Regeneration

King finds that when two posterior pieces are united by their oral ends, regeneration of one or of two heads often takes place at the line of union (Fig. 47, B, Bl, Bz\ as Wetzel had found. If a dark green individual is united to a light green one, it can be seen that in many cases the new heads are formed by both components, as shown in Fig. 47, Bl. Later one of the posterior ends is absorbed, and the halves may then separate (Fig. 47, Blt Bz}. If a number of pieces are united, as indicated in Fig. 47, E, a number of heads may be formed, and one or more of these may have a double origin. No

evidences of separation of the pieces was observed in cases of this sort. In one experiment two posterior pieces were united by oblique surfaces, as shown in Fig. 47, C, and one of the two was afterwards cut across, as indicated by the cross-line. The subsequent regeneration that took place is shown in Fig. 47, C1. A head, com- FIG. 47. — After King. A. Hydra split in two, hanging vertically downwards. Later the halves completely separated. II. Two posterior ends united by oral surfaces. H*. Same; it regenerated two heads, each composed of parts of both pieces, ff1. Absorption of one piece leading to a later separation of halves. C. Two posterior ends united by oblique surfaces. Later one piece partially cut off, as indicated by line. C\ Later still, two heads developed, one at N, the other at M. D. Similar experiment in which only one head developed, at M. E. Five pieces united as shown by arrows. Four heads regenerated, one being composed of parts of two pieces.

posed of parts of both pieces, developed at the cut-surface M, and another in the region N'm Fig. 47, C, composed of material of one component. In another ca'se, shown in Fig. 47, D, a head developed only at the cut-edge, but it was made up of material from both components. A series of grafting experiments of another sort has been made by Rand. A part of one hydra is grafted upon the side of another one in the following way. A groove is scratched in a film of soft paraffine covering the bottom of a dish filled with water. Another groove is made at right angles to the first one, and opening into it. A hydra (the stock) is placed in the first groove, and a wound made in its side with a knife. Another hydra is cut in two, and one piece (the graft) placed in the other groove, and its cut-surface brought into contact with the wound in the side of the first individual. If the operation is successful the exposed surfaces of the two hydras

FIG. 48. —After Rand. A. Head of Hydra cut off. After eight days. A1. Same after thirteen days. Three tentacles misplaced. A2. Same after eighteen days. A8. Same after twentyone days. Misplaced tentacles absorbed. B. Anterior end of Hydra fusca, grafted upon side of body of another individual. Half an hour after operation, /fl. Same after four days. B1. Same after thirty-eight days. £fi. Same, foot-region after forty-nine days. B*. Same after separating. Fifty-second day.

quickly unite, and the combination may be taken out of the groove. If the piece grafted on the stock included about the anterior half of a hydra, a two-headed animal results, as shown in Fig. 48, B. Although the graft has been united to the side of the stock, it soon assumes an apparently terminal position (Fig. 48, B1}. This is due to the graft sharing with the anterior end of the stock the common basal portion of the stock. A slow process of separation of the two anterior ends now begins, brought about by a deepening of the angle between the halves (Fig. 48, 2?2). This leads ultimately to a com-

plete separation of the two individuals (Fig. 48, Bs, B*}. Each may get a part of the original foot, or a new foot may arise on the graft as the division approaches the base. In other experiments only a small part of the foot-end was cut from the animal that served as the graft. The long anterior piece was grafted as before upon the side of the stock. After the two had united, the graft was cut in two, leaving a part of the graft attached to the stock. The part regenerated tentacles, and in two cases subsequently separated from the stock as in the first experiment. In a third case the graft was absorbed by the stock as far as the circle of new tentacles, but its subsequent fate was not determined. In a fourth case the graft did not regenerate its tentacles, and was completely absorbed into the wall of the stock. The smaller the piece that is grafted on the stock the greater the chance that it will be absorbed, and furthermore short, broad rings are more likely to be absorbed than long, tubular pieces of the same volume.1

Rand's results show in general that when hydras are grafted together they regain the typical form in one of two ways, — either by separation into two individuals, or by the absorption of the smaller into the larger component. In the former case the result is brought about in the same way as when the anterior end is partially split in two and the halves subsequently separate. When the graft is absorbed it is not clear whether the absorbed piece disappears or, as seems not improbable, forms a part of the wall of the stock.

It is important to notice the difference between lateral buds and lateral grafts. The buds separate in the course of four or five days by constricting at the base, but this never happens in lateral grafts. Rand has also made some experiments with buds. He cut off the outer oral end of a bud, and grafted it back upon the stock in a new place. It did not separate from the stock as does a bud, but by a slow process of division it was set free in the same way as are lateral grafts. The proximal end of the bud, which was left attached, developed tentacles at its free end, constricted at its base, and was set free. The separation was, however, somewhat delayed. In another experiment a bud was split in two lengthwise, and the cut was extended so that the body of the parent was separated into two pieces. Twenty-four hours later it was found that each half-bud had closed in, and was much larger than when first cut. The halfbud, that was attached to the posterior end of the anterior piece, was constricting at its base, and subsequently it separated at its point of

1 Rand found that when a posterior piece was grafted by its cut, oral end to the side of another hydra that it was absorbed into the stock. In one case it moved down the whole length of the body of the stock and finally disappeared by absorption into the foot of the stock. attachment. The other half of the bud, that had been left attached to the anterior end of the posterior piece, had swung around, so that its long axis corresponded to that of the posterior, parental piece. At first a slight constriction indicated the line of union of the two,

FIG. 49. — After Peebles. A. Grafting in Tubulaiia mesenbryanthemum, A small piece of the stock taken from the region near the base, and grafted in a reversed direction on the oral end of a long piece. B. Same with distal tentacles in small piece, and proximal tentacles in large piece (modified from Peebles). C. Same. Formation of hydranth (original). D. Like A. Both pieces produce hydranths. E. Protrusion of hydranths of last. F. Piece of oral end cut off, turned around and grafted on oral end of long piece. A single hydranth produced. Distal tentacle from both components. G. A short piece from distal (oral) end of long piece cut off, and grafted by its proximal end to proximal end of the same long piece.

but later this disappeared and a single hydra resulted. Whether the difference in the fate of the two half-buds is connected with their different polar relations to the parts of the parent, or is due to some other difference in the absorbing power of the anterior and posterior pieces, is not known. Tubularia is not so well suited as hydra to show the influence of grafting on the united parts, since pieces of tubularia produce hydranths, both at the oral and aboral ends, although the latter hydranths take longer to develop. Peebles has shown, nevertheless, that grafting has an influence on the behavior of a piece. In order to show that the polarity of a small piece could be affected by a larger piece, the following experiment was carried out. After cutting off the old hydranth from the end of a stem, a short piece was then cut from the distal end of the same stem, turned around, and its oral end brought in contact with the oral end of the original piece, as indicated in Fig. 49, F. The two pieces, being held together for a few minutes, stuck together and subsequently united perfectly. From eighty-eight pieces united in this way the following results were obtained. Thirty-six formed a single hydranth at the end at which the grafting had been made. The distal row of tentacles appeared in the smaller reversed component, the proximal row in the larger piece (Fig. 49, B}. The new hydranth pushed out later through the perisarc of the smaller piece (Fig. 49, C). In this experiment the smaller component was shorter than the average length of the hydranth-forming region. In two cases, in which the smaller component was larger, both circles of tentacles appeared in this piece. In six of the experiments the tips of the proximal tentacles arose from a part of the wall of the smaller piece, hence these tentacles had a double origin (Fig. 49, F). In five of the unions the smaller as well as the larger component produced a hydranth ; the two were stuck together by their oral ends (Fig. 49, D, E}. The remaining four unions gave somewhat different results.

In three of these the smaller piece produced only a part of a hydranth that remained sticking to the end of the hydranth formed by the larger component. In the thirty-six cases in which the minor component took part in the formation of the single hydranth, the influence of the larger component was shown not only in reversing the polarity of the smaller component, although this might in part be accounted for by the closing of the oral end of the smaller piece, but also in the time of development, since the hydranth appeared sooner than does the aboral hydranth and at the same time as does the oral hydranth.

In another series of experiments, a short piece was cut from the basal end of a long piece (three to four centimetres) and brought forward and grafted in a reversed position on the anterior end of the same long piece (Fig. 49, A). Of five unions of this sort, one produced a hydranth in each component, neither being reversed. Another of the pieces produced a hydranth partly out of each component (and at the same time another at the aboral end of the large piece). The other two pieces produced a single hydranth, a part of

which came from the minor component and appeared before the aboral hydranth on the aboral end of the larger piece. This last result shows that the small piece from the basal end has been affected by the oral end in such a way that it develops more rapidly than it would have done had it remained a part of the basal end. In a third series of experiments a short piece (about a half of a millimetre) was cut from the anterior end of a long piece (one and five-tenths to two centimetres) and grafted in a reversed position on the posterior end of the same long piece (Fig. 49, G\ In four cases a hydranth developed only at the oral end of the long piece and none from the aboral end or from the short piece. Eight unions produced, however, in the region of the graft, a hydranth formed partly by each component. Later another hydranth developed at the oral end of the larger piece. The latter results are not convincing, but they may show that the small piece has hastened the development of the hydranth at the aboral end.

Peebles has also made some experiments in grafting pieces of different members of the colonies of hydractinia and podocoryne. The colony of the former is made up of three different kinds of individuals : the nutritive, the reproductive, and the protective hydroids. A series of preliminary experiments showed that if these individuals are cut into a number of pieces each piece regenerates the same kind of individual as that of which it had been a part. It was also observed that if pieces of the nutritive individuals were allowed to remain quietly on the bottom of the dish they sent out branching stolons, which stuck to the bottom of the dish, and from these stolons there arose later nutritive hydranths that stood at right angles to the surface. When pieces of the same kind of individuals are grafted together, the results are essentially the same as with tubularia. If pieces of different kinds of individuals are united, the opportunity is given of testing the possible influence of one kind on the other. Peebles united a nutritive and a protective polyp by the cut, aboral ends (Fig. 46, E\ and after they had grown together one of the polyps was cut off near the region of union, so that a small piece of a nutritive polyp was left attached to a protective polyp. When the piece of the nutritive polyp regenerated, it made a new nutritive polyp. The influence of the protective polyp was not apparent. If a nutritive and a reproductive polyp are united in the same way, and the latter cut in two near the line of union, a new reproductive polyp develops from the piece left attached to the nutritive polyp. Again there is shown no influence of the one on the other kind of polyp.

Hargitt has also made a number of grafting experiments on other hydroids. His most interesting results are those in which parts of two medusae were united by holding their cut-surfaces together by means of bristles passing through the individuals. Hargitt also finds that while in certain hydroids it is possible to bring about a union of oral with oral end, or aboral with aboral, or oral with aboral end of the same species,1 yet a permanent union between different species cannot be brought about. These results are in agreement with those of a number of writers who have recorded the difficulty or impossibility of uniting parts of different species of hydra. In a few instances it has been possible to unite temporarily a piece of a brown hydra with a piece of a green one, — as I have also seen accomplished, — yet the pieces subsequently separate. Wetzel succeeded in obtaining better results with two species of brown hydras, Hydra fusca and Hydra grisea. In one experiment the head of Hydra grisea was grafted on the body (from which the head had been cut off) of Hydra fusca. After five hours the pieces seemed to have united. Later a constriction appeared at the place of union, and the head-piece produced a foot near the line of union, and the posterior piece produced a circle of tentacles at its anterior end. Eight days later, when the animal was being killed, it fell apart into two pieces. It was observed that during the period of union a stimulus to one piece was not carried over to the other. Wetzel's results seem to show that pieces of these two species of hydra unite at first, when brought together, as perfectly as do pieces of the same species, but the union never becomes permanent, a constriction appearing later at the line of union, and the pieces separating in this region. These results indicate, it seems to me, that the factors that bring about the first union are different from those that make the grafted pieces one organic whole.

Other results indicate that the union of oral to oral end, or aboral to aboral end, while at first as perfect as between unlike surfaces, nevertheless is less permanent than when unlike surfaces are united ; at least, subsequent regeneration is more likely to occur in the former than in the latter, and after this occurs the separation of the individuals often takes place. It seems, moreover, not improbable that a more permanent union results when similar regions are united by unlike surfaces, than when the union is at different levels. If, for instance, the anterior half of one hydra is united to the posterior half of another individual, the union is generally permanent ; but if one or both of the pieces are longer than half the length, so that a " long animal" results, new tentacles are more often formed at the oral end of one component, and the parts subsequently separate. It may be that, at present, the data are insufficient to establish this general rule, and no doubt other modifying influences must be also taken into account ; but it is important that attention should be drawn to this side of the subject.

1 Pieces from male and female colonies of the same species also unite. Grafting experiments in planarians have so far been carried out in only the two cases which I have described. In one of these the anterior ends of two short pieces of Bipalium kewense were united (Fig. 50, A). Neither piece produced a head at the region of union. Later the pieces were cut apart by an oblique cut that passed across the line of union (Fig. 50, C), so that each piece retained at its most anterior end (at one side) a piece of

FIG. 51. — Two pieces of Bipalium kewense united by posterior ends. Each regenerated a head at anterior end. the other individual in a reversed position. A head developed at the anterior (and lateral) end of each piece, in such a way that a part at least of the small reversed piece was contained in the new head FIG. 5o. — A. Two pieces ot tiipaJtum tewense /p- rO n\ In the other case two stages of same. Line in C indicates how pieces of bipalium were United by

piece produced a new head at its free end, and the pieces greatly elongated, but remained sticking together (Fig. 51). A large number of experiments have been made by Joest in grafting pieces of earthworms. The cut-surfaces were held in contact by means of two or three threads passing through the body wall of each piece and tied across, so that the pieces were drawn together and held firmly in that position. Joest found that pieces of the same or of different individuals could be united in various ways, and the union become permanent. If the anterior end of one worm is united to the posterior end of the same, or of another worm, a perfect union is

formed, and no subsequent regeneration takes place (Fig. 52, A). Long worms can be made by uniting two pieces, each more than half the length of a worm, or by uniting three pieces, as shown in Fig. 52, C. Short worms can be formed by cutting a middle piece from a worm, and uniting the anterior and posterior pieces (Fig. 53, D). Joest found that when a short worm is made in this way, so that no reproductive region is present, the new worm does not produce new reproductive organs. It is conceivable that new reproductive organs might

FIG. 52. — After Joest. A. Union of two pieces of Allolobophora terrestris in normal position. Twenty-two months after operation. B. Union of two pieces Lumbricus rubellus. Pieces turned 180° with respect to each other. C. Union of three pieces of A. terrestris to make a " long worm." D. Union of two worms (by anterior ends) from each of which eight anterior segments had been removed. After three months. Regenerating two new heads. £. A small piece of Lumbricus rubellus grafted upon Allolobophora teirestris. Former regenerated an anterior end.

have been produced either in the old segments, or by the formation of a new reproductive region between the two united pieces, but neither process takes place. In the long worms two sets of reproductive organs, etc., are present. This sort of union is, however, less permanent, as the worms often pull apart. Joest also united two posterior ends by their anterior surfaces. In many cases no regeneration took place, and, in the absence of a head, the combination is destined to die, although it may remain alive, without food, for several months. When two very long pieces

were united by their anterior ends, — only eight segments being removed from each worm, — although perfect union took place at first, later one or two new heads generally developed at the region of union (Fig. 52, D). When only one head developed it did not seem to belong to one of the components rather than to the other, and originated in the new tissue that appeared between the two pieces. These experiments, in which the anterior surfaces of two pieces are united, show also that the new head arises between the two pieces most often, if not exclusively, when the union is in the anterior ends of the worms. This corresponds with what is now known in regard to the development of new heads by isolated pieces, since there is less tendency to produce a head the farther posteriorly the cut has been made. At more posterior levels a tail and not a head is often regenerated, as has been stated, on the anterior cut-surface. This formation of a heteromorphic tail seems to have been suppressed in the pieces united in this region, except in one case,1 in which it appears, •from Joest's account, that a tail probably regenerated, although Joest speaks of it as a head.

It is more difficult to unite two anterior ends by their posterior cut-surfaces, not because the surfaces refuse to unite, but because the two pieces crawl away from each other and pull apart. In one case, however, union of this sort was brought about. In all the combinations that have been so far described, the dorsal and ventral surfaces of both components were kept in the same direction, so that the ventral nerve-cord of one piece came in contact and fused with the nerve-cord in the other piece. Sometimes it may happen that the components are not quite in the same position, and the end of one nerve-cord may fail to abut against the other one. In such cases Joest thinks that regeneration is more apt to take place in the region of union, and he has carried out a series of experiments in which the pieces were intentionally united, so that they are not in corresponding positions. It is found that if one piece is turned so that the nervous system lies 90 degrees, or even 180 degrees (Fig. 52, B), from that of the other piece, the union takes place just as when the pieces have the same orientation, except that the ends of the nerve-cords do not unite. Subsequent regeneration from one or from both components generally takes place in the region of union.

It is more difficult to unite pieces of different species of worms, yet Joest has succeeded also in making combinations of this sort. One union between the anterior end of Lnmbricns rubellus and the posterior end of AllolobopJiora terrestris was permanent, and the new worm reacted as a single individual, and lived for eight months. Each piece retained its specific characters, and showed no influence of the other component. By means of a similar experiment we have a way of rinding out if one component can influence regeneration taking place from the other piece. Although Joest made only a few observations of this sort, the results show that no such influence is manifested.

By means of grafting it is possible to keep alive small pieces of a worm that would otherwise perish. For instance, pieces of a worm FIG. 53. — After Joest. A. Small ! piece of Allolobophora terrestris from posterior end grafted upon anterior end of another individual. Oral end free. Four weeks after grafting eight new segments formed, /i. Same fourteen days later. A new part of thirty-seven segments had appeared at end of former eight segments. C. A piece of the body wall of Allolobophora terrestris grafted upon the cut-end (anterior) of Lumbricus rubellui. Two months later, as shown in figure, a head had grown on major component. D. Anterior and posterior ends of A. terrestris united to make a " short worm." E. A piece of body wall of A. cyanea grafted on side of body of Lumbricus rubellus. f. Piece of L. rubellui grafted on side of body of another individual to produce a double-tailed worm.

containing only three segments are not capable of independent existence, except for a short time, and even pieces of from four to eight segments die in most cases. It is not possible to unite small pieces of this size directly upon larger pieces, since they will die, ordinarily, as a result of the operation, but larger pieces can be united and then after union has been effected, one of them may be cut off near the place of union. The same result is sometimes brought

about accidentally by the worms themselves pulling apart and leaving a small piece of one component attached to the other. Joest found that in several cases these small, attached pieces regenerated. In one case, after two long pieces had pulled apart, a small piece, left by one of the two, regenerated a single new segment with a mouth at its end. In another case, after one of the components had been cut off, leaving two segments attached, a new part of seven segments regenerated.1 Especially interesting is the case in which two individuals (A. terrestris} had been united to form a long worm. The anterior component extended to within two centimetres of the anus ; the posterior piece had had the first four segments removed. Three days later the anterior piece was cut off three segments in front of the region of union. About a month later a small part of eight segments had regenerated from the cut-end (Fig. 53, A). Fifteen days later another new part of thirty-seven segments developed at the end of the first new part (Fig. 53, B). Joest speaks of the first eight segments as a head, and the second simply as a regenerative product. There can be little doubt, I think, that both parts represent a heteromorphic tail. The region from which the regeneration took place would make this interpretation highly probable, and Joest's figures also indicate that the structure is a tail. The result is very interesting, if my interpretation is correct, as it shows that the major component did not influence the kind of regeneration, although the surface of regeneration was separated by only three tail-segments from the anterior end of the major component.

In another experiment a long animal was made by uniting Lumbricus rubellus (whose posterior third had been cut off) and Allolobophora terrestris (whose first six segments had been cut off). Four days later the two components had torn apart, but a small piece of the anterior worm remained attached to the 'anterior end of the posterior component. The small piece consisted of the dorsal part of two and a half segments without any ventral part, so that the anterior end of the posterior component was partially exposed. The small piece of lumbricus was much lighter in color, and this difference made it easy to distinguish between the two. In less than a month the small transplanted piece had replaced its -missing ventral part, so that the entire anterior surface of the larger component was covered over. The small piece, in addition to regenerating its ventral part of four segments, had also begun to make new segments. After a month and a half six new segments were present (Fig. 52, £), with a mouth at the anterior end.2 Even after ten months the color of