FISSION AND REGENERATION I N STYLBRZd

Piece constitution.

Anterior regeneration.

Posterior regeneration.

Cases obseri ed.

229

+ + 14

'-Iv.

+ -

15 41 21

Further Experiments on the Relation between Natural Fission and Regeneration in Xtylaria fossularis. By J. CHU*, D-pir tment of Biology, National University. of Chekiang, Meitan, Kweichow, China.

[Communicated by THE SCIENTIFIC DIRECTOR-Received December 14, 1945.1

(With 7 figures in the test.)

After the demonstration of the presence of the double regeneration gradients in Stylaria fossularis, we have attempted to correlate the phenonienon of natural fission with that of regeneration. In a previous paper (Chu and Pai, 1945, in the press), we have suggested that the fission may occur a t any body- level at random or a t only one definite level representing probably R region with equal anterior and posterior regeneration poten~ialiti-s. Which of these two alternatives is correct is very difficult to answer a t the present time, but in this work we endeavour to show that experiments on piece iegenerntion with different modes of cutting may throw some light on the question.

EXPERIMENTAL. Six sets of experiments have been performed. They will now bs described

in succession. 1. In the first set of experiments, worms without fission zone were klected

as donors. Pieces, each consisting of four trunk segments, wcre obtained following two consecutive cuttings. The level of the two sections may be varied at will so as t o give pieces having different combinations of trunk segments. I n Table I there are listed 125 cases of regeneration of pieces taken from the

TABLE 1.-Data showing the difference between the anterior and posterior regeneration potencies among different pieces taken from the anterior region of the worm, each of them consisting of four trunk segments.

v-VIII. VI-XI. I - + I + + t i

11 I 13

anterior region of the worm. The Roman numerals represent the progressire number of trunk segments in antero-posterior direction. Thr plus s i p indi- cates the success of regeneration, and the minus sign the failure of it. The data show that, while anterior regeneration could take place in any pieco, posteri r regeneration could proceed normally only whrn the level of section fell a t the 7th trunk segment or thereafter. A piece including four (fiom 3rd to 6th) trunk segments may or may not have posterior regmeration. These results agree closely with those given in our earlier report (Cliu and Pai). In that paper anterior and posterior regeneration were subjectc d to separate analysis after a single cutting, and it was noticed that posterior regeneration

* Here T gratefully aclmowledge my indebtedness to Dr. S. Pai, Chairman of the Department, for his helpful suggestions in the progiess of the work and for his criticism of the manuscript.

PROC. ZOOL. SOC. LOND.-VOL. 116. 16

230 J. CHU

Piece constitution.

Regeneration rate in days.

Relative */P rate of -

regenera- tion. P/A

Mean value of relative rate.

will take place wibhout any difficulty when the cutting-level is beyond the 6th trunk segment. The data given here further indicates the existence of a gradat,ion of posterior regeneration capacity along the body axis.

Table I1 presents another set of data with pieces taken from the middle trunk region. Since this region was bipotent for head and tail formation,

TABLE 11.-Piece regeneration with trunk segments taken from middle region of the worms, showing the rate gradation of anterior (A) and posterior (P) regeneration.

XXIV. XXVIII.

XX1- 1 A T

XVII- XX . v-VIII. IX-XII. $I$;;

A . ~ P . A . ( P . A . ~ P . A . I P . A I P . 12.7 43.1 15.4 22.0 19.3 41.2 23.3 21.1 28.7 120.0 39.9 142.0 I I I I

O.55 0.70 0.9 I 1 .10 1.44

1.82 1.43 1.10 IP91 0.w 0.55

1.07 1.01 1.01 1.07 1 .18 ,,In

anterior and pobterior regeneration could be carried out with fair success. When t h e data were expressed in terms of days necessary to complete the regeneration process, the axial difference in the rate of anterior regeneration was clearly shown. The difference in the rate of posterior regeneration was, however, not seemingly significant among different pieces. But if the rate of regeneration is converted into the relative rate, as described previously (Chu and Pai), we can see that the axial difference in posterior regeneration rate is just as marked as the anterior regeneration. Moreover, the mean value of relative rate between anterior and posterior regeneration of any piece always approaches unity, as was also discussed in the previous paper.

2. From the above experiments, it seems to be out of the question that posterior regeneration could be carried on with a p’ece consisting of the 4th trunk segment as its lowest level. Later, we found that this was not the case if experimental conditions were made differently. Suppose we first took a piece having eight (1st to 8th) trunk segments (fig. 1, piece-sectioned between planes C, and C,) and let i t start a certain degree of anterior as well as posterior regeneration, which was generally brought out by culturing it to a stage when head and thorax were well regenerated. Xow, if cutting was again made at the level of the 4th trunk segment in this regenerating worm (fig. 1 , Cs), the unexpected result was obtained that the anterior piece could regenerate posteriorly. In a somewhat similar way, if a piece consisting of its original head and thorax, besides the eight trunk segments, was used as a r( gin8 ate, ~t secondary cut a t the 4th trunk segment would also give pos’tive Fosterior regeneration, provided that a tail-bud had been formed at the posterior end of the piece before the act of sectioning. These and the above r( sults from piece regeneration experiments were not consistent in themselves u n l c ss one assumes that a redistribution of regenerative potencies occurs dur.hg rcge cra- tion, a hypothesis which will be discussed in later sections. For the present, it may sinrply be mentioned that whether the head and thorax ofa r( g nl rating piece was a newly-formed one, or only the remainder of the original worm, did not affect the pyocess of redistribution in general.

Attempts have been made to test to what degree of anterior and posterior regeneration a regenerating piece should attain before it can have a successful

FISSION AND REGENERATION IN STYLARIA 231

Figure 1.

Showing the manner of sectioning described in o w experiment 2. C , , C,, the primary cutting planesin adonor as to give rise t o a piece consisting of eight (I-VIII) trunk segments. C,, plane of secondary cutting after considerable regeneration had taken place in thie piece. Newly formed head and tail are marked by dotted lines:

Figure 2 .

c, --

c2-

- - V V I V I I

. V I I I

Similar to fig. 1, showing the manner of sect,ioning described in our experiment 3. P h e 8 c, and c, represent. the levels of primary and secondary cutting respectively.

posterior regeneration after secondary section at the 4th trunk segment. Xo satisfactory results were obtained, due to the absence of visible criteria of differentiation, Tt. can only be concluded that a well-reconstitilted head and

16*

232 J. OHU

thorax in the anterior end and a well-defined tail in the posterior end were the required conditions.

3. Similarly, posterior regeneration could also be conditionally blocked at the level of the 8th trunk segment by the preliminary cutting of a worm at its 4th trunk segment (fig. 2, CJ, and using the posterior piece as experimental material. When this piece had completed a certain extent of anterior regenera- tion and posterior growth, cutting was again made at the original 8th segment (fig. 2 , CJ. It is quite surprising that-in contrast to the expected result shown in Table I-the piece, while possessing materially the same V-VIII trunk Negments, could not regenerate posteriorly. These contradictory events again point to the possible occurrence of a redistribution of regenerative potencies as regeneration goes on.

Figure 3 a. Figure 3 b.

n f ission - zone

d - t : 1:-

Showing the constitution of the two kinds of regenerate used in our experiment 4 to test the differentiation of fission zone.

4. Experiments were further performcd to investigate the differentiation of the fission zone as well as the effect of the prcscnce of the fission zone on the fate of a regenerating piece. In these experiments we uscd only worms having a single fission zone without any visible difftrrntiation of proboscis and eye spots. Cutting was made at two segmtnts either before or bthind the fission zone (figr. 3 a and 3 b ) . The respecrive anterior and posterior regcneration of thc pieces thus obtained were closely followed.

In the case of posterior regeneration, the results may be classified into three groups :-

(a ) The fission zone gradually disappeared and the continuous posterior regeneration at the cut surface led to the production of a single perfect worm.

(b ) Tl-e fission zone continued its differentiation, forming head and thorax but giving no posterior regeneration. The fragment, which now consisted of a well-recognized proboscis, eye spots and two or three thorax segments, finally fissured down and disintegrated. The piece originally antexior to the fission zone developed into a new worm through its posterior differentiation.

FISSION AND REBINERATION IN STYLARIA 233

(c) The fission zone neither disappeared nor continued its differentiation. The two segments behind the fission zone underwent a slight extent of wound- healing, and finally disintegrated after their separation from the anterior piece, which, as in case (b) , also gave rise to a new worm.

If the worms used for cutting had their fission zone fully developed, the results were always the same as group ( h ) just mentioned.

For anterior regeneration, the results may be similarly grouped as follows :- (a) The disappearance of the fission zone and the success of anterior regenera-

tion at the cut surface led to the production of a single perfect worm. (b ) The piece finally gave rise to two worms as a result of anterior regenera-

tion at the cut surface and the continuous development of the fission zone. (c) No regeneration at the cut surface occurred save for wound-healing or

the formation of an out-growth, the extent of which depended upon the exact condition of the worm at the time of cutting. The two segments anterior to the fission zone fissured down and died away. The remaining posterior part developed into a. single individual.

If worms used for cutting were all provided with well-differentiated proboscis and eye spots, cutting at two segments before the fission zone produced results id-ntical to case (b) .

5. The next cutting experiment was made with worms provided with a chain of two or three fission zones. Cutting was set a t the segment just prior to the youngest fission zone. New primary worms fissured down one after the other at successive intervals, according to the usual manner of natural fission (Chu, 1945). This fact suggests that, although the younger fission zone had no visible morphological differentiation at the time of cutting, it was already provided with the total potentialities to develop into a complete individual. Sometimes the disappearance of the fission zone occurred after cutting. As this was only confined to the youngest ones, it seems safe to conclude that the differentiation of fission zone is unstable during its early phase of development. No case has yet been obtained showing simultaneous reduction of two successive fission zones.

6. The last set of experiments was undertaken to test the effect of starvation on the fission zone. 54 worms, each with a single incipient fission zone, were individually cultured in sterilized water without any feeding. 5 of themshowed the reduction of fission zone after 10 to 15 days.

From experiments 6 6 it is obvious that the determination of a fission zone can be revealed either by cutting or by starvation. The complete reduc- tion and the complete or incomplete development of a fission zone may serve as 8. guide to detect the various degrees of differentiation within it which are otherwise non-detectable. Child (1903) and Van Cleave (1929) have reported a similar phenomenon in the reconstitution of Stenostomum. They referred the resorption of the anterior zooid by the posterior one in a chain of zooids to Child's theory of physiological dominance. Working with Stylaria lacustris, Eckert (1927,1930 and 1934) has already demonstrated that in the development of a fission zone two distinct phases can be separated by their reactions toward a lack of food. In the first or '' reversible " phase, starvation or decapitation was followed by an immediate cessation of growth in the fission zone and by its complete resorption within a few days. Similar interferences had, however, no effect on the development of fission zones which were in the second " refractory " phase. Eckert had also mmtioned in his paper that no visible histological distinctions can be found between these two phases. The data of cutting experiments presented in this paper confirm the results obtained by Eckert.

DISCUSSION. As regards the posterior regeneration in annelids, Hyman (1916) reported

that the head of oligochaetes would not regenerate a tail unless a certain number of trunk segments were associated with it. In Clymenella torquata, Sayles (1932, 1934 and 1936) found that the power of posterior regeneration

,

234 J. CHU

dropped progressively as the cutting was made at more and more anterior levels. All these references indicate the presence of gradations in the rate of posterior regeneration in annelids. That the potency of posterior regenera- tion in Stylaria .fossularis increases along the antero-posterior axis has already been shown in the former paper. Axial differences in the rate of anterior regeneration were thereby also described. Additional evidence illustrating the existence of a double regeneration gradient can be obtained from this paper. The fact naturally suggests that these two gradations of regeneration potency are independent of each other. It is very probable that each of them has its own material basis, but what these regeneration substances are is a very suggestive problem that stimulates further studies.

Although axial gradation of various kinds (Hyman, 1916 ; Hyman and Galligher, 1921; Moore and Kellog, 1916; Hyman and Bellamy, 1922; Watanabe, 1931 ; Hatai, 1924; Kopenhaver, 1937, and others) has been found

Figure 4. Figure 5.

Q- b I

I I I I I I I I I

I I I I I I I I I I I I I I I I I I I I

C' d Fig. 4.-Graphic representation of the distribution of anterior and posterior regenerative

Fig. .',.-Graphic representation of fig. 1 , showing the redistribution of regenerative potencies. Explanation in the text.

in many annelids, whether there is any correlation between these gradient activities and double regeneration gradients has not yet been determined.

In order to discuss the data more fully, it is necessary to present the distri- bution of two regeneration potencies by a diagram shown in fig. 4. The area included in the triangle abc denotes the distribution of the anterior regeneration potency, while that of triangle bcd the posterior regeneration potency. The diagonal bc of the rectangle a.bcd then indicates the line of demarcation between these two potencies. If this rectangle was divided transversely into a seriea of many small rectangles, each representing a trunk segment of Stylaria Jossularis, it would be evident that the anterior trunk segments have a rela- tively higher anterior regenerative potency but a lower posterior regenerative potency, and that t h e reverse is true with posterior segments. The middle part of this diagram then includes those trunk segments which are bipoknt for both anterior and posterior regeneration, as has already been shorn by piece regeneration experiments.

potencies in Stylaria fossularis. Explanation in the text.

FISSION AND REOENERATION IN STYLARLA 236

Cutting is generally recognized as a process of physical isolation. As anterior and posterior regeneration were going on in such an isolated piece, there should occup a process of rearrangement of the pre-existing physiological gradients within the regenerate, during which a redistribution of the two regeneration potencies should also take pl~ce. Fig. 5 is a diagrammatic repre- sentation of fig. 1. The broken line bc of the rectangle abul marks the original distributing gradients of the two regeneration potencies in antero-posterior direction in the worm used for cutting. At the time of first cutting, the original condition of potency distribution in a piece consisting of I-VIII trunk segments is indicated by the rectangle a'b'c'dl, with the areas a'c'OP and b'd'OP serving as indexes of its anterior and posterior regeneration potencies respectively. After a certain degree of anterior and posterior regeneration has occurred in this piece, regensration potencies become redistributed into a condition as represented by the rectangle abc"d". A secondary cut a t 4th trunk segment (plane C, in the figure) will lead to a successful and otherwise impossible posterior regeneration, since the potencj for posterior regeneration within a piece consisting of the first four trunk segments and the newly- regenerated head and thorax will be increased by an amount represented by the difference between the areas bwz and byz. This explains why the posterior regeneration at the 4th trunk segment c m be made possible merely through a preliminary allowance for regeneration followed by secondary cutting. On the other hand, if the piece was permitted to undergo its full extent of regenera- tion, secondary cutting at the 4th trunk segment will again lead to the result found in our first set of experiments.

The failure of posterior regeneration of a piece with V-VIII trunk segments, given in our experiment 3, could also be interpreted in the same way. In fig. 6, which is the corresponding diagram to fig. 2, the original distribution system of the worm is shown by the rectangle abcd. When the posterior part of a worm obtained from cutting at t h e 4th trunk segment (plane C,) is allowed to regenerate, and a certain length of time necessary for the redistribution process has elapsed, the new distribution system of regenerative potencies will now be in the form of the rectangle a'b'c'd'. Accordingly, the potency for posterior regeneration of a piece containing the original V-VIII trunk segments will be decreased by an amount shown by the difference between the areas MLWZ and NLYZ. It should be pointed out, on this occasion, that the areas marked by letters have nothing to do with the actual state within the worm. It is not possible to relate them quantitatively to the regenerative potencies. They serve merely to pick out our idea of potency redistribution.

In Stylaria fossularis, like Stylaria lacustris, the development of a fission zone may also be separated into thrw successive stages. In the undetermined stage the reduction of the fission zone can be achieved by cutting or starvation. In the determined stage similar treatment produces no effect. Between these two stages there is a period of transition or the intermediate stage, which in Stylaria lacustris has been estimated by Eckert t o be about 62-77 hour?. The concept of physiological isolation tells us that during natural fission A part of a worm gradually becomes isolated from the apical domirwice of the parent, and finally it separates from the latter so as to form a new individual. Both the results of our experiments and those of Eckert's indicate that such an isolatiofi process should pass through three successive stages which are generally not very differentiable and overlap one another. From our experiments 2 and 3 it is clear that whether regeneration at the cut surface will succeed or not depends wholly upon the magnitude of the regenerative potency distributed in the piece. Supposing the potency present is already more than the minimum amount required to start the regeneration process ; the rate of regeneration becomes a function of the magnitude of regenerative potency. With this idea in mind, it becomes easier t o qccept the view that fission takes place at R segment possessing equal anterior and posterior regenerative potencies, since only in such a condition could those segments before the fission zone reorganize

236 J. CHU

Figure 6.

Grapliic ropresentation of fig. 2, showing the redistribution of regcneialive potencies. Explanation in the text.

Figure 7 .

Y'

X

Graphic representation of the distribution o€ regenerative potencies in a worm having R fission zone, used for explaining the results of OLW experiment 4.

FISSION AND REGENERATION IN STYLARIA 237

themselves into a tail in a velocity strictly comparable to the regeneration rate of those segments behind the fission zone to form a head and thorax.

Consequently, in the undetermined stage of fission zone development the distribution of regenerative potencies in the neighbourhood of the future fission zone remains in the original parent system. As the development reaches the intermediate stage the regenerative potencies gradually redis- tribute themselves both in the anterior and posterior zooids. The act of redistribution ends in the determined or “refractory” stage. Fig, 7 is a diagrammatic representation serving to explain the results obtained from our experiment 4. In the undetermined stage the anterior regeneration potency of a piece cut a t the second segment before the fission zone (along line a in fig. 7) will be represented by the area X W Z . Similarly, the posterior regeneration potency of a piece cut a t the second segment behind the fission zone (along line b in fig. 7) will be represented by the area X’WZ’ . If worms used in our experiment 4 were in this stage, the results mentioned in case (a) could be expected, for a success either in anterior or posterior regeneration will lead to the production of a single perfect worm after the disappearance of the fission zone. However, if the fission zone should have reaohed its third stage of development, a complete redistribution of regenerative potencies within the two newly-developing systems would thus give a decisive effect on the result of regeneration after cutting. In the case of posterior regeneration, the two segments behind the fission zone are now unable to undergo any posterior regeneration, due to the reduction of regenerative potency from an area OW’Z’P’ to F‘Y‘Z‘. But since the fission zone has already acquired the ability to carry out self-differentiation, a well-recognized proboscis and eye spots can still be formed (case b ) . In case of anterior regeneration, the situation is some- what different, €or here two worms can be obtained a s a result of the success of anterior regeneration at the cut surface (case b) . This requires some explana- tion, as from fig. 7, it is evident that it would be impossible for a marked reduction of anterior regeneration potency from an area FO W Z to F YZ to give any degree of anterior regeneration at the cut surface. This apparently con- tradictory result could be interpreted by the extraordinary power of (,he sep ment just before the determined fission zone to develop into A new worm, as in cases we met with during the formation of succeseivs fission zones. This explana- tion will become more clear after we have considered the nature of this chain formation. With the third possibility, the worms used for cutting may be just in the transition stage of fission zone development. The failure of anterior regeneration at the cut surface of the posterior piece is here also due to a gradual reduct ion of regeneration potency from an area PO WZ to F Y Z in the course of redistribution. The two segments before the fission zone therefore fissured down and died away without showing any self-differentiation except wound-healing or out-growth formation. Whether simple wound-healing or the formation of an out-growth would be found to occur depends mainly on the degree of potency redistribution at the time of cutting. When posterior regeneration is considered under this third possibility, the result happens to be the niaintenanoe of the partially developed fission zone followed by its final collapse (case c).

It seems to be difficult to bring the fact of the formation of successive fission zones in Stylaria fossularis into line with the idea of redistribution of regenerative potencies, if fission takes place only at the segment, having equal anterior and posterior regenerative potencies. In €act, our working hypo- thesis serves as well to explain this apparently inexplicable fact. The results given in our experiment 5 have shown that only the youngest one in a chain of fission zones can be artificially reduced by the stimulstion produced by cutting. All the other relatively older fission zones of the same chain continued their further development into a lot of new worms, the number of which exactly corresponds to the total number of fission zones remaining in the chain afrer the disappearance of the youngest one. Furthermore, the experiennce obtained

238 d CHU

from our experiment 4 informs us that the reduction of the fission zone did not occur after cutting if the zone had already reached its third stage of develop- ment. The non-occurrence of t8he phenomenon of reduction in a chain of fission zones then indicates that even the youngest one must have been already invisibly determined at the time of cut,ting. These facts conjointly show that during the development of successive fission zones a new one can be added to the chain only when the youngest one among the pre-existing fission zones has attained its third uevelopmental stsge. In terms of our conception, this means that the addition of a new fission zone to a chain is possible only when the redistribution of regenerative potencies initiated during the formation of the last but youngest fission zone of the chain has been accomplished. A chain of fission zones is therefore merely the manifestation of a series of successive but independent physiological isolation processes which involves at least a redistribution of regenerative potency. Thus, in the formation of the second fission zone, an isolation process occurred in the anterior zooid which had just been physiologically separated from the posterior zooid after the full develop- ment of the first fission zone. Similarly, the formation of the third fission zone took place in the still more anterior part of the worm, and the whole process was not entirely influenced by the presence of the first two fission wnes. Cutting experiments with worms having consecutive fission zones illustrated that the total potentiality necessary for the development of a new worm could be acquired by a single fission zone after its irreversible determination, even t,liough in structure i t consisted of only one or more still not visibly differentiated segments. This highly potent condition of determined fission zone offers the possibility that successive fissions might still bappen at the segment with equal anterior and posterior regeneration potencies. The only difference is t,hat one of the half-system formed after each physiological isolation process is, here, only represented by one or more potential segments. The proof of this reasoning will never be obtained wit,hout an entirely different method of investigation.

SUMMARY,

The existence of double regeneration gradients in Stylaria fossularis iu further confirmed from the results offered by piece regeneration experiments. It is hypothetically suggested that the two regeneration gradients might have their own material basis. During piece regeneration there occurs within the regenerate a redistribution of these regenerative potencies from a generally unbalanced condition at tbe time of cutting to a balancedone, such as is normally found in any mature but non-fission worm. The act of redistribution is com- pleted when the head, thorax and tail of a regenerate has fully grown. In natural fission a similar phenomenon of potency redistribution is likely to take place in both anterior and posterior zooids, and the redistribution proces8 is accomplished after the fission zone has been irreversibly determined. Natural fission in Stylaria fossularis also proceeds in three successive stages, in the first of which the fission zone is rather unstable, as the stimulation of cutting is sufficient to induce its disappearance. From the analysis of the results of cutting experiments 011 worms with an incipient fission zone or a chain of fission zones, the author is now inclined to believe that the suggestion that fission ta.kes place at the segment possessing equal anterior and posterior regenerativ- potencies appears to be more adequate to explain the various experimental facts. As the evidence presented in this paper is not at all con- clusive, a final decision between them will he reserved for the future. -

LITERATURE CITED.

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A genealogical analysis of natural fission in Stylnria fos,w/nris. SOC., Lonrl. 115, 194.

FISSION AND REGENERATION IN STYLARIA 239

CHU, J., & PAI, S. (1945). The relations between natural fission and regeneration in Stylaria .fossularis. Physiol. Zool., in press.

ECKERT, 1”. (1927). Experimentelle Untersuchungen uber die Teilungszono von Stylaria lacustris L.

ECKERT, F . (1930). Untersuchungen uber die Teilungszone von Stylaria lncwtris L.- I. Die regulatorische Resorption der Zone.

F ~ K E R T , P. (1934). Untersuchungen uber die Teilungszone von Stylaria lacustris L.-II. Der Einfluss der Nahrungsaufnahme auf die Geschwindigkeit den Zonenwachstums. Zool. J b . 54, 89-118.

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HYMAN, L. H., & GALLIOHER, A. E. (1921). Direct demonstration of the existence o f a metabolic gradient in annelids.

KOPENHAVER, M. E. (1937). Axial differences in water absorbing properties of oligo- chaete tissue.

MOORE, A. R., & KELLOG, E. M. (1916). Note on the galvanotropic response of the earthworm. Bwl. Bull. 30, 131.

SAYLES, L. P. (1932). External feature of regeneration in Clynleneh torquala. J. exp. Zool. 62, 237.

SAYLYS, L. P. (1934). Regeneration in the polychaete Clymenella torquata.-II. Effect of level of cut on type of new structures in posterior regeneration.

SAYLYS, L. P. (1936). Regeneration in the polychaete Clynaenella torquata.-III. Effect of level of cut on type of new structures in anterior regeneration. Biol. Bull. 70, 441.

VAN CLEAVE, C. D. (1929). An experimental study of fission and reconstitution ill

Stenostomum. Physiol. 2001. 2, 18. WATANABE, Y. (1931). On the physiological gradients of chaetopod annelids.-11.

Axial gradients of oxidizable substance in earthworm as determined by the Manoilow reaction.

Z . .wiss. 2001. 129, 589-642.

2001. 56. 47, 29-88.

HATAI, S. (1924).

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d . exp. 2001. 20, 99-163.

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