animal, which soon takes place. The chromatin travels to the periphery of the nucleus where it becomes arranged in a thin layer with two to four large cap-like thickenings.1 In optical section these nuclei appear as rings. The cap-like projections are soon cast off into the cytoplasm, where they degenerate, this constituting the first nuclear reduction. Encystment follows, and in the normal course of events the cyst is cast into the water, where a second reduction of chromatin occurs. I will leave the description of the life-cycle at this point, merely noting that each nucleus is now a reduced gamete nucleus, and takes part in the formation of a single ciliated gamete whose destiny is to conjugate in the tadpole’s gut. The essential points in the process just described are (1) formation of chromidia, (2) synthesis of fresh nuclei from these chromidia, (3) reduction of chromatin, and (4) encystment.
Description of Degeneration in Opalina
Having cleared the ground by describing the ordinary course of events preceding gamete formation, I will now pass on to a description of the physiological degeneration of the forms whose usual destiny is to encyst.
Two different kinds of degenerative changes may be distinguished—the one caused by removal from the host, the other within the host. The former is much the more rapid, and is easily induced at any time. Degeneration results from drying, from increase in the number of bacteria, and also doubtless from lack of food, and increase of metabolites. The entire organism simply decomposes, often after first throwing out its nuclei in fragments. It is the second kind of degeneration—that within the host—with which I am here concerned.
In nature, as we have seen already, the encystation of Opalina is contemporary with the sexual activity of its host. The set of degenerative changes which I am about to describe took place when the ordinary activities of the host animals were modified by captivity and starvation. Frogs, as is well known, can endure starvation for many weeks. But the contained Opalinæ, apparently, cannot do so—at all events at their encystation period. Starvation is, I believe, the determining factor in their degeneration. Other causes, which materially influence degeneration in the organisms when removed from their host—such as change in reaction of the medium, drying, increase in the number of bacteria, etc. —do not appear to come into play. For after lengthy starvation the rectal contents of the frogs and toads examined consisted of only a small quantity of a clear, mucous fluid, alkaline in reaction, and containing but few bacteria. It was in cases such as this—where starvation of the host had sometimes lasted for at least two months—that the most advanced stages in the degeneration of Opalina were encountered. My observations extend over a period from the middle of January to the beginning of April, and are based upon the careful examination of the rectal contents of over fifty frogs and toads. The original object of the research was to study the life-histories of the small Protozoa (flagellates and amœbæ) which occur in this situation. My attention was attracted by the curious degeneration forms, although their true nature was not, for some time, understood. However, after a careful study of fresh material, the complete series of degeneration changes here described became apparent.
One of the earliest changes which the Opalinæ undergo is a change of shape. Instead of remaining of a flattened, ovate form, they become modified into all sorts of indefinite shapes. Some of these are shown in text-fig. 1, but a great number of other forms may be seen. The drawings are schematic, and merely indicate the kind of thing which one encounters. These forms do not divide in the normal manner, but simply constrict off pieces, apparently at random, of all shapes and sizes. These again divide until small, irregularly discoidal, or ovoid forms are produced which have a length of about 10 μ to 30 μ. These usually contain from one to four nuclei, but much larger individuals—up to 50 μ, with nine or ten nuclei—are also found in the same condition. Such forms undergo two remarkable changes—(1) they completely lose all cilia, and (2) they give rise to globules of a substance of high refractivity in their cytoplasm. The nature of these globules I am unable to determine. I may mention, however, that they have the following properties :—
In the fresh state they are somewhat greenish, and very highly réfringent. They are coloured a bright pink with eosin, and a bright greenish-yellow with picric acid. With iodine they appear to become slightly more greenish, but the reaction is not well marked. They are insoluble in water, alcohol, and weak acids and alkalies. Heidenhain’s iron haematoxylin colours them a dark greyish-or brownish-black —not so dark, however, as the chromatin. Delafield’s hæmatoxylin does not colour them—neither does borax-carmine.
From their remarkably vivid coloration with eosin I have termed these globules “eosinophile “bodies, in ignorance of their chemical constitution. Although they first appear as separate globules of small size, they ultimately run together, forming large masses lying in the cells. They do not appear to have any connection with the nucleus.
If these degenerate forms be obtained at the right stage, the loss of cilia may be observed in the living animals. It takes some days for all to be completely lost, and all do not seem to disappear in the same way. Apparently some of them actually dissolve, for they become gradually fainter and fainter, and finally disappear. Others are thrown oil entirely, and after moving spontaneously for a short time after detachment, they become motionless and fade away. Still others undergo fusion with one another, and ultimately with the cytoplasm. In this manner many individuals arise which are completely divested of ciliary covering, and contain réfringent eosinophile bodies. The nucleus also undergoes remarkable modifications. I term these forms the atrichous forms.
The nuclear changes which the atrichous forms undergo are as follows, and may be easily seen in the living animal :— The chromatin which was at first evenly distributed through the nucleus becomes massed in granules at the periphery, whilst the nucleus itself increases in size until it becomes sometimes double its original diameter (text-fig. 2, a, b).
The chromatin becomes evenly disposed in a single layer, so that in optical section the nucleus has a very characteristic annular appearance (c), the ring being thickened at various points (see also Plate, figs. 8, 12). A typical atrichous form is therefore distinguished by having no cilia, by possessing “eosinophile globules” and a large ring-like nucleus (or nuclei). When first seen they have a remarkable appearance, and their connection with the ordinary Opalina would hardly be suspected. These forms are quite motionless.
In many of the larger atrichous forms division of the nucleus takes place, followed frequently by division of the cytoplasm. Division may be equal, by a constriction appearing in the middle (text-fig. 2, d), or unequal. In this latter process a blister-like elevation of the chromatin appears, and is finally constricted off. This is shown in text-fig. 2, e. Above is a cap-like outgrowth of the chromatin, whilst below a later stage is seen in optical section (cf. also Plate, figs. 10, 11).
When cytoplasmic division follows the result is either equal bipartition or budding (cf. Plate, figs. 9, 10). The buds so produced are sometimes very small, not reaching a greater diameter than 4—5 μ. Occasionally buds are produced in which no nuclear material whatsoever can be detected, and very commonly small buds are given off which contain an eosinophile globule, but no nucleus. All these enucleate buds appear to die and disintegrate. Finally, a number of uninucleate atrichous forms result, which are of an average diameter of about 20 μ. At this stage they show a marked tendency to attach themselves to one another, thus forming small colonies (cf. Plate, fig. 12). No fusion, as a rule, appears to take place.
The chromatin of the nucleus, which is of a very variable size, but on an average about 8—10 μ. in diameter, is seen to be arranged in lumps peripherally. It soon leaves the nucleus, however, and fills the cytoplasm, where it takes the form of irregular granules and masses of different shapes and sizes. These chromidia, as they may be called, appear to be sometimes in the form of minute hollow spheres, ringlike in optical section. By their formation the original nucleus dwindles away, and finally disappears (see Plate, figs. 1, 2, 7). As a rule most of this chromatin is cast out of the organism, which then dies and breaks up. But occasionally a remarkable thing happens. Only a part of the chromatin is cast out and perishes. The remaining granules run together again, very much as drops of oil might run togethei’ in a watery medium. All the irregular chromidial masses may become aggregated at a single centre, but at other times two such centres are formed, so that finally two nuclei, consisting of solid chromatin, are synthesised. These two solid lumps then approach one another and fuse (see Plate, figs. 3—6). During the chromidial stages a soft cystwall is sometimes formed.
I have been unable to obtain any further stages after this, except such as are disintegrative. Kept under a waxed coverslip or in a hanging drop they always perish by discharging theii’ nuclei in fragments and then breaking up. This also appears to happen in the frog’s gut. It would be exceedingly interesting to know whether a recovery could be made under suitable conditions or not. I have endeavoured to restore some of these degenerate fragments by transferring them into the boiled rectal contents of a normal frog, but without success. I have not been able to obtain sufficient material for more extended experiments in this direction.
The final result then is death ; and with this I finish my description of degenerative changes in Opalina ranarum so far as I have observed them. Before leaving the subject, however, I must draw attention to the extraordinary parallel which exists between these changes and certain so-called “sexual “processes. In many Protozoa gamete nuclei are formed from the original compound nuclei by resynthesis from chromidia, very much in the same way as the solid chromatin nuclei which I have just described in Opalina. In certain autogamie processes the nuclei are formed and fuse in the same cell. Compare, for example, the autogamy of Bodo lacertæ, Grassi, as described by Prowazek. The animal encysts, and inside the cyst the nucleus gives off chromidia into the cytoplasm. From these chromidia a new nucleus is built up, and this divides into two. Each daughter nucleus forms two “polar bodies,” and the reduced nuclei (the gamete nuclei) approach one another and fuse. In Bodo lacertæ heterogamy also occurs, but in Trichomastix lacertæ, Blochmann, and in Entamoeba coli, Loscli, only autogamy is known—no other “sexual “act.
It is possible that the process which I have just described in Opalina is a kind of autogamie attempt on the part of the organism to reconstitute itself. But 1 believe that the sole explanation of this curious set of changes is to be sought in the alteration in chemical and physical properties which living protoplasm undergoes in dying.
In conclusion, mention may be made of certain other’ observations which have been made on degeneration in other Protozoa. But little attention has been bestowed upon the matter, although in a few species degenerative changes have been studied in considerable detail. Among these I may name Actinosphærium (Hertwig), Amoeba (Prandtl), Paramcecium (Maupas, Calkins, etc.), Trichosphærium (Schaudinn), and the sporont of Cyclospora Caryolytica (Schaudinn). In the first three of these increase in the size of the nucleus has frequently been observed as a preliminary occurrence. In Actinosphærium giant nuclei are formed when the animal degenerates owing to overfeeding. Breaking up and discharge of the nucleus usually follows—that is to say, chromidia play a part in the degenerative phenomena. In Trichosphærium, when degeneration is induced by starving the organism, the nuclei clump themselves together at certain points. This agglomeration is not followed by fusion. Stoic has observed a similar condition in starved Pelomyxæ. And I may here recall the observation of Maupas on Paramcecium, that the fragments of the old meganucleus sometimes fuse with the new meganucleus of an exconjugant if it be starved.
In the degenerating sporont of the coccidian Cyclospora the polar bodies divide until eight are formed, and microgametes then attempt to fertilise each of these. In later stages of degeneration pigment is formed ; and Prandtl has shown that pigment appears in a degenerating Amoeba proteus, and is formed from the chromatin of the nucleus. In this particular form there is also a curious tendency for the nucleus to surround food masses in the cytoplasm.
Nuclear fusion sometimes takes place—independently of any sexual process—in multinucleate Protozoa during, or following, encystment: e. g. in Dileptus (Prowazek).
Hyper-regeneration occurs in Stylonychia if mutilated when in a degenerate condition (Prowazek). Loss of appendages has been frequently observed in many different Protozoa undergoing degenerative changes. It is unnecessary to give a number of examples, but Trichosphærium and Paramcecium may be cited as good instances.
Chromidia of the type I have described in Opalina (i. e. bladder-like, or blaschenfôrmig) have only been noticed, so far as I am aware, in one other Protozoon, Bodo lacertæ, Grassi. And here they are formed as a preliminary to gamete formation and autogamy (Prowazek).
Very curious in many other ways is the parallel which exists between degenerative and “sexual “processes. Besides the fact that chromidia are formed in both, we have the observation that an amoeboid condition may occur in degenerating Protozoa, and also sometimes just before conjugation. Fusion also occurs in degenerating forms of various kinds. I have observed it especially in flagellates, e. g. Trichomastix, Trichomonas, etc. Senile Paramcecia enter upon what Calkins calls the “miscible state,” when they tend to adhere to one another. Similarly, Roux has observed that isolated, living blastomeres of frog’s eggs become amoeboid and run together; though, according to Driesch, this is merely due to the capillary forces between the cells. These facts, and many others of a similar nature, are not without interest, both from a pathological and from a zoological point of view. I may mention merely their possible bearing upon the remarkable fusion which appears to take place between leucocytes and cancer cells, and its unknown significance. And since the work of Calkins seems to indicate that chemical change in protoplasmic composition is the chief beneficial effect of conjugation, and there is some proof that Protozoon individuals have chemical compositions differing from one another (cf. Jensen), is it not at least possible that the physico-chemical changes which cause fusion in degenerating cells are of a similar nature to those which gave rise to the first cell-couplings, and which still determine the fusion of one gamete with another ?
Zoological Labobatoby, Cambbidge.
Neresheimer’s full account of the life-cycle of Opalina has appeared since this paper was submitted for publication. It is a very complete description, with full references to the literature, and is to be found in ‘Arch. f. Protistenk Supplement i (Festband für R. Hertwig), 1907, p. 1.
EXPLANATION OF PLATE 38,
Illustrating Mr. C. Clifford Dobell’s paper on “Physiological Degeneration in Opalina.”
[Figs. 7 and 8 are drawn from living specimens, under a 2·5 mm. (apert. 1’25) apochromatic water immersion objective by Zeiss ; compensating ocular 12. The remainder are from permanent preparations : fixed sublimate-alcohol (2 :1). Figs. 1·6, 9 and 12 from specimens stained witli Heidenhain’s iron-hæmatoxylin. Fig. 10, Heidenhain and eosin. Fig. 11, Weigert’s iron-hæmatoxylin and eosin. Drawn under a 2 mm. (apert. 1·40) apochromatic oil immersion (Zeiss), with compensating ocular 12.
All figures are drawn in monochrome for sake of uniformity. In all cases the darker masses are chromatin ; the paler masses surrounded by a light area are the eosinophile bodies.]
FIG. 1.—Atrichous form in which the nucleus has largely broken up into chromidia. The remains of the nucleus are seen near the middle, with a single large cap-like mass of chromatin. Beneath is seen one large eosinophile body. Note that many of the chromidia are in the form of hollow spheres—annular in optical section.
FIG. 2.—Smaller specimen, completely filled with chromidia.
FIG. 3.—Specimen in which the chromidia are running together to form two new nuclei. Some indication of the formation of a cyst can be seen.
FIG. 4.—The chromidia have fused to form two new nuclei, which are approaching one another.
FIG. 5.—A cyst-wall lias been formed, and the two solid chromatin nuclei are applied to one another.
FIG. 6.—Specimen showing a still later stage in the fusion of the nuclei. A thick, soft cyst has been formed.
FIG. 7.—Fresh preparation in which a cyst has been formed and the nucleus has broken up into chromidia.
FIG. 8.—Atrichous form in fresh condition, showing nucleus with peripherally placed chromatin masses (annular in optical section).
FIG. 9.—Large multinucleate atrichous form breaking up to form smaller ones. At least one nucleus is dividing.
FIG. 10.—An individual in the act of forming buds. Small blister-like nuclei are budded off, and the cytoplasm has become constricted round one of these, forming a complete bud.
FIG. 11.—Similar form, in which nucleus is seen in optical section. One nucleus has been completely separated off, and another is almost so.
FIG. 12.—Association of eleven typical atrichous forms. Noue of these have as yet formed chromidia, though one appears to be enucleate.
First described by Loewenthal as “microfiucleus-like structures,”