THE UNIVERSITY OF ILLINOIS LIBRARY NATURAL HISTORY SURVEY 5705 ILL v3cop.4 fU, %> %> Y2 and % of the tail. Four of these levels, 1 / 10 , %, ^ and %, had at least five individuals each for each regeneration. The other two levels, 1 / 18 and %, had less than five individuals per regeneration but are included in the tables though their averages are not as reliable as those of the others. The method as described agrees in principle with that pursued in Experiment I. It has a decided advantage over a direct comparison within a single individual because it eliminates the age factor as well as the effects of change in external conditions such as temperature and food. Data The results of the experiment are given in Tables 21 to 30 an<^ in Figures 2 and 3. The data show on the whole a tendency for the second regeneration to remain in advance of the first for eight or ten days after the operation. The first regeneration then catches up and even slightly surpasses the other; this is apparent both when the regenerated lengths are taken directly and when they are corrected for difference in level of the cut and put in terms of specific regenerated length or the length regenerated per unit of removed length. In making the comparisons certain general features must be borne in mind. The maximum rate of regeneration is reached on or near the seventh day, earlier for the smaller removals and later for the larger removals. The whole regeneration, in so far as it is completed, is finished in nearly all cases at 12^ days, again somewhat earlier for the smaller and somewhat later for the larger removals. In the tadpoles used in the present experiment about four-tenths in length of the re- moved tail is replaced before regeneration stops. This was found to be 33] RATE OF REGENERATION— ZELENY 33 generally true of tadpoles of this size in Rana clamitans. The percent regenerated is somewhat greater for the smallest removals than for the others. After the maximum is reached there is a tendency toward de- crease of the regenerated region though this is hard to determine with accuracy because the boundary between old and new tissue becomes more and more obscure as time goes on. For this reason the data for 56 days of regeneration are not as reliable as the others. mm. .400 to 8 o s "3 i .300 .200 .100 4 6 8 10 12y 2 18 — >■ Days Figure 2. Specific regenerated lengths during the regenerative period for both first and second regenerations. Tadpole tail of Rana clamitans. Series 3676-3765. Broken line = second regeneration. Unbroken line = first regeneration. '3 B 03 n * 9 a a * I o .2 § CO t., oi d M S, si 1 8 •*-> 01 ca w (h 0) T3 e m bo ■d to [S ■g * s* +j 01 w o s a td o> 1=1 S O H A 3 o o 5 J •o IS § E N co CO tH rH O O + O 4- o . 1 o 1 o + O 1 + + a c o t> o 60 C/3 ^ £ 60 to g rH © . + t- © © + rH © + r-i © © + co © 00 © © 1 1-j 1 1 cm © © © 1 © o 1 C4 © + ** r © © 1 rH © + 00 © © + +0.1 0.00 1 © 1 1 CO © © 1 © © i o © © co © 1 CM © © 1 o + © ©' 1 o CO 00 o CO 00 © © © o t4 rH © CO »-H f © o © o © © © © © © © © + + + 1 1 ] 1 1 I + + r^ o •"* CM rH CO © © rH © y-i © t- cq r © © © © © © © © O © © © © | + + 1 + + 1 1 + + i + + 1 + + J rH rH CO cm ' ■r-i CO 00 © o © © y-i © t- © © + © + o + © + © 1 © 1 © 1 o © + © + © + © 1 rH CM © CO 00 © CO CO © © © © rH © © © © © J 1 © 1 © + o i + © 1 © © © © © © © 1 a 1 s a a A a -i-> a A I a bfi d a OX) d a bo d a bJD a bO d a b« p d 9 d d cu d o> d 0> A o td A o td o td ,d o td J3 o td o td bfi o bo o bO o bO o bO o bO o d d 0> d cu o> Pi Ol n B P. 9 P< 0) Pi o> o< ►J m A w J OQ J m rJ 02 1 hJ W 111 5 ! x - 8.2 TJ o«l a rt 1st 3rd regen. = 1st 3rd regen. < 1st 2nd regen. > 1st 2nd regen. = 1st 2nd regen. < 1st 3rd regen. > 2nd 3rd regen. = 2nd 3rd regen. < 2nd Two Four Six Bight Ten Fourteen days days days days days days 2 3 3 4 5 3 1 1 1 1 1 2 2 3 4 3 3 1 1 1 1 1 2 1 1 2 1 2 3 3 1 1 2 1 1 2 1 3 Totals 20 1 4 17 3 5 12 4 8 Experiment IV Amblystoma punctatum Series 3962-3999 The series used for Experiment III furnishes another set of data for the effect of successive removal. When the third operation was made the removed regenerated tails of the first thirty individuals represented an eleven-day second regeneration and those of the second thirty-indi- viduals an eleven-day first regeneration. A direct comparison is thus possible between the first and the second regenerations. It is not possi- ble to make a cut exactly at the border line between old and new tissue and therefore the measurement of the removed regenerating tail is not as accurate a determination as is the direct measurement of a regener- ating unremoved tail. The data are shown in Table 33. Twenty-five individuals are avail- able for each regeneration. The average of the first regenerations is 4.55 ^.11 and of the second regenerations 4.50 ± 0.10. The first regen- eration is ahead of the second in ten cases, the second is ahead of the first in twelve cases and three cases are equal. The first comparison shows a slight difference in favor of the first regeneration but this is so much less than the probable error that it can not be considered as significant. The second comparison shows a slight advantage in favor of the second regeneration. On the whole the data indicate essential equality between the first and the second regenerations at eleven days. 50 ILLINOIS BIOLOGICAL MONOGRAPHS [50 TABLE 33 Amblystoma punctatum Series 3962-3999 Age factor eliminated Comparison of first and second regenerations Eleven days First Second First Second First Catalog regen. regen. ahead ahead and second number mm. mm. of second of first equal 3967 4.0 4.4 0.4 3968 3.7 3.5 0.2 3969 4.9 5.1 0.2 3970 4.5 4.7 0.2 3972 4.7 4.7 * 3973 3.9 4.3 0.4 3975 4.5 5.7 1.2 3976 4.9 4.9 * 3977 3.8 3.7 0.1 3978 4.1 4.5 0.4 3980 4.9 5.0 0.1 3981 3.5 4.4 1.1 3982 5.0 4.7 0.3 3984 5.1 4.0 1.1 3985 5.8 4.3 1.5 3986 3.8 4.1 0.3 3989 4.8 5.5 0.7 3990 •5.5 4.3 1.2 3991 4.1 4.6 0.5 3992 4.6 4.1 0.5 3993 4.5 4.5 * 3994 4.9 5.0 0.1 3995 4.5 4.0 0.5 3997 5.1 4.2 0.9 3998 4.8 4.3 0.5 4.55±0.11 4.50±0.10 ten times twelve times three times Experiment V Amblystoma punctatum Series 6042-6100F This series was devised for a study of the effect of repeated removal of the tail upon the rate of metamorphosis. The removed tails were preserved and they give some data on the comparison of successive regenerations. The interest of the results lies in the fact that the suc- cessive regenerations are compared within single individuals. Thus the effect of the age factor is not eliminated. Environmental differences such as those of temperature may also be factors. The eggs were hatched on March 25 to 29, 1915. Approxi- mately one-half in length of the tail was removed in each of the indi- 51] RATE OF REGENERATION— ZELENY 51 viduals on April 5. The new tissue was removed on April 17 and again on May 1, May 10 and May 19, making five removals in all. The second removal gives the first regeneration, the third the second, and so on. The regenerated lengths were therefore determined by measurement of removed parts. This does not give as accurate a deter- mination as does direct measurement without removal because the cut can not in ordinary practice be made exactly at the border line between old and new tissue. The data are given in Table 34. The first regeneration covers a twelve-day period, the second fourteen days and the third and fourth each nine days. The third and fourth regenerations are the only ones that have the same time interval. Ten individuals are available for this comparison. The average for the third regeneration for these ten is 1.30 mm. and of the fourth regeneration 1.17 mm. When all individuals are taken without regard to representation of both regenerations the average for the third regeneration is 1.28 and for the fourth 1.17. In seven of the ten former cases the third is ahead of the fourth regeneration, in two they are tied and in one the fourth is ahead of the third. The data therefore show an advantage of the third over the fourth regeneration. The first regeneration ran twelve days and the second fourteen days. The maximum rate of regeneration comes on or near the ninth day and the rate has declined to a low point by the fourteenth day. However it is not possible to make the necessary correction because of lack of data on the rate curve for this particular set of larvae. Some facts may however be obtained by a comparison. Sixteen individuals for each of the two regenerations are available for comparison. The average for the first regeneration in these is 2.06 mm. and for the sec- ond 2.01 mm. In seven the first is ahead of the second, in seven the second is ahead of the first, and two are tied. When all individuals are taken without regard to representation of both regenerations the average for the first regeneration is 1.99 ± 0.03 mm. for a twelve-day period and for the second regeneration 2.01 for a fourteen-day period. The difference between the two values is not significant, but when the longer time interval taken by the second regeneration is considered the conclusion is reached that the first regeneration is more rapid than the second. The data thus indicate a progressive decrease in rate from the first to the fourth regenerations. This result taken in connection with the results obtained from the experiments in which the age factor is eliminated makes it highly probable that the decrease in rate of regen- eration observed here is due to increase in age and not to the effect of successive removal. 52 ILLINOIS BIOLOGICAL MONOGRAPHS [52 TABLE 34 Amblystoma punctatum Series 6042-6100 F Age factor eliminated Successive regenerations in single individuals First Second Third Fourth regeneration regeneration regeneration regeneration Catalog mm. mm. mm. mm. number Twelve Fourteen Nine Nine days days days days 6042 2.0 2.4 1.6 6043 1.8 2.5 1.5 1.4 6044 2.2 6046 2.2 2.2 1.3 1.3 6047 2.2 2.1 1.4 1.3 6048 2.0 2.2 6049 2.3 1.8 1.5 1.0 6050 1.8 6052 2.0 6053 1.9 6055 1.9 6056 1.7 6057 2.0 6058 2.0 6059 1.9 6061 1.5 6062 2.3 6065 1.5 6067 1.6 6068 1.6 6071 2.0 6072 1.9 . 6076 2.1 6077 2.0 6079 2.0 6080 1.8 6081 1.9 6082 1.9 2.0 1.0 1.1 6083 2.0 2.1 1.3 1.1 6084 1.9 6085 2.0 2.0 1.5 1.5 6086 2.2 6087 1.8 1.0 0.8 6088 2.1 2.4 1.4 1.2 6090 2.5 6093 2.3* 2.2 1.0 0.8 6094 2.1 53] RATE OF REGENERATION— ZELENY TABLE 34 (Continued) 53 First Second Third Fourth regeneration regeneration regeneration regeneration Catalog mm. mm. mm. mm. number Twelve Fourteen Nine Nine days days days days 6096 2.1 6097 2.5 1.6 6098 1.8 2.1 6099 2.2 ' 6100D 2.0 6100E 1.8 1.6 6100F 2.2 2.0 1.1 1.0 Average 1.99 ±0.03 2.01 1.28 1.17 Rate per day 0.166 0.144 0.142 0.130 Experiment VI Bufo americanus Series 6283-6323 This series was designed for the study of the effect of successive removal of the tail upon the rate of metamorphosis. The lengths of the removed regenerating tails however are of some value in a com- parison of successive regenerations though here as in Experiment V age and external factors are not eliminated. The eggs were laid on April 20-21, 1915. The tadpoles were col- lected on April 27 and the first removals were made on April 28. The first metamorphosis was completed on June 11. The average total length at the time of the first removal was 10.9 mm. and the average tail length 6.4 mm. The average removed length was 3.8 mm., which is approximately 60 per cent of the tail length. The second removal was made on May 7 and gives a nine-day period for the first regeneration. The third removal of May 17 gives a ten-day period for the second regeneration. The fourth removal on May 26 gives a nine-day period for the third regeneration. As in the case of Experi- ment V the cuts could not in practice be made to come exactly at the border line between old and new tissue and the accuracy of the meas- urements is therefore not as great as in those cases in which the lengths were taken directly from the animal without removal of the tail. The data are shown in Table 35. The first, second and third re- generation lengths are given for sixty individuals. The first and third regenerations have the same time interval and are therefore directly comparable. The average for the first regeneration is 1.94 ^0.02 mm. 54 ILLINOIS BIOLOGICAL MONOGRAPHS [54 and for the third 1.80 ^0.03 mm., a difference in favor of the first re- generation of 0.14 ± 0.05 mm. This represents a regeneration of 0.51 mm. per unit of removed length in the first regeneration and 0.47 mm. per unit in the third regeneration. A comparison of individual cases shows that the first regeneration is ahead of the third in 36 individuals, the third is ahead of the first in 18 individuals and 5 are tied. The differ- ence between the two regenerations is thus probably significant. As in Experiment V the decrease is probably due to the age factor. The second regeneration has a time interval of ten days, one day more than the first and third regenerations. In the absence of know- ledge concerning the rate curve for toad tadpoles of this age no cor- rection can be applied. The rates per day for the three regenerations are however given in the table. TABLE 35 Bufo americanus Series 6283-6323 Age factor not eliminated Successive regenerations of tail First Second Third Catalog regeneration regeneration regeneration number Nine days Ten days Nine days Length in mm. Length in mm. Length in mm. 6283 a 2.1 2.0 1.9 b 1.5 2.1 1.6 c 1.7 2.1 1.7 6285 a 2.3 1.9 2.0 b 2.3 2.1 2.0 c 2.0 2.4 2.4 6287 a 1.9 2.1 2.1 b 2.1 2.0 1.9 c 1.8 2.3 1.8 6289 a 1.9 2.3 2.2 b 2.1 2.0 1.7 c 2.2 2.1 2.0 6291 a 2.1 2.2 2.0 b 1.9 1.9 1.8 c 2.0 1.9 1.4 6295 a 2.0 Z.O 2.U b 2.1 2.1 1.8 c 1.8 1.9 1.9 6297 a 1.9 2.0 2.0 b 1.9 2.1 1.8 c 1.9 2.0 1.8 6299 a 1.8 2.0 1.5 b 2.0 2.0 1.7 55] RATE OF REGENERATION— ZELENY TABLE 35 (Continued) 55 First Second Third Catalog number regeneration Nine days Length in mm. regeneration Ten days Length in mm. regeneration Nine days Length in mm. c 1.8 1.9 1.3 6301 a 2.0 1.5 1.4 b 1.8 1.9 1.2 c 1.7 1.8 1.4 6303 a 1.9 1.9 1.3 b 1.9 1.9 1.5 c 1.8 2.0 1.6 6305 a 1.9 2.0 1.5 b 1.7 1.8 2.0 c 2.1 2.2 1.9 6307 a 2.1 1.8 2.0 b 1.9 2.0 2.1 c 1.8 1.9 1.8 6309 a 2.0 1.9 1.9 b 1.9 1.9 2.0 c 2.0 2.0 1.7 6311 a 1.7 2.4 1.8 b. 1.8 1.9 2.1 c 2.0 2.1 2.0 6313 a 2.0 2.3 1.7 b 1.8 1.7 2.0 c 1.7 1.7 1.5 6315 'a 1.9 2.4 2.0 b 2.0 2.1 1.6 c 1.9 1.9 6317 a 1.9 2.0 1.3 b 1.8 2.2 2.0 c 1.9 2.1 1.8 6319 a 2.2 2.0 2.0 b 2.1 2.0 2.0 c 1.9 1.9 2.0 6321 a • 2.3 2.0 1.8 b 1.7 2.0 1.9 c 2.0 2.3 1.3 6323 a 2.1 2.1 1.7 b 1.8 2.3 2.0 c 1.9 2.2 2.0 Average 1.94±0.02 2.02±0.02 1.80 ±0.03 Rate per day 0.216 0.202 0.200 56 ILLINOIS BIOLOGICAL MONOGRAPHS [56 Experiment" VII Rana clamitans Series 3557-3624 This experiment deals with the relative completeness of regenera- tion after successive removals within single individuals. Age and ex- ternal factors are not eliminated. A more complete description of the experiment is given under "Completeness of Regeneration." The tail length averaged approximately 17.0 mm. About one-half of the tail was removed at the first operation. At succeeding operations the cut came as near as possible to the border line between old and new tissue. The first removals came on October 23, 1911, the second on November 28, the third January 3, the fourth February 9, the fifth March 16 and the sixth April 4. At the time of the last removal the hind legs were just starting to grow. The data are given in Table 36. The first regeneration interval is 36 days, the second 36, the third 37, the fourth 36 and the fifth 39 days. Each one of these is more than sufficient for the completion of the regenerative process. The individuals are divided into three sets, A, B, and C. A, with seven individuals, has no record for the first regen- eration; the second regeneration is 9.8 mm., the third 9.3, the fourth 8.5 and the fifth 8.6. B, also with seven individuals, has no record for the first regeneration; the second is 9.1 mm., the third 8.9, the fourth 7.2 and the fifth 7.8. C, with nineteen individuals, has a first regen- eration average of 8.6 mm., a second of 8.0, a third of 7.5, a fourth of 5.5 and a fifth of 6.4. In all the cases there is a decrease in the amount regenerated with successive removal except for the fifth regeneration, which has in each case an increase over the fourth. It is probable that TABLE 36 Rana clamitans Series 3564-3624 Age factor not eliminated Completed successive regenerations compared Set Catalog number Number of individ- uals First regenera- tion 36 Days Second regenera- tion 36 Days Third regenera- tion 37 Days Fourth regenera- tion 36 Days Fifth regenera- tion 39 Days A 3564 to 3570 7 •• 9.8 9.3 8.5 8.6 B 3578 to 3584 7 •■ 9.1 8.9 7.2 7.8 C 3586 to 3624 19 8.6 8.0 7.5 5.5 6.4 57] RATE OF REGEXERATION—ZELENY 57 the decrease is due to increase in age. The increase from the fourth tG the fifth regeneration may be due to some special characteristic of the stage immediately preceding metamorphosis or it may merely indicate the existence of some uncontrolled external factor such as food or temperature. Discussion The evidence shows clearly that when the age factor is eliminated there is no decrease in rate of regeneration with successive removal. On the contrary the second regeneration is more rapid than the first up to the period of maximum rate. The second regeneration however passes its maximum sooner than does the first and after the tenth day the latter therefore catches up to the former in total amount regener- ated. There is no striking difference between the second and the third regenerations but in each comparison the third has a slight advantage. When the successive regenerations in single individuals are com- pared, the rate decreases with successive removal. This decrease is undoubtedly due to the age factor. The possibility has suggested itself that the second regeneration starts out at a more rapid rate than the first because the cells at the cut surface were undergoing regenerative changes at the time of the new operation and can therefore start the process much faster than can the old cells at the first surface of regeneration. Following a first removal there is a considerable degree of reorganization of the cells at the cut surface, accompanied by active migration. During this period, which in Bana clamitans lasts two or three days, there is little or no mitotic cell division. Then follows a division period which reaches its maximum at seven to ten days. Its decline is associated with the oncoming of tissue differentiation (Sutherland 1915, Metcalf 1915). A special study has been made of the relative rates of second regenerations from old cells following a cut inside of the first removal level and from new cells following a cut outside of the first level. This comparison shows only a very slight difference in favor of the new cells and this is largely confined to the early stages, the period of cell migration. The period of increase in rate is the period of active cell multipli- cation and the decline in rate is associated with cell differentiation. The second regeneration therefore reaches the period of differentiation slightly in advance of the first regeneration. Apart from the slowing due to age there is no indication of a limitation of the amount of new material that may be produced by regeneration. The actual limitation comes not from the using up of 58 ILLINOIS BIOLOGICAL MONOGRAPHS [58 regenerative or developmental energy or of determiners by repeated regeneration but from changes in the non-regenerating part associated with age. In another place there is a discussion of the possibility that there may be an effect upon the rate of developmental processes in the organism as a whole due to continued regeneration of a part. This is studied particularly in connection with the effect of regeneration upon rate of metamorphosis in Amphibia. Regeneration studies in general and those on successive regener- ation in particular make it improbable that there is a definite number of cell generations between the fertilized egg and the end product, the differentiated cells. The possibility that certain cells may remain in an early cell generation can not be wholly excluded as an explanation of at least a part of first regeneration phenomena. Under suitable stimu- lation such cells may be postulated to take up development where it had left off. The definite descriptions of de-differentiations of cells as well as other facts of regeneration argue against this conclusion. The view that there can be no such definite number of cell generations is strengthened by the facts of successive regeneration. It does not seem probable that embryonic cells of an early cell generation can be held in reserve through repeated regenerations. The explanation of regeneration by the theory of duplicate sets of determiners meets difficulties in undiminished successive regenerations. The greater the number of repeated regenerations the greater the diffi- culties of explanation on this basis. Of course the difficulty does not hold for the hypothesis that every cell or nearly every cell contains a full set of determiners. The earlier appearance of the maximum rate in the second than in the first regeneration may be due to the more rapid progress of the cells in the early cell migration period alone or it may be due to the acceleration of the whole developmental cycle. Summary 1. The age factor was eliminated in Experiments I to IV. Ex- periments I and II deal with tadpoles of Rana clamitans and Experi- ments III and IV with larvae of Amblystoma punctatum. 2. In Experiment I approximately one-half of the tail was re- moved. At six days the average first regeneration length is 2.01 mm. and the average second regeneration length 2.18 mm. In five cases the first exceeds the second and in six the second exceeds the first. The corresponding specific lengths are 0.194 and 0.205. The first regen- eration exceeds the second in two sets, the second exceeds the first in eight and one is tied. The second regeneration has the advantage in all the comparisons. 59] RATE OF REGENERATION— ZELENY 59 3. At eight days in Experiment I the average first regeneration length is 3.06 mm., and the second 3.42 mm. The first exceeds the sec- ond in three sets and the second exceeds the first in seven. The corre- sponding average specific lengths are 0.298 and 0.323. In four sets the first regeneration exceeds the second and in six the second exceeds the first. The second regeneration has the advantage in all the com- parisons. 4. The advantage of the second regeneration over the first in Experiment I holds true of second regenerations from both old tissue and new tissue levels. 5. In Experiment II observations were made at the 1/10, 1/3, 1/2 and 2/3 levels in a sufficient number of individuals to yield valid data. ^Regeneration measurements were made at each of these levels 4, 6, 8, 10, 12^, 18 and 56 days after the operations. The second regen- eration at all of them tends to be ahead of the first until the tenth day, after which the first regeneration catches up. The maximum rate for both regenerations is reached before this time and earlier for the second than for the first regeneration. 6. In Experiment III two-thirds of the tail was removed. A comparison of the first, second and third regenerations was made at 2, 4, 6, 8, 10 and 14 days.. At two days the first, second and third regenerations average respectively 0.22, 0.25 and 0.26 mm. The cor- responding values at four days are 0.66, 0.75 and 1.00; at six days 1.36, 1.40 and 1.46; at eight days 2.18, 2.68 and 2.68; at ten days 3.55, 3.82 and 4.20; at fourteen days 5.34, 6.12 and 6.08. The advantage is in favor of the second and third regenerations as opposed to the first and of the third as opposed to the second. Individual comparisons at each of the different times as well as in the experiment as a whole show the same results. 7. The removed tails in the preliminary procedure of Experi- ment III furnish the data of Experiment IV and allow a comparison of the first and second regenerations at eleven days. The procedure is however subject to greater error than that of Experiments I to III. Twenty-five individuals for each regeneration give an average of 4.55 ± 0.11 mm. for the first regeneration and 4.50 4 0.10 mm. for the second regeneration. The first regeneration is ahead of the second in ten cases, the second ahead of the first in twelve cases and three are equal. The two regenerations must be considered as essentially equal. 8. In Experiments V and VI the age factor is not eliminated. Successive regenerations in single individuals are compared. In Ex- periment V one-half of the tail in Amblystoma larvae was removed. In Experiment VI 60 per cent of the tail of toad tadpoles was removed. 60 ILLINOIS BIOLOGICAL MONOGRAPHS [60 The time intervals vary somewhat in each set but it is evident in both cases that there is a decrease in rate of regeneration from the first to the third and fourth regenerations. This decrease is undoubtedly due to increase in age and not to successive removal. 9. In Experiment VII a comparison of the completeness of re- generation in single individuals of Rana clamitans shows a progressive decrease in amount regenerated from the first to the fourth regener- ation and an increase from the fourth to the fifth. In this experiment also the age factor is not eliminated and the decrease is probably due to increase in age. 61] RATE OF REGENERATION— ZELENY 61 PART III THE EFFECT OF LEVEL OF THE CUT UPON THE RATE AND COMPLETENESS OF REGENERATION The present study gives a description of some experiments made to define more accurately than has been done the exact relation between the level of the cut and rate of regeneration and especially the relation of this factor to the other factors affecting rate and completeness of regeneration. The factor is one of great interest because if it is true that the ratio between length regenerated per unit time and length removed is a constant it follows that no matter how much material is removed regeneration is always completed in the same time. It is therefore of great interest to determine the extent to which this statement is true, to analyze the elements of the level factor and to determine its relation to other factors. Experiment I Rana clamitans Series 3676-3765 The tadpoles were collected on December 9, 1911, and first removals were made in two-thirds of the individuals on December 22. A second removal was made in these individuals on January 8, and at the same time a first removal in the other one-third. Measurements were made four, six, eight, ten, twelve and a half, eighteen and fifty-six days after the operations of January 8. The first and second regenerations are treated separately and the second regenerations are taken up first because they have a larger number of individuals and therefore give the more uniform results. Second Regenerations The different amounts removed approximate 6, 10, 18, 31, 49 and 67 per cent of the tail length. There are four individuals at the lowest removal, averaging 1.5 mm., seven at the next, averaging 2.8 mm., five at the third with an average of 4.9 mm., ten at the fourth with 8.4 mm., eight at the fifth with 13.1 mm., and ten at the sixth with 18.1 mm. The data are given in tables 37 to 40 and in graphic form in figures 4 to 17. The regenerated lengths at ten days will be taken up first because at this time the period of maximum rate has been passed and its full effect is represented. Differentiation of the tissues has begun but there 62 ILLINOIS BIOLOGICAL MONOGRAPHS [62 is still a considerable production of new cells by mitotic division except in the individuals with the two shortest removals in which the process is completed. The regenerated lengths for the six levels beginning with the shortest removal are respectively 1.0, 1.3, 1.4, 2.3, 3.7 and 5.1 mm. The data are given in the last two columns of table 37. There is very dis- tinctly an increase in regenerated length with increase in removed length. Dividing the regenerated length by the removed length at each level, the fractions obtained are 1.0 1.3 1.4 2.3 3.7 5.1 » . . , , and , 1.5 2.8 4.9 8.4 13.1 18.1 which give the specific regenerated lengths or lengths regenerated per unit of removed lengths. These values are 0.67, 0.46, 0.29, 0.28, 0.28 and 0.28. They show a remarkable constancy for removed lengths of 4.9 mm. and over. The relations between removed lengths and regenerated lengths are further shown in figure 4 which gives the removed lengths along the horizontal axis and the regenerated lengths parallel to the vertical axis. The plotted line of correlation between the two values is straight except for the two lowest removed lengths. The specific lengths are given in Figure 5 in which the removal lengths again are along the horizontal axis and the lengths regenerated per unit of removed length parallel to the vertical axis. The line of correlation is straight and parallel to the horizontal axis for the four highest removals. For these therefore the regenerated length is directly proportional to the removed length or in other words within these limits the same percent- age of the removed length is regenerated in each within the given time of ten days. The two lowest removed lengths give a higher specific rate than the others. They regenerate a higher percentage of the removed length within the given time. The ten day period is chosen as the first example because it is the first one to receive the full benefit of the periods of maximum rate of regeneration, the periods during which rapid multiplication of cells takes place. The other periods give results which agree in general features after the first few days with those at ten days but depart from them in certain respects. The remaining periods will now be taken up in turn beginning with the shortest. During the first four days after the operation the rate of regenera- tion is slow, the new tissue being derived largely from migration of cells over the cut surface. Measurements of regeneration at this time are especially subject to error because of the small amount regenerated 63] RATE OF REGENERATION— ZELENY 63 and because of irregularity in the outer edge of the regenerating tissue. The regenerated lengths at four days are respectively 0.22, 0.39, 0.24, 0.42, 0.50 and 0.52 mm. These data are given in table 37 and are rep- mm 5.0 -d 40 O) +j a u o> 3.0 O) M) 4> t* m 2.0 X) tt c 1.0 1.5 2.8 4.9 8.4 13.1 — >■ Lengths removed in mm. Figure 4 Rana clamitans Second regenerations Ten days 18.1 bo a - o ■ Lengths removed in mm. Rana clamitans Second regenerations Specific lengths 18.1 Ten days resented graphically in figure 6. Dividing the regenerated lengths by the removed lengths the fractions obtained are 0.22 0.39 0.24 0.42 0.50 0.52 L50 2~!80 4~!90 8A~6 1310 1810 giving specific lengths of 0.15, 0.14, 0.05, 0.05, 0.04 and 0.03. These relations are represented graphically in figure 7. There is on the whole a slight increase in regenerated length with increase in removed length but this increase is not proportional to the amount removed so that the proportion regenerated decreases with increase in removed length. The 64 ILLINOIS BIOLOGICAL MONOGRAPHS [64 approach to equality in regeneration at this time is probably due to the fact that the new tissue is largely made up of migrating cells and there is not a striking difference in the extent of the migration at the different levels. The specific length of material regenerated after the smallest removals is greater than that regenerated after the larger removals not only at four days but also later. It is probable that the factors involved during the first few days of regeneration are quite different from those during later days. Following the injury there is a disintegration of injured cells associated with an active migration of the epidermal cells 00 a a> 1.0 Figure 6 1.5 2.8 4.9 8.4 13.1 — ► Lengths removed in mm. Rana clamitans Second regenerations Four days 18.1 "8 bfi a A3 8 0.20 0.10 13.1 18.1 o, Figure 7 CO 1.5 2.8 4.9 8.4 — >- Lengths removed in mm. Rana clamitans Second regenerations Specific lengths Four days over the cut surface. There is practically no mitotic cell division. The rapid multiplication of cells comes later. These processes of cell migra- tion apparently are not essentially different at the different levels. They are local responses of the cells at the cut surface. With the appearance of rapid cell multiplication there is a marked difference at different levels though the shortest removals still show a greater specific length than the others probably because in their ease the migrated cells make up a large percent of the total material of the new part. Between the end of the fourth and the end of the sixth day after the operation mitotic cell division becomes very rapid and the rate of regeneration for second regenerations reaches its maximum at a majority of the levels on the sixth day. At six days the regeneration for the six levels is respectively 0.62, 0.80, 0.70, 1.1, 1.7 and 2.3 mm., as shown in 65] RATE OF REGENERATION— ZELENY 65 Table 37. A graphic representation is given in Figure 8. There is a grad- ual increase with increase in removed length. The fractions obtained by dividing by the removed lengths are : 0.62 0.80 0.70 1.1 1.7 2.3 , , , , , and 1.5 2.8 4.9 8.4 13.1 18.1 They give specific lengths of 0.42, 0.30, 0.14, 0.13, 0.13 and 0.13. The smaller removals still have the larger specific lengths but with removals of 4.9 mm. and more there is an approach to constancy. The relations are shown graphically in Figure 9. Tj mm. to a ■ Lengths removed in mm. Rana clamitans Second regenerations 13.1 18.1 Specific lengths Six days The rate of regeneration between the sixth and the eighth day for second regenerations is not quite as high as for the preceding period, but mitotic divisions are still very numerous and differentiation of the cells is just beginning. At eight days the regenerated lengths are respectively 0.9, 1.1, 1.2, 1.8, 2.7 and 3.7 mm. as shown in table 37. The increase in regeneration with increase in removed length is evident. The relations are shown in Figure 10. Dividing by the removed lengths the fractions obtained are 0.9 1.1 1.2 1.8 2.7 3.7 1.5 2.8 4.9 8.4 13.1 and 18.1 66 ILLINOIS BIOLOGICAL MONOGRAPHS [66 giving the specific regenerations 0.60, 0.39, 0.24, 0.22, 0.21, 0.20. There is a graphic representation in Figure 11. As before, the two shortest removals give the highest specific rates but beyond these there is an approach to constancy though there is still a slight decrease with increase in removal. The ten day values have already been given. Between ten and twelve and a half days after the operation there is no further growth in the case of the two shortest removals. In the two medium removals the process is completed at twelve and a half days. In the two longest removals there is still a small amount of mm. 4.0 3.0 2.0 to 10 c e Figure 10 1.5 2.8 4.9 8.4 13.1 — '—>■ Lengths removed in mm. Rana clamitans Second regenerations Eight days 18.1 - 0.60 0.50 0.40 0.30 0.20 0.10 Figure 11 13.1 18.1 1.5 2.8 4.9 8.4 — >■ Lengths removed in mm. Rana clamitans Second regenerations Specific lengths Eight days proliferation after this time. At twelve and a half days the regenerated lengths are 1.0, 1.3, 1.6, 2.6, 4.4 and 6.2 mm. as shown in Table 38. The increase with increase in removed length is continuous. This is shown 67] RATE OF REGENERATION— ZELENY 67 Dividing by the removed lengths the in graphic form in figure 12. fractions obtained are 1.0 1.3 1.6 2.6 4.4 6.2 » , , , and 1.5 2.8 4.9 8.4 13.1 18.1 giving specific lengths of 0.67, 0.46, 0.33, 0.31, 0.34 and 0.34. graph for specific lengths is shown in figure 13. The There is still a fair § mm. 6.0 5.0 4.0 3.0 2.0 1.0 Figure 12 - mm. I 0.70 fe 0.60 | 0.50 C 0.40 | 0.30 bo S 0.20 B ~ 0.10 Figure 13 1.5 2.8 4.9 8.4 — >- Lengths removed in mm. Rana clamitans Second regenerations 13.1 18.1 Twelve and a half days 1.5 2.8 4.9 8.4 — y Lengths removed In mm. Rana clamitans Second regenerations 13.1 18.1 Specific lengths Twelve and a half days approach to constancy with removals of 4.9 mm. and above. The rela- tive increase in the case of the higher removals is due to the fact that regeneration is continuing in them after it has stopped in the others. 68 ILLINOIS BIOLOGICAL MONOGRAPHS [68 Therefore the data after this time are values for the completeness of regeneration rather than for the rate. Between twelve and a half and eighteen days after the operation there is no further regeneration in the tails with the four shortest re- movals. Two of them even exhibit a decrease in size. The two longest removals show only a slight increase. At eighteen days the regenerated lengths are respectively 1.0, 1.2, 1.5, 2.6, 4.8 and 7.0 mm. as given in Table 38. The same data are represented in graphic form in Figure 14. Dividing by the removed lengths the fractions obtained are 1.0 1.2 1.5 2.6 4.8 7.0 » • . 1 » and 1.5 2.8 4.9 8.4 13.1 18.1 giving specific lengths of 0.67, 0.43, 0.31, 0.31, 0.37 and 0.39. The graph is shown in Figure 15. bo c 7.0 6.0 ■g 5.0 4.0 3.0 2.0 1.0 Figure 14 1.5 2.8 4.9 8.4 13.1 — >■ Lengths removed in mm. Rana clamitans Second regenerations Eighteen days 18.1 At eighteen days there is very little regeneration at any of the levels and at some of them, especially the shorter removals, a consider- able absorption of regenerated material. Regeneration may therefore be considered as completed at this time. However the measurements for 56 days are given in order to show the changes. The regenerated lengths at that time are 0.9, 0.7, 1.6, 2.5, 5.2 and 7.1 mm. These data 69] RATE OF REGENERATION— ZELENY 69 — 2 E mm. 0.70 E 0.60 0.50 m bo a 0.40 0.30 0.20 o 2 o 0.10 Fig ure 15 een days 1.5 2.8 4.9 8.4 — ►■ Lengths removed in mm. Ram clamitans Second regenerations 13.1 18.1 Specific lengths Eight- are given in table 38 and are represented in graphic form in figure 16. Dividing by the average removed lengths, which differ somewhat from the previous ones because of the death of certain individuals, the frac- tions obtained are 0.9 0.7 1.6 2.5 5.2 7.1 1 2 j 0/ o> 3.0 2.0 1.0 1.5 2.6 4.9 8.2 13.2 and 17.9 mm. 7.0 6.0 5.0 4.0 Figure 16 1.5 2.6 4.9 8.2 13.2 — >■ Lengths removed in mm. Ram clamitans Second regenerations Fifty-six days 17.9 70 ILLINOIS BIOLOGICAL MONOGRAPHS [70 giving specific regenerations of 0.60, 0.27, 0.33, 0.31, 0.39 and 0.40. These are shown in the graph given in figure 17. Because of the absorption of regenerated tissue in the shorter removals and a slight growth in the longer ones the latter show a comparative increase in specific lengths. 0.60 i 0.50 <0 t- 040 0.30 bl) a © 0.20 o 0.10 o 0) a GO 1.6 2.6 4.9 8.2 — >■ Lengths removed in mm. 13.2 17.9 Figure 17 ' Rana clamitans Second regenerations Specific lengths Fifty- six days For a comparison of completeness of regeneration it is better to take the greatest lengths regenerated at each level rather than the amounts regenerated at any particular time because the shorter levels complete regeneration and begin to absorb the tissues sooner than do the longer ones. On this basis the greatest regenerated lengths at each of the six levels are, for the 1.5 mm. level 1.0 mm. reached at ten days, for the 2.8 level 1.3 mm. reached at ten days, for the 4.9 level 1.6 mm. reached at twelve and a. half days, for the 8.4 level 2.6 mm. reached at twelve and a half days, for the 13.2 level 5.2 mm. reached at fifty-six days, and for the 17.9 level 7.1 mm. reached at fifty-six days. These data are given in tables 37, 38, 39 and 40 and in graphic form in figure 18. At the last two levels there was a slight increase from eighteen to fifty-six days but this almost certainly came during the early part of the period and the values are therefore completed values. Dividing by the removed lengths the fractions obtained are 1.0 1.3 1.6 2.6 5.2 1.5 2.8 4.9 8.4 13.2 and 7.1 i7~9 giving specific lengths of 0.67, 0.46, 0.33, 0.31, 0.39 and 0.40. The graph is given in Figure 19. The high values for the two short levels are probably due to the fact that the cells migrating to the cut surface form 71] RATE OF REGEXERATIOX —ZELENY 71 7.0 6.0 © 5.0 a E § 4.0 M f 3.0 2.0 1.0 Figure 18 mm. 0.70 d I 8 0.60 0.50 IB 3 ■«-> H 0.40 0.30 .2 0.20 Q 2 o B D. 02 0.10 ni:". Figure 19 ness 1.5 2.8 4.9 8.4 — >- Lengths removed in mm. Rana clamitans Second regenerations 13.2 Completeness 17.9 1.5 2.8 4.9 8.4 — >■ Lengths removed in mm. Rana clamitans Second regenerations 13.2 17.9 Specific lengths Complete- a large proportion of the total mass of the regenerated organ. Since apparently the length of this mass of cells is very much alike at all levels as indicated by the facts of the four day regenerations, the specific lengths for these short removals are greater than for the others. The high values of the two longest removals are due to a continuation of 72 ILLINOIS BIOLOGICAL MONOGRAPHS [72 regeneration at these levels after it has ceased at the others. At ten days the specific lengths regenerated are very nearly the same at all the levels except the first two. Rana clamitans TABLE 37 Series 3676-3765 Second regenerations Catalog number Removed length in mm. 4 Days 6 Days 8 Days 10 Days Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length 3676 1.3 0.27 0.60 0.9 1.0 3682 1.6 0.18 0.60 0.9 1.0 3730 1.6 0.39 0.75 0.9 0.9 6 3754 1.6 0.06 0.55 0.9 1.1 Average 1.5 0.22 0.15 0.62 0.42 0.9 0.60 1.0 0.67 3677 2.0 0.30 0.6 0.9 0.9 3696 2.1 0.48 0.8 1.0 1.1 3701 3.2 0.42 0.8 1.1 1.2 3713 2.8 0.36 0.8 0.9 0.9 10 3719 3.1 0.30 0.8 1.1 1.4 3749 2.8 0.48 0.6 1.2 1.4 3750 3.5 0.42 0.39 1.3 0.30 1.7 1.1 1.9 1.3 Average 2.8 0.14 0.80 0.39 0.46 3678 5.0 0.20 0.5 0.9 1.0 3684 5.5 0.15 0.7 1.2 1.4 3702 4.7 0.42 0.8 1.1 1.3 18 3720 4.6 0.06 0.3 1.3 1.8 3756 4.8 0.36 0.24 1.0 1.3 1.2 1.7 1.4 Average 4.9 0.05 0.70 0.14 0.24 0.29 1 ' 73] RATE OF REGENERATION— ZELENY 73 Rana clamitans TABLE 37 (Continued) Series 3676-3765 Second regenerations Catalog number Removed length in mm. 4 Days 6 Days 8 Days 10 Days Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen • erated length mm. 1.4 Specific length Regen- erated length mm. Specific length 3679 8.4 0.30 0.7 1.9 3685 9.3 0.60 1.2 1.9 2.3 3697 7.3 0.48 1.2 1.7 2.2 3703 9.3 0.45 1.3 2.0 2.5 3715 7.9 0.24 0.9 1.7 2.3 31 3721 8.7 0.57 1.3 1.9 2.3 3733 8.5 0.36 1.0 1.9 2.4 3739 9.6 0.36 1.0 1.8 2.4 3751 6.7 0.45 1.1 1.7 2.1 3757 8.0 0.42 1.2 2.1 2.8 Average 8.4 0.42 0.05 1.1 0.13 1.8 . 0.22 2.3 0.28 3686 14.5 0.60 2.1 3.4 4.8 3698 14.9 0.50 1.5 3.3 4.3 3704 14.5 0.45 2.2 3.3 4.4 3716 12.7 0.39 1.7 2.4 3.4 3722 12.5 0.60 1.6 2.6 3.6 49 3740 13.9 0.30 1.1 2.1 3.0 3752 11.2 0.54 1.7 2.5 3.4 3758 11.0 0.60 1.5 2.2 0.21 2.9 Average 13.1 0.50 0.04 1.7 0.13 2.7 3.7 0.28 3680 16.0 0.60 1.9 3.0 4.2 3681 21.2 0.84 3.0 4.0 5.6 3687 19.7 0.54 3.6 5.6 6.0 3699 21.0 0.54 2.2 4.3 5.9 3705 17.6 0.72 2.0 3.6 4.8 3717 17.6 0.42 2.6 3.6 5.2 67 3723 18.4 0.30 2.3 3.7 5.3 3735 16.5 0.48 2.0 3.4 5.5 3741 16.0 0.30 1.9 3.0 5.4 3753 16.8 0.42 0.03 2.0 2.5 3.8 Average 18.1 0.52 2.3 0.13 3.7 0.20 5.1 0.28 74 ILLIXOIS BIOLOGICAL MONOGRAPHS [74 Rana clamitans TABLE 38 Series 3676-3765 Second regenerations Catalog number Removed length in mm. 12 J Days 18 Days 56 Days Highest values Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length 3676 1.3 1.0 1.0 0.7 1.0 3682 1.6 1.0 1.0 1.1 1.1 3730 1.6 0.9 0.9 0.7 0.9 6 3754 1.6 1.2 0.67 1.2 1.1 1.2 1.0 Average 1.5 1.0 1.0 0.67 0.9 0.60 0.67 3677 2.0 0.9 0.9 0.7 0.9 3696 2.1 1.0 1.0 0.7 1.1 3701 3.2 0.9 0.9 0.5 1.2 3713 2.8 1.4 1.3 1.4 10 3719 3749 3.1 2.8 1.4 2.0 1.3 2.0 0.9 1.4 2.0 3750 3.5 1.3 1.3 0.7 1.9 1.3 Average 2.8 1.3 0.46 1.2 0.43 0.27 0.46 3678 .5.0 1.1 1.1 1.1 3684 5.5 1.4 1.5 1.4 1.5 3702 4.7 1.3 1.3 1.3 1.3 18 3720 4.6 2.3 1.9 2.0 2.3 3756 4.8 1.8 1.6 1.6 1.8 Average 4.9 1.6 0.33 1.5 0.31 0.33 1.6 0.33 3679 8.4 1.9 2.1 2.1 3685 9.3 2.8 3.0 2.6 3.0 3697 7.3 2.4 2.3 2.1 2.4 3703 9.3 2.6 2.5 2.5 2.5 3715 7.9 2.6 2.6 2.8 2.8 3721 8.7 2.6 2.3 2.2 2.6 31 3733 3739 8.5 9.6 2.6 3.0 2.6 2.9 2.8 2.8 3.0 3751 6.7 2.4 2.5 2.3 2.5 3757 8.0 3.1 3.2 3.1 2.5 3.2 2.6 Average 8.4 2.6 0.31 2.6 0-31 0.31 0.31 75] RATE OF REGENERATION— ZELENY TABLE 38 (Continued) 75 Catalog number Removed length in mm. 12* Days 18 Days 56 Days Highest values Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length 3686 14.5 5.3 5.2 5.3 3698 14.9 5.0 5.4 5.4 5.4 3704 14.5 5.2 5.5 5.4 5.5 3716 12.7 4.2 5.1 4.4 5.1 3722 12.5 3.9 3.5 4.2 4.2 49 3740 3752 13.9 11.2 4.6 4.1 5.6 4.0 6.8 6.8 4.1 3758 11.0 3.6 4.4 4.1 4.9 5.2 4.9 5.2 Average 13.1 0.34 4.8 0.37 0.39 0.39 3680 16.0 5.2 6.4 6.6 6.6 3681 21.2 6.3 7.3 7.2 7.3 3687 19.7 6.6 7.0 7.0 3699 21.0 7.1 7.5 7.2 7.5 3705 17.6 6.4 6.2 6.0 6.4 67 3717 17.6 6.0 6.7 6.4 6.7 3723 18.4 6.5 8.1 8.3 8.3 3735 16.5 6.5 7.8 8.0 8.0 3741 16.0 5.8 6.9 7.0 7.0 3753 16.8 5.2 6.4 7.1 7.1 7.1 7.1 Average 18.1 6.2 0.34 7.0 0-39 0.40 0.40 TABLE 39 Rana clamitans Series 3676-3765 Summary Second regenerations Lengths regenerated at different levels at different times Percent of tail length removed Length removed in mm. Number of indi- viduals Days after operation 4 6 8 10 12/* 18 56 6 1.5 4 0.22 0.6 0.9 1.0 1.3 1.4 2.3 1.0 1.0 0.9 10 2.8 7 0.39 0.8 1.1 1.3 1.2 0.7 18 4.9 5 0.24 0.7 1.1 1.2 1.6 1.5 1.6 31 8.4 10 0.42 1.8 2.6 2.6 2.5 49 13.1 8 0.50 1.7 2.7 3.7 3.7 5.1 4.4 6.2 4.8 5.2 67 1*.1 10 0.52 2.3 7.0 7.1 76 ILLINOIS BIOLOGICAL MONOGRAPHS [76 TABLE 40 Rana clamitans .Series 3676-3765 Summary Second regenerations Lengths regenerated at different levels at different times Percent of tail length removed I Length removed in mm. Number of indi- viduals Days after operation 4 6 8 10 12!/ 2 18 56 6 1.5 4 0.15 0.42 0.60 0.67 0.67 0.67 0.60 10 2.8 7 0.14 0.30 0.39 0.46 0.46 0-43 0.27 18 4.9 5 0.05 0.14 0.24 0.29 0.33 0.31 0.33 31 8.4 10 0.05 0.13 0.22 0.28 0.31 0.34 0-31 0.31 49 13.1 8 0.04 0.13 0.21 0.28 0-37 0.39 67 18.1 10 0.03 0.13 0.20 0.28 0.34 0.39 0.40 First Regenerations The data for first regenerations are from a different set of indi- viduals than those for second regenerations. The two kinds of opera- tions were made on the same day. The general results obtained from the first regenerations are in full agreement with those obtained from the second regenerations but there is greater variability because of the smaller number of individuals. The average per cents of the tail length removed are respectively 6, 10, 17, 30, 48 and 62 for the six levels. The first of these has two individuals averaging 1.5 mm. of removed tail, the second five individuals with an average of 2.6 mm., the third three individuals with an average of 4.6, the fourth eight with an average of 8.2, the fifth five with an average of 13.0 and the sixth five with an average of 16.7. The data for these experiments are given in Tables 41, 42, 43 and 44 and in Figures 20 to 35. The progress of a first regeneration is similar to that of a second except that the maximum is reached later in the case of first regenera- tions. In the present series the maximum specific rate for first regenera- tions comes between the sixth and the eighth day after the operation. A comparison of the two regenerations is made in the section on the effect of successive removal. The change in rate during the process of regeneration is also discussed in a separate section. 77] RATE OF REGENERATION— ZELENY 77 The lengths regenerated during the first four days are respectively 0.27, 0.15, 0.51, 0.45, 0.46 and 0.51 for the six levels. There is no regular increase with removed length. The data are given in Table 41 and in Figure 20. The specific lengths regenerated are 0.17, 0.06, 0.11, 0.05, 0.03 and 0.03. They are shown in Table 41 and in Figure 21. As in the case of the second regeneration the shortest removals have the largest proportional amounts. This first period being the period of cell 1.0 Figure 20 1.5 13.0 2.6 4.6 8.2 — y Lengths removed in mm. Rana clamitans First regenerations Four days 16.7 migration with very little cell division it is probable that the length of the material furnished in this way, as measured along the main axis of the individual, is independent of the level of the cut. The area of the cut surface of course is greater at the more proximal than at the more distal levels so that the actual total mass of regenerated material is greater at the deeper levels. At six days the rate of first regenerations is rapidly increasing, the maximum rate coming between six and eight days. The lengths regenerated at six days are respectively 0.47, 0.5, 1.0, 1.1, 1.6 and 1.6 mm. mm. 0.20 0.10 13.0 16.7 1.5 2.6 4.6 8.2 — >■ Lengths removed in mm. Figure 21 Rana clamitans First regenerations Specific lengths Four days 0Q 78 ILLINOIS BIOLOGICAL MONOGRAPHS [78 They are shown in Table 41 and in Figure 22. There is in general an increase with increase in removed length but the first is not proportional to the second. The specific lengths are 0.30, 0.18, 0.22, 0.13, 0.12 and 0.09 as shown in Table 41 and Figure 23. The shorter removals still have proportionately the greater regenerations. •g mm. « 2.0 1.0 1.5 2.6 4.6 8.2 13.0 — > Lengths removed in mm. Figure 22 Rana clamitans. First regenerations Six days 16.7 0.30 0.20 0.10 M a ■ Lengths removed in mm. Rana clamitans First regenerations 13.0 16.7 Specific lengths Six days The maximum rate of regeneration is reached between the sixth and the eighth day. The regenerated lengths at eight days are 0.8, 0.7, 1.5, 1.7, 2.6 and 3.0 mm. The data are shown in Table 41 and Figure 24. With one exception there is increase in regenerated length with increase in removed length. The specific regenerated lengths are 0.53, 0.27, 0.33, 0.21, 0.20 and 0.18 as shown in Table 41 and Figure 25. The shortest removals have the greatest specific regenerations but at 8.2 mm. and above there is an approach to constancy. 79] RATE OF REGENERATION— ZELENY 79 f - 3.0 2.0 1.0 Figure 24 1.5 2.6 4.6 8.2 13.0 — >■ Lengths removed in mm. Rana clamitans First regenerations Eight days 16.1 mm. 0.60 0.50 0.40 0.30 0.20 0.10 1.5 13.0 2.6 4.6 8.2 — >- Lengths removed in mm. Figure 25 Rana clamitans. First regenerations Specific lengths 16.7 Eight days Between the eighth and the tenth day there is a rapid decrease in rate associated with tissue differentiation. The regenerated lengths at ten days are 0.9, 1.0, 1.7, 2.3, 3.8 and 4.5 mm. as shown in Table 41 and Figure 26. There is an uninterrupted increase in regeneration with increase in removed length. The specific lengths are 0.58, 0.38, 0.37, 0.28, 0.29 and 0.27 as shown in Table 41 and Figure 27. ILLINOIS BIOLOGICAL MONOGRAPHS [80 Between ten and twelve and a half days regeneration is slow, reach- ing its end for the two shortest removals at the latter day. The regen- erated lengths at twelve and a half days are 0.9, 1.2, 1.8, 2.6, 4.7 and mm 5.0 •a 4.0 ••-> «j E a> § 3.0 M 4> U an J3 2.0 ■*-> ttf) S3 a> 1.0 Figure 26 1.5 13.0 2.6 4.6 8.2 — >- Lengths removed in mm. Rana clamitans. First regenerations Ten days 16.7 •a mm. d 0.60 u 1$ - 1.5 2.6 4.6 8.2 — >- Lengths removed in mm. 13.0 16.7 Figure 27 Rana clamitans First regenerations Specific lengths Ten days 5.8 mm. as shown in Table 42 and Figure 28. There is a steady increase with increase in removed length. The specific lengths are 0.61, 0.46, 0.39, 0.31, 0.36 and 0.35 as shown in Table 42 and Figure 29. There is an approach to constancy in the four largest removals. 81] RATE OF REGENERATION —ZELENY 81 Between twelve and a half and eighteen days the regenerated material is decreasing in the case of the two shortest removals, has made no progress in the third and in the three longest removals the increase is very slight. The data therefore are of value more particularly in con- nection with the problem of the relative completeness of regeneration from the different levels. The regenerated lengths at eighteen days are mm. 6.0 5.0 4.0 3.0 2.0 1.0 Figure 28 *o mm. ■ Lengths removed in mm. Rana clamitans First regenerations 13.0 16.7 Twelve and a half days 1.5 13.0 2.6 4.6 8.2 — y Lengths removed in mm. Figure 29 Rana clamitans First regenerations Specific lengths a half days 16.7 Twelve and 82 ILLINOIS BIOLOGICAL MONOGRAPHS [82 0.9, 1.1, 1.8, 2.7, 5.5 and 6.8 mm. as shown in Table 42 and Figure 30. There is a regular increase with increase in removed length. The specific lengths are 0.60, 0.42, 0.39, 0.33, 0.42 and 0.40 as shown in Table 42 and Figure 31. Though there are irregularities the specific lengths approach constancy at all levels except the most distal one. Between eighteen and fifty-six days there is practically no increase ■8 mm. 7.0 6.0 5.0 4.0 3.0 2.0 1.0 Figure 30 1.5 2.6 4.6 8.2 13.0 — > Lengths removed in mm. Rana clamitans. First regenerations Eighteen days 16.7 - mm. 0.70 0.60 0.50 0.40 0.30 0.20 0.10 Figure 31 days 1.5 2.6 4.6 8.2 — >- Lengths removed in mm. Rana clamitans First regenerations 13.0 16.7 Specific lengths Eighteen 83] RATE OF REGENERATION— ZELENY 83 in regenerated length and some absorption of material especially with the shorter removals. The regenerated lengths at fifty-six days are 0.7, 1.1, 1.5, 2.5, 5.5 and 6.9 mm. as shown in Table 42 and Figure 32. There is a regular increase in regeneration from the shortest to the longest removal. The specific lengths are 0.45, 0.42, 0.34, 0.30, 0.42 and 0.41 as shown in Table 42 and Figure 33. These data are of value only for mm. 7.0 6.0 -% 5.0 to e 4.0 3.0 2.0 1.0 Figure 32 •o A -u mm. V a 0.50 ■ Lengths removed in mm. Ram clamitans First regenerations Fifty-six days 16.7 1.5 13.0 2.6 4.6 8.2 — y Lengths removed in mm. Rana clamitans First regenerations Specific lengths 16.7 Fifty-six days 84 ILLINOIS BIOLOGICAL MONOGRAPHS [84 a comparison of completeness of regeneration but for such a comparison it is better in some ways to compare the regenerations at the time when absorption has not begun. The greatest average regenerated length attained for the 1.5 mm. level is 0.9 mm. at ten days, for the 2.6 level 1.2 at twelve and a half days, for the 4.6 level 1.8 at twelve and a half days, for the 8.2 level 2.7 at eighteen days, for the 13.0 level 5.5 at eighteen days and for the 16.7 level 6.9 at fifty-six days. There is an uninterrupted increase from the shortest to the longest removal in com- plete amount regenerated. This is shown graphically in Figure 34. The mm. 7.0 6.0 5.0 4.0 3.0 2.0 1.0 Figure 34 1.5 2.6 4.6 8.2 13.0 — >- Lengths removed in mm. Rana clamitans First regenerations Completeness 16.7 p. x mm. 0.70 0.60 0.50 0.40 0.30 0.20 0.10 1.5 2.6 4.6 8.2 13.0 16.7 — >- Lengths removed in mm. Figure 35 Rana clamitans First regenerations Specific lengths Completeness 85] RATE OF REGENERATION— ZELENY 85 completed regenerations are less than the removed lengths. The specific completed regenerated lengths obtained as before by dividing by the removed lengths are 0.61, 0.46, 0.39, 0.33, 0.42 and 0.41, as shown in table 42 and figure 35. The greater specific lengths from the shortest removals are probably due as in the case of the second regenerations to the fact that a greater proportion of their substance is made up of cells that have migrated over the cut surface during the first stages of regeneration. This migrated material is not essentially different in axial length at the different levels. The largest removals have a greater spe- cific length than the medium ones because regeneration continues at the former levels after it has ceased at the latter. On the whole there is no essential difference between the results obtained from first regenerations and those obtained from second regen- erations. The latter give the more regular data because the averages are taken from a larger number of individuals. Rana clamitans TABLE 41 Series 3676-3765 First regenerations Catalog number Removed length in mm. 4 Days 6 Days 8 Days 10 Days Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length 3706 1.4 0.24 0.54 0.9 1.0 6 3742 1.7 0.30 0.40 0.7 0.8 0.8 0.9 Average 1.5 0.27 0.17 0.47 0.30 0.53 0.58 3688 2.5 0.12 0.3 0.3 0.7 3707 3.2 0.24 0.8 1.1 1.4 3724 2.6 0.06 0.5 0.8 1.1 10 3743 2.5 0.03 0.1 0.4 0.8 3760 3.1 0.30 0.6 0.9 0.7 1.1 1.0 Average 2.6 0.15 0.06 0.5 0.18 0.27 0.38 86 ILLINOIS BIOLOGICAL MONOGRAPHS [86 TABLE 41 (Continued) Catalog number Removed length in mm. 4 Days 6 Days 8 Days 10 Regen- erated length mm. Days Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length Specific length 3708 5.3 0.54 1.2 1.9 2.1 3726 4.3 0.42 0.9 1.3 1.4 17 3762 4.1 0.57 1.0 1.2 1.5 1.7 Average 4.6 0.51 0.11 1.0 0.22 1.5 0.33 0.37 3690 9.7 0.48 1.0 1.7 2.4 3709 8.8 0.48 1.3 2.0 2.6 3727 8.3 0.48 1.1 1.6 2.0 3745 10.0 0.54 1.8 2.4 3.8 3744 6.0 0.36 1.0 1.3 1.7 30 3761 6.6 0.39 1.0 1.5 1.9 3763 8.5 0.57 1.1 1.8 2.4 3689 6.3 0.30 0.05 0.7 1.2 1.7 1.5 2.3 ~ Average '3.2 0.45 1.1 0.13 0.21 0.28 3710 12.3 0.42 1.8 2.9 3.7 3728 12.8 0.60 1.7 2.8 3.9 3746 13.3 0.54 1.7 2.4 4.1 48 3764 14.6 0.42 1.3 2.5 4.2 3765 12.2 0.30 1.5 2.3 2.6 3.2 3.8 Average 13.0 0.46 0.03 1.6 0.12 0.20 0.29 3692 16.8 0.51 1.1 2.2 3.2 3693 17.2 0.48 1.8 3.3 5.0 3711 17.0 0.54 1.8 3.6 5.6 62 3729 16.1 0.48 1.9 3.3 4.6 3749 16.2 0.54 1.2 2.7 3.0 0.18 4.2 4.5 Average 16.7 0.51 0.03 1.6 0.09 0.27 87] RATE OF REGENERATION —ZELENY 87 TABLE 42 Rana clamitans Series 3676-3765 First regenerations \ Catalog number Re- moved length in mm. 124 Days 18 Days 56 Days Percent removed Average Regen- erated length mm. Specific length Regen- erated length mm. Specific length Regen- erated length mm. Specific length 3706 1.4 1.0 0.9 0.7 6 3742 1.7 1.5 0.9 0.9 0.7 Average 0.9 0.61 0.9 0.60 0.7 0.45 3688 2.5 0.9 0.9 0.7 3707 3.2 1.4 1.3 0.7 3724 2.6 1.4 1.4 1.2 10 3743 2.5 1.0 1.0 1.7 3760 3.1 2.6 1.2 1.1 1.1 Average 1.2 0.46 1.1 0.42 1.1 0.42 3708 5.3 2.3 2.3 1.8 3726 4.3 1.4 1.4 1.4 17 3762 4.1 1.7 1.8 1.4 Average 4.6 1.8 0.39 1.8 0.39 1.5 0.34 3690 9.7 2.6 2.7 2.2 3709 8.8 3.2 3.4 3.3 3727 8.3 2.2 2.2 2.2 3745 10.0 4.4 4.8 4.2 3744 6.0 1.8 1.7 30 3761 3763 6.6 8.5 2.2 2.9 2.3 3.1 1.8 3689 6.3 1.6 1.7 1.4 Average 8.2 2.6 0.31 2.7 0.33 2.5 0.30 3710 12.3 3.9 3.9 3.9 3728 12.8 4.8 5.4 5.8 3746 13.3 5.7 7.0 6.8 48 3764 14.6 5.3 6.8 6.5 3765 12.2 3.9 4.5 4.5 Average 13.0 4.7 0.36 5.5 0.42 5.5 0.42 3692 16.8 4.3 5.0 5.2 3693 17.2 6.5 7.3 6.6 3711 17.0 7.0 7.7 8.3 62 3729 16.1 5.5 6.7 6.4 3749 16.2 5.6 7.1 7.8 Average 16.7 5.8 0.35 6.8 0.40 6.9 0.41 88 ILLINOIS BIOLOGICAL MOXOGRAPHS TABLE 43 Rana clamitans Series 3676-3765 Summary First regenerations Lengths regenerated at different levels at different times Percent of tail length removed Length removed in mm. Number of indi- viduals Days after operation 4 6 8 10 12J4 18 56 6 1.5 2 0.27 0.5 0.8 0.9 0.9 0.9 0.7 10 2.6 5 0.15 0.5 0.7 1.0 1.2 1.1 1.1 17 4.6 3 0.51 1.0 1.5 1.7 1.8 1.8 1.5 30 8.2 8 0.45 1.1 1.7 2.3 2.6 2.7 2.5 48 13.0 5 0.46 1.6 2.6 3.8 4.7 5.5 5.5 62 16.7 5 0.51 1.6 3.0 4.5 5.8 6.8 6.9 TABLE 44 Rana clamitans Series 3676-3765 Summary First regenerations Specific lengths regenerated at different levels at different times Percent of tail length removed Length removed in mm. Number of indi- viduals Days after operation 4 6 8 10 \V/z 18 56 6 1.5 2 0.17 0.30 0.53 0.58 0.61 0.60 0.45 10 2.6 5 0.06 0.18 0.27 0.38 0.46 0.42 0.42 17 4.6 3 0.11 0.22 0.33 0.37 0.39 0.39 0.34 30 8.2 8 0.05 0.13 0.21 0.28 0.31 0.33 0.30 48 13.0 5 0.03 0.12 0.20 0.29 0.36 0.42 0.42 62 16.7 5 0.03 0.09 0.18 0.27 0.35 0.40 0.41 Experiment II Amblystoma punctatum Series 4600-5052 The eggs were hatched on March 29 to April 4, 1913. Opera- tions on the tail were made on May 7 in numbers 4600-4752 and on May 10 in numbers 4800-5052. The removed lengths were approximately Vio> Vs* Vs> Vs ^d 3 A of the tail length. Measurements of the regen- erated tissue were made at 2, 4, 6, 8-9, 10-11, 13, 15-16 and 17-18 days after the operation. The data are given in Tables 45 to 54 and in Figures 36 to 51. The salamander larvae are much more irregular in their regeneration as well as in ordinary growth than frog tadpoles. The measurements 89] RATE OF REGENERATION— ZELENY 89 in the present experiment were made on killed individuals so that only a single regeneration measurement is made in a single individual. This procedure also tends toward a greater variability in the data. The number of individuals in any particular measurement also is less than for the second regeneration of frog tadpoles. Notwithstanding all these unwelcome factors the general features of regeneration are similar to those for the tadpole experiment. The regenerated length at any time is approximately proportional to the removed length. It is true even in the earliest measurements. As for frog tadpoles the shorter removals have proportionately a larger regeneration than the others at practically each time of measurement. The approach to equality in specific lengths is true only of the lengths of removal equal to one-fifth or more of the tail length. At two days the regenerated lengths are respectively 0.10, 0.15, 0.15, 0.47 and 0.53 mm. for the five levels of removal. They give specific lengths of 0.07, 0.07, 0.04, 0.08 and 0.06 as shown Table 45 and Figures 36 and 37. At four days the regenerated lengths are 0.12, 0.15, 0.30, 0.41 and 0.40 mm. and the specific lengths 0.11, 0.07, 0.07, 0.07 and 0.05 as shown in Table 46 and Figures 38 and 39. At six days the regenerated lengths are 0.32, 0.47, 0.62, 0.70 and 1.02 mm. and the specific lengths 0.30, 0.20, 0.16, 0.12 and 0.11 as shown in Table 47 and Figures 40 and 41. At eight to nine days the regenerated lengths are 0.40, 0.65, 0.80, 1.40 and 1.52 mm. and the specific lengths 0.44, 0.28, 0.23, 0.23 and 0.19 as shown in Table 48 and Figures 42 and 43. At ten to eleven days the regenerated lengths are 0.50, 0.63, 1.54, 2.22 and 2.22 mm. and the specific lengths 0.62, 0.26, 0.43, 0.41 and 0.27 as shown in Table 49 and Figures 44 and 45. At thirteen days the regenerated lengths are 0.78, 0.92,- 1.74, 2.40 and 3.60 mm and the specific lengths 0.74, 0.43, 0.48, 0.44 and 0.48 as shown in Table 50 and Figures 46 and 47. At fifteen to sixteen days the regenerated lengths are 0.80, 1.30, 1.37, 2.80 and 3.80 mm. and the specific lengths 0.67, 0.61, 0.40, 0.48 and 0.54 as shown in Table 51 and Figures 48 and 49. At seventeen to eighteen days the regenerated lengths are 0.70, 1.40, 1.60, 3.80 and 4.67 mm. and the specific lengths 0.67, 0.62, 0.41, 0.66 and 0.57 as shown in Table 52 and Figures 50 and 51. A summary of regenerated lengths is given in Table 53 and of specific regenerated lengths in Table 54. Since the experiment was closed at eighteen days and since the measurements at different times were made on different individuals it is not possible to make as accurate a comparison of completeness of 90 ILLINOIS BIOLOGICAL MONOGRAPHS [90 regeneration as in the case of the frog tadpoles. For the three shortest removals regeneration is probably completed at this time but this is not true for the two longest ones. In this respect as in others there is an agreement with the former experiment. The percent of the removed tail that is regenerated is greater for all levels than in the frog tadpoles. It is probable also that if the longest removals had been allowed to com- plete their regenerations their specific regenerations as in the case of the frog tadpoles would have been shown to be greater than those for medium levels. TABLE 45 Amblystoma punctatum. Series 4600-5052. Average tail length =10.9 mm. Regeneration: 2 days Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 14 5022 1.5 0.1 Average . 1.5 0.10 0.07 4641 2.3 0.2 4741 1.9 0.1 20 4841 2.2 0.1 5050b 2.3 0.2 Average 2.2 0.15 0.07 4811 3.9 0.3 4911 3.3 0.1 32 5012 3.3 0.05 Average 3.5 0.15 0.04 4601 6.0 0.3 4801 6.0 0.7 53 5001 5.4 0.4 Average 5.8 0.47 0.08 4631 9.5 0.7 4831 9.1 0.6 81 5032 7.9 0.3 Average 8.8 0.53 0.06 91] RATE OF REGENERATION— ZELENY 91 The data from both experiments show that except for very short removals the length regenerated in a given time is approximately pro- portional to the length removed. TABLE 46 Amblystoma punctatum. Series 4600-5052. Average tail length— 10.9 mm. Regeneration: 4 days Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4622 0.9 0.1 4722 0.8 0.2 4822 1.5 0.1 10 4922 0.8 0.1 5024 1.6 0.1 Average 1.1 0.12 0.11 4742 2.4 0.1 21 4842 2.3 0.2 I Average 2.3 0.15 0.07 4612 3.7 0.3 4712 3.6 0.2 37 4812 4.5 0.4 5012 4.2 0.3 Average 4.0 0.30 0.07 4602 6.0 0.2 4702 6.0 0.05 4802 6.2 0.7 55 4902 6.0 0.5 5004 5.9 0.6 Average 6.0 0.41 0.07 4632 9.3 0.4 4732 8.5 0.2 4832 10.9 0.6 76 4932 5.9 0.4 5034 7.1 0.4 Average 8.3 0.40 0.05 92 ILLINOIS BIOLOGICAL MONOGRAPHS [92 Amblystoma punctatum. TABLE 47 Series 4600-5052. Average tail length=10.9 mm. Regeneration: 6 days Percent of tail length Catalog Removed Regenerated Specific . removed number length length length Average mm. mm. regenerated 4623 1.0 0.3 4723 0.8 0.2 10 4923 1.6 0.6 1.1 0.2 Average 1.1 0.32 0.30 4643 2.1 0.7 4743 2.2 0.2 21 4843 2.1 0.4 4943 2.8 0.6 Average 2.3 0.47 0.20 4613 3.6 0.6 4713 2.9 0.6 4820b 4.4 0.8 35 4913 4.5 0.6 5013 3.6 0.5 Average 3.8 0.62 0.16 4603 6.1 0.4 4703 6.7 0.4 54 4803 5.3 1.4 5003 5.6 0.6 Average 5.9 0.70 0.12 4633 8.8 0.6 4733 8.2 0.9 82 4833 10.6 1.8 5033 7.9 0.8 Average 8.9 1.02 0.11 93] RATE OF REGENERATION — ZELENY 93 TABLE 48 Amblystoma punctatum Series 4600-5052 Average tail length— 10.9 Regeneration: 8-9 days (8 for 4800-5052, 9 for 4600-4752) Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4724 0.7 0.2 4824 1.1 0.7 8 5026 1.0 0.3 Average 0.9 0.40 0.44 4644 2.3 0.6 4844 2.3 1.0 21 4944 2.2 0.7 - 5045 2.3 0.3 Average 2.3 0.65 0.28 4614 3.0 0.8 4624 3.0 0.4 4714 3.4 1.2 31 4814 3.7 1.0 4914 3.2 0.6 5016 4.3 0.8 Average 3.4 0.80 0.23 4604 6.0 1.8 4704 6.3 1.7 4804 5.9 1.4 56 4904 5.4 0.9 5006 6.8 1.2 Average 6.1 1.40 0.23 4634 8.3 2.1 4734 8.0 1.3 4834 9.0 1.6 75 4934 7.5 1.1 Average 8.2 1.52 0.19 94 ILLINOIS BIOLOGICAL MONOGRAPHS [94 TABLE 49 Amblystoma punctatum Series 4600-5052 Average tail length = 10.9 Regeneration: 10-11 days (10 for 4800-5052, 11 for 4600-4752) Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4725 0.6 0.3 4825 0.8 0.6 7 5027 1.0 0.6 Average 0.8 0.50 0.62 4746 2.6 0.4 4845 2.6 1.0 23 5046 2.2 0.5 Average 2.5 0.63 0.26 4620b 3.0 1.9 4715 3.0 1.8 4815 4.3 2.0 33 4920 3.6 1.2 5017 3.9 0.8 Average 3.6 1.54 0.43 4605 5.2 2.8 4705 4.6 1.4 4805 6.3 2.8 50 4910b 5.0 1.9 5007 6.0 2.2 Average 5.4.. 2.22 0.41 4735 7.5 2.5 4835 9.4 2.9 74 4935 7.6 1.3 5037 8.1 2.2 Average 8.1 2.22 0.27 95] RATE OF REGENERATION— ZELENY 95 Ambly.stoma punctatum. TABLE 50 Series 4600-5052. Average tail length=10.9 mm. Regeneration: 13 days Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4626 1.4 0.8 4726 1.0 0.6 4830b 1.0 0.7 10 4926 1.0 0.9 5028 0.9 0.9 Average 1.1 0.78 0.74 4646 1.9 1.4 4745 2.2 0.8 19 4846 2.6 1.0 4946 1.9 0.5 Average 2.1 0.92 0.43 4616 3.6 1.8 4716 3.2 2.0 4816 3.9 2.5 32 4916 3.7 1.6 5018 3.7 0.8 Average 3.6 1.74 0.48 4706 6.4 2.3 4806 5.8 2.9 50 4910 5.0 2.5 5008 4.5 1.9 Average 5.4 2.40 0.44 4636 8.1 3.4 4740b 7.7 4.7 69 4936 7.4 3.5 5038 6.7 2.8 Average 7.5 3.60 0.48 96 ILLINOIS BIOLOGICAL MONOGRAPHS [96 TABLE 51 Amblystoma punctatum. Series 4600-5052. Average tail length=10.9 mm. Regeneration: 15-16 days (15 for 4800-5052, 16 for 4600-47g2) Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4927 1.2 0.8 11 5029 1.2 0.8 Average 1.2 0.80 0.67 4647 1.9 1.5 4747 2.7 1.7 4847 2.4 1.0 19 4950b 1.9 1.2 5049 1.8 1.1 Average 2.1 1.30 0.61 4617 3.1 1.4 4717 3.5 1.3 32 4917 3.2 1.8 5019 4.1 1.0 Average 3.5 1.37 0.40 4607 5.7 2.7 4807 5.7 2.9 53 4817 5.2 2.6 5009 6.6 3.0 Average 5.8 2.80 0.48 64 4937 7.0 3.8 Average 7.0 3.80 0.54 97] RATE OF REGENERATION —ZELENY 97 TABLE 52 Amblystoma punctatum. Series 4600-5052. Average tail length=10.9 mm. ' Regeneration: 17-18 days (18 for 4800-5052, 17 for 4600-4752) Percent of tail length Catalog Removed Regenerated Specific removed number length length length Average mm. mm. regenerated 4828 1.0 0.8 10 4929 1.1 0.6 Average 1.0 0.70 0.67 4648 2.1 1.5 4749 2.6 2.1 4848 2.1 1.1 20 4949 2.2 1.3 5050 2.2 1.0 Average 2.2 1.40 0.62 4718 3.9 1.6 36 Average 3.9 1.60 0.41 4608 5.4 4.2 4708 6.6 4.1 53 4808 5.4 3.1 Average 5.8 3.80 0.66 4838 9.4 5.0 4939 6.4 4.5 76 5040 9.0 4.5 Average 8-3 4.67 0.57 98 ILLINOIS BIOLOGICAL MONOGRAPHS [98 TABLE 53 Amblystoma punctatum Series 4600-5052 Summary Regenerated lengths (Tables 45 to 52) Percent of tail length removed Average Length removed mm. Average Ave •age le ngth regenerated in mm. 2 Days 4 Days 6 Days 8-9 Days 10-11 Days 13 Days 15-16 Days 17-18 Days 10 1.1 0.10 0.12 0.32 0.40 0.50 0.63 0.78 0.92 0.80 0.70 21 2.2 0.15 0.15 0.47 0.65 0.80 1.40 1.30 1.40 34 3.7 0.15 0.30 0.62 1.54 1.74 1.37 1.60 53 5.8 0.47 0.41 0.70 2.22 2.40 3.60 2.80 3.80 74 8.1 0.53 0.40 0.94 1.52 2.22 3.80 4.70 TABLE 54 Amblystoma punctatum Series 4600-5052 Summary Specific lengths regenerated (Tables 45 to 52) Percent of tail length removed Average Length removed mm. Average Ave rage specific regenerated len gths 2 Days 4 Days 6 Days 8-9 Days 10-11 Days 13 Days 15-16 Days 17-18 Days 10 1.1 0.07 0.11 0.30 0.43 0.62 0.74 0.67 0.67 21 2.2 0.07 0.04 0.06 0.20 0.28 0.26 0.43 0.61 0.62 34 3.7 0.07 0.16 0.23 0.43 0.48 0.40 0.41 53 5.8 0.08 007 0.12 0.11 0.23 0.41 0.44 0.48 0.48 0.66 74 8.1 0.06 0.15 0.19 0.27 0-54 0.57 99] RATE OF REGENERATION— ZELENY 99 mm. 0.67 1 g Figure 36 1.5 2.2 3.5 5.8 — >■ Lengths removed in mm. Amblystoma punctatum Lengths regenerated 8.8 Two days mm. 0.10 « Figure 37 1.5 2.2 3.5 5.8 8.8 — >- Lengths removed in mm. Amblystoma punctatum Specific lengths regenerated Two days _ .2 mm. I 0.67 i © M i 13 1.1 J — Figure 38 Amblv stoma 2.3 4.0 6.0 ■>- Lengths removed in mm. punctatum Lengths regenerated 8.3 Four days § mm. 0.10 1.1 6.0 2 Figure 39 2.3 4.0 — >- Lengths removed in mm. Amblystoma punctatum Specific lengths regenerated 8.3 Four days - 100 ILLINOIS BIOLOGICAL MONOGRAPHS 100 bo a © mm. 0.67 Figure 40 1.1 2.3 3.8 5.9 — >- Lengths removed in mm. Amblystoma punctatum Lengths regenerated 8.9 Six days to s o mm. 0.30 0.20 0.10 » Figure 41 to mm. 1.33 1.1 2.3 3.8 5.9 8.9 — >- Lengths removed in mm. Amblystoma punctatum Specific lengths regenerated Six days 0.67 s 0.9 6.1 8.2 2.3 3.4 — >■ Lengths removed in mm. Figure 42 Amblystoma punctatum Lengths regenerated Eight to nine days % mm. g 0.50 g 0.40 bfl Z 0.30 3 0.20 M g 0.10 0.9 6.1 2.3 3.4 — >■ Lengths removed in mm. Figure 43 Amblystoma punctatum Specific lengths regenerated nine days 8.2 Eight to 101] RATE OF REGENERATION— ZELENY 101 2.00 1.33 0.67 0.8 2.5 3.6 5.4 — >■ Lengths removed in mm. Figure 44 Amblystoma punctatum Lengths regenerated 8.1 Ten to eleven days — 0.60 0.50 to 0.40 ■- 10 0.30 i2 o 0.20 0.10 - X 0.8 2.5 3.6 5.4 8.1 — >■ Lengths removed in mm. Figure 45 Amblystoma punctatum Specific lengths regenerated Ten to eleven days 102 ILLINOIS BIOLOGICAL MONOGRAPHS [102 fee d 5 mm. 3.33 2.67 2.00 1.33 0.67 1.1 2.1 3.6 5.4 7.5 — y Lengths removed in mm. Figure 46 Amblystoma punctatum Lengths regenerated Thirteen days mm T) 0.70 a 0.60 a 0.50 0> l-> OS 0.40 .3 0.30 o ""* 0.20 o

- Lengths removed in mm. Figure 48 Amblystoma punctatum Lengths regenerated days Fifteen to sixteen 0.70 09"0 0.50 0.40 0.30 i> 0.20 0.10 1.2 5.8 2.1 3.5 — >■ Lengths removed in mm. Figure 49 Amblystoma punctatum Specific lengths regenerated sixteen days 7.0 Fifteen to 104 ILLINOIS BIOLOGICAL MONOGRAPHS [104 5 a mm. 4.67 4.00 3.33 2.67 2.00 1.33 0.67 1-0 2.2 3.9 5.8 8.3 — >- Lengths removed in mm. Figure 50 Amblystoma punctatum Lengths regenerated Seventeen to eight- een days mm 0.70 — cti 0.60 s bo u 0.50 00 0.40 J3 fi 0.30 a 0.20 o S 0.10 00 1.0 5.8 2.2 3.9 — ►■ Lengths removed in mm. Figure 51 Amblystoma punctatum Specific lengths regenerated to eighteen days 8.3 Seventeen 105] RATE OF REGENERATION— ZELENY 105 Discussion That the level of the cut has an important influence upon the rate of regeneration has been made out by a number of investigators (Spal- lanzani 1768, King 1898, Morgan 1906, Stockard 1908, Ellis 1909, Morgu- lis 1909a, b, and others). Their work indicates that regenerations from deeper levels are on the whole more rapid that from more superficial ones. The data obtained from the present experiments confirm this conclusion and make possible a further analysis of the relation. They show that in the regeneration of the tail of amphibian larvae there is a striking relation between the level of the cut and the rate of regeneration. Within wide limits the length regenerated is directly proportional to the distance of the cut surface from the original tip of the tail. Within these limits therefore regeneration at any particular time after the operation has the same degree of completeness from all levels of injury. An analysis of the progress of the regeneration brings out the fact that two distinct periods are to be recognized in rate of regeneration in its relation to level of the cut. During the first two to four days after the operation regeneration is confined to cell migration from the old tissues without cell division. During this period in the frog tad- poles there is no essential difference in length regenerated at the differ- ent levels and the specific rate is therefore much greater after shorter than after longer removals. In the second period with the initiation of rapid cell multiplication the rate of regeneration is greater the deeper the level and furthermore is directly proportional to the length removed. As soon as the bulk of material produced by cell division is considerably greater than that which was produced by cell migration there is an approach to constancy in specific length regenerated. This holds for all except the shortest removals. After the shortest removals the total regeneration is so small in amount that a large part of it is made up of the original migrated material. Therefore from these levels the spe- cific regenerated lengths are greater than from the deeper levels even at a late period of regeneration. A further complication is introduced by the fact that regeneration is not complete. Only a certain per cent of the removed length is re- placed and the end of the process is reached sooner after the shorter than after the longer removals. From the deepest levels regeneration is still proceeding when it has stopped from the medium and shallowest ones. When the process is completed in all cases the specific length is therefore slightly greater after both the longest and the shortest re- movals than after medium ones. As to the cause of the difference in rate at the different levels 106 ILLINOIS BIOLOGICAL MONOGRAPHS [106 little more can be said than that it does not seem to be due to inherent differences in the cells at the different levels. If differentiation in the tail proceeded from the tip toward the base, the more rapid rate from the more basal levels might be explained by the more embryonic char- acter of the cells at these levels. As the tip is approached the material would become more and more inert. There is however no evidence that differentiation proceeds in this way in this case. The progressive increase in rate with depth of level of the cut is undoubtedly due to reactions which involve a more central control, a co-ordination of the functional activity as a whole. The period of cell migration probably is only slightly subject to such control. It is a period in which the response is largely local in character and there is correspondingly little if any difference at the different levels. The rate of cell division which is the important factor during the period of rapid increase in length is however undoubtedy under central control. Summary 1. In frog and salamander larvae with removed tail lengths of one-fifth to two-thirds, the general rule holds that the length regenerated in a given time is proportional to the length removed, or in other words the length regenerated per unit of removed length is a constant. 2. An analysis of the data shows however that this applies only to the material produced by active cell division. 3. During the first four days, in frog tadpoles, when the regener- ating part is made up almost entirely of cells that have migrated from the old tissues without division there is no such relation between length removed and length regenerated. The length of new material at this time is not strikingly different for the different levels and the process seems to be a local response of the cells to the injury. The length regenerated per unit of removed length is greater at this time for the shorter than for the longer removals. 4. Since comparatively a large part of the regenerating material after the shorter removals is made up of migrated cells even at the later periods it follows that the specific regenerations from these levels are greater than from the deeper ones. 5. During the later periods the specific regenerated lengths tend to be higher after both the shortest and the longest removals than after medium ones. In the case of the shortest ones this is due to the rela- tively large part of the whole regenerated tail that is made up of mi- grated cells. In the case of the longest removals it is due to the fact that regeneration continues for a time after it has stopped in the medium ones. 107] RATE OF REGENERATION— ZELENY 107 6. It does not seem probable that the differences in length regener- ated at different levels can be due to differences in the original character of the cells involved in the process. Such a well graduated difference in cell capacities is difficult to conceive. The process must be under a more central control, probably connected with general functional activity. 108 ILLINOIS BIOLOGICAL MONOGRAPHS [108 PART IV THE CHANGE IN RATE OF REGENERATION DURING THE REGENERATIVE PROCESS The present experiments were undertaken in extension of previous studies on the change in rate throughout the regenerative cycle. This previous work showed that the increase in amount of material during regeneration follows the general rule of increase during an ordinary life cycle. The rate is at first very slow, then increases very rapidly to a maximum, then declines rapidly at first and then more and more slowly as zero is approached. Frog tadpoles and salamander larvae were used in the present study. Large tadpoles of Rana clamitans which remained fairly con- stant in size during the course of the experiments were found to be the most satisfactory. The results obtained from them were uniform enough for an analysis of the change in rate. The salamander larvae showed a great variation in rate from day to day apparently associated with external factors such as food and temperature. The data obtained from them are however of interest in comparison with the frog tadpole results. The experiments will be taken up in turn beginning with the series containing the largest number of individuals and giving the most uni- form results. Experiment I Rana clamitans Second regenerations of the tail Series 3676-3765 The tadpoles were collected on December 9, 1911 and first remov- als were made on December 22 and second removals on January 8. Measurements were taken 4, 6, 8, 10, 12y 2 and 56 days after the opera- tion. The operations were made at six different levels, the removals approximating 6, 10, 18, 31, 49 and 67 per cent of the tail length. The first of these removals averaged 1.5 mm. and four individuals with completed measurements are available, the next averaged 2.8 mm. with seven individuals, the third 4.9 mm. with five, the fourth 8.4 mm. with ten, the fifth 13.1 mm. with eight and the sixth 18.1 mm. with ten individuals. The rates per day for each level during each period are given in table 55 and in graphic form in figure 52. The maximum 109] RATE OF REGENERATION— ZELENY 109 mm. 0.20 0.10 0.20 0.10 0.20 0.10 0.30 0.20 0.10 2 57 9 11% 15% 37 — >- Days after the operation Figure 52 Rates of second regenerations of the tail per day at different times after the operation for six different levels Rana clamitans The removed lengths are 1.5, 2.8, 4.9, 8.4, 13.1 and 18.1 mm. 110 ILLINOIS BIOLOGICAL MONOGRAPHS [110 rate is reached during the period between four and six days at three of the levels and between six and eight days at the other three. The rise in rate is very rapid and the decline also rapid. As discussed in the preceding section on the effect of the level of the cut, the rate of regeneration increases with depth of the level and the increase is such that in general the specific length or length regener- ated per unit of removed length is approximately a constant. A reduc- tion of the rates to specific rates therefore gives an opportunity for averaging the different levels together. The resultant average is based upon a sufficiently large number of individuals to give a considerable degree of smoothness in the curve of rate. The data for specific rate 2 nd 5 7 9 11% 15% — >■ Days after the operation Figure 53. Specific rates of first and second regenerations at different times after the operation Rana clamitans Tail regeneration Upper figure, second regenerations; lower, first regenerations. are given in Table 56. The average specific rates for all six levels to- gether are 0.019 mm. during the to 4 day period, 0.066 during the 4 to 6 day period, 0.051 for 6 to 8 days, 0.033 for 8 to 10 days, 0.017 for 10 to 12i/ 2 days, 0.001 for 12y 2 to 18 days and —0.001 for 18 to 56 days. This change in rate is represented graphically in the upper part of Figure 53. For the four deepest levels the averages are given in a separate column of Table 56. They exclude the two lowest levels which Ill] RATE OF REGEXERATIOX—ZELENY 111 depart considerably from the others in specific rate. There is how- ever no essential difference in the two sets of values as regards the form of the rate curve. The change in rate of regeneration or acceleration of rate from any period to the succeeding one is shown in Table 57 in which the period of change is represented by the middle days of the two periods which are being compared. The average of all the levels shows the acceler- ation to be +0.095 mm. from the 2 to the 5 day period, — 0.015 for 5 to 7 days, —0.030 for 7 to 9 days, —0.058 for 9 to 11% days, —0.028 for 11% to 1514 days and — 0.001 for 15% to 37 days. It is only between the first two periods that acceleration of rate is a plus quantity. Dur- ing all the others it is minus, the most rapid rate of decrease coming between 9 and 11% days. The accelerations of specific rate are more reliable measures for obtaining averages including the different periods. Such values are given in Table 58 and in graphic form in Figure 54. They give a result in the relation of the periods to each other essentially similar to that above. The average accelerations of specific rate are -f 0.014 for the 6 8 10 13% 26 — >■ Days after the operation Figure 54 . Acceleration of specific rate First and second regenerations of the tail in Rana clamitans Unbroken line=First regeneration Broken line= Second regeneration. 2 to 5 day periods, — 0.004 for 5 to 7 days, — 0.009 for 7 to 9 days, —0.0085 for 9 to 11% days, —0.003 for 11% to 15% days and 0.000 for 15% to 37 days. The first period is the only one with a plus accel- eration. The greatest minus acceleration comes between the 7 and the 9 day periods instead of 9 to 11% days. Averaging only the regener- ations for the four deepest levels which show a constant specific rate 112 ILLINOIS BIOLOGICAL MONOGRAPHS [112 the values are respectively +0.011, 0.000, —0.005, —0.006, —0.004 and 0.000, putting the greatest rate of decrease between the 9 and the 11*4 day periods. An examination of the curves of specific rate and a comparison with the facts of histogenesis shows that acceleration of rate is a plus quantity only during the period before active differentiation of the cells, i. e. until the end of the fifth or seventh day. As soon as tissue differentiation is fairly begun the retarding influence is apparent and by the ninth to eleventh days when muscle fibres and other cells are in full process of differentiation the negative acceleration is at its height. Following the percentage increment method used by Minot (1908) for ordinary growth and using length instead of weight because the latter could not be determined with sufficient accuracy the results given in Table 59 are obtained. The values for the six periods excluding the first one are 106, 28, 12, 5 and 0. The regenerated material present at the end of four days is made up almost wholly of cells that have migrated from the old tissues and have not as yet undergone division. After the fourth day the additions to regenerated material are almost wholly the result of cell division. From the end of the fourth to the end of the sixth day the material is on the average more than doubled in length each day. After this time the percentage increment decreases rapidly. The change from period to period is represented in graphic form in Figure 55. The curve is a logarithmic one quite similar to that obtained eS •a 9 M eS 4-> C 9 O $~ 9 a* 100 80 60 40 20 7 9 11*4 15% — ►■ Days after the operation Figure 55 Percentage increment per day at different periods after the opera- tion First and second regenerations of the tail of Rana clamitans Un- broken line=first regeneration. Broken line=second regeneration. 113] RATE OF REGENERATION —ZELENY 113 by Minot for growth. It should however be pointed out that both regeueration and ordinary growth undoubtedly have a very rapidly ascending branch of the curve if the very beginnings of the processes are included. TABLE 55 Rana clamitans Series 3676-3765 Second regenerations Rate of regeneration of tail per day at different times during the regenerative process for six different levels Percent of tail length removed 6 10 18 31 49 67 Length removed in mm. 1.5 2.8 4.9 8.4 13.1 18.1 No. of in- dividuals 4 7 5 10 8 10 Days 0- 4 0.05 0.10 0.06 0.10 j 0.12 0.13 4- 6 0.20* 0.20* 0.23 0.34 0.40 0.91* 6- 8 0.14 0.15 0.25* 0.35* 0.50* 0.70 8-10 0.05 0.10 0.10 0.25 0.50* 0.70 10-12% 0.00 0.00 0.08 0.12 0.28 0.44 12Ms-18 0.00 —0.02 —0.02 0.00 0.07 0.15 18-56 0.00 —0.01 0.00 0.00 0.01 000 114 ILLIXOIS BIOLOGICAL MONOGRAPHS [114 TABLE 56 Rana clamitans Series 3676-3765 Second regenerations Specific rates at different levels at different times Percent of tail length removed 6 10 18 31 49 67 Average of all levels Length removed in mm. 1.5 2.8 * 4.9 8.4 13.1 18.1 Average of four longest remov- No. of in- dividuals 4 7 5 10 8 10 als. . Days 0- 4 0.037 0.Q35 0.012 0.012 0.010 0.007 0.019 0.010 4- 6 0.135* 0.080* 0.045 0.040 0.045* 0.050* 0.066* 0.045* 8-10 0.035 0.035 0.025 0.030 0.035 0.035 0.051 0.042 6- 8 0.090 0.045 0.050* 0.045* 0.040 0.040 0.033 0.032 10-12% 0.000 0.000 0.025 0.015 0.030 0.030 0.017 0.025 12%-18 0.000 —0.005 —0.004 0.000 0.005 0.009 0.001 0.002 18-56 —0.002 —0.004 0.001 0.000 0.001 0.000 —0.001 0.000 115] RATE OF REGENERATION— ZELENY 115 TABLE 57 Rana clamitans Series 3676-3765 Second regenerations Acceleration of rate of regeneration of tail per day at different times during the regenerative process for six different levels Percent of tail length removed 6 10 Length removed in mm. 1.5 2.8 No. of in- dividuals 4 7 Middle of periods Days 2- 5 +0.05* +0.03* 5- 7 —0.03 —0.02 7- 9 —0.04* —0.02 9-11% —0.02 —0.04* 11%-15% 0.00 —0.00 15^-37 —0.00 —0.00 18 4.9 +0.06* +0.01 -0.07* -0.01 -0.02 +0.00 31 8.4 10 + 0.08* +0.00 —0.05 —0.06* —0.03 -0.00 49 13.1 +0.09* +0.05 0.00 -0.10 s1 —0.05 —0.00 67 18.1 10 +0.26* —0.10 0.00 —0.12* —0.07 -0.01 Average of all levels +0.095* .015 —0.030 —0.058* —0.028 .001 116 ILLINOIS BIOLOGICAL MONOGRAPHS [116 TABLE 58 Rana clamitans Series 3676-3765 First regenerations Acceleration of specific rate of regeneration of the tail Percent of tail length removed 6 10 18 31 49 67, Average of . all levels Average of four deepest levels Length removed In mm. 1.5 2.8 4.9 8.4 13.1 18.1 No. of in- dividuals 4 7 5 10 8 10 Days 2- 5 +0.033* +0.011* +0.012* +0.010* +0.007* •fO.014* +0.014* +0.011* 5- 7 —0.020 —0.007 +0.002 0.000 +0.004 —0.006 —0.004 0.000 7- 9 —0.027* —0.007 —0.014* —0.006 0.000 0.000 —0.009* —0.005 9-11% —0.013 —0.014* —0.002 —0.007* —0.008* —0.007 —0.008 —0.006* 11*4-15% 0.000 0.000 —0.004 —0.004 —0.004 —0.004 —0.003 —0.004 15*4-37 0.000 0.000 0.000 0.000 0.000 —0.001 0.000 0.000 117] RATE OF REGENERATION— ZELENY 117 TABLE 59 Rana clamitans Series 3676-3765 First regenerations Percentage increment of regenerating tail per day during each time period for six different levels Percent of tail length removed 6 10 18 31 49 67 Average of all levels Length removed in mm. 1.5 2.8 4.9 8.4 13.1 18.1 No. of in- dividuals 4 7 5 10 8 10 Days 4- 6 91 53 96 142 80 175 106 6- 8 23 19 36 32 29 30 28 8-10 5 9 8 14 19 19 12 10-12^ 6 5 8 9 5 12%-18 —2 —1 2 2 18-56 —0 —1 +0 —0 +0 ( +0 118 ILLINOIS BIOLOGICAL MONOGRAPHS [118 Experiment II Rana clamitans First regenerations of the tail Series 3676-3765 The tadpoles were collected on December 9, 1911, and the tail remov- als were made on January 8. Measurements were taken 4, 6, 8, 10, 12^2, 18 and 56 days after the operations. The operations were at six levels approximating 6, 10, 17, 30, 48 and 62 per cent of the original tail length. For the first of these levels only two individuals with an average removal of 1.5 mm. are available, for the second five individuals with 2.6 mm., for the third three with 4.6 mm., for the fourth eight with 8.2 mm., for the fifth five with 13.0 mm. and for the sixth five with 16.7 mm. The rates of regeneration per day are given in table 60 and the graphs for the rates in Figure 56. The specific rates are given in Table 61. Averaging these values so as to include all the different levels for each period the specific rates are 0.018 for to 4 days, 0.046 for 4 to 6 days, 0.057 for 6 to 8 days, 0.037 for 8 to 10 days, 0.026 for 10 to 12y 2 days, 0.002 for 12i/ 2 to 18 days and — 0.001 for 18 to 56 days. The graph is shown in the unbroken line in Figure 53. Using only the four deepest levels the average specific rates are respectively 0.013, 0.042, 0.045, 0.036, 0.025, 0.006 and 0.000 giving essentially the same form of curve as for the average of all levels. The accelerations of rate are shown in Table 62 and the accelerations of specific rate in Table 63 and in the unbroken line of Figure 54. The average accelerations of rate per day are respectively -j-0.078, 0.000, —0.022, —0.042, —0.025 and 0.000 mm. The average accelerations of specific rate including all levels are respectively -4-0.011, — 0.001, —0.007, —0.0075, —0.003 and 0.000 and including only the four deepest levels, 4-0.009, 0.000. —0.004, —0.005, —0.003 and 0.000. As for sec- ond regenerations the only plus acceleration is between 2 and 5 days and the most rapid decrease takes place between 9 and lli/4 days. The percentage increments per day are shown in Table 64 and in the unbroken line of Figure 55. The values are respectively 98, 29, 17, 6, 1 and percent per day giving approximately the same form of curve as for second regenerations. In general the first regenerations agree with the second but on the whole the second regenerations reach their maximum earlier and are more rapid than the first up to the time of maximum rate. The first are more rapid than the second after the maximum. 119] RATE OF REGENERATIOS —ZELENY 119 2 5 7 9 11% 15% — > Days after the operation Figure 56 Rates of first regenerations of the tail per day at different times after the operation for six different levels Rana clamitans The removed lengths are 1.5, 2.6, 4.6, 8.2, 13.0 and 16.7 mm. 120 ILLINOIS BIOLOGICAL MONOGRAPHS [120 TABLE 60 Rana clamitans Series 3676-3765 First regenerations Rate of regeneration of tail per day at different times during the regenerative process for six different levels Percent of tail length removed 6 10 17 30 48 62 Length removed in mm. 1.5 2.6 4.6 8.2 13.0 16.7 No. of individuals 2 5 3 8 5 5 Days 0- 4 0.07 0.02 0.12 0.10 0.12 0.12 4- 6 0.10 0.20* 0.25* 0.35* 0.55* 0.55 6- 8 0.15* 0.10 0.25* 0.30 0.50 0.70 8-10 0.05 0.15 0.10 0.30 0.40 0.75* I 10-12% 0.00 0.08 0.04 0.12 0.36 0.52 12%-18 0.00 —0.02 0.00 0.02 0.15 0.18 18-56 -0.01 0.00 —0.01 —0.01 0.00 0.00 121] RATE OF REGENERATION— ZELENY 121 TABLE 61. Rana clamitans Series 3676-3765 Second regenerations Specific rates at different levels at different times Percent of tail length removed 6 10 17 30 Length removed in mm. 1.5 2.6 4.6 8.2 No. of in- dividuals 2 5 3 8 Days 0- 4 0.042 0.015 0.027 0.012 4- 6 0.065 0.040 0.055* 0.040* 6- 8 0.115* 0.045 0.055* 0.040* 8-10 0.025 0.015 0.055* 0.020 0.035 10-12% 0.040 0.010 0.015 12%-18 —0.002 —0.007 0.000 0.004 18-56 —0.004 —0.001 —0.001 —0.001 48 13.0 0.000 62 16.7 5 5 0.007 « 0.007 0.045* 0.030 0.040 0.045* 0.045* 0.045* 0.035 0.040 0.011 0.009 0.000 —0.001 Average of all levels Average of four longest remov- als 0.018 0.013 0.046 0.042 0.057* 0-045* 0.037 0.036 0.026 0.025 0.002 0.006 0.000 122 ILLIX01S BIOLOGICAL MOXOGRAPHS [122 TABLE 62 Rana clamitans Series 3676-3765 First regenerations Acceleration of rate of regeneration of tail per day at different times during the regenerative process for six different levels Percent of tail length removed 6 10 17 30 48 62 Average of all levels Length re- moved in mm. 1.5 2.6 4.6 8.2 13.0 16.7 No. of in- dividuals 2 5 3 8 5 5 Middle of periods Days. 2- 5 +0.01 +0.06* + 0.04* + 0.08* +0.14* +0.14* +0.078* 5- 7 +0.02* —0.05* 0.00 —0.02 —0.02 +0.07 0.000 7- 9 —0.05* +0.02 —0.07* 0.00 —0.05* +0.02 —0.022 9-11% —0.02 —0.03 —0.02 —0.07* —0.02 —0.09* —0-042* ll^-lo^i 0.00 -0.02 —0.01 —0.02 —0.04 —0.06 —0.025 . 15%-37 —0.00 0.00 —0.00 —0.00 0.00 +0.00 -O.000 123] RATE OF REGENERATION— ZELENY 123 TABLE 63 Rana clamitans Series 3676-3765 First regenerations Acceleration of specific rate of regeneration of the tail Percent of tail length removed 6 10 18 31 49 67 Average of . all levels Average of four deepest levels Length removed in mm. 1.5 2.8 4.9 8.4 13.1 18.1 No. of in- dividuals 4 7 5 10 8 10 Days 2- 5 4-0.007 +0.023* f0.009* +0.010* -fO.011* + 0.008* -1-0.011* +0.009* 5- 7 f0.013* —0.019* 0.000 —0.002 -0.002 +0.004 —0.001 0.000 7- 9 —0.033* -1-0.008 —0.015* 0.000 —0.004* +0.001 —0.007 —0.004 9-11% —0.013 —0.012 —0.004 —0.002 —0.009* —0.002 —0.005* —0.007* —0.005* 11^-15% 0.000 —0.008 —0.002 —0.003 -0.004 —0.003 —0.003 15^-37 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 124 ILLINOIS BIOLOGICAL MONOGRAPHS [124 TABLE 64 Rana clamitans Series 3676-3765 First regenerations Percentage increment of regenerating tail per day during each time period for six different levels Percent of tail length removed 6 10 17 30 48 62 Average of all levels Length re- moved in mm. 1.5 2.6 4.6 8.2 13.0 16.7 No. of in- dividuals 2 5 3 8 5 5 Days 4- 6 33 200 50 87 110 110 " 98 6- 8 30 20 25 27 31 44 29 8-10 6 21 7 18 23 25 17 10-12% 8 2 5 9 12 6 12%-18 ' —2 1 3 3 1 18-56 . —0 —0 " —0 +0 Experiment III Rana clamitans . First and second regenerations op the tail. Series 3628-3675 For comparison with the data of experiments I and II it is of interest to note the results obtained from this entirely different series of the same species which was designed primarily for the comparison of first and second regenerations. A full description of the experiment is given in the section on the effect of successive removal upon the rate of regeneration. The data of specific value for present purposes are given in Table 65. Measurements were made only at six and at eight days after the operation. Fifty percent in length of the tail was removed in both first and second regenerations. Twenty- one individuals are available for first and sixteen for second regenerations. The rates per day are 0.52 mm. for 6 to 8 days for first regener- ations as compared with 0.50 for the same period in Experiment II and 0.62 for second regenerations as compared with 0.50 in Experiment I. The specific rate per day for 6 to 8 days for first regenerations is 0.049 125] RATE OF REGENERATION— ZELENY 125 and for second regenerations 0.057 as compared with 0.050 for forty eight percent removals in the first regenerations of Experiment II and 0.050 for forty nine percent removals in the second regenerations of Experiment I. The percentage increments per day are 26 for first regenerations as compared with 29 in Experiment II and 28 for second regenerations as compared with 28 in Experiment I. The close agreement of these values taken from a comparatively large number of individuals strengthens the conclusion as to the validity of the comparisons at different periods and levels in experiments I and II. TABLE 65 Rana clamitans Series 3628-3765 First and second regenerations of the tail Six and eight days No. of individ- uals Total length mm. Tail length mm. Percent of length re- moved Length re- moved Regen- erated length Six days Regen-i erated length Eight days Rate per day Spe- cific rate Percent- age in- crement per day First regeneration 21 32.7 21.4 50 10.6 2.01 3.06 0.52 0.049 26 Second regeneration 16 33.4 21.8 50 10.9 2.18 3.42 0.64 0.057 28 Experiment IV Amblystoma punctatum Tail Series 4600-5052 Operations were made at five levels approximating 10, 21, 34, 53 and 74 per cent of the original tail length. The removed lengths average respectively 1.1, 2.2, 3.7, 5.8 and 8.1 mm. Measurements were made 2, 4, 6, 8-9, 10-11, 13, 14-15 and 16-17 days after the operation. The rates per day for each of the levels at each of the different times are given in Table 66. The specific rates are shown in Table 67. The averages for all the levels at each of the time periods are respectively 0.032 mm. for to 2 days, 0.004 for 2 to 4 days, 0.053 for 4 to 6 days, 0.039 for 6 to 8 days, 0.064 for 8.4 to 10.3 days, 0.043 for 10.3 to 13.0 days, 0.012 for 13.0 to 15.2 days and 0.019 for 15.2 to 17.3 days. As in the case of other salamander experiments the data are more irregular than those for the frog tadpoles because of the susceptibility of the salamander larvae to factors which have not so far been brought under control. The character of the food is probably an important factor. The greatest rate comes between 8.4 and 10.3 days after the operation for three of the five levels and also for the average of all levels. This is later than 126 ILLINOIS BIOLOGICAL MONOGRAPHS [126 the maximum for the frog tadpole which comes between four and six days for second regenerations and between six and eight days for first regenerations. The period of decline in rate is also more extended in these salamander larvae than in the frog tadpoles of Experiments I and II. On account of the irregularity of the data it is not possible to study the acceleration of rate for the present data. The percentage increments per day are given in Table 68. The values for the seven time periods are respectively 8, 71, 20, 23, 14, 4 and 7. The greatest percentage increment comes between 4 and 6 days as in the case of the frog tadpoles. An earlier period, that be- tween two and four days is represented here. During this period the percentage increment is low. If this value can be accepted the curve here includes the very steep ascending portion discussed above. The irregularities in rate to which the salamander larvae are subject and the fact that the low value during this period does not appear in all the salamander experiments however makes the interpretation doubtful. TABLE 66 Amblystoma punctatum Series 4600-5052 Rate of regeneration of tail per day at different times during the regenerative process for five different levels Percent of tail , removed 10 21 34 53 74 Length removed, in mm. 1.1 2.2 3.7 .5.8 8.1 Days 0-2 0.05 0.07 0.07 0.23 0.26 2-4 0.01 0.00 0.07 —0.03 —0.06 4-6 0.10* 0.16 0.16 0.14 0.27 6-8.4 0.03 0.07 0.07 0.29 0.21 8.4-10.3 0.05 —0.01 0.39* 0.43 0.37 10.3-13.0 0.10* 0.11 0.07 0.07 0.51* 13.0-15.2 0.01 0.17* —0.17 0.18 0.09 15.2-17.3 —0.05 0.05 0.11 0.48* 0.41 127] RATE OF REGENERATION— ZELENY 127 TABLE 67 Amblystoma punctatum Series 4600-5052 Specific rates of regeneration of the tail at different levels at different times after the operation Percent of tail removed 10 21 34 53 74 Average of all levels Length re- moved in mm. 1.1 2.2 3.7 5.8 8.1 Days 0-2 0.035 0.035 0.020 0.040 0.030 0.032 2-4 0.020 —0.005 0.015 —0.005 —0.005 0.004 4-6 0.095 0.070 0.045 0.025 0.030 0.053 6-8.4 0.054 0.033 0.029 0.046 0.033 0.039 8.4-10.3 0.100* —0.011 0.105* 0.095* 0.042 0.064* 10.3-13.0 0.044 0.063 0.019 0.011 0.078* 0.043 13.0-15.2 —0.032 0.081* —0.036 0.018 0.027 0.012 15.2-17.3 0.000 0.005 005 0.069 0.014 0.019 128 ILLINOIS BIOLOGICAL MONOGRAPHS [128 TABLE 68 Amblystoma punctatum Series 4600-5052 Percentage increment of regenerating tail per day during each time period for five different levels Percent of tail removed 10 21 34 53 74 Average of all .levels Length re- moved in mm. 1.1 2.2 3.7 5.8 8.1 Days 2-4 10 50 —6 —12 8 4-6 83* 107* 53* 35 77* 71* 6-8.4 10 16 12 42* 20 20 8.4-10.3 13 —2 49 31 24 23 10.3-13.0 27 17 5 3 23 14 13.0-15.2 1 19 —10 8 3 4 15.2-17.3 —6 4 8 17 11 7 Experiment V Amblystoma punctatum Tail Series 4101-4540 The experiment consists of the regenerations of removed halves of the tail without additional injury in some individuals and with an additional removal of the two forelegs in others. Measurements were made at nine periods, 2, 4, 6, 8, 10, 12, 14, 16 and 19 days after the operation. The rates of regeneration are given in Table 69. The number of individuals for most of the levels is five. The full data are discussed in the section on the effect of degree of injury. The average rate for each of the different times shows that the maximum comes during the eight to ten day period. The high value for the greater degree of injury at 14 to 16 days is due to the death during that period of the two individuals with the lowest values. The result agrees very well with the maximum rate in Experiment IV. The percentage increments are given in Table 70. The highest value comes during the two to four day period followed by decrease with but little irregularity. 129] RATE OF REGENERATION— ZELENY 129 Experiment VI Amblystoma punctatum Tail Series 3962-4004 First, second and third regenerations after removal of approximately one-half of the tail were studied. The complete data are given in the section on the effect of successive removals. Measurements were made at 2, 4, 6, 8, 10 and 14 days. The rates per day are given in Table 71. The maximum rate comes between 8 and 10 days agreeing with the other data for regeneration of the tail in salamander larvae. The percentage increments are given in Table 72. The highest rate comes at the earliest period, between two and four days, and is followed by a rapid and then a slower decrease. TABLE 69 Amblystoma punctatum Series 4101-4540 Rate of regeneration per day of tail at different times during the regenerative process for two degrees of injury Period of regeneration Days 0-2 2-4 4-6 6-8 8-10 10-12 12-14 14-16 16-19 Middle of period Days after operation 11 13 15 17% Rate of regeneration per day for each period One-half tail 0.17 0.19 0.29 0.37 0.69* 0.46 0.23 0.37 0.16 One-half tail .+ fore-legs 0.13 0.27 0.25 0.47 0.50 0.46 0.37 0.53* 0.03 Average rate 0.15 0.23 0.27 0.42 0.59* 0.46 0.30 0.45 0.09 130 ILLINOIS BIOLOGICAL MONOGRAPHS [130 TABLE 70 Amblystoma punctatum Series 4101-4540 Percentage increment per day of regenerating tail at different times during the regenerative process for two degrees of injury Days Percentage increment per day during each period Average One-half tail One-half tail+ fore-legs 2 to 4 54* 100* 77 4 to 6 40 31 35 6 to 8 28 36 32 8 to 10 33 22 27 10 to 12 13 14 13 12 to 14 5 9 7 14 to 16 8 11 9 16 to '19 3 1 TABLE 71 Amblystoma punctatum Series 4101-4540 Rate of regeneration per day at different times during the regenerative process Period of regeneration Days Middle of period Days after operation Rate of regeneration per day during each period Average First Second Third 0-2 1 0.11 0.12 0.13 0.12 2-4 3 0.22 0.25 0.37 0.28 4-6 5 0.35 0.32 0.18 0.28 6-8 7 0.41 0.64* 0.66 0.57 8-10 9 0.68* 0.57 0.76* 0.67* 10-14 12 0.45 0.57 0.47 0.50 131] RATE OF REGENERATION —ZELENY 131 TABLE 72 Amblystoma punctatum Series 3962-4004 Percentage increment per day at different times during the regenerative process Days Percentage increment per day during each period Average First Second Third 2 to 4 100 100 142 114* 4 to 6 53 43 18 38 6 to 8 30 45 48 41 8 to 10 31 21 28 27 10 to 14 12 15 11 13 Experiment VII Amblystoma punctatum Series 4101-4540 Forelegs The experiment consists of the study of the rate of regeneration of single completely removed fore-legs under three degrees of injury to the individual : without additional injury, with the other fore-leg re- moved at the same time and with the other fore-leg plus one-half of the tail removed . Measurements were made at 2, 4, 6, 8, 10, 12, 14, 16 and 19 days. The rates of regeneration are given in Table 73. The maxi- mum rate does not come until the 14 to 16 period. The percentage increments are given in Table 74. The highest value comes during the 2 to 4 day period. There is a gradual decrease from this time. On the whole the data for the leg regeneration show a more extended period than do the tail regenerations^ 132 ILLINOIS BIOLOGICAL MONOGRAPHS [132 TABLE 73 Amblystoma punctatum Series 4101-4540 Rate of regeneration per day of fore-leg at different times during the regener- ative process for three degrees of injury Period of regeneration Days Middle of period Days after operation Rate of regeneration per day for each period Average One fore-leg Both fore-legs Both fore- legs + one-half tail 0-2 1 0.06 0.08 0.07 0.07 2-4 3 0.04 0.10 0.07 0.07 4-6 5 0.10 0.08 0.13 0.10 6-8 7 0.12 0.15 0.09 0.12 8-10 9 0.12 0.25 0.25 0.21 10-12 11 0.28 0.18 0.18 0.21 12-14 13 0.25 0.29 0.34 0.29 14-16 15 0.52* 0.41* 0.39* 0.44* 16-19 17% 0.27 0.21 0.27 0.25 133] RATE OF REGENERATION— ZELENY 133 TABLE 74 Amblystoma punctatum Series 4101-4540 Percentage increment per day of regenerating fore-leg at different periods for three degrees of injury Days Percentage increment per day during each period Average One fore-leg Two fore-legs Both fore- legs + one-half tail 2-4 34 62* 46* 47* 4-6 45* 23 45 38 6-8 28 28 16 24 8-10 19 30 35 28 10-12 31 9 15 18 12-14 17 18 21 19 14-16 26 19 17 21 16-19 14 10 13 12 Discussion The results obtained from the present study show that with certain material it is possible to control disturbing factors so as to get data of a sufficiently uniform nature for an analysis of the change in rate. Such material was found in the tails of the tadpoles of Rana clamitans. The analysis has yielded results which should be of value in a determination of the factors involved in the stimulation of growth and more particu- larly those concerned in slowing it down and finally bringing it to a stop. The characteristics of the change in rate have been studied by means of the curves of rate, of acceleration of rate and of percentage increments. The rate is slow at first, increases rapidly until it is near a maximum at about eight days; then decreases, at first rapidly and then more and more slowly as zero is approached. The acceleration of rate is plus only between the first two periods, i. e., up to the fifth day. After that it is minus, reaching its lowest point at ten days. The percentage increment 134 ILLINOIS BIOLOGICAL MONOGRAPHS [134 is very high between the first and second periods but decreases very rapidly at first and then more slowly. It is evident that there is a close similarity between the change in rate of growth during the regeneration cycle and the change in rate during an ordinary developmental cycle and there is every reason to believe that the factors controlling the one are similar to those controlling the other. The problem of the factors is particularly interesting when it is noted that for widely different levels the rates of regeneration differ in such a way that length regenerated in a given time is proportional to the length removed. The process of regeneration apparently is initiated in a similar manner at each level but is kept under such control that only a certain per cent of the length is regenerated in a given time. Knowledge of the process is at present insufficient to enable one to discuss with profit the nature of the control of rate of regeneration. All that can be done is to point out the relations of certain phenomena. The initial slow period is coincident with the period of cell migration without cell division, the period of rapidly increasing rate is coincident with the period of rapid cell multiplication without pronounced cell differentiation and the period of rapidly decreasing rate is associated with the appearance of pronounced differentiation in the cells. There is certainly some causal relation between these phenomena. Summary 1. In second regenerations of the tail in Rana clamitans the average specific rates are 0.019 mm. for the to 4 day period, 0.066 for the 4 to 6 day period, 0.051 for 6 to 8 days, 0.033 for 8 to 10 days, 0.017 for 10 to 12y 2 days, 0.001 for 12y 2 to 18 days and —0.001 for 18 to 56 days. 2. The average accelerations of rate are -f-0.095 mm. per day from the first to the second period, — 0.015 from the second to the third, —0.030 from the third to the fourth, —0.058 from the fourth to the fifth, — 0.028 from the fifth to the sixth and — 0.001 from the sixth to the seventh. 3. The average percentage increments between the same periods are respectively 106, 28, 12, 5, and 0. 4. The average accelerations of specific rate for the four deepest levels between the same periods are respectively -4-0.011 mm., 0.000, —0.005, —0.006, —0.004 and 0.000. 5. In first regenerations of the tail in Rana clamitans the average specific rates are 0.018 mm. for to 4 days, 0.046 for 4 to 6 days, 0.057 for 6 to 8 days, 0.037 for 8 to 10 days, 0.026 for 10 to 12y 2 days, 0.002 for 12y 2 to 18 days and —0.001 for 18 to 56 days. 6. The average accelerations of rate are +0.078 mm. per day from 135] RATE OF REGENERATION— ZELENY 135 the first to the second period, 0.000 from the second to the third, — 0.022 from the third to the fourth, —0.042 from the fourth to the fifth, —0.025 from the fifth to the sixth and 0.000 from the sixth to the seventh. 7. The average accelerations of specific rate for the four deepest levels between the same periods are respectively +0.009, 0.000, — 0.004, —0.005, —0.003 and 0.000. 8. The average percentage increments between the same periods are respectively 98, 29, 17, 6, 1 and 0. 9. The experiments on salamander larvae show a similar change in rate of regeneration during the process but the number of individuals is too small to allow an analysis of the data. 10. The changes in rate that have been noted bear a definite relation to the histological changes that have been observed during the regener- ation of the tail. 136 ILLINOIS BIOLOGICAL MONOGRAPHS [136 PART V THE EFFECT OF DEGREE OF INJURY UPON THE RATE OF REGENERATION In a former series of papers the writer gave the results of experi- ments on the effect of degree of injury upon the rate of regeneration. A number of different species of animals and various combinations of injuries were involved. The results then obtained tend on the whole to show that within certain limits the rate of regeneration from an injured surface is not retarded by simultaneous regeneration in other parts of the body. Where a difference exists between the rates with and without additional injury there is usually an advantage in favor of the part with additional injury. The differences are however often slight and in some of the cases come within the limits of probable error. It is only when the data as a whole are taken that it is possible to judge of the correctness of the general conclusion that within fairly wide limits of additional injury there is certainly no decrease in rate of regener- ation but rather a tendency toward an increase. Some additional data on these points have been obtained in connec- tion with the present study of the factors of regeneration. On the whole they confirm the previous results. The principal experiment (Experi- ment I) was planned with a view to further analysis of the problem, especially the determination of the effect of additional injury to a like organ as compared with additional injury to an unlike organ. Experiment I Amblystoma punctatum Series 4101-4540 The young were hatched on March 29-April 4, 1913, and the oper- ations were made on May 4 and 5. The measurements of the control individuals at the time of the operations are given in Table 75. The average total length is 31.3 mm., the tail length 14.4 mm., the average length of the fore-legs 3.6 mm. and the average of the hind-legs 1.5 mm. The measurements of control individuals at the end of the experi- ment on May 23 are given in Table 76. The total average length is 42.7 mm., the tail length 20.0, the average of the fore-legs 6.2 and the average of the hind-legs 4.5 mm. The experiment consisted in the determination of the regenerated length of the right fore-leg under three degrees of injury: when the 137] RATE OF REGENERATION— ZELENY . 137 right fore-leg alone is removed, when its mate is also removed and finally when its mate and one-half of the tail are removed. In the last two cases the average of the two fore-legs is taken as the proper value for the regeneration 'of a fore-leg. A large number of individuals, all hatched from the same lot of eggs, were used and a selection of larvae was made so as to make the experimental animals as nearly alike as possible in this respect. In each of the five sets an individual for each degree of injury was killed at two days after the operation, and also at four, six, eight, ten, twelve, fourteen, sixteen and nineteen days. The data are given in Tables 77 to 88. The three degrees of injury may be represented by (1) R, (2) R+L, (3) R+L+y 2 T, in which R=right fore-leg removed, L=left fore-leg removed and 1 / 2 T=one-half of the tail removed. The second involves the removal of some additional material of the same kind as that removed in the first. The third as compared with the first involves the removal of some of the same kind of material and some of another kind. In every case it is the regeneration of the fore-leg that is used as the basis of comparison. The additional simultaneous injury and regeneration does not de- crease the regeneration of the individual fore-leg. At two days the average regenerated lengths of a fore-leg are respectively 0.13, 0.16 and 0.15 mm. for the three degrees of additional injury; at four days the corresponding values are 0.22, 0.36 and 0.29 ; at six days 0.42, 0.53 and 0.55; at eight days 0.66, 0.83 and 0.73; at ten days 0.91, 1.34 and 1.24; at twelve days 1.48, 1.60 and 1.61 ; at fourteen days 1.98, 2.19 and 2.29 ; at sixteen days 3.02, 3.01 and 3.08 ; at nineteen days 3.84, 3.64 and 3.90. At only two of the nine periods is the regeneration of the fore-leg without additional injury as rapid as that of a fore-leg with additional injury and at these two times it is less rapid than one of the two other groups. In seven of the nine cases the regeneration of the fore-leg without additional injury is less than either of the two with such injury. Among the forty individual comparisons in which all three degrees are present the degree with no additional injury has 6% firsts, the degree with an additional fore-leg 15% firsts and the degree with an additional fore-leg plus one-half of the tail has 17% firsts. Among the nine time groups the degree with no additional injury has IV3 firsts and each of the additional injury combinations has 3% firsts. Taking up the lowest positions in the three degrees in the same way, among the forty individual comparisons the degree with no additional injury gives the lowest regeneration in 21i/ 3 cases while the additional injury combinations each have only 9% lowest regenerations. Among the nine time groups the degree with no additional injury has the lowest value 6 times, the one with an additional removal of the other fore-leg 138 ILLINOIS BIOLOGICAL MONOGRAPHS [138 2y 2 times while the one with the highest degree of injury gives the lowest regeneration for the fore-leg only y 2 times. These comparisons show very clearly that the regeneration of a fore- leg is not as rapid when the individual is regenerating no other part at the same time as it is when the other fore-leg is being regenerated at the same time. The additional removal of one-half of the tail does not seem to accelerate the regeneration any further because there is no essen- tial difference between the effect of an additional injury of a fore-leg and an additional injury of a fore-leg plus one-half of the tail. It may be that the effect of additional removal is confined to removal of a similar part, the tail removal in this case involving a different kind of organ. Or it may be that the accelerating effect is found only within certain degrees of injury the limit being exceeding by the highest of the three degrees. TABLE 75 Amblystoma punctatum Series 4101-4540 Experiment I Controls at beginning of experiment Date Cata- log number Total length mm. Tail length mm. Fore legs Hind legs Right Left Av'age. Right Left A.v'age. 5/4/13 4110 3 o.O 16.4 4.0 4.0 4.0 3.0 3.1 3.05 5/4/13 4210 31.8 14.8 3.6 3.8 3.7 1.4 1.5 1.45 5/4/13 T4310 28.1 11.9 3.3 3.3 3.3 1.0 0.9 0.95 I 5/4/13 [4320 33.8 15.3 3.3 3.6 3.1 3.2 1.1 1.0 1.05 J 5/5/13 4410 30.2 13.8 3.6 3.6 1.0 1.0 1.0 5/5/13 4510 28.7 14.0 3.9 3.8 3.85 1.4 1.2 1.3 Average 31.3 14.4 3.6 1.5 139] RATE OF REGENERATION— ZELENY 139 TABLE 76 Amblystoma punctatum Series 4101-4540 Experiment I Controls at end of experiment Date Cata- log number Total length mm. Tail length mm. Fore legs Hind legs Right Left Av'age. Right Left Av'age. 5/23 4120 4130 4140 46.7 44.7 44.5 24.3 21.7 20.1 6.1 6.5 6.5 6.1 6.6 6.4 6.1 6.55 6.45 5.0 5.2 5.2 5.0 5.1 5.1 5.0 5.15 5.15 Average 45.3 22.0 6.4 5.1 5/23 4220 4230 4240 43.1 45.5 43.7 20.1 20.6 20.4 6.1 6.0 6.0 6.0 6.0 5.5 6.05 6.0 5.75 4.1 4.0 4.0 4.1 5.0 4.4 4.1 4.5 4.2 Average 44.1 20.4 5.9 4.3 5/23 4330 4340 47.2 41.5 21.9 19.5 7.1 6.1 7.2 6.2 7.15 6.15 5.3 4.0 5.2 4.1 5.25 4.05 Average 44.3 20.7 6.6 4.6 5/23 4420 4430 4440 41.0 40.5 40.4 18.2 19.4 18.9 7.0 5.6 6.0 7.0 5.6 6.0 7.0 5.6 6.0 4.9 4.0 4.1 4.8 4.1 4.0 4.85 4.05 4.05 Average 40.6 18.8 6.2 4.3 5/23 4520 4530 4540 36.5 40.9 40.6 16.0 18.5 19.1 5.6 6.7 5.0 5.6 6.8 5.0 5.6 6.75 5.0 4.0 4.9 4.0 4.0 4.8 4.0 4.0 4.85 4.0 Average 39.3 17.9 5.8 4.3 Grand average 42.7 20.0 6.2 4.5 140 ILLINOIS BIOLOGICAL MONOGRAPHS [140 TABLE 77 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Two days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs 4- one-half tail 4101-11-21 0.10 0.22 0.22 4201-11-21 0.10 0.15* 0.11 4301-11-21 0.10 0.15 0.17* 4401-11-21 0.20 0.17 0.20 4501-11-21 0.15* 0.10 0.07 Average 0.13 0.16* 0.15 TABLE 78 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injurj Four days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs + one-half tail 4102-12-22 0.25 0.25 0.52 4202-12-22 — 0.52 0.22 4302-12-22 0.15 0.37* 0.22 4402-12-22 0.40* 0.30 0.27 4502-12-22 0.10 — 0.20 Average 0.22 0.36* 0.29 141] RATE OF REGENERATION— ZELENY 141 TABLE 79 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Six days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs + one-half tail 4103-13-23 0.40 0.20 0.92* 4203-13-23 0.50 0.87* 0.52 4303-13-23 0.45 0.65* 0.42 4403-13-23 0.45 0.60* 0.47 4503-13-33 0.30 0.35 0.42* Average 0.42 0.53 0.55* TABLE 80 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury. Eight days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs ■f one-half tail 4104-14-24 0.50 0.75 0.97* 4204-14-24 0.80 0.80 0.80 4304-14-24 0.85 0.87* 0.62 4404-14-24 0.43 0.95* 0.75 4504-14-24 0.70 0.80* 0.52 Average 0.66 0.83* 0.89* 142 ILLINOIS BIOLOGICAL MONOGRAPHS [142 TABLE 81 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Ten days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs -f one-half tail 4105-15-25 0.25 1.82* 1.60 4205-15-25 0.95 1.10* 1.07 4305-15-25 1.05 1.22 1.32* 4405-15-25 1.20 1.37* 1.07 4505-15-25 1.10 1.20* 1.12 Average 0.91 1.34* 1.24 TABLE 82 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Twelve days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs + one-half tail 4106-16-26 1.45 1.50 1.77* 4206-16-26 1.35 1.47* 1.44 4306-16-26 1.80* 1.60 1.65 4406-16-26 1.00 1.70* 1.50 4506-16-26 1.80* 1.72 1.67 Average 1.48 1.60 1.61* 143] RATE OF REGENERATION— ZELENY 143 TABLE 83 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Fourteen days Degree of injury Both Catalog One Both fore-legs number fore-leg fore-legs + one-half tail 4107-17-27 2.60 2.25 4207-17-27 1.70 1.97 2.22* 4307-17-27 1.45 1.87 1.95* 4407-17-27 2.25 2.72 2.90* 4507-17-27 1.90 2.12 Average 1.98 2.19 2.29* TABLE 84 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Sixteen days Degree of injury Catalog number One fore-leg Both fore-legs Both fore-legs -f one-half tail 4108-18-28 4208-18-28 4308-18-28 4408-18-28 4508-18-28 2.60 2.40 2.80 3.60 3.70 2.60 2.62* 2.67 3.57 3.57 2.70* 2.22 2.85* 3.65* 3.97* Average 3.02 3.01 3.08* 144 ILLINOIS BIOLOGICAL MONOGRAPHS [144 TABLE 85 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Nineteen days Degree of injury Catalog number One fore-leg Both fore-legs Both fore-legs + one-half tail 4109-19-29 4209-19-29 4309-19-29 4409-19-29 4.00* 3.65 3.60 4.10* 3.72 4.05 2.85 3.95 3.95 4.25* 3.60 3.80 Average 3.84 3.64 3.90* TABLE 86 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg in millimeters for different degrees of injury Summary Two to nineteen days 1 Degree of injury Days One fore-leg Both fore-legs Both fore-legs + one-half tail 2 0.13 0.16* 0.15 4 0.22 0.36* 0.29 6 0.42 0.53 0.55* 8 0.66 0.83* 0.73 10 0.91 1.34* 1.24 12 1.48 1.60 1.61* 14 1.98 2.19 2.29* 16 3.02 3.01 3.08* 19 3.84 3.64 3.90* Groups first Groups last 7 4 2 5 145] RATE OF REGENERATION— ZELENY 145 TABLE 87 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg for different degrees of injury Tabulation of firsts for individual comparisons Injury Days One fore-leg Both fore-legs Both fore-legs + one-half tail 2 1% 1% 2* 4 1 1 1 6 3* 2 8 y 3 3H* 1* 10 4* 1 12 2 2 1 14 3* 16 1 4* 19 2 2 Total firsts 6Ve 15% 17J* Groups first w 3% 3% 146 ILLINOIS BIOLOGICAL MONOGRAPHS [146 TABLE 88 Amblystoma punctatum Series 4101-4540 Length of regenerated fore-leg for different degrees of injury Tabulation of lowest values for individual comparisons Injury Days One fore-leg Both fore-legs Both fore-legs + one-half tail 2 3 1 1 4 iy 2 1% 1 6 3 1 1 8 2H Vs 2^ 10 4 1 12 3 1 1 14 3 ' 16 % 3% 1 19 i 2 1 Total lasts 21^ 9H m Groups last 6 2y 2 Vz Experiment II Amblystoma punctatum Series 4101-4540 This experiment deals with the same series of individuals as Experi- ment I. The comparison in this case however is one between the regen- eration of the removed half of the tail when it alone is removed and its regeneration when there is an additional removal of the two fore-legs. The data are given in Tables 89 to 99. At two days the regeneration of the tail without an additional injury is 0.35 mm. and with an additional injury 0.27. The corresponding values at 4 days are 0.73 and 0.81, at 6 days 1.32 and 1.31, at 8 days 2.06 and 2.26, at ten days 3.44 and 3.27, at twelve days 4.36 and 4.20, at fourteen days 4.82 and 4.94, at sixteen days 5.57 and 6.00 and at nineteen days 5.90 and 6.06. The regenerating tail with no additional injury is ahead at four times and the one with additional injury is ahead five times. In thirty three individual com- 147] RATE OF REGENERATION— ZELENY 147 parisons the group with no additional injury is ahead seventeen times and the additional injury group sixteen times. Taking the individual cases by time groups the individuals with no additional injury are ahead 5y 2 times and those with an additional injury 3y 2 times. These comparisons show no advantage of one combination over the other. The additional removal of the fore-legs does not retard nor does it accelerate the regeneration of the tail. This result strengthens the view that the acceleration in Experiment I is probably due to the addi- tional removal of material similar to that whose rate is being studied. TABLE 89 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Two days Degree of injury Catalog number 4131-21 4231-21 4331-21 4431-21 4531-21 Average One-half tail + fore-legs 0.15 0.35 0.25 0.30 0.30< 0.27 TABLE 90 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Four days Catalog number 4132-22 4232-22 4332-22 4432-22 4532-22 Average Degree of injury One-half tail 4- fore-legs 1.00* 0.90 0.60* 0.95 0.60 0.81* 148 ILLINOIS BIOLOGICAL MONOGRAPHS [148 TABLE 91 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Six days Degree 3f injury Catalog One-half One-half number tail tail + fore-legs 4133-23 1.60 4233-23 0.90 1.00* 4333-23 1.70* 1.65 4433-23 1.10 1.50* 4533-23 1.10 Average 1.32* 1.31 TABLE 92 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Eight days Degree of injury Catalog number One-half tail One-half tail + fore-legs 4134-24 4234-24 4334-24 4434-24 4534-24 2.40 1.80 1.80 2.70* 1.60 2.60* 1.90* 2.26* 2.30 Average 2.06 2.26* 149] RATE OF REGENERATION— ZELENY 149 TABLE 93 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Ten days Degree of injury Catalog One-half One-half number tail tail + fore-legs 4135-25 3.65* 3.20 4235-25 2.55 4335-25 3.20 1.46 4435-25 3.65* 3.20 4535-25 3.25 4.15* Average 3.44* 3.27 TABLE 94 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Twelve days Catalog number 4136-26 4236-26 4336-26 4436-26 4536-26 Average injury One-half tail -f fore-legs 4.20* 3.55 3.50 4.50 5.25* 4.20 ISO ILLINOIS BIOLOGICAL MONOGRAPHS [ISO TABLE 95 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Fourteen days Catalog number 4137-27 4237-27 4337-27 4437-27 4537-27 Average Degree of injury One-half tail + fore-legs 6.00* 4.70 4.95 4.00 5.05* 4.94* TABLE 96 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Sixteen days Catalog number 4138-28 4238-28 4338-28 4438-28 4538-28 Average Degree of injury One-half tail -f- fore-legs 5.50 6.40* 6.10 6.00* 151] RATE OF REGENERATION— ZELENY 151 TABLE 97 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Nineteen days Degree of injury TABLE 98 Amblystoma punctatum Series 4101-4540 Length of regenerated tail in millimeters for different degrees of injury Summary Two to nineteen days Degree of injury Days One-half tail One-half tail + fore-legs 2 0-35* 0.27 4 0.73 0.81* 6 1.32* 1.31 8 2.06 2.26* 10 3.44* 3.27 12 4.36* 4.20 14 4.82 4.94* 16 5.57 6.00* 19 5.90 6.06* Groups first 4 5 152 ILLINOIS BIOLOGICAL MONOGRAPHS [152 TABLE 99 Amblystoma punctatum Series 4101-4540 Length of regenerated tail for different degrees of injury Tabulation of firsts for individual comparisons Injury Days One-half One-half tail tail + fore-legs 2 2%* 1% 4 2% 2% 6 1 2* 8 1 3* 10 2* 1 12 3* 2 14 3* 2 16 1 1 19 1 1 Total firsts 17 16 Groups first 5«/ 2 3J/ 2 Experiment III Amblystoma punctatum Series 4005-4008 Experiments III, IV, V and VI comprise merely a few individual comparisons obtained from experiments devised principally for the study of other factors. They are included here under the rule that no valid data on the matter at hand are to be excluded. In Experiment III the regeneration of the hind-leg is compared under the four conditions of (1) no additional injury, (2) removal of the other hind-leg, (3) removal of the other hind-leg and one fore-leg and (4) removal of the other hind-leg and both fore-legs. The data are given in Table 100. Three sets of comparisons were made at twelve days after the oper- ations, each with a single individual for each degree of injury. The regenerating hind-leg with no additional injury is distinctly behind the cases with additional injury. The greatest regenerated length comes in one case with an additional injury of one hind-leg plus one fore-leg and in two cases with one hind-leg plus two fore-legs. The averages begin- 153] RATE OF REGENERATION— ZELENY 153 ning with the lowest degree of injury are respectively 1.50, 1.73, 1.86 and 1.88 mm. The additional removals are in every ease removals of leg material and the result agrees with that of experiment I in giving an increased rate of regeneration of a part when similar organs are removed at the same time. TABLE 100 Amblystoma punctatum Series 4005-4008 Length of regenerated hind leg in millimeters for different degrees of injury- Twelve days Catalog number 4005 4006 4008 Average One hind-leg 1.35 1.65 1.50 1.50 Degree of injury Both hind-legs 1.90 1.80 1.50 1.73 Both hind- legs+one fore-leg 1.95" 1.82 1.80 1.86* Both hind- legs+both fore-legs 1.75 1.92" 1.85" 1.84 TABLE 101 Amblystoma punctatum Series 4005-4008 Length of regenerated fore-leg in millimeters for different degrees of injury Twelve days Degree of injury Catalog number One Both fore-leg fore-legs + both + both hind-legs hind-legs 4005 3.0* 2.8 4006 3.1* 3.0 4008 3.0 3.15* Average 3.07* 2.98 Experiment IV Amblystoma punctatum Series 4005-4008 In this experiment the regeneration of the right fore-leg is compared under conditions of differing degrees of additional injury. In one com- bination there is an additional removal of the two hind legs and in the 154 ILLINOIS BIOLOGICAL MONOGRAPHS [154 other of both hind-legs plus the remaining fore-leg. The data are given in Table 101. In two of the three cases the smaller additional degree of injury shows the greater regeneration of the fore-leg. The average is 3.07 mm. for the lesser degree and 2.98 for the greater degree, an advantage in favor of the lesser degree. It should be noted that this is not strictly comparable with the main issue of Experiments I, II and III. Aside from the small number of cases it is a comparison between two degrees of injury each of which is of considerable extent. It may be that the removal of three of the four legs is near the degree of injury yielding the maximum rate for each removed leg. Experiment V Ambltstoma punctatum Series 4010-4025 A comparison is made between the regeneration of a half of the tail when it alone is removed and when both fore-legs are removed at the same time. Four individual comparisons are made at fourteen days. The data are given in Table 102. The regenerated lengths and specific lengths regenerated are ahead in two of the four cases for each of the degrees of injury. The average regenerated length with no additional injury is 5.1 mm. and with additional injury 5.0 mm. The specific regenerated length is 0.65 with no additional injury and 0.68 with addi- TABLE 102 Amblystoma punctatum Series 4010-4025 Regeneration of tail for different degrees of injury Fourteen days Degree of injury One-half tail One-half tail + both fore-legs Catalog number Length removed Length regener- ated Specific amt. re- generated Length removed Length regener- ated Specific amt. re- generated 4014-13 7.7 4.9 0.64 7.0 5.2* 0.74* 4018-17 8.8 5.2* 0.59* 8.0 4.3 0.54 4022-21 8.0 5.3* 0.66* 8.0 5.1 0.64 4025-24 7.0 4.9 0.70 6.6 5.3* 0.80* Average 5.1 0.65 5.0 0.68 155] RATE OF REGENERATION— ZELENY 155 tional injury. The data show essential equality between the rates of regeneration under the two conditions of the experiment. This agrees with the data in Experiments I and II which show no increase or decrease in rate of regeneration when unlike material is removed simultaneously with the removal of the organ whose rate is being studied. Experiment VI Amblystoma punctatum Series 4010-4025 Three individual comparisons were made at fourteen days of the right fore-leg, when it alone is removed, when the other fore-leg is also removed and when the other fore-leg plus one half of the tail is removed. The data are given in Table 103. In two of the three cases the individuals TABLE 103 Amblystoma punctatum Series 4010-4025 Length of regenerated fore-leg in millimeters for different degrees of injury Fourteen days Catalog number 4011, 12, 13 4015, 16, 17 4019, 20, 21 4023, — , 24 Average Degree of injury One fore-leg 2.00* 2.00* 1.95 2.00 1.99* Both fore-legs 1.77 1.60 1.82 1.73 Both fore-legs -f- one-half tail 1.65 1.80 2.22* 2.00 1.92 with no additional regeneration are ahead of the others. The greater injury gives the greater rate in one of the three. The average regener- ated lengths beginning with the lowest degree of injury are respectively 1.99, 1.73 and 1.92 mm. The few cases may be a sufficient explanation of the lack of agreement with the more extended series of Experiment I. Discussion The experiments as a whole show that a part regenerates slightly more rapidly when additional material of the same kind is removed than when the part alone is removed. Simultaneous removal of tail material does not accelerate the regeneration of a leg nor does simultaneous re- moval of a leg accelerate the regeneration of the tail. The rate in these cases however is not decreased by the additional injury. The state- 156 ILLINOIS BIOLOGICAL MONOGRAPHS [156 ment may therefore be made that within limits the regeneration of a part is not retarded by simultaneous removal and regeneration of material in other parts of the body. When this additional material is of the same kind as that whose rate is being studied there may even be an acceler- ation of regeneration. In comparison with such a factor as level of the cut this difference in rate is slight and no such quantitative relation as in that case can be made out. It must however be considered that the principal object of the original experiments was to show that additional injury within the given limits tends to increase rather than decrease the rate of regeneration. This has been proved for these experiments. The evidence in favor of a definite increase in rate with any certain increase in degree of injury is not so conclusive. It is obvious that in many series of experiments factors whose influence is greater than that of the factor under discus- sion may obscure the result. Emphasis should again be placed on the fact that all data obtained by the writer are included. That some of the series, especially those with a few individuals, diverge from the general result is to be expected by anyone in similar work who has attempted to eliminate entirely all of the factors except the one under observation at a particular time. Summaky 1. A comparison was made of the rate of regeneration of a leg or of the tail of an Amblystoma larva when the part alone is removed with its rate when similar or dissimilar parts of the individual are removed at the same time. The data are derived from two principal Experiments, I and II, and from a few scattered observations listed as Experiments III to VI. 2. In Experiment I a comparison was made of the rate of regener- ation of the right fore-leg when it alone is removed with its rate when the other fore-leg is removed at the same time and when the other fore- leg and one half of the tail are removed. The result obtained from forty individual comparisons made at different times shows that the rate of regeneration of the right fore-leg in each of the series with additional injury is greater than in the series without additional injury. 3. The rate of regeneration of a right fore-leg when its mate plus one-half of the tail is removed is not essentially different from the rate when its mate alone is removed. The addition of the injury to a dissimilar organ, the tail, does not alter the rate of regeneration of the fore-legs. 4. In Experiment II it is shown that there is no significant differ- ence between the rate of regeneration of a tail one-half of which has been 157] RATE OF REGENERATION— ZELENY 157 removed without additional injury to the individual and the rate after the same injury plus a removal of both fore-legs. 5. The data of Experiments III to VI show some departures from the general rule probably because they deal with few individuals. On the whole however they bear out the results obtained from the principal experiments. 158 ILLINOIS BIOLOGICAL MONOGRAPHS [158 PART VI THE COMPLETENESS OF REGENERATION One of the striking facts in connection with amphibian regeneration as made out in the present studies is the lack of completeness of the process. When a part of the tail is removed the lost part is never com- pletely restored. Data on this problem are to be found in a number of sets of experiments one of which (Experiment V) was devised especially for the present purpose. Experiment I Rana clamitans Series 3557-3624 One-half of the tail was removed in the individuals of three groups, A, B and C. After 35 to 39 days, which was sufficiently long so that regeneration had stopped, another removal was made and so on until each individual had undergone five regenerations. The data are given in Table 104. The average removed length as estimated from the measure- ment of a few individuals was 17.0 mm. The average length of the com- pleted first regeneration is 8.6 mm. or 51 per cent of the removed length, of the second regeneration 8.0 mm. or 53 per cent, of the third 7.5 mm. or 51 per cent, of the fourth 5.5 mm. or 42 per cent and of the fifth 6.4 mm. of 45 per cent. On the average about one-half of the removed length is replaced when one-half of the tail length is removed. Experiment II Rana clamitans Series 3628-3675 One-half of the tail length was removed in the individuals of this experiment and regeneration was allowed to proceed for twenty days, a sufficient time for bringing it to a stop. The data are given in Table 105. The average original tail .length was 21.8 mm., of the removed length 10.6 mm. and of the regenerated length 5.4 mm. The completed regenerated length is thus 51 per cent of the removed length. Experiment III Rana clamitans First regenerations Series 3676-3765 The data are given in Table 106. The tails were removed at different levels approximating 6, 10, 17, 30, 48 and 62 per cent of the tail lengths. Regeneration was completed at these levels at 12^2, 12%, 12V2> 18, 18 159] RATE OF REGENERATION— ZELENY 159 and 56 days respectively. The regenerated lengths at these times of completion are respectively 61, 46, 39, 33, 42 and 41 per cent of the removed lengths. It will be noted that the two shortest removals give the highest per cents and the two medium ones the lowest per cents. This difference is discussed in Part III on the effect of level of the cut. Experiment IV Kana clamitans Second regenerations Series 3676-3765 The data are given in Table 107. The tail was removed at different levels approximating 6, 10, 18, 31, 49 and 67 per cent of the removed lengths. Regeneration was completed for these levels at 10, 10, 12%, TABLE 104 Rana clamitans Series 3557-3624 Completeness of regeneration Successive regenerations in single individuals One-half of tail removed ■= 17 mm. on the average First operation Oct. 23, 1911 Second operation Groups A and B Nov. 18 Group C Nov. 28 First Second Third Fourth Fifth regener- regener- regener- regener- regener- Catalog ation ation ation ation ation number Nov. 28 Jan. 3 Feb. 9 Mar. 16 April 24 3564 9.5 8.5 — — 3565 9.8 11.4 7.3 8.2 3566 10.0 9.3 8.1 6.3 Group 3567 11.9 9.5 8.0 11.9 A 3568 8.4 9.9 11.0 8.5 3569 10.0 8.7 8.0 8.1 3570 8.7 8.1 — — Average 9.8 9.3 8.5 8.6 3578 8.3 9.0 5.8 8.2 3579 8.2 8.1 11.3 7.0 3580 11.9 7.3 6.9 8.1 Group 3581 9.7 8.0 7.6 11.6 B 3582 8.3 12.8 7.4 5.4 3583 8.8 7.4 5.0 6.8 3584 8.8 9.5 6.4 — Average 9.1 8.9 7.2 7.8 160 ILLINOIS BIOLOGICAL MONOGRAPHS [160 TABLE 104 (Continued) First Second Third Fourth Fifth regener- regener- regener- regener- regener- Catalog ation ation ation ation ation number Nov. 28 Jan. 3 Feb. 9 Mar. 16 April 24 3586 8.1 — — — — 3588 6.1 7.5 7.3 5.7 7.2 3590 8.5 6.6 7.5 5.1 6.5 3592 7.1 8.0 5.7 4.9 6.5 3594 8.6 7.8 2.0 5.0 6.1 3596 9.0 8.6 9.4 6.7 6.8 3598 10.7 9.7 9.3 6.1 6.2 3600 8.2 8.0 6.9 5.8 5.9 3602 9.9 7.7 6.8 4.4. 4.7 Group 3604 9.6 7.6 6.6 4.9 5.7 C 3606 7.4 7.8 8.0 5.0 5.9 3608 9.0 8.0 9.0 5.5 6.9 3610 8.5 8.9 8.3 5.4 7.1 3612 7.4 7.0 7.1 4.8 5.5 3614 8.3 6.6 6.2 4.5 5.2 3616 8.0 8.3 7.9 6.1 7.9 3618 9.7 9.5 9.7 7.3 8.0 3619 — 8.0 7.5 6.6 6.1 3622 10.2 8.5 — — — 3624 9.3 7.5 — Average 8.6 8.0 7.5 5.5 6.4 Percent Df removed length r egen. Av. 51 53 51 42 45 1214, 56 and 56 days respectively. The regenerated lengths at these times of completion are respectively 67, 46, 33, 31, 40 and 39 per cent of the removed lengths. As in the case of the first regenerations the two shortest removals give the highest per cent of regeneration and the two medium removals the lowest per cent. Experiment V Amblystoma punctatum Series 6212-6281 The experiments on tadpoles of Rana clamitans having shown that only a half or less of the removed length on the average is completed dur- ing regeneration it became a matter of interest to see if this might not have been due to the age of the tadpoles, which were obtained in the fall. Accordingly a series of Amblystoma larvae was operated upon within a 161] RATE OF REGENERATION— ZELENY 161 few days after they had left the egg envelopes and was kept until the salamanders were well advanced in their metamorphosis. Since in young salamander larvae the border line between old and regenerated tissue is soon obliterated it became necessary to devise another method of testing completeness of regeneration than the direct measurement of the regen- TABLE 105 Rana clamitans Series 3628-3675 Regenerated Tail Removed length length length Twenty days 24.1 13.1 5.9 24.6 13.2 5.2 22.1 11.0 4.9 23.2 11.1 5.5 23.1 11.7 5.9 25.0 12.5 5.8 20.4 9.9 5.6 20.8 10.0 5.2 29.2 15.5 5.9 23.8 10.5 5.6 23.3 10.9 5.6 25.6 10.9 5.9 20.8 10.1 4.7 19.2 9.8 4.6 21.1 11.5 5.5 22.0 11.8 6.1 17.0 8.2 5.6 19.0 9.7 5.5 22.4 9.8 4.3 19.8 10.1 5.2 20.8 8.4 5.1 21.8 9.1 5.0 15.4 7.3 6.0 18.1 9.0 6.0 21.8 10.6 5.4 Percent removed 49 Percent of removed part rege derated 51 162 ILLINOIS BIOLOGICAL MONOGRAPHS [162 TABLE 106 Rana clamitans Series 3676-3765 First regenerations Average Percent Tail maximum Average Days after Number of tail length regeneration maximum operation of length removed in regeneration when maxi- cases removed in mm. percent of in mm. mum is Average Average removed length reached Average 2 6 1.5 61 0.9 12% 5 10 2.6 46 1.2 12% 3 17 4.6 39 1.8 12% 8 30 8.2 33 2.7 18 5 48 13.0 42 5.5 18 5 62 16.7 41 6.9 56 TABLE 107 Rana clamitans Series 3676-3765 Second regenerations Average Percent Tail maximum Average Days after Number of tail length regeneration maximum operation of length removed in regeneration when maxi- cases removed in mm. percent of in mm. mum is Average Average removed length reached Average 4 6 1.5 67 1.0 10 7 10 2.8 46 1.3 10 5 18 4.9 33 1.6 12% 10 31 8.4 31 2.6 12% 8 49 13.1 40 5.2 56 10 67 18.1 39 7.1 56 163] RATE OF REGENERATION— ZELENY 163 erated material. This consisted in a comparison of the ratio between tail length and body length in the operated individuals with that in control unoperated individuals. This was done after regeneration had been tail going on during the whole larval period. If the } r~T~ period is the same in operated as in unoperated individuals it is proper to suppose that regeneration has been complete. If however the ratio is lower the conclusion that regeneration is incomplete is very probably correct though absolute certainty can not be assumed because of the possibility of the changed ratio being due to regulatory changes in other parts of the individual. The experiment consists of a comparison of the relative degree of completeness of regeneration of the tail in four groups, (1) with no operation, (2) with one-fourth of the tail removed, (3) with one-half of the tail removed and (4) with three-fourths removed. The operations were made as soon as possible after the animals left the egg envelopes and the experiment proceeded until all four legs were well developed and absorption of the gills had begun. This allowed practically the entire larval period for regeneration. There were seventy individuals at the start but a high mortality reduced the number very considerably. Lim- nodrilus was used as food. The data are given in Tables 108 to 112. The average ratio between tail and body length in control individuals at the end of the experiment is 1.09, in individuals with one-fourth of the tail removed it is 1.01, in those with one-half removed 0.93 and with three-fourths removed 0.86. This progressive relative decrease in the tail length as compared with the body length is very probably due to lack of completeness of regeneration even though the whole larval period has been allowed for such completion. Discussion Apart from the starting stimulus in regeneration the most interesting problem is undoubtedly that of the stopping stimulus. With the growth once started what are the factors involved in checking it 1 In general it has been assumed that regeneration goes on until the removed organ is entirely replaced and that over- and under-regeneration occur but rarely. The present data make it probable that incompleteness is more general than has been supposed. The factors at work in bringing regeneration to a close tend to overdo rather than underdo their function. A further investigation of the problem of completeness of regener- ation would be of interest. 164 ILLINOIS BIOLOGICAL MONOGRAPHS [164 T. 4_9 ,_ a B *3 o ^ n os M r~. O o 00 U5 o rH os a •S3 o iH o rH rH © T- — o q *n i-i H 1-i tA rH — rH T-i rH !• t» ei eo" d — i r~ rH o d cq N CO eo CO eo > -a o "E H m 1-1 oo oo iH cnj OS 00 eo ■* N. & x 6 X t-; t- oo r- EC t^ i- 00 fs. © d © c= d O d o o d 3 « 3 d ■d H | bO a © •■* 00 35 rH t« b; © eo M CO os eo CO 2 > 3 Eh a tJ* r? M L~ ■*« re ■J -» id bO fis V 5 a 00 N eo CO t» CO t- CO 00 ra rH •M ■M DC eo ■* >♦ CO CO cn ri M M B4 ■M •M ^] e o d < 165] RATE OF REGENERATION —ZELENY 165 ii -•-> © _ >> iH 00 ■* CNI T- •o T-l O) © OS o a a o PQ 1-i © T- 1 o o © «M IS >> CM CD © ■*. CO o 1— 1 OS o PQ bo id CO ■*< ■*' ^t t3 iH (3 0> " iH IH 1-1 T ~ OS T3 H oa a p i © > O a 43 00 CO io w ■O 43 •-» "3 bO d CO •* CO ■* © bO H a tH rH iH 7-t , ~ ^ H = "3 0) , " H O .2 "3 43 © OS US t~ CO *-> IS O on CN* -.- od t~ 00 M 3 © CO CI :i CNI CM S3 O © i i a ? 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X V 5 s CO CO CO CO CM T3 •" 5? a c *" ■" 11 " r " 4) ^ co M t>- o 5 ■? re Ol m ■^r CM 1- en H c T " T " T " " r " c • T3 "re CM 00 CM CO — 4* O) O ai CO CO ^ h c CO CM CM CM ■a I 1 ' > en O "* E CM io r^ — O o o V re *> OC 1- E 4) E T3 > *d r^ eo co a E O) •- eo ^J- Q. a> X OC H- o i > ■o N 00 i- en .E S re o fs i-- CO N- 1- CQ O o o O .E en o> ,_ V 00 E >» *• en t- CO a. < 73 O 03 O) c <> CO r^ CO N. £ *> D) e o _ CM «- CO CM re O) cc to co co en c 1- u > -1 £ -M cr T— N. O o o o> B r* ^ * >* 1- V • ^ • re • *> c o -t-" .~ CO o 7 re «t re re v re «* ^ a c o ro 7, O o -t- * . V c V t. c c C £ ! c o O H 167] RATE OF REGENERATION— ZELENY 167 BIBLIOGRAPHY Abel M. 1902. Beitrage zur Kenntnis der Regenerationsvorgange bei den limicolen Oligochaeten. Z. f. wiss. zool., 63:1-74. Allen, W. E. 191 1. A Study of the Relation of Tissue Differentiation to Rate of Growth during Regeneration. Biol Bull., 21 : 187-206. Barfurth, D. 1903. Die Erscheinungen der Regeneration bei Wirbeltierembryonen. O. Hertwig's Handbuch der Entwickelungslehre der Wirbeltiere. Bonnet, C. 1745. Traite d'insectologie. Seconde partie. Observations sur quelques especes de vers d'eau douce, qui, coupe par morceaux deviennent autant d'animaux complets. Paris. Davenport, C. B. 1899. Experimental Morphology, Part II, New York. Driesch, Hans 1897. Studien iiber das Regulationsvermogen der Organismen, I. Von den regulativen Wachstums-und Differenzirungs-fahigkeiten der Tubularia. Arch. f. Entw. Mech., 5:389-418. Durbin, Marion L. 1909. An Analysis of the Rate of Regeneration Throughout the Regener- ative Process. J. Exp. Zool., 7 :397-420. Ellis, M. M. 1909. The Relation of the Amount of Tail Regenerated to the Amount Removed in Tadpoles of Rana clamitans. J. Exp. Zool., 7 :42i-456. Em mel, V. E. 1006. The Relation of Regeneration to the Molting Process in the Lobster. Thirty-sixth Annual Report of the Commissioners of Inland-Fisheries of Rhode Island. Special paper, no. 27:257-313. Kam merer, Paul 1905. Ueber die Abhangigkeit des Regenerationsvermogens der Amphib- ienlarven von Alter, Entwicklungsstadium und spezifischer Grosze. Arch, f. Entw. Mech., 19:148-180. King, Helen D. 1898. Regeneration in Asterias vulgaris. Arch. f. Entw. Mech., 7 :35i-363. Minot, C. S. 1908. Age, Growth and Death. New York. 168 ILLINOIS BIOLOGICAL MONOGRAPHS [168 Morgan, T. H. 1902. Further Experiments on the Regeneration of the Tail of Fishes. Arch. f. Entw. Mech., 14:539-561. 1906. The Physiology of Regeneration. J. Exp. Zool., 3 :457-50o. 1909. The Dynamic Factor in Regeneration. Biol. Bull., 16:265-276. Morgulis, S. 1907. Observations and Experiments on Regeneration in Lumbriculus. J. Exp. Zool, 4:549-574. 1909a. Regeneration in the Brittle-Star Ophiocoma pumila, with Special Ref- erence to the Influence of the Nervous System. Proc. Amer. Acad, of Arts and Sc, 44:655-659. 1909b. Contributions to the Physiology of Regeneration. I. Experiments on Podarke obscura. J. Exp. Zool., 7:595-642. 1909c. Contributions to the Physiology of Regeneration. II. Experiments on Lumbriculus. Arch. f. Entw. Mech., 28:396-439. Przibram, Hans 1906. Aufzucht, Farbwechsel und Regeneration einer agyptischen Gottesan- beterin (Sphodromantis bioculata Burm). Arch. f. Entw. Mech., 22: 149-192. Scott, G. G. 1907. Further Notes on the Regeneration of the Fins of Fundulus heterocli- tus. Biol. Bull., 12:385-400. 1909. Regeneration in Fundulus and its Relation to the Size of the Fish. Biol. Bull., i7:343-353- Spallanzani, Lazarq Abbe 1769. An Essay on Animal Reproductions. Translated by M. Maty. London. Stockard, C. R. 1908. Studies of Tissue Growth, I. An Experimental Study of the Rate of Regeneration in Cassiopea xamachana. Carnegie Institution Publication No. 103:63-102. 1909a. Studies of Tissue Growth, II. Functional Activity, Form Regulation, Level of the Cut, and Degree of Injury as Factors in Determining the Rate of Regeneration. The Reaction of Regenerating Tissue in the Old Body. J. Exp. Zool., 6:433-471. 1909b. Studies of Tissue Growth, IV. The Influence of Regenerating Tissue on the Animal Body. Arch. f. Entw. Mech., 29:15-32. Ubisch, Leopold v. 1915. Tiber den Einflusz von Gleichgewichtsstorungen auf die Regenerations- geschwindigkeit. Arch. f. Entw. Mech., 41 :237-25o. Vanlair, C. 1894. Recherches chronometriques sur la regeneration des nerfs. Archives de physiologie normale et pathologique. 5 e Serie., 6:217-231. Zeleny, C. 1902. A Case of Compensatory Regulation in the Regeneration of Hydroides dianthus. Arch. f. Entw. Mech., 13 :597-6o9. 1903. A Study of the Rate of Regeneration of the Arms in the Brittle-Star, Ophioglypha lacertosa. Biol. Bull., 6:12-17. 169] RATE OF REGENERATION— ZELENY 169 1905a. Compensatory Regulation. J. Exp. Zool., 2:1-102. 1905b. The Relation of the Degree of Injury to the Rate of Regeneration. J. Exp. Zool., 2:347-369. 1907, The Effect of Degree of Injury, Successive Injury and Functional Activity upon Regeneration in the Scyphomedusan, Cassiopea xamachana. J. Exp. Zool., 5:265-273. 1908. Some Internal Factors Concerned with the Regeneration of the Chelae of the Gulf -Weed Crab (Portunus sayi). Carnegie Institution Publica- tion No. 103:103-138. 1909a. The Effect of Successive Removal upon the Rate of Regeneration. J. Exp. Zool., 7:477-512. 1909b. The Relation between Degree of Injury and Rate of Regeneration — Additional Observations and General Discussion. J. Exp. Zool., 7 :5i3-562. 1909c. Some Experiments on the Effect of Age upon the Rate of Regener- ation. J. Exp. Zool., 7:563-593- 1912. The Quantitative Study of the Internal Factors Controlling Regenera- tion. Proc. Seventh Intern. Zool. Congress 1907:491-494. Zuelzer, M. 1907. tJber den Einflusz der Regeneration auf die Wachstumsgeschwindig- keit von Asellus aquaticus. Arch. f. Entw. Mech.» 25 :36i-397. 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