BIOLOGICAL BULLETIN OF THE fIDarine Biological laboratory WOODS HOLL, MASS. Editorial Staff. E. G. CON KLIN — The University of Pennsylvania. JACQUES LOEB — The University of California. T. H. MORGAN — Bryn Mawr College. W „ M. WHEELER — American Museum of Natura/ History. C. O. WHITMAN — The University of Chicago. E. B. WILSON — Columbia University. FRANK R. LILLIE — The University of Chicago, VOLUME V WOODS HOLL, MASS. JUNE, 1903, TO NOVEMBER, 1903. BIOLOGICAL BULLETIN OF THE flDarine Biological Xaboratcr\> WOODS HOLL, MASS. Editorial Staff, E. G. CONKLIN — The University of Pennsylvania. JACQUES LOEB — The University of California. T. H. MORGAN — Bryn Mawr College. W. M. WHEELER — The University of Texas. C. O. WHITMAN — The University of Chicago. E. B. WILSON — Columbia University. FRANK R. LILLIE — The University of Chicago VOLUME VI. WOODS HOLL, MASS. DECEMBER, 1903, TO MAY, 1904. PRESS of HE NEW ERA PRINTING COMPANV LANCASTER, PA. CONTENTS OF VOL VI. No. i. DECEMBER, 1903 PAGE. C. M. CHILD : Form Regulation in Cerianthits, II. The Effect of Position, Size and Other Factors upon Regeneration, II. Discussion of Results i CHARLES ZELENY : A Study of the Rate of Regeneration of the Arms in tJie Brittle-star, Ophioglypha lacertosa 12 D. B. CASTEEL AND E. F. PHILLIPS : Comparative Variability of Drones and Workers of the Honey Bee 1 8 HENRY H. LANE : The Ovarian Structures of the Viviparous Blind Fishes, Luc if it ga and Stygicola 3^ No. 2. JANUARY, 1904 C. M. CHILD : Form Regulation in Cerianthus, III. The Initia- v tion of Regeneration 55 E. A. ANDREWS: An Aberrant Limb in a Cray-fish 75 ROBERT M. YERKES : The Reaction-time of Gonionemus murbachii to Electric and Photic Stimuli 84 No. 3. FEBRUARY, 1904 E. G . SPAULDING : The Special Physics of Segmentation as shown by the Synthesis, from the Standpoint of Universally Valid Dynamic Principles, of all the Artificial Parthenogenetic Methods 97 N. YATSU : Experiments on the Development of Egg Fragments in Cerebratulus 123 THOS. H. MONTGOMERY, JR.: Some Observations and Considera- tions upon the Maturation Phenomena of the Germ Cells... 137 No. 4. MARCH, 1904 T. H. MORGAN: Notes on Regeneration 159 E. H. HARPER: Notes on Regulation in Sty/aria lacustris 173 CARL HARTMANN : Variability in the Number of Teeth on the C/aws of Spiders, showing their Unreliability for Systematic Description 19* E. P. LYON : A Biological Examination of Distilled Water 198 iii IV CONTENTS PAGE. No. 5. APRIL, 1904 HARRY BEAL TORREY : On the Habits and Reactions of Sagartia davisi 203 FRANK E. LUTZ : Variation in Bees 217 ALBERT M. REESE: The Sexual Elements of the Giant Salamander, Crypto 1) ranch us allegheniensis 220 v E. G. SPAULDING : The Rhythm of Immunity and Susceptibility of Fertilized Sea-urchin Eggs to Ether, to HCl, and to Some Salts 224 GEORGE T. HARGITT : Budding Tentacles of Gonionemus 241 No. 6. MAY, 1904 WILLIAM MORTON WHEELER: A Crustacean-eating Ant (Lepto- genys elongata) Buckley 251 WM. S. MARSHALL: The Marching of the Larva of the Maia Moth, Hemileiica maia 260 C. M. CHILD: Form- Regulation in Cerianthiis, IV. 7 he Role of Water-Pressure in Regeneration 266 HELEN DEAN KING : Notes on Regeneration in Tubularia crocea.. 287 Research Seminar of the Marine Biological Laboratory for the Year ipoj 307 Vol. VI. December, / FIG. i. The abscissae, represent the disk diameters in millimeters and the ordinates the regenerated arm lengths, also in millimeters. The unbroken line gives the aver- age of Series I. -II. (the specimens with one and two arms removed). The broken line gives the average of Series III. -IV. (the specimens with three and four arms re- moved). The uppermost curves show the conditions 22 days, the middle curves 33 days, and the lowermost curves 46 days after the operation. ^^HS REGENERATION OF ARMS IN BRITTLE-STAR. I 5 urements taken 33 days and 46 days after the operation. Thus in the 3 3 -day measurement for Series I. and II. the regenerated length increases from 1.07 mm. for a disk diameter of 7 mm. to a maximum of 2.37 mm. for a disk diameter of 14 mm. and then goes down to .21 mm. for a iQ-mm. diameter. Also for the Series III. and IV. at the same time the length increases from 2.04 mm. at a diameter of 7 mm. to a maximum of 3.45 mm. at a 12-mm. diameter and down again to 1.36 mm. at a diameter of 1 8 mm. The medium sized individuals tints have the maximum rate of regeneration. 2. More striking still is the very constant difference between the average of Series I. and II. as compared with Series III. and IV. This shows a very decided advantage in favor of the ani- mals with the greater number of removed arms. The difference is evident in the upper curves of the figure from measurements taken 22 days after the operation but becomes more striking in the 33-day and 46-day curves. For example, in the 33-day curve for a 1 2-mm. diameter (the diameter at which we have the maximum rate of regeneration of Series III. and IV.) we get a regenerated length of 2.08 mm. for Series I. -II. and of 3.45 mm. for Series III. -IV., an advantage of 1.37 mm. or 66 per cent, in favor of the latter. Likewise at a diameter of 14 mm. (where the Series I.-II. has its maximum regeneration) we get 2.37 mm. for Series I.— II. and 2.77 mm. for Series III. -IV., an advantage of .4mm. or 17 per cent, in favor of Series III.— IV. In a similar manner in the curves obtained from the 46-day measure- ments we get at a 12 -mm. disk diameter a regenerated length of 2.46 mm. for Series I.-II. and 5.42 mm. for Series III. -IV., and at a 15 -mm. diameter 3.14 mm. for Series I.-II. and 3.72 mm. for Series 1 1 1. -IV. which represents an advantage for the group with the greater number of removed arms of respectively 2.96 mm. (= 1 20 per cent.) and .58 mm. ( = 18 per cent.) for the two points named. We must therefore conclude that when more than one arm is removed the regenerative energy as expressed in the replacement of the lost arms is greatly increased. Not only is the total re- generative energy greater in this case but the energy expressed in each arm is greater than the total energy when only one is removed. I 6 CHARLES ZELENY. Expressing this in mathematical form, if El represents the re- generative energy exhibited in the replacement of the lost arm when only one is removed, assuming that increase in length is a measure of such energy, and En represents the energy exhibited in regeneration when more than one arm is removed, # being the number of absent arms, then not only is E > E, but also E ln~>E, J n~^ \ n' -^ 1 or En>nEr Therefore when we remove n arms we increase the total regenerative energy by more than n times the amount ex- hibited when only one is removed. The force of this statement is made especially strong when we consider that throughout the experiments the animals received no food supply whatever. Expressing the relation in still another way, let us take a brittle-star with arms A, B, C, D and E, in which ar bv cv d^ and el represent the respective lengths these arms will attain after a definite period of regeneration, supposing that one alone is cut off in each case. Now let us suppose instead that the first four are cut off, then after this same period of time we get for the regener- ated lengths a4 > av b4 > blt c4 > cv d± > dr Now in the first case mentioned we cannot assume that the stimulus of removal and the resultant reaction of regeneration are purely local and con- cern only the tissues in the immediate vicinity of the cut surface for we then get into difficulty as soon as we try to explain the cases where four arms are simultaneously removed. Here we find we must add a considerable quantity (/-4) to each of the ori- ginal single regeneration lengths, e. g., 4 -f c4 + d4 —R4. But whether we consider the influence of the organism as a whole to be one of acceleration or one of retardation we must recognize in either case that the regeneration rate is not a matter which involves only the local conditions at the wounded surface REGENERATION OF ARMS IN BRITTLE-STAR. I / as determined by the direct action of the operation. It seems, on the other hand, to be bound up with intricate reactions affecting the whole character of the activities and organization of the ani- mal. A more direct application of the above statements to the special theories of regeneration would be out of place at the present time. We may sum up my results on the rate of regeneration of the arms of the brittle-star, Ophioglypha lacertosa^ as follows : 1. There is a definite relation between the size (/. c., age (?) ) of the animal and the rate of regeneration of its arms. The maxi- mum rate is exhibited by individuals of medium size (with a disk diameter of 12 to 15 mm.). Both the smaller and the larger ones give a diminishing rate as we go away from this point. 2. The greater the number of removed arms (excepting the case where all are removed) the greater is the rate of regenera- tion of each arm. HULL ZOOLOGICAL LABORATORY, THE UNIVERSITY OF CHICAGO, October 12, 1903. COMPARATIVE VARIABILITY OF DRONES AND WORKERS OF THE HONEY BEE.1 D. B. CASTEEL AND E. F. PHILLIPS. INTRODUCTION. According to the theory of germinal variation it would be concluded that the workers of the honey bee, Apis uuilifica, being produced from fertilized eggs, would show more variation than would the drones which come from parthenogenetic eggs. This variation would be manifested by coloration and by relative size of parts, and it might be expected that a series of measure- ments made on like parts of drones and workers would show a smaller degree of variability for drones than for workers. To test this fact a series of measurements have been made and the results tabulated. Owing to the difficulty of measuring the extent of coloration on the segments of the abdomen this could not well be used for this work, and so a series of measurements on the wings were chosen although coloration is practically the only difference usually observed between the varieties of Apis mcllifica. The wings are also desirable for other reasons. They are of classificatory importance in systematic work, do not shrink when preserved in alcohol and are easily examined with a microscope by simply clipping off the wing and mounting in alcohol on a slide. They also give more accurate results since the extent of coloration would vary according to the retraction of the seg- ments of the abdomen in preserving and it would be practically impossible to get the individuals normally extended in all cases. The reason for taking up this work was rather indirect and should perhaps be stated since the results throw some light on a widely separated line of work. Perez2 (1878) took an Italian queen fertilized by a French black drone, and after some time examined 300 drones from this queen. As the queen was pure 1 Contribution from the Zoological Laboratory of the University of Pennsylvania. 2 Perez, J., " Memoire sur la ponte de 1'abeille reine et la theorie de Dzierzon. Ann. Sci. Nat., 6 Ser., Zool., T. 7, 1878. 18 COMPARATIVE VARIABILITY IN THE HONEY BEE. 1 9 Italian her drones would also be pure Italian, since they are pro- duced from parthenogenetic eggs, and the fact that she was mated with a black drone should make no difference. He found, however, that 149 of the drones did show some markings which he thought indicated hybridism, and from these observations rejected the theory of Dzierzon. His results were criticised severely and all manner of arguments were used against them, atavism, impurity of the queen and other reasons being given in explanation. Weighing the arguments of Perez and those pre- sented in opposition to them, however, would lead one to believe that Perez had the best of the argument. If then we accept the theory of Dzierzon, and it is well established, we must account for the results of Perez. An examination of a large number of hives has shown us that the coloration of the drones cannot be used as a test of their purity, and that, therefore, Perez' work is inaccurate, since he used this test as the basis of his argument. Drones from an Italian queen fertilized by an Italian drone show gradations in amount of coloration of the segments of the abdomen which would easily lead one to conclude that some of them were not pure, provided the evidence for their purity was not so strong ; while at the same time the workers from the same queen show a uniformity of marking which is very striking. In the face of these facts it is evident that extent of coloration could not be used as a basis for investigation in relation to parthenogenetic development in the case of the bee. It might also be added that the fact of the irregularity of coloration of the drones is well known to most bee-keepers, and a number of these men have stated to us that they do not consider the coloration of the drones as in any sense a test of purity. This little investigation led to the conclusion that possibly the drones showed more variation in other ways than did the work- ers, and to test this the measurements here recorded were made. We wish to express our appreciation to Mr. E. L. Pratt, of Swarthmore, Pa., for material furnished, and especially to Mr. E. R. Root, of Medina, Ohio, for material and for many courtesies shown during investigations carried on by one of us in his apiary. 2O D. B. CASTEEL AND E. F. PHILLIPS. MEASUREMENTS. For the measurements taken in this work we have chosen veins and cells which in the bee differ from the typical hymen- opterous wing and which are to a certain extent typical of the bee in their direction and extent. In this choice we have fol- lowed the discussion of the venation of the Hymenoptera of Corrlstock and Needham.1 The measurements were : (i) Length of vein radius (7?) ; (2) diagonal length of cell radius-four (7?4) ; (3) length of vein media-two (^/,) ; (4) length of medial cross- vein (;«) ; (5) ratio between m and M2, and (6) number of hooks or hamuli on the hind wing. An attempt was made to measure the angles formed by the union of veins radius-four (7?4) and radius-sector (Rs\ and veins radius-five (7?5) and radius- sector (RsJ, but on account of the difficulty of getting the exact angle at which the veins branch these measurements were dis- continued through fear of inaccuracy. In all cases right wings c R FIG. i. were measured. The measurements were made from camera- lucida sketches, Leitz ocular 2, objective 3 with lower lens re- moved and sketch made at table level, this giving a -magnification of forty-two diameters. In all cases this magnification is retained in the tables. The choice of the veins and cells measured perhaps needs some explanation since each one has certain peculiarities in direc- tion or extent. It should be stated, however, that we do not think it makes much difference what veins are chosen for a com- parison of variations in this case since all our observations show ^omstock, J. H., and Needham, J. G., " The Wings of Insects," Chap. III. (continued), IX., "The Venation of the Wings of Hymenoptera." Amer. Nat., Vol. 32, pp. 413-424, 12 figs., 1898. COMPARATIVE VARIABILITY IN THE HONEY BEE. 21 about the same degrees of variability of the two sexes. Any other veins or cells would no doubt show like variations. Length of Vein R. — In the typical hymenopterous wing the media (M) branches from the vein R + J/ at a1 point nearer the base of the wing than in Apis. The length of the vein R + M would therefore be desirable for measurement, but from the diffi- culty of getting exact measurements this was discarded, and in its place we took the measurement from the point where M branches off from R + M to the point where R divides into Rl and Rs or the length of R, which is therefore shorter than in the typical hymenopterous wing. Diagonal Length Cell R4. — In the typical hymenopterous wing veins R4 and R5 are nearly at right angles to the vein from which they branch, while in the bee, R4 is bent out to about 135° and R5 to 160°. This makes the cell R4 considerably longer, and the diagonal length varies according as the angle R4 — Rs varies. The measurements were made from the proximal side of R5 — Rs to the anterior angle of R4 — J/,. Length of Veins M, and in. — In the bee's wing there is a bend- ing in of the veins M4 and Ms toward the base of the wing with a corresponding lengthening and shifting of vein m. This vein gives a convenient measurement for the relative length of wing since it varies almost directly as the length increases. J/9 was chosen because it is correlated in its length with ;//, and forms a convenient relative measurement for the breadth of the wing. Ratio between m and M.r — As stated above, the lengths of m and M.y are correlated in their variation, so in order to test the relative variabilities of the two veins in drones and workers, we computed the ratios between the two — Mz : m \ : i : x ; x in every case being carried to two decimal places. From these computations it was found that the variation of m is in inverse proportion to that of J/9 as will be shown later. Number of Hooks on Hind Wing. — This count was taken to see whether the hind wing varied as did the fore wing, and the number of hooks served as a conservative test. Besides these measurements and calculations we looked for cases of abnormal wings in which the subcostal (Sc], radius- two (R.2] and cubital-two (Cu.^) veins might be present, these 22 D. B. CASTEEL AND E. F. PHILLIPS. being absent normally in the bee, and also for all other cases of abnormalities in the venation. In none of the wings observed were S<~, R2 or Cn.2 present. The other abnormalities will be discussed later. THE CHOICE OF MATERIAL. The individuals used were not all taken from the same hive, since observations show that all colonies do not vary to the same extent, at least in coloration. For this reason it appeared best to use individuals from different hives and different strains in order to get a more correct idea of the natural variations. In all cases individuals were selected at random. The material used was as follows : DRONES. I. Fifty individuals from Medina, Ohio, May 16, 1903. Hybrids, Italian and black. II. One hundred individuals from Medina, Ohio, May 23, 1903. Italians (?) from a peculiar strain bred by F. A. Hooper, Jamaica, very light in color. III. One hundred individuals from same hive as I. May 25, 1903. IV. One hundred individuals from Medina, Ohio. V. Fifty individuals from Medina, Ohio, May 9, 1903. Italians. VI. One hundred individuals from Swarthmore, Pa., August 20, 1903. Italians from a peculiar strain bred by E. L. Pratt, the queen having an entirely yellow abdomen. WORKERS. I. Fifty individuals from same hive as Drones V., May 9, 1903. II. Three hundred and fifty individuals from Philadelphia, Pa., August 10, 1903. Italians. III. One hundred individuals from Philadelphia, Pa., May 15, 1902. Hybrids, Italian and black. LENGTH OF VEIN R. The measurement of the length of this vein was found to be somewhat difficult owing to the hairs covering the angles and to the difficulty of getting the exact middle of the curve at the place where R{ and ^separate. However, with considerable care and COMPARATIVE VARIABILITY IN THE HONEY BEE. the reexamination of cases showing the greatest variation we think the figures are nearly correct. Since the drones varied over 5 mm. on an average more than the workers and any error in measurement could scarcely be more than I or 2 mm. we feel justified in concluding that in this case the drones vary consider- ably more than the workers. The greatest variations were in Lot III. of the workers, where the variation was from 32 mm. to 42 mm., and in Lot VI. of the drones, where the variation was from 35 mm. to 48 mm. Lots I. of the workers and V. of the drones taken from the same hive at the same time show a range of variability in the ratio 8: I I. TABLE I. VEIN R. Drones. Lot. 3° 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Av. I. I I 2 2 3 12 6 12 i 5 3 2 40.16 II. I I I 7 8 22 19 21 ii 6 3 35-99 III. 2 3 I 2 5 13 13 12 10 14 12 3 6 3 I 41.42 IV. 2 2 H 16 23 15 13 8 5 i i 34.38 V. I 3 3 5 II 8 7 5 3 3 i 36.92 VI. 3 9 ii 18 H 10 14 8 8 3 i I 40.42 3 3 16 26 37 49 46 5° 42 55 40 37 26 27 23 8 6 4 2 Workers. I. 2 i 3 4 ii 17 9 3 3746 II. 4 13 21 65 78 70 7i 17 9 2 38.42 III. I I 5 ii 10 26 19 12 10 2 3 37-40 I 3 10 27 35 102 114 91 84 J9 12 2 DIAGONAL MEASUREMENT OF CELL R±. This measurement being taken in one of the most variable regions of the wing shows a remarkable difference in the range of variability of the two sexes. The measurement of this distance was quite easy since the limits are well marked and easily distin- quishable and in no case do we think there was room for doubt in the length to more than i mm. This fact taken with the length makes these measurements a very good test of the relative variability. In this region of the wing a very large part of the abnormalities were found, so that we conclude that this portion shows more variation than any other ; but in spite of this tendency D. B. CASTEEL AND E. F. PHILLIPS. to vary, the workers' wings were quite constant. The greatest range of variation in workers was found to be 13 mm., while the least range in drones was 19 mm. The fact that the average length of the cell in drones was between 96 mm. and 102 mm., while the average length for workers was about 75 mm., will however account for part of this greater range since, with a given range of variability, the greater the length, the less the actual variation. This, however, does not account for the extreme differ- ence which we find and in this case again, as in the first set of measurements, we find a much greater variability in drones than in workers. TABLE II. DIAGONAL CELL J?t. Drones. Lot. 75' So1 87' 89 90 91 92 93 94 95 96 97 98 99 IOO I. i I 2 2 2 2 3 4 6 II. i 2 6 10 9 6 10 6 s 9 0 13 III. I I i 3 6 7 IV. 7 6 5 8 5 13 13 ii 8 5 7 V. i 2 i 3 i 5 4 VI. I I I i i 2 12 II 4 6 12 13 I I i 2 9 13 18 19 IS 39 34 28 27 40 5° Lot 101 102 103 104 i°5 106 107 108 109 no in 112 "31 "5 Average. I. 2 2 3 3 4 I 5 3 3 I 101.68 II. 9 4 i i 96.49 III. 7 8 9 9 it 10 5 8 4 6 i I I J 104. 14 IV. 2 4 3 i i i 96.15 V. 3 6 6 8 4 2 3 I lOI.gS VI. II 6 8 2 3 2 I i I 98.77 34 30 29 24 23 13 14 16 8 7 I 2 I I 1 No individuals found in columns omitted. Workers. Lot. 69 70 71 72 73 74 75 76 77 78 79 So 81 82 Average. I. I I 3 6 6 12 7 3 6 3 I I 76.36 II. I 10 18 20 42 37 7i 62 38 24 16 4 4 3 75.08 III. I i 3 5 8 19 20 12 12 16 2 i 76.24 2 12 22 29 53 57 97 85 56 39 33 7 5 3 LENGTH OF VEIN Mz. This is not a specialized vein, and while it has for its anterior boundary the edge of the cell Rv it shows comparatively little abnormality. A few cases of extra veins thrown in in the region of this vein will be discussed later, but it is located out- COMPARATIVE VARIABILITY IN THE HONEY BEE. side the portion of greatest abnormality of the wing. The prin- cipal reason for the measurement of this vein was to get a ratio with the vein ;//, but as the actual measurements add to the evi- dence, it seems advisable to give the table. The greatest range of variability in workers was 9 mm., the least for drones 1 1 mm., or, if we drop the two quite abnormal cases in Lot V., 9 mm. Taking into consideration the relative lengths of the average veins, 34 5 mm. and 45 mm., this makes the greatest variation in workers about equal to the least variation in drones, while the greatest range in drones is over twice that of the greatest range in workers. TABLE III. VEIN M Drones. Lot. 32 33 34 35 36 37 38 39 40 41 42 43 44 I. 3 2 II. 6 8 12 6 21 16 17 III. I IV. I 3 3 15 9 V. 2 4 VI. I I 2 i 2 I 6 II 13 II 28 33 33 Lot. 45 46 47 48 49 So 51 S* S3 54 55 56 Average. I. 2 8 2 ,5 15 8 2 2 I 48.82 II. 6 5 2 I 42.^6 III. 3 6 7 17 is 2.S ii 8 I 3 2 I 49-43 IV. IS 13 IS 14 6 3 2 I 45-72 V. 8 S 6 10 ii 2 I I 46.96 VI. 2 3 7 II 14 13 IO 15 13 2 3 48.64 36 32 45 55 5i 58 32 27 16 6 5 i Workers. 32 33 34 35 36 37 3S 39 40 I. 7 12 19 9 I 2 33-82 II. 8 40 64 134 59 31 10 3 i 34.83 III. 4 IO 42 22 10 I I I 34.6i 19 62 125 165 70 44 II 3 i LENGTH OF CROSS- VEIN in. This vein shows no abnormalities and may therefore be con- sidered as entirely outside the region so well marked about the cells R± and R5, which shows so much abnormal variation. If it be argued that, since the more anterior veins tend to be abnormal, 26 D. B. CASTEEL AND E. F. PHILLIPS. they are not therefore good tests of the comparative variability, then, since vein m is entirely outside this area, this measurement would serve as an answer to that argument and is in itself a sufficient test. As in the case of vein M2, the measurements of this vein were made principally for the sake of getting a ratio of variation between two veins which meet at nearly a right angle, but as the drones show greater variation than the workers, al- though not to so great a degree as in some other cases, and as this is a constant vein, we add the table. The greatest range in workers is 16 mm., the least in drones 15 mm.; the average for workers almost 13 mm., the average for drones 20 mm.; the greatest for drones 23 mm. The average lengths in the two cases (72 mm. and 97 mm.) reduces this difference about one third, so that the least variable drones are about as constant as the least variable workers, but the average range for drones still remains considerably greater than for workers. TABLE IV. VEIN m. Drones. Lot. 78 79 80 81 82 83 84 85 86 87 88 89 9o 91 92 93 94 95 96 I. I I I 3 3 I 6 II. i 4 3 7 12 8 17 8 III. 2 2 IV. I i 4 I 2 3 2 ii II 6 16 5 12 7 8 5 3 V. 2 2 3 4 2 4 VI. I I i 2 2 5 5 6 10 12 14 13 I i I 4 2 3 3 2 13 H 8 27 13 28 35 35 41 36 Lot. | 97 98 99 too 101 IO2 103 104 105 106 107 108 109 no in 112 "3 114 "5 Avg. I. I 4 2 7 5 2 3 3 3 4 IOO.O4 II. 14 8 5 6 3 2 2 95-65 III. 4 9 S 16 4 12 8 ii 10 4 2 4 2 2 I i I 102.43 IV. 2 88.84 V. 9 13 i 3 2 3 i i 97.08 VI. 6 7 7 5 2 i 94-63 34 43 20 37 16 20 H IS 13 8 2 4 2 2 I i I Workers. 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 Avg. I. I 2 2 4 6 13 7 8 4 2 I 72.30 II. I 2 3 IS 12 69 35 73 42 43 40 I I 3 i 72.00 III. i 2 1 10 16 18 27 13 8 2 2 72.43 I 1 2 5 I.I IS 83 57 104 76 64 5^ 15 6 i COMPARATIVE VARIABILITY IN THE HONEY BEE. RATIO BETWEEN M, AND ;;/. Z To get this ratio the length of the vein m was divided by the length of the vein M2 and the division carried to two decimal points giving a ratio in the form M^.in: : i : x. To get the com- parative variability of the two sexes the individuals were tabulated according to the values of x and in this tabulation the values of x to two decimal points were used, but owing to the length of such a table we have combined these values and give them ac- cording to x carried to one decimal point. This is advisable also for another reason ; owing to the relative smallness of the num- bers in the first part of the proportion certain columns remained empty since, for example, no combination of figures between 32 and 57 and between 63 and 1 15 can give a ratio of i : 1.99. In order then to get a series of numbers which represents what is no doubt more nearly the true scale of ratios, the first table has been reduced to a table with x carried to but one decimal point. In tabulating these ratios the individuals were not grouped ac- cording to lots as in the other cases since we found practically little difference between the lots in any one sex. The average ratio for drones is 1 : 2.06 and for workers i : 2.08 so that no ac- count need here be taken of the difference in averages since it is so slight. The range of the values of x for workers is from 1.70 to 2.34 or .64 ; that for drones from 1.68 to 2.90, or, omitting one wing with a very abnormal ratio, from 1.68 to 2.57 or .89 show- ing .25 greater range in drones than in workers. It might also be said, since our long table cannot be used here, that this greater range is not caused by a few abnormal cases but that the variations are shown by a gradually decreasing number of indi- viduals in each value of x in both drones and workers, barring, of course, certain values of x which as above mentioned are im- possible with the figures worked upon. TABLE V. RATIO BETWEEN M AND m. Value of j-. i-7 1.8 1.9 2 2.1 2.2 2-3 ,4 -.3 2.6 Drones. 13 51 81 126 89 70 36 19 12 3 Workers. I II 53 119 217 Si 17 I 28 D. B. CASTEEL AND E. F. PHILLIPS. The working out of these ratios brought to light another point which explains this greater range in the values of x in the propor- tion. It was found that a certain inverse ratio exists between the lengths of ;;/ and M2 so that the area of the cell istM2 bounded distally and posteriorly by these veins remains more nearly con- stant for wings of the same area than do the bounding veins. This does not mean that the area of the cell istM2 does not vary, for a little calculation shows that it does vary considerably. The ratios of the wings for each length of M0 were gathered together and an average taken of the ratios in each case, with the result that we found a relatively constantly decreasing ratio with the increase in the length of M2. The range of these ratios for drones was from 1.80 to 2.57, omitting again the one abnormal wing mentioned above, and for workers from 1.77 to 2.21, a difference in this case of .33 in the ranges of the two sexes. This would indicate seventy-five per cent, more range of variation for the drones than for the workers and this difference is directly corre- lated with the great difference in the size of M2 found to exist. TABLE VI. RATIOS FOR LENGTHS OF VEIN M0. mm. 32 33 34 35 36 37 38 39 40 4i 42 43 44 Drones. Workers. 2.21 2.19 2.05 2.13 2.06 2.01 1-93 2.57 1.90 2.43 1.86 2-35 1-77 2.25 2.28 2.16 2.14 mm. 45 46 47 48 49 5° 51 52 53 54 55 56 A r. Drones. Workers. 2.09 2.O6 2.O2 2.03 1.98 2.OO 1.91 1.88 1.83 1. 80 1.79 1. 80 2.1 2. D6 38 HOOKS ON THE HIND WING. The number of hamuli or hooks on the hind wing which are used to fasten the two wings together during flight were used as a means of testing the variability of this wing. The examination of 1,000 wings has shown that this is not the most variable feature of this wing, but that far more variation occurs in the breadth of the wing and in the angles of the veins which form the cross sup- ports. As the area of the hind wing increases the increase takes place principally by a widening of the wing although by no means entirely by that method. The drones especially show COMPARATIVE VARIABILITY IN THE HONEY BEE. 29 this widened wing, in some cases the width equalling the length/ It may safely be stated, although no measurements were made on this point, that the area of the back wing is more variable than that of the fore wing. Since the number of hooks is correlated with the length of the wing they do not, therefore, show as much variation as would be found on the other parts of the hind wing. The least range of variation in number of hooks in workers was over seven points, the greatest, ten ; the least for drones, nine, the greatest eighteen, or omitting one very abnormal wing with but twelve hooks, twelve. The relative amounts of variation are FIG. 2. shown far more clearly here by examining the numbers of indi- viduals in each case which have the same number of hooks. For the workers the greatest number is 139 individuals which have 21 hooks while for drones the greatest number is 98 with 22 hooks, the average number for drones being a little higher than for workers. In workers the descent in numbers of individuals from 21 is far more sudden than that for drones, which is really TABLE VII. HOOKS ON HIND WING. Drones. 12 16 r? 18 19 20 21 22 23 24 25 26 27 28 29 Avg. I. 4 4 8 II 9 4 6 i i 2 21. s6 II. 2 I 12 21 23 2O 17 2 2 20.12 III. I 7 12 15 28 14 14 6 2 I 22.C9 IV. 4 10 17 22 27 12 2 5 i 20-33 V. 4 2 II IO 9 7 i 4 I i 21-54 VI. I 3 3 7 6 23 23 19 6 6 I i 22.42 I 2 5 34 54 83 89 98 52 47 18 10 4 i i Workers. I. 6 9 12 ii 6 5 I 21.42 II. 4 13 42 74 94 61 45 ii 4 2 21. 08 III. 2 ii 18 18 33 8 6 4 20-37 6 24 66 101 139 80 57 20 5 2 3O D. B. CASTEEL AND E. F. PHILLIPS. the true test of the relative variability far more than is the range of variation. So far we have discussed the range of variability in each case, but a far more important test of the comparative variability is the relative centralization of individuals about the average point in the table. If, for example, the drones showed exactly the same range as did the workers, the fact that in every one of the tables the workers show a greater number of individuals with the average dimensions, together with a rapidly decreasing number in each direction, while the drones show a more nearly equal number of individuals over several dimensions on each side of the average point, would lead one to conclude that the drones show far more variability than do the workers. It is not necessary to go over all the tables to point out this fact, but even a hurried examina- FIG. 3. tion will show the facts as above stated. If, for example, the tables were put in the form of curves of variation the workers would show a longer and narrower curve in every case than the drones, and were these curves expressed in the form of mathe- matical formulae the fact would be brought out still more strongly that the males in this case show more variation than do the abortive females. It may be well here to offer some explanation as to why curves were not used in this work, since that is the usual method for work on variation. It was not the purpose of this investigation to work out the law of variation for the two sexes, but merely to show which sex did vary to the greater extent. This purpose has been fulfilled and is shown in the tables. In order to get a true curve of comparative variability it would be necessary to COMPARATIVE VARIABILITY IN THE HONEY BEE. 31 measure many times the number of individuals which were used and to extend the observations over a far greater range of varie- ties. No curve made with 500 individuals would express the true law of variation, nor would ten times that number be suffi- cient, and since the formulation of the law of variation for par- thenogenetic and fertilized forms for this particular kind of par- thenogenesis, arrenotoky, is too important a matter to be based on an inaccurate mathematical formula, it seems better to us to state simply the fact that greater variation does occur in the males and leave the formulation of the law to be worked out with a far greater range of observations and measurements. And then, too, it is by no means certain that this variation follows any fixed law. If the variation were caused entirely by germinal variation, or by any other one factor, then it might be assumed that the law of this variation could be stated in the form of a mathematical for- mula, but as will be shown later, it appears probable that a large part of the greater variability of the drones is due to chance and is therefore not in accordance with any law. It may be argued that variation according to chance is but a way of stating our ignorance of the true law, but if there is a law for this variation it is certainly very obscure, and the working out of this law would require an extremely large number of measurements taken from individuals, each one with its life history known together with a high degree of mathematical ability in its formulation. ABNORMAL WINGS. As noted previously, in all wings examined, record was made of wings having veins which are not typically found in the bee. Fig. 4 shows, in dotted lines, where these abnormalities occur most frequently. It is very difficult to record these irregularities in any kind of a table, since the irregular veins vary widely in ex- tent and do not arise at exactly the same place in many cases. An attempt was made to classify these according to the veins from which they branch, their extent and direction. Manifestly any tabulation must be considered as merely a matter of convenience in examination. In this we have recorded cases where a vein bends (b in table) from its true course, showing but a tendency toward abnormality, as well as the well-marked cases. The ex- D. B. CASTEEL AND E. F. PHILLIPS. tent of the abnormality is expressed roughly in the terms, very small (vs), small (s), almost complete (ac) and complete (c). The letters used to designate these abnormalities are in no way connected with the naming of the normal veins, but are chosen merely as a convenient means of marking the irregularities. It will be understood that it is impossible to draw at all times the same line between, for example, the terms small and very small, FIG. 4. and the table is of no value except to give an idea of the number of cases of abnormalities. MALES. a. 82 b, 32 vs, 109 s, I ac, 2 irregular. b. 7 s, I double s, 4 ac, 6 c. c. 3 vs, 18 s, 2 ac, 13 c, 2 irregular. d. lib, 27 s, I double s, 7 ac, 2 c. e. 9 s, I ac, 5 c. f, g. 8 irregular at anterior end. h. 5 s, 2 c, I very irregular. i. none. Other irregularities, 14. FEMALES. 2 b. 2 b. I b, 3 TS, 4 s. 3 b, 6 vs, 9 s. I b, I vs, 3 s. 2 irregular at anterior end. none. 2 lost veins ; I almost lost. none. Deducting the cases where more than one irregularity occurs on one wing we have 271 irregular drone wings and 37 worker wings. Leaving out of consideration those cases in which merely a bend is recorded there are 206 irregular drone wings and 30 irregular worker wings or almost seven times as many for drones as for workers. The figure and these tabulations make clear what has been said previously about an area of irregularity in the wing. This region is rather well defined and no irregularities in venation were seen outside it. A point which may well be recorded is that none of these veins COMPARATIVE VARIABILITY IN THE HONEY BEE. 33 could be considered as in any sense mutations since there are in all cases varying degrees of abnormality indicating a gradual variation in the directions indicated. No attempt has been made to correlate definitely these ab- normalities with any lengths of the neighboring veins, but in a general way it can be said that the largest wings of each sex are the most abnormal, and this fact explains to some extent why the drone wings are so much more abnormal than those of the workers. From this it would appear that the cause of these abnormalities was the need of extra strengthening of the cells as they became larger, and that the irregularities are the result of extra growth energy, which has a chance to show itself when room is allowed for its manifestation. SOME EXPLANATIONS OF THESE RESULTS. With theseYacts before us it seems desirable to find, if possible, some explanations for these peculiar results. As stated in the introduction the theory of germinal variation would lead one to believe that the parthenogenetic individuals of the bee would show less variation than those from fertilized eggs and if there were no complicating factors it may be supposed that this would be true, but there are certain factors which modify this result so that the ratio of variation is exactly reversed. In the first place that kind of parthenogenesis in which males only are produced (arrenotoky), is not such a specialized form of agamic reproduction as are those kinds of parthenogenesis in which females or both males and females are produced. When females only result from parthenogenetic eggs (thelytoky], a crossing of two lines of heredity seldom occurs and amphimixis can bring about little variation. In the case of the Aphids and Daphnids we have a similar condition except that once in a year or once in some life cycle males also are produced parthenoge- netically and then there is an opportunity for the blending of hered- itary traits through fertilization. To this last form of partheno- genesis the name amphoterotoky is applied. It is thus seen that in arrenotoky this mixing of hereditary traits is not dispensed with to such an extent as in the cases of thelytoky and ampho- terotoky. On the other hand, the production of females par- 34 D. B. CASTEEL AND E. F. PHILLIPS. thenogenetically is of far more use to a species where gamic reproduction is unnecessary, since fertilization is not necessary to give a stimulus to the egg so that it may develop, and what the species loses in lack of cross fertilization is more than made up in the advantage it has through its parthenogenetic power. Since then arrenotoky is the least specialized form of partheno- genesis, and since a crossing does occur at every second genera- tion in species with this power, it follows that according to the theory of germinal variation we would find more variation in arrenotoky than in either thelytoky or amphoterotoky. In fact the decrease in variability would not be very great, since in every case but one half the crossing is dispensed with. Variation in parthenogenetic forms has been observed previ- ously. Weismann l found that the parthenogenetic ostracod, Cyprus reptans, showed variation, and Warren 2 found consider- able variation in Daphnids. Both of these cases fall under amphoterotoky, so that on a priori grounds we would expect still more variation in arrentoky. It is also held by some that males tend to vary more than females, and perhaps this tendency has something to do with what we find in the case examined. Davenport and Bullard,3 found 2^4 per cent, more variation in males than in females in swine. Darwin 4 gives a considerable number of cases showing the same tendency, and others have observed similar facts. On the other hand there are cases in the Odonata (Gouiphus and Macrothemis, Calvert5) and in the Lepidoptera (Thy re us abbotii, Field6), in which the females are more variable than the males so, that we must not assume too much on this ground. This still leaves considerable variation in drones to be accounted 1 Weismann, A., "The Germ-plasm," 1893. 2 Warren, E., "An Observation on Inheritance in Parthenogenesis," P. R. Soc. Lond., Vol. LXV., pp. 154-8, 1899. 3 Davenport, C. B., and Bullard, C., " Studies in Morphogenesis," VI. "A con- tribution to the quantitative study of correlated variation and the comparative varia- bility of the sexes." Proc. Am. Soc., Vol. 32, pp. 85-97, 1897. 4 Darwin, Charles, "The Descent of Man." London, 1871. 5 Calvert, P. P., "The Odonate Genus Macrothemis and Its Allies," Proc. Boston Soc. Nat. Hist., Vol. 28, pp. 301-332, 1898. "On Gomphus fraternus, externns and crassus (Order Odonata)," Entomol. Areios, March, 1901. 6 Field, W. L. W., " A Contribution to the Study of Individual Variation in the Wings of the Lepidoptera," Proc. Am. Ac. Sc , Vol. 38, pp. 389-396, 1898. COMPARATIVE VARIABILITY IN THE HONEY BEE. 35 for and the following facts seem to us to help in this. The workers in a hive are hatched from cells one fifth of an inch in width and the size of these cells is remarkably uniform. On the other hand the drones hatch from cells which are generally one fourth of an inch in width, but often hatch in worker cells and from cells of all intermediate sizes. In the making of the comb under natural conditions there are a great many irregular cells formed which are transition cells between the worker and drone cells, and from these, if used for brood at all, drones are produced. It is true that sometimes a worker is produced in a drone cell, but this is very rare, provided there are any worker cells in the hive. Drone pupae, on the contrary, are frequently seen in worker cells and are very noticeable on account of the exception- ally high arched cap which the workers put on when the larva is sealed up. This then gives to drones a greater amount of variation in the room provided for their growth while in the plastic state. A bee larva will grow until it fills the cell in which it is placed and the young bee which emerges will be the size of the cell from which it came, within certain limits. This is shown in the production of queens by the modern methods of queen rearing used in apiculture. A young worker larva, less than one day old, is lifted from its cell and put into a cell cup of queen size. The workers complete this cup and form a queen cell and the larva in this cell grows to a much larger size than would be possible if it had remained in its original cell. Once in a while the bees will attempt to make a queen from a drone larva and while, of course, this is a failure yet the result is a very large drone. Generally, however, the drone under these conditions dies before reaching the imago stage. These facts show that the growth of an individual is limited by the size of the cell and also undoubtedly by the amount of food received during the unsealed larval stage. Then it follows that since the cells from which o drones hatch vary from one fifth of an inch to over one fourth of an inch in width, while those from which workers hatch are quite uniform, that the variation in size will be^ considerably greater for drones than for workers. This supposition is further strengthened by some of the facts 36 D. B. CASTEEL AND E. F. PHILLIPS. brought out by measuring the wings. Referring again to the table of the ratios varying according to the length of vein M2 we find that the vein m varies inversely as the length of Mv The length of vein /// represents the ratio of the length of the wing while vein M2 represents the ratio of the width. Probably most of the drones which have the shortest vein M2 are those hatched from the smallest cells and the wings could not increase in width and therefore to meet the needs of the animal in making the necessary area of wing for flight the vein ;;/ must be lengthened. On the other hand, those drones hatched from the largest cells would be allowed greater room for the development of vein M2 and vein ;// need not be so long. Another fact which seems to indicate this is that those drone pupae which are developing in worker cells are covered over by a very high cap, making the length of their cell much greater than that of the ordinary drone cell. The drones which hatch from these cells are long and narrow when compared with those from drone cells proper. The drones in Lot II. were taken from a hive in which there were no drone cells except possibly a very few in the corners of the frame or near the top bar of the frame since all the combs were made on what beekeepers call foundation and the cells were uniformly of worker size. These drones show the least variation since they were all hatched under the same conditions. The drones of Lots I. and III. were hatched in old irregular combs and the tables show considerably greater variability. The greatest number of abnormalities were found on the largest drone wings and the throwing in of extra veins is probably caused by the necessity for greater strengthening of the wings. Just how these extra veins arose is not easy to explain. They may be sports, or reversions to an ancestral type, or the result of extra growth-energy or caused by the splitting of normal veins so that it is rather difficult to say just what factors bring about this extra amount of variation. While we speak of these as abnormal veins it must be noted that we do not know whether they are really abnormal or whether they are but the manifestations of a tendency possessed by all bees but which can develop only under certain conditions, just as the ovaries of the workers can develop only when extra room and food are provided. ^•H COMPARATIVE VARIABILITY IN THE HONEY BEE. 37 If then germinal variation will not explain all these variations and if we accept the explanation offered as a partial and possible statement of the cause, then it would appear that the mere chance as to which cell happens to be the receptacle of a drone egg determines its variation. While it is probable that even this " chance " is according to fixed law, the fact remains that in any event this law is beyond the possibility of formulation from any observations except those extending over far more individuals than those here used. On this account we consider ourselves justified in our tabulation of results rather than in the plotting of curves and expression in mathematical formulae, since that would be undesirable except with far more measurements and with material gathered under conditions better controlled. Our tables show the variation as it actually exists in a state of nature and the real laws can be worked out only from observations from control experiments and this it is hoped will be possible in the near future. We do not wish to be considered as advocating the inadequacy of the theory of germinal variation to explain variation, since we have no means of knowing whether these variations can be inherited but simply wish to express the facts as we find them, and leave the explanation of the bearing of germinal variation on this problem for future investigation. October I, 1903. EXPLANATIONS OF FIGURES. FIG. I. Fore wing of honey bee, normal. Cells and veins are named according to Comstock and Needham. FIG. 2. Hind wing of honey bee, normal. FIG. 3. Typical hymenopterous wing according to Comstock and Needham. FIG. 4. Part of fore wing of honey bee showing (in dotted lines) where acces- sory veins were seen to occur in the wings examined. Lettering purely arbitrary as explained in text. THE OVARIAN STRUCTURES OF THE VIVIPAROUS BLIND FISHES, LUCIFUGA AND STYGICOLA.1 HENRY H. LANE, A.M. I. INTRODUCTORY. During the spring of 1902, Dr. C. H. Eigenmann collected a number of specimens of blind fishes in the caves of western Cuba, within a radius of I 30 kilometers of Havana. The fishes belong to the two distinct but closely related genera, Liicifuga and Stygicola. It has been my good fortune to have the oppor- tunity of studying the reproductive organs — more particularly, the ovarian structures --of these blind fishes, with special ref- erence to their method of reproduction. It was discovered upon examination of the specimens that they are viviparous,2 a fact long known in regard to some of their deep-sea rela- tives. Owing to the lateness of the season when they were col- lected, unfortunately but one female was pregnant. This one measured only 65 mm. in length and contained four foetuses — borrowing a term to designate the post-larval stages of the young until birth — 18-20 mm. long. These foetuses were in an ad- vanced stage of development, very probably being within a few days, or possibly hours, of birth, since a number of young only 25 mm. long were caught in the water. No other prenatal speci- mens having been secured, it has been impossible to study the early stages of development. My attention has been particularly directed to the ovarian structures of the mature females secured. A few of the young specimens, evidently taken not long after birth, were also examined. I wish here to express my deep sense of obligation to Dr. C. H. Eigenmann for his assistance and criticism in the preparation of this paper. 1 Contributions from the Zoological Laboratory of Indiana University, No. 58. 2 Eigenmann, '03, p. 236, pi. 21. 33 OVERIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 39 II. SYSTEMATIC POSITION OF LUCIFUGA AND STYGICOLA. Poey1 ('60) described these fishes in 1860, giving them the generic name of Lucifuga, recognizing them, however, as two species, dentatus and subterraneus. Later Gill, '63. separated dentatits from the other and created the genus Stygicola fortit. The two genera are different in that Stygicola has teeth on the palatines, where Liicifitga has none, and the teeth in the jaws of the former are larger than those of the latter. There is also a very noticeable difference in the depth of the head at the nape, in adult individuals. The two species or genera are however so nearly alike that it is only after a prolonged comparison that the above technical differences were made out. The several species of blind cave fishes, found in Indiana, Illi- nois, Kentucky and Missouri, are not related to the Cuban species. The latter are descended from marine forms which have worked their way through underground channels into the Cuban caves. Related genera that still live in the ocean about Cuba are Brotula2 and Ogilbia? in moderate depths and Bassosetus* and Apkyonus5 in deep water. All of these genera belong to the family Brotulidae, a deep-sea group comprising about forty-five genera and one hundred species, living mostly in the tropical seas of both hemispheres. The two genera under consideration in this paper are the only ones found in fresh water. Jordan and Evermann (op. cit., Pt. III., p. 2498) observe very properly that " these fishes are closely related to the Zoarcidae. In spite of various external resem- blances to the Gadidae their affinities are rather with the blen- nioid forms than with the latter." III. HISTORICAL. Numerous contributions to our knowledge of viviparity in fishes have been made from the time of Cuvier to the present. 1 Lucifuga, Poey, " Memorias," II., 95, 1860 (su&ferraneas) ; Lucifuga subter- rannts, Poey, "Memorias," II., 96, 1860; Luiijuga dentatus, Poey, "Memorias," II., 102, 1860; Stygicola dcntata, Gill, Proc. Ac. Nat. Sci. Phil., 252, 1863. 2 Brotula (vid. Bull. 47, U. S. Nat. Mus. , Jordan & Evermann, "fishes of North America," Pt. III., p. 2500). 3 Ogilbia {vid. idem, Pt. III., pp. 2502, 2503). 4 Bassozetns, Gill (viJ. idem, Pt. III., p. 2507). 5 Aphyoniis, Giinther (yiJ. idem, Pt. III., p. 2525). 4O HENRY H. LANE. Among the most important ones are the following, most of which I have consulted in connection with my own investigation : Cuvierand Valenciennes, in their " Histoire Naturelle des Pois- sons," I., Paris, 1828, have a short general account of viviparity in. fishes, and mention is made of it frequently throughout their work in the description of such fishes as bring forth living young. Much of their work has been superseded by the more accurate observations of later investigators. Rathke, in 1833, published his " Bildungs- und Entwickel- ungsgeschichte des Blennius viviparus odes des Schleimfisches." This was long the best paper on the subject. In 1844 (Ann. des Sc. Nat., t. I., 3d series, p. 313) Duvernoy published a paper on Pcecilia snrinamensis, which is frequently referred to by more recent writers, but which I have not had the opportunity of consulting. Tn 1846, Cuvier and Valenciennes described the genus Ana- blcps, one species of which, A. gronovii, formed the subject matter of an important paper by Jeffries Wyman, in the Boston Journal of Natural History, Vol. VI., No. IV., p. 432, 1857. I shall refer to this article more at length below. In 1853, Louis Agassiz (Am. Jour, of Science, XVI., 2d series, Nov., 1853) described a new family of fishes from California — the Embiotocidas, which embraces the genus Cymatogaster. The only species of this genus, C. aggregates, was studied in detail by Dr. Eigenmann, and the results published in the Bulletin of the U. S. Fish Commission, Vol. 12, p. 401, 1892 (1894). Another very important paper, " On the Development of Vi- viparous Osseous Fishes and the Atlantic Salmon," by John A. Ryder (Proc. U. S. Nat. Mus., 1885, pp. 128-162, Pis. VI.-XII.) will be noticed frequently below. In 1887, Dr. Franz Stuhlmann made a detailed study of Zoarccs viviparus, Cuv., the results of which he published under the title, " Zur Kenntnis des Ovariums der Aalmutter (Zoarces vivipariis, Cuv.)." Frequent references to this volume will be made below. IV. VIVIPARITY IN GENERAL. Cuvier and Valenciennes (loc. cit.} give a general account of viviparity in fishes so far as known at that time ; but since their OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 4! statements are now either matters of common knowledge or else not in accordance with the facts as revealed by later investiga- tions, I shall not speak of their work further. Wyman (op. cit.~) classifies viviparous fishes into two groups "according to the position occupied by the embryo during the period of growth. In the first group may be arranged those fishes in which the ovum leaves the ovary in an undeveloped state, and in which the process of eolntion (sic] is not commenced until it readies the lower portion of the oviduct. The fishes which this group comprises are nearly all, if not all, Plagiostomes. The best known are Spinax, Carcharias, Mustcllus, Galeus, and Torpedo. ... II. In the second group those fishes are comprised in which the gestation is wholly or in part ovarian, the last stages only of the process usually occurring in the oviduct. Among the genera included in this division are Sihirns, Blcnnius, Ana- bleps, Ptfcilia and Embiotoca. In all of these genera impregna- tion takes place in the ovary, and, as seems probable, while the ovum is still invested with its original envelopes." Wyman found that each of the foetuses in A. gronovii is envel- oped in a separate sac of vascular tissue, much too large for the foetus enclosed, the extra space being filled up with an albuminous fluid. He seems to regard these foetal sacs simply as extensions of those within which the ova were suspended. Eigenmann, considering only teleosts, found two types of viviparity (op. cit., p. 404) ; he says : " At least two types of viviparity may be distinguished in fishes ; first, those in which the yolk furnishes all the intra- ovarian food ; and second, those in which the greater part of the food is furnished by the ovary. " In the first type the number of young is not less than in re- lated oviparous forms, while the number of young in the second is always greatly reduced. . . . The size and development of the young in this class (type I.) of fishes at the time of birth is of course much less than in the second class of viviparous fishes." As will be seen, Lucifuga and Stygicola belong to the second type of both Wyman and Eigenmann. The number of young is small and they are born in quite a mature condition. 42 HENRY H. LANE. V. GROSS ANATOMY. For the sake of clearness the following terms will be used in the sense here given : Oviduct — the single duct leading from the ovary to the uro- genital pore. Ovisac — the forward continuation of the oviduct which covers the ovary. Ovary — the structure containing the eggs. Stroma — the supporting tissues of the ovary itself. The term ovary is also used in a general way to include the ovisac and the ovarian structures proper. The context in every case will determine what is meant. In Liicifitga and Stygicola the ovary is enclosed between two layers of peritoneum above the posterior portion of the alimentary canal. It may extend so far forward as to lie in part even beside the stomach. The ovary has a bilateral arrangement. Externally it is a Y-shaped, bifurcated, subcylindrical organ, whose greatest diam- eter is immediately posterior to the point at which the division begins (Fig. i). The two horns lie on the right and left sides respectively and may enclose between them the posterior portion of the stomach. Interiorly the stem of the Y is divided by a median partition with which the ovarian structures proper are associated and which extends to near the oviduct, though here only the portion attached to the ventral wall is found (Fig. 2). From the tips of the ovarian horns slender though comparatively strong threads of connective tissue, inclosing blood vessels, run for- ward and fasten to the peritoneal walls, thus very securely hold- ing the ovary in position. Dorsally, the ovary is attached to the peritoneal lining of the body cavity by the mesovarium ; ventrally, there is a corresponding attachment, the mesorectum. The ovi- duct, which opens externally at the urogenital pore, increases gradually in size as it approaches the ovary and finally merges into the ovisac, or outer wall of the ovary. A somewhat immature specimen shows finely those structures connected with the support of the ovary. In it one sees that each horn is supported by its own fold or lamina of peritoneum ; that these two laminae become united at or near*the point of di- vision of the horns and are continued posteriorly as a single though thicker mesovarium supporting the body of the ovary OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 43 and the oviduct. Below there is no sign of a mesorectum in the region of the ovarian horns, except for a short distance near the base of one of them, but posterior to them there is such a mem- brane, inclosing several blood vessels, and itself somewhat thicker than the mesovarium above. It is also clear from an examina- tion of this ovary that, as will be noted more particularly below, the egg-bearing tissue, the ovary proper, forms a thick median partition in the ovisac. The external appearance of the ovary agrees very closely with Eigenmann's description (op. cit., p. 418) of that of Cy matog aster : " The ovary is' a spindle-shaped bag, divided anteriorly into two arms which indicate the bilateral origin of the present struc- ture. One of these arms, the left, is usually smaller than the other. . . . The ovaries of the two sides have evidently been united from behind forward, so that externally only the two an- terior horns show the bilateral structure, and one of these horns seems to be in process of phylogenetic resorption." While there is frequently a difference in the size of the two horns in Lu'cifnga and Stygicola, there is no uniformity in this matter. There seems to be no evidence that the right or left portion of the ovary is "in process of phylogenetic resorption." Ryder found that in Gainlnisia patrnelis " the ovary is a simple unpaired organ, the greater part of which lies on the right side of the body-cavity below the air bladder. . . ." The size of the ovary varies, of course, with the age and size of the female, as well as with the state of development of the ova or embryos contained in it. One female of the genus Litcifiiga, which had a length of 65 mm. and which contained four foetuses nearly ready to be born, had an ovary with a length --measur- ing to the extremity of the longer horn --of 16 mm., and a di- ameter of 8 by 9 mm. As the foetuses were 1 8 to 20 mm. long, it was not surprising to find that their tails were bent over. An- other female of the same genus, 83 mm. long, had evidently given birth to young only a short time before her capture and had an ovary 12 mm. in length. The point of division into the two horns is usually about five twelfths the distance from the anterior tip of the ovary to its pos- terior end. As already stated, the two horns rarely show equal 44 HENRY H. LANE. PlATE ' Fit 3 OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 45 development, there being always a more or less marked differ- ence in size. Between the ovisac and the ovary proper there is a lumen of varying size. When there are larvae present, the ovisac and the oviduct are extremely thin and so stretched, especially near the close of gestation, that their cellular structure cannot be made out with any satisfaction (Fig. 5). Shortly after birth of the young they contract and assume the form and appearance found in the ovaries of mature but non-pregnant females (Fig. 3). The wall of the ovisac is then quite thick, and the lumen very small. The histological structure of the ovisac will be described below. In non-pregnant ovaries, the stroma is a mass, which, in- ternally, has a bilateral arrangement and occupies most of the space within the ovisac (Figs. 2, 3). It is, in general shape, fusiform with its largest diameter just posterior to the division of the ovisac into the two horns down both of which it is con- tinued along their median surfaces, forming the prongs of a Y. In the middle of the ovary the stroma forms a median parti- tion ; somewhat posteriorly this partition is cut across (Fig. 3), and still further back only the ventral part remains (Fig. 2). It has many lobes which are usually somewhat pointed and comparatively large and distinct, the indentations sometimes leav- ing merely a "neck" of tissue to support them. The whole stroma at this time is fully distended by the large amount of lymph contained in the sinuses described below. Where the ova are well advanced they can be seen by the unaided eye in the form of opaque dots. When the ova are surrounded by follicles, they lie some distance below the surface of the stroma and there is a tubular indentation of the epithelial covering of the latter down to the follicle (Fig. 7, /?). In a circular space over the egg the epithelium is apparently continuous with the follicle. It is only on very close inspection that the independence of the follicle can be made out. It is then found to be of only a single cell in thick- ness beneath the epithelial indentation. A similar position of the epithelium was noted by Stuhlmann over the ova in the ovary of Zoarccs and was called " Delle " by him. The pregnant ovary is quite different in appearance from that of a non -pregnant female. A cross-section of the former shows 46 HENRY H. LANE. it to be an almost bilaterally symmetrical organ, but without any folds or pockets in which the embryos are contained as is the case in Cymatogaster1 and numerous other species of Embiot- ocidae. During the intra-ovarian development of the embryos, or rather the development of the foetuses within the oviduct of Liicifjiga, the ovarian structure proper or stroma which forms the thick median partition in non-pregnant ovaries, becomes gradually reduced and compressed into a narrow wall (Fig. 5). The stroma is much thickened both dorsally and ventrally near the oviduct (Fig. 4), where the partition is incomplete, but ante- riorly its greatest thickness is near the median plane (Fig. 5). The arrangement of the stroma in each horn of the ovary is as in non- pregnant ovaries (Fig. 6, ov.st.}. The single oviduct runs from the caudal end of the ovary proper to open at the urogenital pore. In pregnant females it is widely distended for some distance when the foetuses are well advanced, but in the non-pregnant females it is a rather cylin- drical, thick-walled, muscular tube with numerous folds or laminae on its inner surface, covered with a layer of columnar epithelial cells, 12 fj. in depth. It is not materially different, except as to dimensions, from the ovisac described above. Stuhlmann2 sim- ilarly found the oviduct of Zoarces to be a tube composed of the same cell-layers as the ovary, with the exception of the " ger- minal " and follicular epithelia. VI. HlSTOLOGICAL PART. /. The Walls of the Ovary or Ovisac. The following system will be used to facilitate cross references to the descriptions of the various ovaries. Each ovary will be referred to by a letter, A, B, C, etc., the meanings of which are as follows : A represents a female of the genus Stygicola with a length of 95 mm. B represents a female of the same genus, but with a length of 128 mm. C represents a female of the genus Lucifuga, length 87 mm. 1 Eigenmann, op. cit., p. 418. 2 Op. cit., p. 10. OVARIUM STRUCTURES OF VIVIPAROUS BLIND FISHES. 4/ D and E represent females of the same genus with a length of 83 and 65 mm. respectively. E was pregnant; the others were non-pregnant. An examination of the ovisac and oviduct reveals quite a range of variation, depending in the main upon the condition of preg- nancy or upon the length of time that had elapsed since the close of that period. In the ovary of D, which had not been pregnant at all, or at least for so long a time that the ovarian structures had regained their normal form, the wall of the oviduct and the ovisac is from 100 to 150 // in thickness at different places. Structurally, the ovisac consists of at least four cell-layers. The outer, a sinuated, peritoneal layer, immediatly beneath which there is a thicker layer of longitudinal muscle fibers ; below this there is another some- what thicker transverse band of muscle fibers ; on the inner sur- face there is an epithelial layer containing numerous capillaries. This will be described in detail below. The nuclei of the longi- tudinal band are rod-like in appearance ; the nuclei of the second muscle layer appear more nearly round, being evidently the cross- sections of nuclei of the same form as those in the longitudinal band. The innermost layer of epithelial cells has nuclei oval or round in shape, while the peritoneal layer shows few nuclei, but those which do appear are rod-shaped in section. Quite numer- ous capillaries are found between the cells of these several layers and in some places there are large blood vessels. In the ovary A from a female which had evidently given birth to young but a short time previous to her capture, the ovisac measures only 15 to 20 fi in thickness. Structurally it con- sists of four or five thin cell-layers, between which there are anastomosing capillaries. The outermost layer consists of peri- toneum, the cells of which are very much elongated and com- pressed. The muscle fibers beneath are mostly transverse and of the non-striated type. The inner layer is epithelial and is also much compressed. The nuclei of the muscle-fibers are long, narrow, rod-like structures which stain deeply, as would be ex- pected, with haematoxylin : the nuclei of the epithelium are oval in form. The condition of this ovary does not permit me to go into greater detail. PLATE II OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 49 In the ovary of the pregnant female E, whose young were almost ready for birth, the ovisac is thinner than in the case just described. The different cell-layers can scarcely be distinguished, though where the cells themselves are visible, the nuclei in section have the rod-like form mentioned above. The capillaries have mostly disappeared, apparently being closed by the crowding to- gether and stretching of the cellular structure ; but in places one comparatively large vessel appears, containing two or more rows of corpuscles, side by side. At this place the wall is enlarged somewhat to accommodate the vessel. In the ovary of C, which contained ova quite well advanced, the ovisac is very similar to that described for D. But in some portions of this ovary the muscle layer is restricted almost entirely to longitudinal fibers, the transverse layer being much reduced. Capillaries penetrate freely through these muscle layers in all directions ; those in the lining epithelium are larger than those in the other specimens already described. In the ovary of B the ovisac is similar to that in C, but the peri- toneal covering is not so distinct ; there is the same arrangement of muscle-fibers - - the outer longitudinal and the inner transverse, the latter being much the deeper. The innermost epithelial layer is composed of " pavement " cells with quite large distinct nuclei. Numerous capillaries are found in this inner lining. Compare in this connection Eigenmann's description of the ovarian wall in Cymatogaster (op. cit., p. 418) : " The ovarian walls are composed, first, of the thin peritoneal membrane ; second, of a layer of longitudinal muscle fibers ; third, of a layer of circular muscle fibers, inside of which there is, in places, a layer of longitudinal fibers ; fourth, of a very thin layer of cells with flattened, deeply stainable nuclei ; fifth, of a layer of epithelium. This layer is derived from the peritoneum." Stuhlmann found the ovarian wall in Zoarces to have a toler- ably deep, non-striated, muscle layer, the fibers of which were closely packed together next to the peritoneal covering, but toward the lumen they were split apart by numerous sinuses containing blood vessels. The oviduct was similarly composed, except that there were few if any clefts between the fibers and there were fewer blood vessels. 5O HENRY H. LANE. The unique feature in the ovisac as well as in the epithelial •Covering of the stroma of Lucifuga and Stygicola is the presence of capillaries in the lining epithelium (Fig. 8). So far as could be determined this condition has never been observed in the ovary of any other form. So numerous are these capillaries that they attract attention at the first glance. The epithelium itself is often reduced to extreme thinness, sometimes serving merely as a mem- brane to contain the blood. VII. THE OVARY. The ovarian structure itself is highly vascular and much lobed. There is a tendency in some instances for these lobes to be ar- ranged in a bilaterally symmetrical pattern, when seen in cross- section, though this is not equally evident in all ovaries or even in all parts of the same ovary. The ovarian structures of the different specimens examined, while presenting numerous points in common, are yet characteristically different in every case. The ovisac of A had but recently contained young, to judge from its extreme thinness ; the stroma was so large that it gave promise of containing embryos. Instead of that condition, how- ever, it was found that the large size was due to the mass of stroma which is composed in part of highly vascular tissue. Numerous blood vessels penetrate the stroma in all directions — while around the ova themselves there is a network of capillaries. The greater portion of the stroma is split up into numerous sinuses, many of which are larger than any of its blood vessels. These are closely similar in appearance to the " lymph-spaces ): described by Stuhlmann (op. cit., p. 19) for Zoarccs and no doubt serve the same purpose (Fig. 7, /.5.). The ova of A are few in number, less than ten over 60 /J. in diameter appearing in any cross-section. Five or six ova are of quite large size, visible even to the naked eye, and measuring from 300 to 800 11 in diameter. They have a large amount, proportionately of yolk-substance. The smaller ova are about 50 to 60 IJL in diameter, and are of the usual appearance of ova of that size. The cells of the stroma in this ovary are very irregular in shape, indistinct in outline, and usually of inconsid- erable size. The nuclei are round, oval, or elongated, appar- OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 5 I ently influenced as to form and shape by the cell-body. The entire surface of the stroma is covered by a layer of epithelium, with a depth of 10 to I 5 ij.. The nuclei of these epithelial cells appear quite distinct, are of a comparatively large size, and are round or oval in shape. By far the largest amount of space in this epithelial layer is given up to the numerous capillaries contained in it. They are so numerous that, in cross-section, they appear as a row of large per- forations, there being no more than a scant cell thickness between them. The average diameter of these capillaries is less than eight micra, in many instances being only five micra. This con- dition is comparable to that described above for the ovisac and is also unique (Fig. 8). The ovarian stroma of E, which contained the mature foetuses, has been squeezed and crowded into a median position by the young (Fig. 5). The cellular structure resembles that of the ovary just described, except for such variations as would be caused by its closely packed condition. The capillaries of the epithelial layer, covering its surface, are not so numerous as in the ovary of A, but are of larger size. The larger blood vessels are more nearly cylindrical in form and have their walls more thick- ened than have those in the first ovary. The lymph-spaces in this ovary are compressed by the foetuses and temporarily elimi- nated. Quite different in appearence from either of the two just de- scribed, though somewhat intermediate between them in some respects, and more advanced than the first in others, is the ovary of D. In this the ovarian stroma has not so many nor such large lymph-sinuses as A, but on the contrary has more nearly the ap- pearance of that in E, from which it differs conspicuously, how- ever, in not showing a "crowded" appearance, and in having quite numerous ova of various sizes, though none of the latter are so large as those of A, and in many cases are grouped together in " nests " in a way largely unknown in A. The blood vessels are comparatively numerous, large and quite thick walled. The capillaries in the epithelial covering of the ovary — so conspicu- ous is that of A — are so few in this case as to be visible only when carefully searched for. The cellular structure near and 52 HENRY H. LANE. next to the surface is dense and without important sinuses. The outlines of the cells are very indistinct, but the nuclei are alto- gether similar to those of the ovaries previously described. The ovary of C approaches more nearly to the condition of A than has any of the others ; but it differs very characteristically, since in many places it contains " nests " of ova much more con- spicuous than any seen in A, while the largest ova in this speci- men are larger than those in A. The ovarian structure itself, while evidently of the same character as that of A, does not con- tain quite so many lymph-spaces, and the walls of the sinuses are somewhat thicker and denser. The ovary of B is almost exactly in the same stage as that of A. It differs from C in that the egg-nests have given place to single ova of considerable size and greater development than most of those in the latter. It will be noted that the " nests " of ova are conspicuous in Lucifuga, though inconspicuous or lacking in the specimens of Stygicola examined. Whether this is a constant distinction can only be determined by the examination of more material than I have in hand. VIII. BLOOD SUPPLY TO THE OVARY. A small artery, with a diameter, in different ovaries, of 20 to 75 fjt, enters each horn of the ovary and runs back near the inner surface of the horn. In the main portion or body of the ovary, the two arteries occupy parallel courses near the center, separated by perhaps one third the diameter of the ovary. Since none of the specimens at hand were injected, the course of these arteries could not be traced except in a general way. But it is plain that they extend posteriorly in a tortuous course through the ovary and give off numerous branches, which find their way to or toward the surface, where they form the capillaries so dis- tinctly visible in some of the ovaries in the epithelial covering. The blood from the epithelial capillaries of the anterior half of the ovary is collected by veinlets, frequently quite large and dis- tinct in the vicinity of the larger ova, which join to form larger veins that pour their contents into the chief vein of the ovary at the " horseshoe bend" (infra). This largest vein has two branches OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 53 (one going out by either horn) which are united near the point of division of the ovarian horns, forming a single " horseshoe"- shaped vessel. The veinlets which return the blood from the posterior part of the ovary collect into one vessel which joins the right horn of the "horseshoe" at a considerable distance in front of the fork of the ovary, after running above and parallel to the portion with which it unites, for the distance, in one speci- men at least, of nearly 2 mm. It quite frequently occurs that red-blood corpuscles are pres- ent in the ovarian sinuses of Stygicola and Lucifitga, though their presence may be due to accident. As indicated elsewhere, these sinuses are very probably filled with a plasma or lymph. BIBLIOGRAPHY. Agassiz '53 (Embiotoca.) Am. Journ. ofSci., Vol. XVI., 2d ser., Nov., 1853. Cuvier et Valenciennes. '28 Histoire Naturelle des Poissons. Paris, 1828. Duvernoy. '44 Observations pour servir a la connoissance du developpement de la Poecile de Surinam (Poecilia surinamensis). Ann. des Sc. Nat. Zoolog., ser. III., T. I., 1844. Eigenmann. '92 Cymatogaster aggregatus Gibbons ; A Contribution to the Ontogeny of Vi- viparous Fishes. Bull. U. S. Fish Commission, Vol. 12, p. 401, 1892 (1894). Eigenmann. '97 Sex-Differentiation in the Viviparous Teleost Cymatogaster. Archiv f. Ent- wickelungsmechanik der Organism, Bd. IV. Leipzig, 1897. Eigenmann. '03 The Freshwater Fishes of Western Cuba. Bull. U. S. Fish Commission, for 1902 (1903), pp. 213-236 (3 pis.). Gill. '63 Stygicola. Proc. Ac. Nat. Sci., Phila., 1863, 252. Girard. '58 Explorations and Surveys for a Railroad from the Mississippi River to the Pacific Ocean, IV., Fishes, 1858. (Embiotoca.) Jordan & Evermann. '98 Bull. 47, U. S. Nat. Mus., Fishes of North America. 1898, Pt. III. Ludwig. '74 Uber die Eibildung im Tierreich. Arb. Aus dem zoolog-zootom. Inst. Wiirzburg, Bd. I., 1874. Poey. '60 Memorias, II., Havana, 1860. 54 HENRY H. LANE. Rathke. '33 Abhandlungen zur Bildungs- und Entwickelungsgeschichte des Menschen und der Tiere, II. Leipzig, 1833, I., Abth. : Bildungs- und Entwickelungs- geschichte des Blennius viviparus oder des Schleimfisches. 61 pp. Ryder. '85 "On the Development of Viviparous Osseous Fishes, etc." Proc. U. S. Nat. Mus, 1885, pp. 128-162, Pis. VI. -XI. Stuhlmann. '87 "Zur Kenntnis des Ovariums der Aalmutter (Zoarces viviparus, Cuv. ). Tab. IV. (1887). Wyman. '57 Observations on the Development of Aiiableps gionovii. Bost. Journ. of Nat. Hist, Vol. VI., No. IV., 1857. The paper by William Wallace, B.Sc., "Observations on Ovarian Ova and Folli- cles in certain Teleostean and Elasmobranch Fishes," Quart. Jonrn. Micr. Sei., XLVII, (July, 1903), pp. 161-213 (3 pis.), was not seen by me until after my paper was in the Editor's hands. EXPLANATION OF PLATES. FIG. I. External ventral view of the ovary of Stygicola. Portions of the peri- toneal covering are visible along the sides. FIG. 2. Cross-section of ovary near the beginning of the oviduct proper. Two large ova at the sides. FIG. 3. Cross-section of non-pregnant ovary with stroma in two lobes — one dorsal, the other ventral. FlG. 4. Cross-section of pregnant ovary. The section is made through a region corresponding to that of Fig. 3. FIG. 5. Cross-section of pregnant ovary through the middle portion. The ovisac collapsed when the foetuses were removed. FlG. 6. Cross-section of pregnant ovary through the horns. FIG. 7. A portion of a cross-section of a non-pregnant ovary, showing a part of a large ovum (0} surrounded by its follicle (fl.c.} ; the epithelial covering of the stroma dips down, forming a tube to the ovum (D). Bausch and Lornb one sixth objective ; 2-in. ocular; tube length, 160 mm. FIG. 8. Portion of the epithelial covering of a non-pregnant ovary showing the capillaries (r/j. ). Bausch and Lomb one twelfth objective ; i-in. ocular. a. anterior. mr. mesorectum. cps. capillaries. o. ovum. d. dorsal. ovs. ovisac. D. the"Delle." ov.sf. ovarian stroma. fl.c. follicular cells. /. posterior. l.s. lymph sinus. v. ventral. mov. mesovarium. All drawings by the author; outlines made with Abbe camera; details put in free- hand but with the closest possible regard to accuracy. Vol. VI. January, /poy. No. 2 BIOLOGICAL BULLETIN. FORM-REGULATION IN CERIANTHUS, III. THE INITIATION OF REGENERATION. C. M. CIIfLD. In the first paper of this series ('03^) the typical course of re- generation in a cylindrical piece was described ; in the second paper ('03$) some of the factors influencing the process of regener- ation as a whole were discussed ; these papers have served to clear the ground for a detailed analytical study of the process of re- generation in Cerianthus in its various manifestations. In this and following papers of the series various phases of this subject will be considered. CHANGES IN FORM CONSEQUENT UPON SECTION. The reduction in size of the opening at the end of a cut piece by the bending inward of the cut margins was described in its simplest form in the first paper of this series. A somewhat more general consideration of this peculiar process is necessary before proceeding to the discussion of other points. Early in the course of my experiments upon Ccriantlnis it was noted that in nearly every case, however the pieces might be cut, the body-wall became rolled or folded in such a manner that the opening into the enteric cavity resulting from the cut \vas much reduced in size or was closed by approximation or contact between different parts of the body-wall. The usual result of the infold- ing is the complete removal of the entodermal surfaces from con- tact with the external water, /. c\, the piece rolls up or closes in such mariner that the entoderm is on the inside. For conve- nience we may designate inrolling about a transverse axis as transverse inrolling, and inrolling about a longitudinal axis as longitudinal inrolling. At first glance this process appears much like an adaptive re- action. In some cases it is almost as if the animal or part were 55 50 C. M. CHILD. consciously closing the artificial openings. In the following paragraphs the principal forms of inrolling in the cut pieces are described. The case of the closure of the ends of a cylindrical piece which was described in the first paper is the simplest of all. Collapse occurs with the escape of the water from the enteron and within FIG. 2. FIG. 6. FIG. i. FIG. 4. FIG. 8. FIG. i. FIG. 7. FIG. 9. a few moments the cut ends begin to bend inward and finally close the openings except for the small slits between the folds. The diagrams, Figs. I,1 2 and 3, illustrate this case, Fig. I repre- senting the cylindrical piece at the time of section, Fig. 2 the longitudinal section of the ends after the bending in of the cut margins, and Fig. 3 a transverse section, indicating the flattening 1 The diagrams representing the inrolling are much less highly magnified than pre- ceding figures. FORM REGULATION IN CERIANTHUS. 57 of the piece as it lies on a flat surface. In pieces of this kind complete collapse and contact of the body-walls is prevented by the large mass of mesenteries and mesenterial filaments which occupy the enteron. These are not represented in the figures, but they fill the whole enteron after collapse. Any solid mass in the enteron would of course have the same effect. A piece cut from the extreme aboral end of the body (Fig. 4) differs in certain respects from the piece just described. Figs. 5 and 6 show the changes in a piece of this kind. Here the cut end becomes rolled inward to a much greater extent than in the previous case so that the enteron is nearly filled by the inrolled portion and the cut surface is so situated that closure by growth of new tissue from this surface is impossible. The reason for the greater degree of rolling in this piece as compared with the longer piece is undoubtedly to be found in the absence of mesenteries, except a single pair, in the aboral region. Since the enteric cavity is not filled with a mass of mesenterial filaments as in a region further orally the inrolling continues until the entire cavity is practically obliterated by the inrolled parts. If a cut be made in one side of the body or a piece removed as in Fig. 7, the cut edges roll inward as in other cases, but in addi- tion to this the body becomes bent at the level of the cut, so that here also the inrolled edges are brought into contact (Figs. 8 and 9). The widest departures from the typical form are found, how- ever, in those pieces which were cut longitudinally as well as transversely. In these the results differ to some extent accord- ing to the shape and relations of the pieces. Fig. 10 represents a cylindrical piece split longitudinally on one side. One form after collapse and inrolling of cut margins is shown in Fig. n. Fig. 12 represents a transverse section and Fig. 13 a longitudinal section of one end. In Fig. 14 another form of closure is rep- resented ; here the ends fold over to a greater extent so that the opening is entirely on one side of the piece. Fig. I 5 represents a transverse section of this piece and Fig. 16 a longitudinal sec- tion in the plane indicated by the vertical line in Fig. 14. In most cases, however, the right and left longitudinal cut edges do not roll inward with equal rapidity and the result is C. M. CHILD. that the piece rolls up spirally on its longitudinal axis. Such a case is shown in Fig. 17 ; here the inner and outer coils of the spiral are on the same level at the outer end of the piece. Fig. 19 shows a spirally coiled piece in which the inner coils are higher than the outer. Figs. 18 and 20 represent longitudinal sections of one end of these pieces and Fig. 2 i a transverse sec- tion. The Figs. 1 1—21 represent only the chief types resulting from pieces like Fig. 10. All possible intermediate forms and modifi- FIG. 13. FIG. 15. FIG. j i . FIG. 14- FIG. 16. FIG. 10. cations of these different types occur, the differences depending on various conditions, but chiefly on the relative rapidity of the inrolling in the different directions. Semi-cylindrical pieces or longitudinal strips may roll either longitudinally or transversely. The greater the length and the less the breadth of the strip the more likely it is to roll trans- versely. Figs. 22—24 show a strip and two forms of trans- verse rolling which it may undergo. The longitudinal strips FORM REGULATION IN CERIANTHUS. 59 often roll longitudinally soon after section, but by gradual inroll- ing of the ends finally become rolled transversely. Loeb ('91) has suggested that the cut edges roll inward be- cause the inner layers of the body- wall are stretched to a greater degree than the outer layers ; this view assumes that all layers are more or less similar in elasticity and therefore that the layer that is most stretched will undergo the greatest con- traction when the tension ceases. It is difficult to understand why one layer of the body should be more stretched than another, since all have been subjected to the same conditions, viz., the tension resulting from the fluid pressure on the walls of the enteron. FIG. 17. FIG. 1 8. FIG. 19. FIG. 20. FIG. 21. FIG. 22. FIG. 23. FIG. 24. I am inclined to believe that this remarkable capacity for roll- ing which the pieces exhibit is due primarily to a difference in elasticity between the different layers of the body-wall, though it may be increased or modified by other factors. The succession of layers in the body-wall is as follows : ectoderm', longitudinal muscles, mesogloea, entoderm. The mesoglceal layer is fibrillar in appearance and, while not as thick as the muscular layer, is well developed. Judging from the fact that this layer is not folded or wrinkled, even in strongly contracted animals, the inference that it possesses 60 C. M. CHILD. a considerable degree of elasticity appears justifiable. Under normal conditions the body-wall is subjected to tension. By sec- tion of the body at any point the internal pressure is removed and collapse occurs ; the body-wall is no longer under tension and contraction of the elastic layer begins. If the ectoderm and muscles are to a large extent passive in this elastic contraction the result will be not simply a reduction in surface area, but an inrolling of the body-wall, since the mesoglcea is situated near its inner surface. The fact that the region near the cut surface is always more strongly rolled than other parts may perhaps be the result of the direct injury to the tissues in this region, causing contraction, but here as elsewhere the contraction must be greater in the inner portions of the body-wall than in the outer, otherwise inroll- ing could not occur. More probably, however, the greater degree of inrolling near the cut surface is largely, if not wholly due to the fact that the physical obstacles to the inrolling offered by resistance of other tissues, etc., are much less near a free end or cut surface than elsewhere and the effect of elasticity is there- fore greater. It appears probable from the preceding consider- ations that the mesoglcea plays the chief part in the inrolling about the cut surface as well as in regions distant from it. Objection to this view may, however, be made on the ground that a tonic muscular contraction resulting from the injury is not only a possible but a much more probable cause of the inrolling. A brief consideration of the facts is sufficient to show that the inrolling cannot be explained as the result of muscular contraction. This is evident first from the fact that it occurs in all directions, longitudinally and obliquely as well as transversely. If it were the result of muscular contraction we should expect it to occur only transversely since the body-wall contains only longitudinal muscles. It is difficult to understand how the contraction of longitudinal muscles could account for the inrolling of a longi- tudinal cut margin, since the muscle fibers are parallel to the cut. Moreover, there is no apparent reason why the inner por- tions of the muscular layer should contract more strongly than the outer, since all must be equally affected by the injury. And finally, observation renders it very evident that the inrolling is FORM REGULATION IN CERIANTHUS. 6 1 not due to muscular contraction, for although strong contraction occurs at the time of section the muscles relax within a few moments and before inrolling begins. Moreover, the muscular contraction consequent upon stimulation of pieces after inrolling has occurred causes in most cases a more or less complete un- rolling, provided the inrolling was in the transverse direction but does not affect inrolling in the longitudinal direction. In the light of these facts it is difficult to escape the conclusion that the inrolling is caused by some part of the body-wall axial to the muscular layer, viz., either entoderm or mesogloea. The delicate cellular layer of entoderm cannot be supposed to possess any such elasticity ; there remains therefore only the mesoglcea. It now remains to consider whether the different forms of in- rolling described and figured above are all explicable on the basis of elasticity of the mesogloea. For this purpose we may regard the tension as resolved into longitudinal and transverse components. As regards the inrolling of cylindrical pieces with transverse cut margins (cf. No. I of these studies, '03^, also Figs. I and 2 of the present paper), it is easy to see that it can proceed only a certain distance. Since the elastic tension is present in all parts of the cylindrical piece reduction in size may occur, but the cut ends are the only regions where marked change of form can take place. These are bent inward until the more prominent folds come into contact and the size of the opening is reduced. Beyond this the inrolling cannot go since contact between dif- ferent parts of the margin and the radial folds into which the contracting margin is thrown both oppose further change. The appearance of the radial folds requires a word of explanation. Their presence would seem at first glance to indicate that elastic tension exists only in the longitudinal direction. A brief con- sideration will show, however, that this is not the case. After escape of the water from the enteron and collapse of the body, reduction of the circumference occurs throughout the whole piece, undoubtedly in consequence of the elasticity of the body- wall. There is, however, no physical ground for greater con- traction in the transverse direction at the ends than elsewhere since there is no break in the transverse continuity of the body- 62 C. M. CHILD. wall. The longitudinal component must cause inrolling at the cut end until either the local tension due to the formation of radial folds in consequence of the in rolling or the mutual contact of appressed portions of the inrolled wall opposes a resistance equal to the elastic tension. It is probable that in the oral half of the body, the mass of mesenteries and mesenterial filaments also oppose more or less resistance to the inrolling margins. The flattening of the piece which often occurs (Fig. 3) is simply the result of gravity. If the collapsed piece lies on one side during several hours the weight of the body-wall is sufficient to bring about the flattening to a greater or less extent. In con- sequence of the flattening the openings at the ends are frequently elongated in the plane of flattening and slit-like, the inrolling occurring chiefly on the two margins of the slit. In pieces from the extreme aboral end the inrolling at the cut margin may proceed much further (Figs. 4-6). Here the body- wall and especially the muscular layer is much thinner and must offer much less resistance to the elastic tension. Moreover, the enteron is practically empty in this part of the body. In con- sequence of these conditions the inrolling may proceed so far that portions of the margin are directed orally (Fig. 6). The closure of a lateral cut by bending of the whole piece (Figs. 7—9) especially resembles a definite adaptive reaction, but can be explained as the result of elastic contraction. A cylindri- cal piece such as Fig. I does not become bent or curved so long as elastic tension on opposing sides of the body is equal. If in any way the tension on one side be reduced in effectiveness the body must bend toward that side. A transverse cut through the body-wall on one side, or the removal of a piece as in Fig. 7 interrupts the continuity of the body-wall. The longitudinal component of the elastic tension acts on the parts above and below the cut and causes contraction and inrolling of their edges. But by removal of a piece of the body-wall an open space is left and the longitudinal component of tension on the opposite side of the body causes bending of the piece so that the concave sur- face is on the side of the cut. The larger the piece cut out from the one side the greater will be the bending since it will continue until contact between the cut margins affords a resistance equal to the opposing tension. FORM REGULATION IN CERIANTHUS. 63 Figs. 10-21 require little explanation. Here transverse con- tinuity is interrupted by a longitudinal cut on one side. The form of the piece after inrolling is at least in large part a matter of chance, being dependent upon the relative rapidity with which the different margins roll inward. In Fig. 1 1 the inrolling at the ends has been less than in Fig. 14 and the resulting form is different. In Figs. 17—21 the spiral form is due simply to the fact that one longitudinal cut margin rolled inward somewhat more rapidly than the other. An oblique spiral results from more rapid inrolling of the longi- tudinal margin near one end. It is clear that various conditions such as the degree of contraction of the muscles of a certain part of the body, the resistance offered by the mesenteries, the posi- tion of the piece in the aquarium, etc., may constitute conditions affecting the result. The frequent rolling about a transverse axis of longitudinal strips cut from the body is clearly the result of the predominance of the longitudinal component of tension. It is interesting to note that this transverse rolling occurs only when the muscles are fully relaxed. If the piece be stimulated sufficiently to cause strong muscular contraction more or less complete unrolling often occurs. Pieces of this sort frequently roll about a longi- tudinal axis after cutting while the muscles are more or less con- tracted and then as the muscles relax after a longer or shorter time begin to roll transversely and continue until completely rolled up in a single or double spiral. In cases of spiral or transverse inrolling (Figs. 17, 19, 23, 24) there is little resemblance to an adaptive reaction. As will appear, typical regeneration is impossible in these cases. Since it is scarcely to be supposed that in pieces of a certain form the reaction is adaptive in nature while in pieces of other forms it is due merely to elasticity it is preferable at least to attempt to analyze the apparently adaptive reaction. In the present case I think the analysis has demonstrated that the various methods of inrolling are all explicable on the basis of elastic contraction of the mesoglcea. The apparently adaptive character of the inroll- ing in cylindrical pieces where it results in more or less perfect closure of the ends is due to the particular physical conditions 64 C. M. CHILD. present in such pieces. The inrolling of pieces after section is not then a definite reaction adapted to close the wound, except in so far as we may regard the presence of an elastic layer in the body-wall as an adaptation. After the inrolling is completed gradual reduction in the size of the whole piece continues until the artificial openings are closed by new tissue or otherwise and the water pressure is again estab- lished in the enteron. This reduction in size can scarcely be due to the loss of tissue in the absence of food, for that is much less rapid. The piece appears to contract continuously after collapse and closure and if the closure and distention with water is pre- vented in any way, becomes much reduced within a few days. Frequently new wrinkles or folds appear as the contraction pro- gresses, indicating that it is not due to actual loss of material but to some other cause. There can be no doubt that this reduction in size of collapsed pieces is simply a continued reduction in the surface area of the tissues resulting from mechanical conditions. It is due at least in part to the elasticity of the body-wall (or especially of the mesoglcea). This being effective in all directions must cause gradual reduction in size of the whole after the inrolling of the margins is completed, unless it is counterbalanced in some way, which is not usually the case. This quality of the body-wall is remarkable ; pieces kept under conditions where distention with water is impossible often contract to half the size after section and collapse. If they are then permitted to close and become distended with water they may again attain in two to three days almost the original size, provided the period during which they remained contracted was not too long. The longer the period of collapse the slower and less complete is the return to the original size. These facts indicate that in the absence of the ten- sion due to internal water-pressure the tissues gradually rearrange themselves in accordance with the altered physical conditions. There is no return to the " normal " form unless mechanical con- ditions once more become normal. In his study of Ccriantlius, Loeb ('91) has attempted to explain the collapse of tentacles and other phenomena by loss of turgor in the cells. As I shall show in a later paper, this explanation is FORM REGULATION IN CERIANTHUS. 65 wholly incorrect. The question as to whether osmotic phenom- ena play a part in the changes above described requires, however, a moment's consideration. As regards the inrolling after section and the reduction in size of the collapsed pieces there is certainly no reason for supposing that it is due to changed osmotic condi- tions. It is difficult to understand how, in a form like CcriantJius section of the body-wall at one level should cause changes in turgor in the cells of the whole piece or of those at a distant region unless we suppose that special stimuli producing these changes arise from the region of the cut. If this be the case then the change is not primarily osmotic but reactive. More- over, the phenomena are so obviously due to elasticity that the search for any other explanation is clearly unnecessary. THE ROLE OF THE SLIME SECRETION IN THE CLOSURE OF THE ENTERIC CAVITY AFTER SECTION. As has been shown, the inrolling of the margins of the piece under ordinary conditions approximates the various parts of the cut surface, and thus reduces the size of the opening. The radi- ating wrinkles and folds into which the inrolling portions are thrown and the frequent protrusion of parts of the mesenteries through the opening render the closure by contact imperfect. There are always slits and angles between the various parts, through which the enteric cavity is in communication with the exterior. In spite of this fact I have often found pieces distended with water, before any closure of the ends by new tissue has occurred. A series of experiments in which the body-wall was sectioned transversely at some level and the oral portion, still bearing ten- tacles and disc intact, was used, will serve to illustrate this point. In every case collapse of the tentacles and body occurred imme- diately after section, owing to the escape of water from the en- teron, but very frequently the whole oral piece including the ten- tacles was again distended with water in less than an hour. O Examination of the aboral cut end in such cases showed that inrolling and approximation of the margins had occurred, but frequently distinct spaces between the wrinkles could be observed opening into the enteron. If the end were spread open with 66 C. M. CHILD. needles collapse occurred at once, but was followed by renewed extension in a short time. Pieces with tentacles and disc intact show these changes much better than others, since the phenom- ena of distention and collapse are especially conspicuous in the tentacles. Moreover, in these pieces the presence of the mouth permits much more rapid entrance of water than is possible in pieces with oral end removed, since in these latter there is no apparatus for forcing the water into the enteron. The rapid distention of pieces under the conditions described is made pos- sible only by the ectodermal slime secretion which under normal conditions forms the tube. The manipulation incidental to section of the body and the stimulus of the cut itself cause a rapid secretion of this slime during the operation and for some time after. The secretion is tenacious even when first formed and clings closely to the body. After inrolling has occurred at a cut surface only ectoderm is visible from without and where different parts of the inrolled margins are in contact the contact is usually in part ectodermal. The slime is secreted over this inrolled portion and forms a tenacious coating which closes all the crevices between the in- rolled portions of the body-wall. Thus, so far as the escape o water in appreciable quantities is concerned, the cut end may be closed within a short time after section, almost as soon, in fact, as the inrolling is completed. If the piece is left undisturbed the slime accumulates and the closure becomes more and more complete until finally the thin membrane of new tissue constitutes the definitive closure. If at any time before the definitive closure the slime be carefully removed with needles without causing violent contraction or changes in form of the piece, collapse will occur at once, showing clearly that the slime alone prevented the escape of the water. THE GROWTH OF NEW TISSUE FROM THE CUT SURFACE. The closure of the ends by new tissue was briefly described in the first paper of the present series, but the conditions which determine it were not discussed. As described, the course of the process at both oral and aboral ends in typical regeneration is as follows : First the appearance of FORM REGULATION IN CERIANTHUS. 6/ a thin membrane of new tissue between those regions of the cut surface which are sufficiently approximated ; the growth of this membrane until the whole opening is closed ; the increase in size of the area of new tissue as the piece becomes distended. During the course of my experiments it was found that certain definite conditions are necessary for this growth of new tissue. Mention was made of the fact that the new tissue appears first in the folds and wrinkles where two cut surfaces are most closely in contact and that from these regions it extends until closure is complete. In pieces rolled spirally (Figs. 17, 19, 24) or in any such manner that the cut surfaces are not brought into contact no appreciable growth of new tissue occurs ; the cut edges heal, but may remain without further change for months. Thus, in these spirally or transversely rolled pieces typical regeneration of new tissue from the cut surface does not occur. Moreover, this is true of all cases in which there is no approxi- mation or contact of two cut surfaces or parts of a cut surface. Never is a thin membrane of new tissue found growing out from a cut surface and without other connections. When present it always connects two cut surfaces or the two sides of a fold where different regions of the cut surface have been approximated. This is a point of considerable importance ; indicating as it does that there is nothing in the cut surface itself which initiates re- generation, the necessary condition being found rather in the relations of different cut surfaces or their parts. Never do we find regeneration of the body-wall occurring in the manner represented in the diagram, Fig. 25, as a continuation with free margin of the old tissue. New tissue arising from cut surfaces always appears between two cut surfaces which are in contact or closely approximated as in Fig. 2. These surfaces become united by new tissue which then increases in amount under cer- tain conditions, thus forming a thin membrane connecting the two parts of the cut surface. In the ordinary closure of the end of a cylindrical piece the new tissue first appears, as has been noted, in the folds and wrinkles where parts of the cut surface are closely approximated (Figs. 6—8, '03^), but from this it spreads rapidly until the whole space is covered and the end closed (Fig. 9, '03«). 68 C. M. CHILD. The process is briefly as follows : After exposure of a cut sur- face some slight proliferation occurs which results in healing unless another cut surface be so near that the cells arising from o both are in contact ; if this is the case then organic union be- tween the two cut surfaces is rapidly established. In the closure of the ends of cylindrical pieces this process is usually completed in a few days, but in certain other cases it may proceed much more slowly. For example, in pieces which are split down one side (Fig. 10) and in which both of the longitudinal cut margins roll inward as in Fig. 11, the process of closure often requires two months or more for completion. In such cases the longitu- dinal cut margins usually roll inward so far that they are not in contact. At one or both of the ends, however, the closure may occur in nearly the typical manner. From the end the new tissue begins to grow along the longitudinal cut, and as it grows actually draws the cut edges together to a certain extent. The process may be compared for the sake of illustration to that of sewing up the longitudinal slit in the piece from one or both ends. If we take, for example, a case where two cut surfaces are in con- tact at one point and diverge at an acute angle from this point, we find that the growth of new tissue always begins at the point of contact. From this point growth and the formation of a thin membrane continue for a certain distance along the diverging cut surfaces, the extent of the membrane depending in a given species on the angle of divergence of the surfaces. This thin membrane is itself somewhat elastic and so tends to approximate the cut surfaces in greater or less degree unless opposed by other conditions. The approximation of the surfaces renders possible a further extension of the membrane between them, and so the process continues unless at some point the cut surfaces are so situated that the elasticity of the new tissues is insufficient to bring them into contact, or near enough to permit the extension of the thin membrane between them. In such a case the process of closure must cease, as often occurs. That this is actually what occurs I have convinced myself by repeated examination of speci- mens cut in such manner that at least some parts of the cut sur- faces were not in contact while others were. The diagrammatic Figs. 26—30 will serve to illustrate the process. Fig. 26 shows FORM REGULATION IN CERIANTHUS. 69 a cylindrical piece slit down one side and represented as cut across obliquely to show the separation of the cut surfaces ; the cut surfaces have rolled inward and the oblique section at the lower end of the figure shows that the inrolling along the longi- tudinal cut is so great that the cut surfaces are not in contact. The growth of new tissue and closure begins at the oral end (it may begin at the aboral end also) where the cut surfaces are much more closely approximated and the numerous folds afford FIG. 25. FIG. 27. FIG. 30. FIG. 28. FIG. 26. FIG. 29. FIG. 3r. various points of contact. From this region it gradually extends along the longitudinal cut, drawing the cut surfaces together as it proceeds, as is shown by the two transverse sections, Figs. 28 and 29, Fig. 28 from a level where union has already occurred (the upper transverse line in Fig. 27) and Fig. 29 from a level just beyond where the cut surfaces have united (the level of the lower transverse line in Fig. 27). In Fig. 28 the margins have been drawn together and united, in Fig. 29 they are not yet in contact but are nearer together than at the level of the oblique section at the lower end of Fig. 27. In Fig. 30 the closure is 7<3 C. M. CHILD. finally complete. In these figures only one end has been shown ; usually, however, closure proceeds from both ends, and the mid- dle portions are the last to become united. It is evident that in a spirally or transversely rolled piece this process can never occur, for even if it begins in some fold or local approximation of the cut surfaces it cannot continue, because approximation of the cut surfaces to a degree sufficient to permit their union by new tissue is impossible as long as the piece remains spirally or transversely rolled. Description of all possible cases of closure of pieces cannot of course be attempted. Results depend so largely on chance, that every piece affords, it might almost be said, a different solution of the problem. The examination of hundreds of pieces has, how- ever, convinced me that the essential features of the process are those described above. The cut surfaces appear to remain capable of giving rise to new tissue for an indefinite period. Often closure of pieces slit open longitudinally is completed only two or three months after sec- tion ; yet during all this time the cut surface retains the power of producing new tissue under proper conditions, though it has no power to produce anything more than the proliferation con- nected with healing, provided it is not in contact with another surface. Union is as complete and perfect, though perhaps not as rapid, when it occurs two months or more after section as when it occurs within a few days. When we compare C. solitariits and C. membranaceus we find that in the latter species the thin membranous growth of new tis- sue if once begun between cut edges in contact, may continue until it forms a connecting membrane between widely separated cut surfaces ; in C. solitaries , on the other hand, the membrane is incapable of bridging over spaces so wide ; the new tissue ceases to extend long before a point is reached where the cut surfaces are so widely separated. This difference is so marked that it raises the question as to whether there is a fundamental difference in the conditions and method of growth of new tissue in the two species. Figs. 6-9 of paper I. ('03^) and Fig. 31 of the present paper (an aboral end) illustrate the closure of open- ings in C. membranaceus. In Fig. 31 the new tissue which is FORM REGULATION IN CERIANTHUS. /I growing over the opening is in two parts which are advancing to meet near the middle. The concave free margin of both portions is noticeable. The dotted lines represent various stages in the growth of the new tissue. It is evident that it appeared first in the angles where two cut surfaces were in contact or very closely ap- proximated. In C. solitarius closure by new tissue of only very much smaller pieces is possible. I believe the explanation of this difference is to be found in the different quality of the thin mem- brane of new tissue in the two cases. In C. meuibranaceus it is much thicker, more resistant, and less easily ruptured than in C. soli- tarius. The new tissue arises at a region where the cut surfaces are close together and may extend from this to regions wHere they are more widely separated. As was shown above, it exerts a certain degree of tension on the parts connected by it. As the distance between the cut surfaces increases a point may finally be reached where the tension is equal to the cohesive power of the tissue ele- ments. Beyond this the new tissue cannot extend. In C. solitarius this limit is attained with a slight separation of the cut surfaces, while in C. membranaceus the new tissue is capable of resisting much greater tension and so of extending over wider spaces. The membrane extending between the two cut surfaces may be compared with a fluid film bounded by lines diverging at an acute angle. The film extends a certain distance from the apex of the angle, this distance being determined with a given fluid by the size of the angle. The free margin of the film is always concave toward the opening of the angle. So long as the relation between cohesion and adhesion remains the same and the angle does not change the film can never extend beyond a certain point, since the surface-tension will cause rupture. As the angle and surface- tension decrease or as the adhesion increases the film will spread. If the*arms of the angle are sufficiently pliable or capable of movement they may be drawn together by the surface-tension of the fluid, and thus permit further extension of the film. In Cerianthus the thin membrane of new tissue which may be compared to the fluid film, arises at the apex of the angle, /. e., where the two cut surfaces are in contact. The membrane ex- tends along the diverging surfaces to a certain point. Its free margin is always concave (Fig. 31, also Figs. 6—8 ; '03^). The /2 C. M. CHILD. distance to which the membrane spreads between the surfaces depends upon its composition and the degree of divergence of the surfaces, just as in the case of the fluid film. The thicker membrane with greater resistance will grow farther just as the film with less surface-tension will spread farther, other things being equal. Even the elasticity of the newly formed membrane is paralleled by the tension to which the fluid film is subjected. In both cases the margins of the space may be approximated by this tension and thus permit further spreading of the connecting film or membrane. The illustration of the fluid film has been employed primarily as an analogy. It is not to be supposed that the thin membrane growing between two cut surfaces behaves in all respects like a fluid film extending across an angle. Yet the close parallelism between the two series of phenomena must raise the question as to whether after all the growth of new tissue from a cut surface in Cerianthus may not be, at least to a large extent if not entirely, determined by the laws which govern the behavior of fluids. The following facts point toward this conclusion : except so far as heal- ing of a cut surface is concerned new tissue arises from a cut sur- face only when it is in contact with another, the thin membrane of new tissue which is under tension spreads between diverging cut surfaces to a certain point, beyond which no growth occurs, unless the surfaces are brought nearer together ; the point where growth ceases differs in different species, depending on the quality of the membrane ; the cut surfaces may themselves be approxi- mated by the tension of the membrane and so further growth of the membrane made possible ; the free margin of the membrane is always concave in the direction of growth, /. c., the margins of the membrane extend further than its middle region. In all of these respects the thin membrane and the fluid film behave simi- larly. The conclusion is at least probable that the similarity in behavior is due to the fact that similar conditions are present. I think it probable, therefore, that the appearance and growth of new tissue from the cut surfaces of the body of CcriantJnts is governed, at least to a large extent, by the laws of capillarity. Qf course the cellular structure of the tissue may complicate con- ditions, and the thickening and structural differentiation which FORM REGULATION IN CER1ANTHUS. 73 occur in the membrane after its formation bring into play other factors. These need not be considered here, however. Certainly the phenomena are far from being adaptive or teleological in any sense although the closure of the cut might appear at first glance to be an adaptation. It is difficult at present to see how they can be due to anything except simple physical conditions, though it is possible that increased knowledge may afford another explan- ation. Provisionally then we may regard the delicate thin mem- brane which appears in the angles between cut surfaces as pos- sessing some of the properties of a fluid and as subject, at least in large degree, to the laws of capillarity. Whether these suggestions are correct or not, the two facts above mentioned are of great importance, viz., that regeneration of new tissue from cut surfaces occurs only when two surfaces are in contact, and that the new tissue cannot extend indefinitely between diverging cut surfaces but ceases at a certain point de- termined by the angle of divergence of the two surfaces and the (physical) quality of the membrane, /. e., is different in different spe- cies. The only possible inference from these facts is that all con- ditions for regeneration are not given in the living tissues them- selves, nor in these plus the normal environment as a whole, but that the formation of new tissue from a cut 'surface is probably dependent upon certain simple physical conditions similar to those which govern the existence of a liquid film between two diverging boundaries. Healing of the cut surface does not require these conditions ; for this the necessary conditions, which are very probably also primarily due to capillarity, are established by the cut itself. The same conditions are not, however, adequate for the formation of a membrane of new tissue from the cut surface. In this case then the conditions for new growth and closure of a wound are to be found, not in the absence of a certain part, nor in the presence of a special stimulus at the cut surface, but in simple, external, physical relations of parts. Discussion of the bearing of these facts may be postponed to another time. Atten- tion may be called, however, to the difficulty of reconciling these facts with the neo-vitalistic theories of life and especially with that • of Driesch which is based upon the phenomena of form-regula- 74 C. M. CHILD. tion and has adopted a modification of the Aristotelian idea of an entelechy as the basis of organic form. SUMMARY. 1. The inrolling of the margins and the closure of openings by contact of the inrolled margins is the result of the elasticity of the body-wall. This elasticity must be greater in the inner por- tions than in the outer portions, in order to produce the results observed. The facts indicate that the mesogloea plays the most important part in this elastic contraction. 2. Openings between folds of the inrolled body-wall may be stopped by the ectodermal slime secretion. This method of clo- sure often occurs in pieces before the formation of new tissue and permits the existence of considerable water-pressure in the enteron. 3. Contact or close approximation between two cut surfaces or parts of a cut surface is a necessary condition of the growth of new tissue from these surfaces. A single exposed cut surface may heal over but no further growth occurs from it. 4. The new tissue having arisen at a point of contact between two cut surfaces is capable of extending in the form of a thin membrane for a certain distance between diverging cut surfaces ; the distance to which it extends is determined in a given species by the angle of divergence of the cut surfaces, and in different species by the thickness and quality of the membrane. 5. The new tissue rising between two cut surfaces behaves in certain respects as if subject to the laws of capillarity. HULL ZOOLOGICAL LABORATORY, UNIVERSITY OF CHICAGO, September, 1903. BIBLIOGRAPHY. Child, C. M. '033 Form- Regulation in Cerianthus, I. The Typical Course of Regeneration. Biol. Bull., Vol. V., No. 5, 1903. 'O3b Form- Regulation in Cerianthus, II. The Influence of Position, Size and other Factors upon Regeneration. Biol. Bull., Vol. V., No. 6; Vol. VI., No. I, 1903. Loeb, J. '91 Untersuchungen zur physiologischen Morphologic, I. Heteromorphose. Wiirzburg, 1891. AN ABERRANT LIMB IN A CRAY-FISH. E. A. ANDREWS. A striking aberration in the form of a third, left-walking leg of a female Cambarus Bartoni found in class dissection in Febru- ary, 1892, seems of enough interest to warrant its being put on record. A view of the anterior face of the limb (Fig. i) shows a mark- edly forceps-like structure in addition to the usual forceps at the end of the limb, so that there are four instead of the usual two terminal points. The added structure is, however, not a true forceps with one movable finger, but a movable piece with two immobile prongs FIG. I. Camera sketch of anterior face of left third leg of C. Bartoni. Genita opening indicated in black. that otherwise resemble the index and pollex of a forceps. This is evident in the enlarged view, Fig. 2. The real forceps in this limb is nearly normal, but on compar- ing it with an anterior view of the third walking leg of a normal C. Bartoni (Fig. 3), of about the same size, we may note some differences. Thus, in place of the straight-lined articulation of dactyl and propodite, we find the propodite presenting a hollowed, socket-like face where the dactyl articulates. Again, while the dactyl and the index are both normal in form, the dactyl is not a straight continuation of the propodite, but bends down at a noticeable angle, thus increasing the wide divergence of the double set of tips of this limb. The propodite departs from the usual form in being wider dis- tally where it bears, as it were, a large protuberance that is 75 76 E. A. ANDREWS. truncated to articulate with the unusual pronged structure or second claw-like ending of the limb. There is also an abnor- mality in the propodite, indicated in Fig. 2, and suggesting some former injury ; it is a slight indentation upon the middle of the anterior face. The movements possible to the dactyl in the normal, alcoholic specimen (Fig. 3) are a swinging of 4.11 mm. in one plane to FlG. 2. Anterior view of terminal part of Fig. I. Camera. Zeiss 2, a. bring about direct apposition of the tips of the forceps, with no overlapping and also a very slight lateral movement. In the aberrant limb the dactyl is so set that apposition is im- perfect ; the dactyl passes the tip of the index by about .5 mm. while the entire swing is the same as above, 4. 1 1 mm. The actual gape of the forceps is restricted to that same amount, .5 mm. All movement in this forceps is strictly in one plane. Next considering the monstrous, pronged structure we find that, starting from the position shown in Fig. 2, there is a possible AN ABERRANT LIMB IX A CRAY-FISH. 77 movement of 2.5 mm. in the plane of the real forceps, one half of this being towards the forceps and one half away from it. There is also mobility at right angles to the above plane, in a general antero-posterior direction. Anteriorly this is 1.5 mm. and posteriorly about .7 mm., a total swing of 2.2 mm. On moving the abnormal structure and the normal dactyl as far as possible toward one another they came just into contact. The angle of divergence between the index and the most remote part of the abnormal structure is greater than the extreme opening of a normal claw. The form of the pronged structure is remarkable for its sym- metry : the two prongs (Fig. 2) differ but slightly in shape and in size. But the one standing nearer to the claw is slightly FIG. 3. Camera sketch of an anterior view of a terminal part of the third left leg of a normal C. Bctrtoni. thicker and less sharply pointed and it also lacks the clear, per- forated horn-like tip present upon the other prongs as upon normal claws. That this tip was lost by wear or by accident seems evident from the rough surface ending the prong and from the fact that staining liquids easily enter at this point. Since the anterior and posterior faces of the prongs are not alike one could not put either prong into a space of the shape of the other prong ; the two prongs are symmetrical about a plane between them ; they are mirror images of one another, except for the above noted difference in form. This symmetry extends to such details as the distribution of clusters of bristles or hairs and to the arrangement and number of the serrations along the op- posed faces of the prongs. This latter detail deserves special description. These serrations are like those along the opposed faces of the dactyl and the index and they add greatly to the impression that the pronged structure 78 E. A. ANDREWS. is in some sort an imitation claw. On both dactyl and index is a long series of transverse plates closely crowded together and freely projecting to give the serrated appearance noticeable under a low power. Each plate is itself serrated near its tip but these fine serrations are seen only with a higher power. As these plates stand nearer to the posterior than to the anterior face of the claw, they are more readily seen from a posterior view. Each plate, like these in Fig. 4, stands obliquely transverse and is shaped like a scalene triangle with bluntly rounded apex. It is just below this apex that the outer and distal edge bears a series of sharp teeth. This fine serration is on the edge that faces pos- teriorly as well as distally and the plates overlap one another so that the teeth could not be seen from an anterior view, such as Fig. 2, were it not that the plates are so transparent that the teeth can be seen through the next overlapping plate. Each plate has a central canal that passes from the epidermis through the length of the plate and ends at the surface in the blunt apex : it passes by the serrations without any connection with them. Morphologically these plates seem to be flattened setae or hairs. In this claw there are 61 plates on the dactyl and 67 on the index ; three or four are broken. As seen in Fig. 2 the series of plates is longer on the index where the proximal six or seven plates are opposed by a bare space upon the dactyl. With this exception the plates of the index and dactyl correspond, each plate having its duplicate in the opposite series. In the pronged structure there are two series of serrations show- ing this same symmetry ; some of each series are represented in Fig. 4. To save space the two series are drawn as if close together while in reality the rigid prongs always held the two series far apart (Fig. 2). The plate marked T is about the thirty-fifth one from the tip of the prong that has a perfect terminal spine. The edges with teeth are those nearer to the tip and also those farthest away from the serrations of the opposite series.1 The edge turned toward the plate of the opposite series is smooth and free. The third edge of the triangular plate is the line of attachment and is 1 The use of these serrated plates may be to aid in cleaning the animal rather than to aid in preparing food by acting against the opposed series ; they may be like the combs on the legs of certain birds. AN ABERRANT LIMB IN A CRAY-FISH. 79 i somewhat anterior to the free tip of the plate. The terminal plates are smaller than the others and have a smaller number of teeth. This number may be as high as twelve toward the middle of the series. In all these respects the plates of the mon- strous growth agree with those of the real claw. No difference was found between single plates of the dactyl and the index, nor between plates of the two prongs nor between plates of the nor- mal and abnormal growths. The pronged structure, however, differs from the natural claw both in number of plates and in their arrangement at the angle. While the normal claw has 61 and 67 plates the pronged struc- FIG. 4. Camera sketch of posterior view of serrated plates from the abnormal pronged structure. 2-D. ture has 53 on the prong with a broken tip and 54 on the other. At the angle (Fig. 2) the plates have the arrangement shown in Fig. 5 : the terminal plates are crowded together and the two series interfere at the angle. Plate 52 of the imperfect prong steps out of rank and stands partly in between plates 53 and 54 of the series on the perfect prong, which is indicated by the letter T on the fifty-second plate. The angle thus has plates of both series carried into it till they fuse into one curved line. More- over, these plates at the angle are not the same as the terminal plates of the normal claw, nor do they agree in number of teeth with plates at that distance from the tip of the normal claw. They are evidently special terminal plates in their own series but not directly comparable with the normal terminal plates. The prongs are shorter than the index and the dactyl and have not room for a full number of plates. Where they have a free edge So E. A. ANDREWS. it is set with plates at the same rate as on an equal length of claw edge. If the common base of the prongs were to be split for about two fifths of its length and the prongs so lengthened they could bear about seventy plates each and the prongs would be much more like the normal claw ; still it would be necessary to transform the present terminal plates at the angle into plates of the right character in the new series and to make new terminals at the new proximal ends. But little was made out regarding the internal anatomy of this abnormal limb either from preparations cleared and stained or from sections ; but it was evident that the muscles in the propo- FIG. 5- Camera sketch, 2-D, of a posterior view of the plates at the angle between the two prongs. dite were not arranged as in a normal propodite. At the distal end there were two muscle masses that seemed to connect with opposite edges of the articular end of the pronged structure. These would probably move this structure up and down in the plane of the claw. The usual muscles of abduction and adduc- tion seemed to be developed, but attached to the dactyl in an abnormal way in connection with the above extra muscles and with abnormal widening of the distal part of the propodite. The gist of the above description is that this abnormality is a case in which the propodite is to some extent double and bears a AN ABERRANT LIMB IN A CRAY-FISH. 8 I normal claw as well as a pronged structure that simulates a claw even in details and was probably movable after the manner of a dactyl. This pronged structure is remarkable for its symmetry. Comparing this with other described cases we find in the first place that it is unusual in being upon a walking leg. Of the thirty-one cases of abnormal appendages quoted by Bateson,1 two are of antennae, four are of non-chelate legs, and all the rest of chelae except one, which is of a chelate walking leg. Of the eleven additional cases given by Herrick 2 only two are of walking legs. However, this relative infrequency of described abnormalities in walking legs may be due, in part, to the greater ease with which other cases are collected or noted. In the second place, it is unusual in being a monstrosity of the propodite. Bateson found the greater number of cases of repe- tition of parts, in the Crustacea, are repetitions of extra dactyls upon a normal dactylopodite (some fifty cases), and that next in frequency are the cases of extra index upon a normal index (some fifteen cases). In the third place it seems to fall into none of the four cate- gories established by Bateson, but rather to be like the excep- tions, of which he found only two. It has, however, resemblance to the case 815 of Bateson and more to the case shown in Fig. 195 of Herrick and still more to the case shown in Fig. 2 of Faxon, 3 which was described as follows : " This leg is provided with two chelae. One of them has the ordinary form and structure, but is bent at a strong angle with the long axis of the leg. The second appears to have been budded off from an amputated surface of the propodite. It con- sists of two fingers which have the form of the normal dactyl us and index, but neither is articulated with the other at the base. The two fingers together seem to be morphologically equivalent to a single segment, and represent a two-branched supernumerary dactyl us." Though the pronged structure we have described is markedly like a claw in its symmetry yet any tentative attempt to interpret 1 Bateson, " Materials for the Study of Variation," 1894. 2 Herrick, "The American Lobster," Bull. U. S. F. C., 1895. 'Faxon, "On Some Crustacean Deformities," Bull. M. C. Z., VIII., 1880. 82 E. A. ANDREWS. it morphologically would seem to meet with more difficulty in assuming it to represent a claw than in assuming it to represent two fused dactyls or a branched dactyl. Were it a claw with fused articulation of dactyl and index we would have a limb so doubled distally as to have an extra segment and a lack of coin- cidence between the two series of segments. A propodite would spring from propodite instead of from a carpodite ; and if we bear in mind the partial double appearance of the propodite and regard it as a fusion we would have a carpodite and a propodite springing from a carpodite, and so on. Bateson's thorough study led to the conclusion that almost all cases could be interpreted as repetitions of claws in which there was more or less suppression of index or of carpus. The pronged structure would then be regarded as two party fused dactyls placed face to face and we would expect to find some representative of the two indices. On the line of imagined fusion there is a slight eversion of membrane where the pronged struc- ture articulates with the propodite, but there is no reason for regarding this as of any morphological significance. In the case described by Faxon, as quoted above, Bateson thought he had found a representative of the required indices in a small protuberance shown in Faxon's Fig. 2 ; this however was an error for I am informed by Faxon that " the artist un- fortunately represented a protuberance which does not exist." There are thus two cases in which pronged structures have nothing with them to countenance the idea that they represent double dactyls with even traces of double indices. Moreover, it will be seen from the above Fig. 2 that the prong nearer to the dactyl is not a mirror image of that dactyl but that it rep- resents the index and likewise the other prong is not a mirror image of the index but represents the dactyl ; this is true since all have their serrations nearer to the posterior face than to the anterior face. There is thus a departure from Bateson's rule of symmetry and we have to deal with a very unusual abnormality that is not interpretable in the same way as most of those hitherto known. But any morphological interpretation seems somewhat prema- ture and unsatisfactory in the lack of more knowledge of the AN ABERRANT LIMB IN A CRAY-FISH. 83 mode of formation of such structures. The appearance of the limb suggests a new growth following some injury in which the material for claw making was partly severed and displaced. This might happen, we can suppose, not only in the egg and in the young, but in the adult, especially at the periods of shedding when the interior of the claw is soft and the blood peculiar. That limbs may regenerate from a peripheral wound was shown by Herrick for the tips of the claws and by Morgan l for large parts of the limb. Possibly then such a monstrosity as this might arise in regeneration following an injury to the propodite. An attempt to get experimental evidence resulted in failure, but this is what would be expected from the rarity of such mon- strosities and from the difficulties in keeping the material long enough. In that attempt 103 mature Canibanis affinis were oper- ated on in February. In each a deep cut was made in the carpodite of each chelate walking leg at a point corresponding to the pronged structure in this abnormal limb. In ten days many had healed, some could again use the dactyl and some had dropped the parts peripheral to the cut. Subsequently a piece was removed where the cut had been made in order to prevent such rapid healing. The breeding season then came on and after some months all the specimens had died without shedding and no new formations were found. 1 Morgan, " Regeneration of the Appendages of the Hermit Crab and Crayfish," Anat. Anz., XX., 1902. THE REACTION-TIME OF GONIONEMUS MURBACHII TO ELECTRIC AND PHOTIC STIMULI.1 ROBERT M. VERKES. CONTENTS. PAGE. Problems and Methods 84 Reaction-Time to Electric Stimuli 85 Reaction-Time to Photic Stimuli 86 Relation of Reaction-Time to Region Stimulated 88 Reaction-Time of Tentacles 89 Relation of Quality of Stimulus to Time of Reaction 91 Absolute and Relative Variability 92 PROBLEMS AND METHODS. The reaction-time method as applied to the study of the func- tioning of the nervous system has already given us certain im- portant facts in human neuro- physiology, and it promises much more valuable results when its application to representatives of the various animal phyla makes a comparative survey of the time relations of neural processes possible. The value of reaction- time studies lies chiefly in the knowledge which they give us of the biological significance of the nervous system. " Certainly they are not important as giving us knowledge of the time of per- ception, cognition or association, except in so far as we discover the relations of these processes, and the conditions which are most favorable for them. To determine how this or that factor in the environment influences the activities of the nervous system, and in what way system may be adjusted to system or part process to whole is the task of the reaction-time investigator." For the reaction-time measurements which furnish the ma- terial of this paper chronoscopic methods were employed. All reactions to light were measured by means of a stop watch read- able to tenths of a second, but for the reactions to electric stimuli, which were very much quicker, it was necessary to make use of 1 From the Marine Biological Laboratory, Woods Holl, Mass. 2Yerkes, Robert Mearns : "The Instincts, Habits and Reactions of the Frog." Harvard Psychological Studies, Vol. I., 1903, p. 509 {Psychological Review Mono- graph Supplement, Vol. IV.). 84 REACTION-TIME OF GONIONEMUS MURBACHII. 85 an instrument readable to hundredths or thousandths of a second. For this purpose a Hipp chronoscope, readable to thousandths, was placed in circuit with the stimulus electrodes and the reac- tion-key. The electrodes were connected in such a way that the chronoscope circuit was made, and the record thereby started, the instant the stimulus circuit was completed. The motor reaction of the medusa in response to the electric shock served to break the chronoscope circuit, thus stopping the record. The experi- menter was then able to read from the chronoscope dials the time which intervened between stimulus and reaction (reaction- time). Cattell's falling screen served as a regulator for the chronoscope.1 The reaction-key used in these measurements of the time of reaction to electric stimuli consisted of a frame for the support of an easily sliding rod, one end of which carried a cork disk and the other a platinum point by which the circuit was completed. The movement of the medusa against the disk when a stimulus was given, caused the rod to slip upward, thus breaking the chrono- scope circuit. REACTION-TIME TO ELECTRIC STIMULI. Gonionemus reacts to an electric current, indirectly applied, in from one to five seconds, according to the strength of the stimu- lus, and the position of the electrodes. The following averages indicate the facts. Since it was not possible to get more than four or five satisfactory reactions in series with any one animal, the averages, unless otherwise marked, are for five reactions. I. Reactions to a 4-Mesco-cell current, with electrodes on opposite sides of the bell, not in contact with the organism. M. 1.023 sec. ; M.V. 0.168 sec. ; R.V.2 16.0. II. Same, with 2-cell current. M. 1.489 sec.; M. V. 0.199 sec. ; R. V. 13.4. III. Reactions to a 4-cell current, with electrodes 5 mm. apart in contact with the margin of the bell. M. 0.605 sec. ; M. V. 0.128 sec. ; R. V. 21. Repetition of the 4-cell stimulus at intervals of a minute causes 1 For fuller description of the chronoscopic method used see Harvard Psycholog- ical Studies, Vol. I., pp. 601-605. 2 R.V. = (M.V. X I0°) / M- = Relative Variability. 86 ROBERT M. YERKES. a rapid lengthening of the time of reaction. Thus : first reaction, .506 sec. ; second, 1.003 ; third, 3.607. As compared with the reactions of the small medusa Gonione- j/n/s, those of the jelly-fish Cyanca arctica are slow. Some indi- vidual reaction-times of a single individual (Cyaned) to a four-cell current, with electrodes in contact with opposite points on the margin follow. Reaction. Reaction-Time. Deviation from Mean. I .026 sec. .504 sec. 2 .987 •457 3 .324 .206 4 .636 .106 5 .629 .099 6 .760 .230 7 .2OO •330 8 1.328 .202 9 I.SOO .270 10 1.610 .080 Mean. 1.530 Mean variation. .248 Relative Variability 16.2. REACTION-TIME TO PHOTIC STIMULI. The reaction-time of Gonionemus to increase in light intensity, as I have stated in another paper,1 varies with the strength of the stimulus, temperature, condition of the organism, etc., from one to thirty seconds. To daylight the organism usually responds in about seven seconds ; to sunlight the reaction is at first much quicker, but it rapidly lengthens as the organism is exposed to the influence of the intense light. The relation of time of reac- tion to intensity is indicated by the following averages : Weak daylight, 9.4 sec. ; daylight, 7.0 sec. ; sunlight, 5.5 sec. Moreover, the reaction-time varies with the size, sex, and pig- mentation of the individual, as well as with such external condi- tions as temperature, density, and chemical constitution of the medium. Increase in temperature gradually shortens the time from about 8-9 sec. at 19° C., to 2-3 sec. at 32° C. Decrease in temperature lengthens the time, until reactions fail entirely at about 10-12° C. 1 Yerkes, Robert M., with the assistance of James B. Ayer, jr.: "A Study of the Reactions and Reaction-Time of the Medusa Gonionema miirbackii to Photic stimuli." Amer.Journ. Physiol., Vol. 9, 1903, pp. 279-307. REACTfON-TIME OF GONIONEMUS MURBACHII. Between these reaction-times of the Coelenterata and those of most vertebrates, as well as of many invertebrates, there is a strik- ing difference in rapidity. Whereas, the jelty-fish and medusa re- spond to an electric stimulus in from one to four seconds, the fish or frog responds in a fraction of a second, usually not more than one fourth, and sometimes one tenth. Observe the reaction of the fiddler crab to a shadow, and note how quick it is in com- parison with the reaction of Gonionemus to the same change in illumination. Is this difference in reaction-time due to a differ- ence in sensitiveness (/. r., is the latency period of stimulation longer) ; is there a difference in the rate of impulse transmission, of central nerve processes, or of muscle contraction ? Such ques- tions should be answered by means of reaction-time investigations. The rate of impulse transmission (presumably nerve transmission) is much slower in the medusa Gonionevnis than in the vertebrates and most invertebrates thus far studied. Furthermore, it differs for different regions of the medusa ; the margin and the radial Frog Medusa. Electric Stim. Intensity. M. M. V. R. V. Light Intensity. M. M. V. R. V. I 2 4 .301 sec. .231 " .103 " .085 sec. -034 " .012 " 28.2 14-7 ii. 6. Weak daylight. Daylight. Sunlight. 9.4 sec. 7.0 " 5-5 " 3.16 sec. 2.39 " 1. 60 " 33-6 34-1 29.0 canal regions transmit impulses much more rapidly than do the inter-radial regions. The exumbrellar layer of tissue, so far as I have been able to determine, does not transmit impulses at all. * The frog reacts to such an electric stimulus as was applied to Gonionevius in . i 50— .200 sec. In comparison the medusa's reac- tion-time is very long ; but it differs in yet another respect — it is far more variable. The reaction -times and variabilities of the reactions of frogs to three intensities of electric stimulation as determined in an experimental investigation - are here given for comparison with the results given by Goiiioncinus to three inten- sities of light. 1 Yerkes, Robert M. : "A Contribution to the Physiology of the Nervous System of the Medusa Gonionema Murbachii. Part II. — The Physiology of the Central Nervous System,' 'Amer. Jour. Physiol., Vol. 7, 1902, p. 193. 2 Harvard Psychological Studies, Vol. I., 1903, pp. 616-618. 88 ROBERT M. YERKES. This table shows that the relative as well as the absolute vari- ability is higher for the medusa than for the frog. In general it is true that variability increases with increase in the time of reac- tion. Stimuli or .intensities of stimulation which give extremely short reaction-times may be expected to give low indices of vari- ability ; similarly animals which are slow in reacting exhibit high degrees of variability. The reflex reaction is absolutely and rela- tively the least variable among the common types of action ; the instinctive reaction is much more variable, and most variable of all in time of execution as also in form, is the voluntary reaction so-called RELATION OF REACTION-TIME TO REGION STIMULATED. As might be expected the reaction -time of Gonioneinits varies with the region stimulated. When the electrodes are placed in contact with the margin at the bases of the radial canals the re- action is noticeably quicker than when the inter-radial regions or other portions of the bell are stimulated. The average reaction- time to a four-cell current applied to the inter-radial portions of the margin is .605 second ; for the radial canal regions it is .507 second. It is not necessary, however, to make measurements to thousandths or even hundredths of a second to exhibit this fact ; stimulating different regions and simply watching the responses will make clear the differences in reaction-time. Again the time of reaction to light varies according as the light falls upon the subumbrellar or the exumbrellar surfaces. It is much shorter when the subumbrella is exposed to the light (3.4 seconds as compared with 17.4 seconds for the other position).1 It is not at all likely that the differences in reaction-time here noted for electric and photic stimuli are due to the same condi- tions. The quicker reaction to stimulation of the radial canal regions is doubtless due to the higher transmission rate of the differentiated nerve tracts along the radial canals. Stimulation of any other portion of the bell causes reaction less quickly sim- ply because the tissues transmit impulses less rapidly, since they possess less highly specialized nerve tracts. In case of the quicker reaction to light when the medusa is resting with the 1 Anier. Jour. Physiol,, Vol. 9, 1903, p. 301. REACTION-TIME OF GONIONEMUS MURBACHII. 89 subumbrellar, instead of the exumbrellar, surface toward the light, a difference in sensitiveness is apparently the cause of the dif- ference in reaction-time. Certain organs which are especially sensitive to light are found on the subumbrellar surface of the margin, and it is when they are most fully exposed to the action of light that the organism responds most promptly to the stim- ulus. The rate of impulse transmission is probably the same no matter which surface is stimulated by light, but the latency period of stimulation is far greater for stimulation of the exumbrella. These experimentally demonstrable facts clearly prove that the nervous system of the medusa Gonionemus consists of cells which possess irritability and conductivity in high degrees. And they further show that rapidity of reaction is directly dependent upon specially differentiated paths of conduction. REACTION-TIME OF TENTACLES. A study of the reaction-time of the various parts of Gonionemus (tentacles, manubrium, margin, bell), when normally functioning, and when isolated by operation, throws interesting light upon certain problems in the physiology of the nervous system. Cutting off the tentacles close to the bell causes Gonionemus a severe shock. If only one tentacle is cut off the usual response is a contraction of the bell, which may occasionally lead to a swimming bout. The severity of the shock, or as we would commonly say, the strength of the stimulus which Gonionemus receives from tentacle excision, evidently varies directly with the size of the organ. Small tentacles frequently may be cut with- out causing any visible reaction except slight contractions of the adjacent tentacles; large tentacles cause one or more contractions, and in general the larger the organ the greater the number and force of the contractions. Excision of the primary tentacles, those at the bases of the radial canals, apparently causes the most severe shock, for when one of them is suddenly clipped off the animal frequently swims about rapidly for a considerable length of time. In every way its reaction is more vigorous than those caused by excision of smaller tentacles or of large tentacles in other positions. It would appear from this that the radial canal tentacle is of ROBERT M. YERKES. special significance in the life of the medusa. And in support of this belief it is worth noting that they are almost always held dif- ferently from the others. When the majority of the tentacles of a "bell up" (exumbrellar surface uppermost) individual are resting on the bottom of the vessel, the primary tentacles are usually held slightly higher in the water than the others. They are used for attachment and for food seizing sooner than the others. These facts point toward either a specialization or a modification in func- tion which is of interest because of its bearing upon certain neuro-anatomical facts which have been presented by Miss Hyde.1 She finds well-defined cell-fiber tracts along the radial canals. This being the case we should expect the radial canal tentacles to have a more important and direct influence upon the reactions of the organism than have the other tentacles. When the medusa is stimulated to motion by light the tenta- cles contract from . I— .2 second before the bell. At times tentacle reactions occur in the absence of a general bell contraction. As determined with a stop-watch the reaction-time of the normal I. REACTION-TIME OF NORMAL TENTACLES TO DAYLIGHT. Tentacle. M. M. V. R. V. No. i 2.2 sec. 0.22 sec. IO.O " 2 " 3 3-4 " 3.6 " 0.62 " 0.46 " 18.2 12.6 General averages. 3-i- 0.43 + 13.6 II. REACTION-TIME OF TENTACLES OF EXCISED MARGIN TO DAYLIGHT. Tentacle. M. M. V. R. V. No. I " 2 " 3 2.5 sec. 2.7 " 2.9 " o.i 6 sec. 1. 06 " 0.79 " 6.4 39-2 27.2 General averages. 2.4- 0.67 " 24-3 II. REACTION-TIME OF EXCISED TENTACLES TO DAYLIGHT. (AVERAGE OF FIRST THREE REACTIONS.) Tentacle. M. M. V. R. V. No. i " 2 " 3 5.3 sec. 4.2 " 4-4 " 2 30 sec. 1.92 " 1-33 " 43-4 45-2 30.2 General averages. 4-5- 1.85 " 39-6 1 Hyde, Ida H.: " The Nervous System of Gonionema Murbachii," BIOLOGICAL BULLETIN, Vol. IV., 1902, pp. 40-45. REACTION-TIME OF GONIONEMUS MURBACHII. 9! tentacle to the increase in light intensity caused by suddenly uncovering a dish containing the medusa is from two to five seconds. Reaction-time averages for three conditions of the tentacle are given in table on page 90. A fact significant in this connection is that the excised tentacle rapidly loses its power to react to photic stimuli. To the first four or five repetitions of a stimulus it usually reacts quickly, then the time of reaction, as is shown in the series herewith pre- sented, rapidly increases until reaction fails entirely. SERIES OF REACTIONS OF AN EXCISED TENTACLE TO DAYLIGHT. Reaction I 4.6 2 2.5 3 8.7 4 15-6 5 35-o 6 No reaction except to 7 , mechanical stimulation. The reaction-time of the normal tentacle, 3.1 seconds, is con- siderably shorter, as would be expected, than that of the bell. Its variability is low. The reactions of the tentacles of excised margins are slightly quicker, 2.4, according to the results pre- sented, than are those of the normal animal, but they are also more variable. The quickness of these reactions may possibly be due to a temporary increase in the irritability of the margin caused by the operation. Finally, the reactions of excised tentacles are much longer, 4.5, and more variable than are those of either the normal animal or the excised margin. This may mean that the tentacle contraction in response to light is. normally initiated by stimulation of the margin, or that the ability of the organ to react is lessened by its separation from the bell. At any rate there is a marked difference here indicated in the time of reaction of isolated and normally attached organs, a difference which may possibly be an indication of a function of the central nervous system or of the special organs of light stimulation which are in all probability situated in the margin of the bell. RELATION OF QUALITY OF STIMULUS TO TIME OF REACTION. The motor reaction of Gonionemus to increase in light is much slower than that to other forms of stimuli. This is due in part 92 ROBERT M. YERKES. to difference in strength of stimulus, but it is of interest to en- quire whether the quality of the stimulus is not of importance. We may ask, for example, whether the reaction-time to the thres- hold stimulus of all modes of stimulation is the same. If it is not, quality of stimulus is evidently significant. Wundt l presents the following figures in support of his statement that the reaction- time to the threshold intensity of all modes of stimulation is the same. Threshold Stimulus. Mean. Mean Variation. Sound. Light. Touch. •337 sec. •331 -327 0.50 sec. 0-57 0.32 The results which I have gotten with frogs in working with electric and tactual stimuli cause me to question the appli- cability of this statement to the reactions of all organisms. It seems highly probable that the just perceptible stimulus reac- tion-time is by no means the same for different qualities of stimulus. Those modifications of the vital processes which make survival possible appear even in the responses to minimal stimuli. In one case the just perceptible stimulus may cause nothing more than a slight local change in circulation, excretion, muscu- lar action, in another it may produce, just because of the particu- lar significance of the stimulus for the life of the organism, a violent and sudden motor reaction.2 ABSOLUTE AND RELATION VARIABILITY. As already pointed out 3 it is generally useless to compare re- action-times with respect to variability unless the reaction-time value as well as the absolute variability is considered. If, for example, to an electric stimulus Gonionemus reacts in 2.0 seconds, with a variability of 0.5 sec. ; and to a photic stimulus in 6.0 seconds, with a variability of 1.5 sec., it is not correct to say that the reaction-time to light is three times as variable as that to electricity. As a matter of fact the two variabilities as such are 1 Wundt, Wm. : " Grundziige der physiologischen Psychologic," Fiinfte Auflage, Leipzig, 1903, Dritte Band, S. 428-429. 2 Harvard Psychological Studies, Vol. I., 1903, p. 625. 3 Amer. Jour. Phvsiol., Vol. 9, 1903, p. 291. REACTION-TIME OF GONIONEMUS MURBACHII. 93 not equal, but when we consider the reaction-times we find that the ratio of variability to reaction-time is in each case 1:4. • Al- though the absolute variability is in one case three times as great as in the other, it is 25 per cent, of the average reaction-time in both instances. Heretofore I have expressed relative variability as a ratio (M.V. : M.), or as a percentage value of the mean (M.V. = x per cent, of M., in which case x •=• R.V., the relative variability.) Obviously it is always important in comparative reaction-time work to know the relative variability of reactions ; in fact it is often quite impossible to make significant comparisons of results until this value is found. For this reason I have given in every table of this paper the percentage value of the mean variation in terms of the mean. This value I have called the relative vari- * ability (R. V.). It is obtainable by the formula recently used by Myers, ' v.c. = m.v. x loo/av. In this formula, which as inspection shows gives the ratio (in per cent.) of m.v. to av., v.c. is a value called by Myers the variation-coefficient, and av. is the mean (M.). Supposing a reaction-time of .180 second to have an absolute variability of .020 second, then by the formula (.020 x lOO/.iSo) the variation-coefficient (Myers), or what I prefer to call the relative variability (R. V.), is I i.i + . If we chose this value might be written ii.i -(-per cent., thus indi- cating that the absolute variability (M. V.) is ii.i + Per cent, of the average reaction -time (M.). Since Pearson 2 in this statistical work has made use of a quan- tity which he calls the "coefficient of variability," and which is obtained by the formula C. V. = — - - x 100, it seems unwise to use the term variation -coefficient, suggested by Myers, for this new quantity in reaction-time work. As the value which we obtain by Myers' formula is in reality the per- 1 Myers, Chas. S.: "Reports of the Cambridge Anthropological Expedition to Torres Straits." Vol. II., "Physiology and Psychology," Part II., 1903, p. 212. 2 Pearson, Karl : " Mathematical Contributions to the Theory of Evolution, III., Regression, Heredity and Panmixia," Phil. Trans. Roy. Soc. London, Vol. 187, A, pp. 253-318. 94 ROBERT M. YERKES. centage value of the mean variation in terms of the mean, I see no reason why we should not call it the relative variability, in contrast with the absolute variability. Thus the confusion with Pearson's quantity which will inevitably result from the use of variation-coefficient can be avoided. Myers' formula gives us precisely what we need for the direct comparison of reaction-times, with respect to their variableness, either to different stimuli or of different organisms. Strange to say most investigators of the time relajtions of neural processes have paid little or no attention to the variability of their results ; none, so far as I know, have ever determined the relative varia- bility throughout their work. It may be objected that those who have use for the relative variability can find it for themselves since the reaction-time and its mean variation are usually given. But the value is far too important to be left half-way determined ; in fact it is even more useful in most cases than the mean variation. Every one who has had experience in dealing with reaction-time results will admit that the reaction-time to a particular stimulus has different meanings according to its variability, and that it is never possible to compare reaction-times without considering this value. It is clear then that no reaction-time statistics sJiould be published without determinations of the relative variabilities. Conventionally we compare human reaction-times to visual, tactual and auditory stimuli without noticing their variabilities or the strength of the stimulus employed. Jastrow : in a table of results, collected from the papers of many investigators, which is intended to indicate the differences in time of reaction for the dif- ferent senses gives these averages : Visual reaction-time .185 sec- ond ; tactile, .148 ; auditory, .139. Not even the mean variability is given in connection with the averages. Since reaction-time varies with the strength of the stimulus it is possible by varying the stimulus-intensity to get any one of the above reaction-times with any of the qualities of stimulus named. This being true, how are we to make valuable comparisons of reaction-times to different kinds of stimuli ? As before stated the threshold intensities of all modes of stimu- lation may be regarded as directly comparable. No matter what 1 Jastrow, Joseph : "The Time Relations of Mental Phenomena," New York, 1890, p. II. REACTION-TIME OF GONIONEMUS MURBACHII. 95 the form of the stimulus, the threshold gives the longest and most variable reaction-time which can be obtained by the use of that particular quality of stimulus. Now, as the intensity of the stimulus is increased the variability decreases. Why may we not choose equality in relative or in absolute variability as a basis of comparison ? If it should be found — and I am now gathering data for the settlement of the point — that the relative variability is the same for the threshold reaction-time to all qualities of stimuli, equality in relative variability would be the most satisfactory basis ; if, on the other hand, absolute variability is a constant quantity at the threshold, it should be used in preference. NOTE. — Reasons have recently appeared for returning to the original spelling of the name of the medusa. Gonioncnnts there- fore is used instead of Gonionenia. Vol. VI. February, 1904.. No. BIOLOGICAL BULLETIN. THE SPECIAL PHYSICS OF SEGMENTATION AS SHOWN BY THE SYNTHESIS, FROM THE STAND- POINT OF UNIVERSALLY VALID DYNAMIC PRINCIPLES, OF ALL THE ARTIFICIAL PARTHENOGENETIC METHODS. E. G. SPAULDING. The genesis of this paper is a twofold one. In the first place the careful perusal of the literature which has appeared in very recent years on the matter of the obtaining of artificial partheno- genesis in various forms by a number of methods, and which in- cludes also varying theories of the process of segmentation as in- terpretations of these data, this perusal readily convinces one that such a consistent and far-reaching synthetic view of the nature of segmentation as known data would seem to warrant one in try- ing to obtain is quite lacking. In fact no attempt seems to have been made to show that all the methods employed must result in bringing about one and the same series of physical events in the cell preceding and during segmentation, to which the process resulting from normal fertilization is no exception. That vital (?) phenomena can be reduced to a purely physical basis will doubtless be disputed as long as any details connected therewith remain unstudied or in any way ambiguous. The absence of such a complete reduction is in itself, however, no disproof of the correctness of the view as a theoretical standpoint, and suc- cess in it will at least always remain a scientific ideal.1 A clear and detailed demonstration that the effectiveness of the various artificial parthenogenetic methods can be explained if it is held that one series of physical events always occurs in the process of segmentation would seem therefore to go a considerable way in 1 The position that this standpoint is logically necessary for biology as a science is discussed in the author's article, " The Contrary and the Contradictory in Biology; a Study of Vitalism," in The Monist, July, 1903. 97 98 E. G. SPAULDING. the attainment of that ideal. To attempt to do this states ac- cordingly the purpose of this paper. The second genetic element was the desire to get such a uni- tary view as at least a preliminary to and if possible a justifica- tion of the attempt to initiate segmentation by new methods, viz., by the application of the electrical current to the unfertilized eggs of the starfish, and although these experiments were unsuc- cessful, the theory, although based on the experimental work of others, is offered for what it may be worth as an endeavor to gain an end, the value of which in itself will not be denied. i. EXPERIMENTAL DATA. A brief recapitulation of the results already obtained by arti- ficial parthenogenetic methods may, as a preliminary to subse- quent discussion, be pardoned. In the starfish egg parthenogenesis may be produced by : (i) the use of HC1 ; l (2) increasing the osmotic pressure of the surrounding medium ; 2 (3) by lowering the temperature ; 3 (4) by mechanical agitation.4 By the first method it is held that the "parthenogenesis of Asterias eggs is to be produced by means of specific (hydrogen) ions," at least this is the interpretation of the fact that 100 c.c. of sea water plus 3-5 c.c. N/io HC1 acting for from 3 to 20 minutes on the eggs, which are then removed, brings about the desired result.5 In the case of the second method,6 although the results are stated somewhat ambiguously, the maxi- mum number of parthenogenetic eggs seems to have been se- cured by using 15 c.c. of 2*4 N KG + 85 c.c. of sea water at about 23° C. for 15 minutes, then transferring. As for the third, " eggs °f Asterias may be made to develop parthenogenetically by exposing them for a definite length of time to a temperature of i0-/0 C., in sea water, and then raising the temperature." As an interpretation of this, we find it stated that "the produc- 1 Loeb, Fischer u. Neilson, Archiv fiir die geschichlliche Physiologie, Bd. 87, 1901. 2Greeley, A. W., BIOLOGICAL BULLETIN, IV., 3, Feb., 1903, says that Neilson found this method successful. 3Greeley, A. W., Am. Jour, of Physiologv, VI., 1902, p. 296. 4 Mathews, A. P., Am. Jour, of Physiology, VI., II. 5 Loeb, Fischer u. Neilson, loc. cit. 6 Greeley, loc. cit. PHYSICS OF SEGMENTATION. 99 tion of artificial development by lowering the temperature is brought about by an extraction of water from the protoplasm, just as if the eggs had been placed in a solution of higher os- motic pressure than that of the sea water," l though no explana- tion of the reason for this is offered. A suggestion as to this is however made by Mathews in his comments on the fourth method, that " the getting of parthenogenesis by agitation may be due to a dissolution of the nuclear membrane, since the cen- trosome originates close to the nucleus, or it may cause the eggs to lose water like the cells of sensitive plants. The loss of water could be caused only by lowering the osmotic pressure in the cell, and this by decreasing the number of molecules in the cell ; and this in turn by synthetic processes." In other forms artificial parthenogenesis may be obtained by similar or slightly different methods ; c. g., in Arbacia by osmotic pressure, 50 c.c. :2^°- TV MgG2 or NaCl + 50 c.c. sea water,3 and at least a segmentation by lack of oxygen, by heat, or by ex- posure to alcohol, chloroform, or ether;4 in Chcetopterus likewise by the use of KG, KNO3, K2SO4 (2^4 N+ 100 c.c. sea water), NaCl, MgCl2, CaCl2 and sugar,5 in Amphitntns by Ca(NO3)5 (2 c.c. N -\- 99 c.c. sea water) ;'' in Nereis by osmotic pressure, (20 c.c. 2^/2 N KG + 80 c.c. sea water, 30 minutes),6 in Podarke obscura by use of the same solution.7 As theories and interpretations of the results obtained by these factual methods, we find in addition to those already cited the following, which are quoted in abstract : " All that the spermatozoon needs to carry into the egg for the process of fertilization are ions, Mg, K, HO or others, to supplement the lack of the one or counteract the effects of the other class of ions in the sea water, or both. The ions and not the nucleins in the spermatozoon are essential to the process of 1 Greeley, loc. cit. 2 Mathews, loc. cit. 3Loeb, J., Am. Jon?: of Physiology, Vol. III., Nos. III. and IX. and Vol. IV., IV. 4 Mathews, A. P., Am. Jour, of Physiology, IV., VII. 5Loeb, J., Am. Jour, of Physiology, IV., IX. 6Fischer, M., Am. Jour, of Physiology, VII., III. 7Treadwell, BIOLOGICAL BULLETIN, III., 5. IOO E. G. SPAULDING. fertilization ; l or the spermatozoon may carry enzymes." " Either of these two causes affects the most important qualities of life phenomena, /. c., causes the proteids (i) to change their state, or (2) to take up or lose water." 2 Further details as to these two possible events are not given, however, but it is quite evident that the two may be coincident, so that the latter change may take place in any case. To summarize systematically, a cell division can be caused in various forms by one or more of the following classes of stim- uli : (a) mechanical ; (//>) heat (or cold); (c) osmotic ; (V) chem- ical, or, if one will, ionic ; the third for the reason that either of the electrolytes MgCl, or NaCl, or the non-electrolyte sugar, maybe used ior Arbacia ;* therefore no specific chemical effect is to be accepted here. The fourth, a distinctly chemical effect, is evident, for HC1 is effective for Asterias eggs and KC1 is not ; so also only the Ca ion for Amphitritus. Here then it is the kation that is considered to cause the segmentation, but that a fundamental chemical effect different to an ionic, i. c., electrical / charge effect is present is shown by the fact that, keeping the osmotic pressure and the number of charges on the kation the same, but changing the ions, the effect is different. This is con- firmed by the comparison of the action of KC1 and NaCl on muscle.4 A specific chemical effect is therefore not done away with even if the difference in effect is reduced to a difference in the path of the charge moving around the atom. For the cause of this latter difference must in turn be a fundamental difference in the atoms themselves. The same kind of proof of an irredu- cible and ultimate chemical difference is found in the results of Lillie's work on the effect of Na, K, Ca and Mg salts on Aren- icola and Polygordius, and of Mat hews on the different stimu- lating effects on the nerve of NaCl, NaBr, Nal and NaFl. This fundamental chemical difference is related to the difference in so- lution tension, as Mathew's work this past summer has shown. However, not alone the stimuli, the external agents initiating , J. , Am. Jour, of Physiology, III., III. 2Loeb, J., Am. Jour, of Physiology, III., IX. 3Loeb, Am. Jour, of Physiology, IV., IV. and III., IX. 4Loeb, Am. Jour, of Physiology, III., VIII. PHYSICS OF SEGMENTATION. IOI segmentation are to be put into the above classes, but also, with the addition of the class, surface energy, which is of special im- portance here, the phenomena taking place within the cell itself, preceding and during cleavage. We accordingly consider the cell to be a physico-chemical object, whatever else it may be, and subject therefore to general physical principles. The special na- ture of the physical processes that occur in it is to be demon- strated by showing that the effectiveness of the physical methods used for causing segmentation implies that, or at least can be ex- plained, if by each method only one and the same series of events is made to take place. The bringing together of results in this way exemplifies what we have termed synthesis, and the internal agreement with which it is identical makes for the probable cor- rectness of our theory. 2. GENERAL PHYSICAL PRINCIPLES TO BE OBSERVED IN THE INTERPRETATION OF THESE DAT A.1 If the energies both within and without the cell belong to the classes named, we must in our endeavor to get at the meaning of the data at hand be guided strictly by the most general funda- mental chemical and physical principles valid for those. These principles, some of which are of course well known, may be stated as follows : I. The " first law " of energetics, that of the conservation of energy. This is considered to have an experimental basis in the fact that, e. g., a weight of one kilogram falling 424 mecers raises the temperature of one kilogram of water I ° C. as indicated on an arbitrarily selected scale. This is interpreted to mean that the kinetic energy of the falling body is quantitatively equal to the heat energy gained in the rise in temperature. However this cannot be strictly proven, for the two energies are qualitatively different, and have no common factor. It would therefore be 1 The principles as stated are to be found in no one author, but are with their criticism the result of the study of the works of Planck, Mach, Ostwald, Helm, Wald, Riecke, and others; some of these are as follows: Planck, " Prin. d. Erh. d. Energie " ; Ostwald, " Vorlesungen iiber Naturphilosophie," " Allgemeine Chemie," and other writings; Helm, " Die Energetik nach ihrer gesch. Entw. " ; Rankine, Philos. Alag., 1867 (4); Mach, " History of Mechanics," " Warmelehre," "Pop. Lect," "An- alyse der Empf" ; Riecke, " Lehrbuch d. Physik." IO2 E. G. SPAULDING. quite as logical, though not as practical to interpret the two as quantitatively different. To interpret as equal is therefore to base the law of conservation on an assumption not proven, yet not disproven.1 In the second place the law is based upon the impossibility of a system's continuing to do work unless energy is received from without. In this the assumption is implicitly contained that work cannot be created ex niliilo. Were it possible, however, for a system to receive from without the work (energy) which it itself does, a perpetual motion would be possible. II. The second law prevents this. In every " Ausgleichung" or transformation (Umgleichung) of energy, some heat is pro- duced, only part of which at best is again available, for the reason that it tends to " dissipate " ; it cannot pass from the body of the lower to that of the higher potential (temperature) but only con- versely. The entropy of the universe is accordingly said to increase. This characteristic of heat energy is a special case of a law (the second) valid for all the energies. Its meaning is that all events have a definite direction. According to the first law then if one form of energy disappears another form or forms held to be quantitatively equal to it must appear. Energy may therefore be defined as that which in changing conserves itself. Implicit in both the first and second laws is the definition of it as that which does work, but that there are objections to this is evident from the facts stated in the second, that some energy in the form of heat with no difference of potential (7~) cannot do work. III. The factors of energy. Already present though unre- cognized in the early development of mechanics, but made ex- plicit first in thermodynamics, and later extended to all forms of energy is the view that each is made up of the product of two factors, a potential or intensity, on the one hand, and an extensity or capacity factor, on the other. In heat energy these factors are respectively temperature and entropy Qj T (specific heat), in kinetic energy f^and MV, in volume energy (gases and solutions) 1 This procedure illustrates the necessary dogmatism of all science, and the superi- ority of a pragmatic to a logical justification, a subject which will be treated at length in another paper. PHYSICS OF SEGMENTATION. 103 pressure and volume, in surface energy surface tension and sur- face, in chemical energy chemical intensity (avidity) and mass. IV. The law of events or action. Eveiy event, i. e., the going over of energy from one body to another or the transformation into another form is conditioned () a side view is given. The condition shown in (l>) and (f) followed seventeen days after the cut. NOTES ON REGULATION IN STYLARIA LACUSTRIS. 189 FIG. 13. Oblique growth of bud of prostomium in (/>), and slightly incomplete regeneration. Stages (b, c, d) were 4, 8 and 18 days after the operation, respec- tively. The eye was not regenerated. The other eye increased in size. FIG. 14. Failure of regeneration in a sexual individual after removal of two seg- ments. This condition existed nineteen days after cut was made. FIG. 15. Dwarfed anterior regeneration from a sexual individual after removal of fifteen segments. The prostomium is not regenerated. A pharyngeal region of five short segments is produced, as shown by the number of setae bundles. In (c) a side view is given. The level at which the cut was made is shown in (a). FIG. 1 6. Regeneration from posterior end of a sexual individual. The part pro- duced is of less diameter. enough to determine whether the regeneration of the sexual or- gans was complete. The cut was made just behind the clitellum. CONCLUSIONS. 1. The formation of a regenerating region will under certain conditions inhibit the process of asexual multiplication and cause the disappearance of the zone of fission. This effect may be pro- duced by a cut anterior to the zone of fission, less often by a cut posterior to it, and occurs only when the zone is embryonic. The zone is also more likely to disappear if the cut is near to it. The band of transparent embryonic tissue redifferentiates and the energy of growth is transferred to the regenerating region. 2. There is evidence of disorganization and a return to embry- onic conditions in organs just behind a cut surface. This effect does not appear to be a direct mechanical result, /. c., due to crushing. Fragmentation of the pigmented portion of the eye is one case adduced. 3. Internal conditions favorable to proliferation, such as the exposure of cut surfaces of intestine and blood-vessels, are pres- ent in nearly all possible experiments. But if a corner of the head segment be removed, including the prostomium, without injuring the pharynx, the ectoderm may close over the surface and regeneration may fail to take place. 4. Short posterior pieces often fail to regenerate anteriorly, but no cases of heteromorphosis, such as individuals with tails on the anterior end, have been obtained. Short posterior pieces which failed to regenerate within the time of observation may elongate and show some differentiation in the budding region. 5. The middle portion of the body has the greatest power of regeneration, the specialized pharyngeal region has the least. IQO E. H. HARPER. 6. Growth takes place at right angles to a cut surface, and it the cut is oblique the bud will grow out at an angle to the axis of the body. Straightening is affected after the penetration of the lumen of the pharynx into the region, probably under the influence of the tension produced by the peristaltic motions of the intestine. 7. Sexual individuals lose the power of regeneration to a large extent just as they do their power of budding. In an advanced sexual stage regeneration may fail to occur or be incomplete. I wish to express my thanks to Dr. C. M. Child for suggesting to me the subject of this paper and for further suggestions rela- tive to some of the results described. HULL ZOOLOGICAL LABORATORY, UNIVERSITY OF CHICAGO, 1903. REFERENCES. Bourne, A. G. '91. Notes on the Naidiform Oligochseta. Quart. Jour. Micr. Sci., vol. 32, pp. 335-356- Child, C. M. 'oo. A specimen of Nai's with bifurcated Prostomium. Anat. Anz., Bd. 17, pp. 311-312. Galloway, T. W. Observations on Non-Sexual Reproduction in Dero vaga. Bull. Mus. Comp. Zool., Vol. XXV, No. 5. Harper, E. H. '01. Abstract in "Science," N. S., Vol. XIV, No. 340, pp. 28-29, Juty 5- Hepke, P. '97. Uber histo- und organogenetische Vorgange bei den Regenerationsprozessen der Naiden. Zeit. \yiss. Zool., LXV. Mayer, C. '59. Reproductionsvermogen und Anatomic der Naiden. Ver. Nat. Vereins, Rheinlande XVI. Morgan, T. H. '97. Regeneration in Allolobophora foetida. Arch. f. Entw.-mech. V. Rievel. '96. Die Regeneration des Vorderdarmes und Enddarmes bei Einigen Anneliden. Zeit. f. Wiss. Zool., Bd. 62. Schultze, Max. '49. Ueber die Fortpflanzung durch Theilung bei Nais proboscidea. Arch. f. Naturgesch., Bd. I., Jahrg. 15, pp. 293-304. VARIABILITY IN THE NUMBER OF TEETH ON THE CLAWS OF ADULT SPIDERS, SHOWING THEIR UNRELIABILITY FOR SYSTEMATIC DESCRIPTION.1 CARL HARTMANN. Since the number of teeth on the claws of spiders is often used as a specific character, the need of testing its constancy sug- gested itself. It has been pointed out by W. Wagner 2 that the number of ungual teeth varies with each moult. In the present study of the variations in the adult this fact was well taken into consideration, great care being exercised in choosing fully mature individuals that had undergone the last moult. To my knowl- edge no one has ever tested the constancy of the number in fully mature individuals. The study was made on the claws of the right legs of 70 females and 40 males and comprises, therefore, observations on nearly 1,320 claws of 440 legs. Representatives of a number of different families were chosen as follows : Dictynidae {Dictyna volupis, Keys., West Chester, Pa.), Theridiidae (Theridium tepi- dariorum, Koch, Philadelphia), Pholcidae (Spermaphora sp .? Hentz, Austin, Texas), Epeiridae (Epeira marmorea Clerck ; E. benjaiiiini, Walck.; Acrosoma reduvianum, (Walck), West Chester, Pa.), Lycosidae (Lycosa nidicola, Emerton, Austin, Tex.). The counting of the claws was easy except in the case of Dictyna volupis and of Spennophora ; but even here, if any mis- takes in the counting are recorded, they are extremely few, for I never left a claw until convinced that my count was correct ; or in a few cases where this seemed impossible the individual was entirely discarded. To count the teeth the three claws of each leg, if they were large, were snipped off with a needle (keeping the foot on a slide in a drop of alcohol) and pressed flat with a cover-glass. If the claws were too small, the whole tarsus was 1 Contributions from the Zoology Department of the University of Texas, No. 55. 2 W. Wagner, " La Mue des Aragnees," Ann. Sc. Nat., 1888, p. 363. 191 192 C. HARTMANN. TABLE I. FEMALES. in (*- No. of the Individual. 'o ' V V 0. ^ i 2 3 4 5 6 7 8 9 10 13,9 13,12 I3,I1 12,11 12,11 12,10 14,12 14,12 12,12 13," ^ s 5 5 5 5 5 5 5 5 5 1 2 12,10 12,12 12, JO ii,- 12,12 12,12 12,11 13,13 11,12 1 5 5 5 5 4 5 5 5 5 1 9,10 10,9 -,9 9,9 9,9 8,9 9,10 10,10 10,10 9,9 * 4 4 4 4 3 4 3 4 3 4 c5 9,10 9,10 10,10 9,10 9,10 9,9 11,10 9,10 10,11 10,10 4 4 4 4 3 4 4 4 4 4 i 6,6 6,5 5,8 6,5 6,5 6,6 4,54,5 6,5 6,5 6,6 2 2 2 2 2 2 2 2 - 2 2 ll 2 6,6 6,5 6,5 6,5 6,6 6,5 5,5 -,6 7,5 6,6 6,6 *S-2 2 2 3,2 2 2 2 2 2 2 2 2 V « 5,6 5,6 5,5 5,6 5,6 5,6 ->5 -.6 5,6 5,6 6,6 s^, 2 2 2 2 2 2 2 2 2 2 i •fc* 4,2 3,2 4,2 4,2 4,2 4,1 3,2 3,2 4,2 3,1 4,2 4 2 I I 2 2 i I I I 2 i i 11,10 10,10 11,11 10,10 10,10 10,10 11,10 11,10 9,9 11,10 . I I I I I I I I i I k 2 10,10 10,10 11,10 12,10 10,10 11,10 II, II 9,9 10,10 •§, I I I I I I I i I s 9,10 10,10 II, II 10,10 II, II 9,9 10,9 10,10 9,9 9,12 %• 2 I I I I i i I i I A 7,9 7,9 -,9 9,10 7,9 8,10 8,10 8,9 7,8 10,10 T- o i 2 i i i i i i I I 7,8 8,8 7,8 8,9 9,7 7,8 7,8 8,8 8,8 9,9 2 V 2 2 2 2 2 3 3 2 3 3 i 2 7-8 6,7 7,8 9,9 8,8 8,7 7,8 7,8 8,8 8,8 K. 2 2 2 2 2 2 3 2 2 3 i u 6,6 -,7 6,7 7,6 6,5 6,6 6,7 6,6 8,7 .5 * 2 2 2 2 2 2 i 2 2 § A 5,6 6,5 6,8 6,6 5,6 6,5 6,5 6,6 6,7 ^ 2 i 2 2 2 2 2 2 2 I 4,4 4,4 4,4 4,4 4,4 5,4 5,4 4,5 4,4 4,4 J o o o 0 o o o 0 0 o *N» •§ 2 7,6 7,6 5,4 5,4 5,4 6,5 5,4 5,6 5,5 4,5 .12 o o o o 0 i o o o o 8 3 7 7,6 6,6 7,6 6,5 7,7 7,6 7,7 7,7 6,6 2 •J o o o 0 o o o o 0 4 A " 6,5 9,9 6,7 6,7 5,7 8,8 7,7 8,8 7,7 7,7 o o 0 o o o 0 o o 0 i 5,8 5,9 8,- 8,8 9,8 8,8 10,8 8,9 7,8 6,8 *«•« .8 2 2 3 2 3 2 3 2 2 2 i 2 5,9 6,9 5,6 7,9 7,9 8,8 8,9 7,8 6,8 7,8 «? 2 2 3 2 3 2 2 2 2 2 "« •J 3,6 8,7 7,6 7,7 8,7 7,7 6,11 7,6 7,7 6,6 *1S, j 2 2 2 2 2 2 2 2 2 2 § d. 6,5 7,6 7,6 4,5 6,5 5,5 6,5 6,5 6,5 6,5 T^ 2 2 2 2 2 i 2 2 2 2 TEETH ON CLAWS OF ADULT SPIDERS. 193 TABLE I. FEMALES. — Continued. (fl « V D. C/3 1*4 No. of Individuals. ' 2 3 4 5 6 7 8 9 10 i 7,6 7,6 7,7 7,6 7,7 7,6 8,7 7,6 7,7 7,7 2 2 2 2 2 2 2 2 2 2 r * ? 7,6 7,6 7,6 7,- 6,6 6,7 7,7 7,7 6,7 § 5 2 2 2 2 2 2 2 2 2 t i 7 5,6 5,5 6,5 6,5 6,6 5,5 6,6 5,5 6,6 5,5 g< * 2 2 2 2 2 2 2 2 2 2 A 3,2 3;4 3,4 4,4 4,5 5,4 4,2 4,4 4,3 I 2 2 2 2 2 I 2 i covered, placed under the compound microscope and pressure applied until all the teeth came into full view. The results of the counting of the teeth are recorded in the accompanying tables, Table I. containing the figures for the fe- males and Table II. for the males. The three claws were always distinguished from one another and the number of teeth on each recorded after the formula ----- , where « represents the number of teeth on the anterior, / on the posterior and z on the inferior claw. The reduction in the number of teeth seems to take place at the proximal end of the claw because, firstly, the distal teeth usually maintain the size and form characteristic of the species, and secondly because the proximal tooth (or teeth) in some cases becomes so small as to merit the name tubercle in place of tooth. This latter fact forced me to establish a criterion to de- termine what to count as a tooth and I decided to call the structure a tooth if it had attained a length at least half as great as its width at the base.. In Dictyna volupis the two distal teeth are small and are closely approximated to the claw for nearly their entire length. One of these was counted in some four or five cases where it was unusually large and stood out from the claw for at least half its length. In order to reduce the tables to percentages so as to get at a simple set of figures for comparison I have adopted what may be called the " percentage of constancy " method, which may be illustrated as follows : In Table I. the anterior claw of the first leg of Lycosa nidicola 194 C. HARTMANN. TABLE II. MALES. en V *»- No of the Individual 'o • a? 1) p< O 1 i 2 3 4 s 6 7 8 9 IO 12,11 12,11 12,10 12,11 12,9 II, II II, II 10,10 II, II 11,10 .3 I 5 5 5 5 5 5 5 4 7 6 1 12,12 12,11 13,12 n, ii 12,11 12,11 ii,- II, II 12,11 II, IO 2 6 5 5 5 5 5 5 5 6 6 s n, ii 11,10 9,9 9,10 9,9 9,9 10, IO 9,9 -, IO IO, IO •£> .Vj 3 4 4 4 4 4 4 5 4 4 5 3 ii, 1 1 10,10 9,9 10,10 10,9 10,10 11,10 9,10 10,11 10,10 4 5 4 4 4 4 4 4 4 5 5 4,4 5,5 5,4 5,5 6,4 -.4 5,- 5,5 5,5 5,5 i i 2 2 2 2 2 2 2 2 ~ s ft s V J^ 2 4,4 5,4 5,4 5A 5,4 5,4 5,4 5,5 5,5 5,5 It i 2 2 2 2 2 I 2 2 2 5-2 -A 4,5 5,3 4,5 3,5 4,3 4,5 4,3 4,4 5,5 *-;§• i 2 2 2 2 2 2 2 2 2 2,1 3, i 3,1 3,1 4,1 3,- 4,1 4,1 3-1 4,1 4 I i i i i 1 i 1 I I 12,11 12,- 12.11 12,10 11,10 10,9 10,10 ii, 10 11,10 12,11 I I 1 i I i i i i I 1 2 12,10 11,10 12,11 11,10 12,11 10,9 n, 10 10,10 ii, 10 11,10 ^s 1 I 1 i I i i I I I i 10,11 II, II II, IO -,IO II, II 9,9 10,10 9,9 10,9 10,10 4 3 1 I I I I I i i I to 9,10 8,10 8,10 8,10 9,IO 8,9 8,9 8,9 9,10 4 i i i i I i i I I i 7,9 10,11 9,8 8,9 8,9 2 2 2 2 2 2 1 8,8 7,9 8,8 8,8 3 3 5 3 SP D D D _> .> a >, ctf cd c3 o 3 w Js , >— > "O >« >-• o — C C S ^ < S S G £ . .*-» 0 0 •d 2 D u E S D j, ^ ^ rt ^3 i-i a fc -b £ ^ 1 1 » 1.1 ' ~ J CS D 1 = = J Sod -| .1 S3 < in S< S G[JH< = § 1 od -g S * c£ 3 .5 d "3 VQ *" «« qj CJ w ^ ttJ . 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Q<<: o M B'l }| S M ^ H i c • c •— H <~ M tn tn *£> rt O o o "3 «) ™^ 15 p. > b-o"o 6 J2 • "^^i^l I ^"^ 2' -2-2 2' U -^ £ .^K 3 D "^ 0 C l- ^H^ Q PQ^i Q QQQ ^ 5 E *" S u £ J i -2 ^ - c c 3 y, ^co^s 'S1*,^ 5 « g 1 M -D"§O uT 0 S _S a1? |'s«j. j^ | o 1 M !> 0 'S l*"C'OO. c«"~ C «• (/j1^ D^1^-'.'"'^ a o "O i3--;ni-i'r5:^si.* . rj ^3 ^3 C ca 13 tuo to w (J 0 O O • • • '"" « • ^ ri *"^ f *~ ' o • cj y o ^_. u *^ c . *o .^ r— • ^ §(J O CJ -— "TJ ^ 1 D D ^ O J 'ft O o S3 .y ui o — <->'3'S5'!n u i3 ^f"> u-j '^ _n J^1 i^> CX cd L'^ 3 F~- '-*-• o = § £ 2 N "S ^ S^^^23^^'5^^ o 6 i 4 HH t/l D O -a c CS ex I) rt BIOLOGICAL EXAMINATION OF DISTILLED WATER. 2O I From the above and other experiments I can make the state- ments enumerated below, which apply, of course, to Arbacia only but point to the necessity of caution in using distilled water on other organisms. It is true also that the results are most appli- cable to Woods Hole conditions and to the tap-water used as a basis of the distilled water there. It is probable that the toxicity is due to ammonia although this was not proven. It is certain from experiments made that Arbacia larvae are very sensitive to that substance. 1. Tap-water is decidedly, although variably, toxic. The toxicity is not lost by sterilization but is greatly reduced by boil- ing the water for a long time, say until one third has been boiled away. The residue in such cases is less toxic than some distilled waters, particularly commercial brands and that from automatic stills. 2. Water from ordinary automatic stills, whether metal or en- tirely glass, is toxic. 3. The commercial distilled waters used by me were toxic, often in high degree. 4. In distilling water in the ordinary way from glass, the first one tenth distilled over is decidedly toxic, the second tenth less so, the third tenth still less so. The fourth tenth is of good quality. 5. The best distilled water used was produced by double dis- tilling in glass, the first fourth distilled over in each distillation being rejected. 6. Nearly as good water was produced by single distillation from tap-water to which H2SO4 and K2Cr2O7 had been added. 7. If a little H2SO4 is added to the tap-water to start with, an excellent quality of distilled water may be produced from an automatic still consisting of a copper vessel and glass condenser, the arrangement being such that none of the condensed water touches the metal. This water is practically free from ions or toxic volatile substances. It is much better than water double distilled in glass in the ordinary way, unless in the later case a large proportion of the product be thrown away. Such an auto- matic still is recommended for use at Woods Hole. 8. It was noted in a number of cases that Arbacia lived longer 2O2 E. P. LYON. in an artificial sea-water prepared from a good quality of distilled water than in natural sea-water. It is probable that the volatile toxic substance (ammonia ?) exists in sufficient quantity in sea- water to have an appreciable effect. HULL PHYSIOLOGICAL LABORATORY, UNIVERSITY OF CHICAGO. Vol. VI. April, 1904. No. BIOLOGICAL BULLETIN ON THE HABITS AND REACTIONS OF SAGARTIA DAVISI. HARRY BEAL TORREY. Sagartia davisi1 is the Pacific representative of the 5. luciic of the Atlantic coast of the United States. The two species appear to differ only in the absence, in 5. davisi, of the narrow yellow stripes on the column which are so characteristic of the eastern form. Both species commonly reproduce by fission, and it is probable that the following descriptions of some of the other habits of 5. davisi may be applicable to J>. lucicc also. 5. davisi was first discovered clustered on the valves of a spe- cies of the bivalve Chione, common in the harbor of San Pedro, Cal.2 The clam dwells near or on the surface of the sand. As it plows its way along, the uppermost regions of the shell are about the hinge and the siphons. To these regions, invariably, the polyps cling. The deeper the clam goes the more do their small, thin-walled bodies lengthen, supported by the surrounding sand, to a degree I have never seen in an erect and unsupported form. vS. davisi is not, however, a burrower, nor is the association with Chione a case of commensalism, as might be concluded at first sight. It fastens readily to any object which can give it a foot- hold and keep it out of the sand. On the sand flats at San Pedro 1 Description. Column of largest polyps about I cm. in diameter; spread of ten- tacles about 2. cm. Foot disk very extensible ; body wall everywhere quite thin and semitransparent ; a distinct capitulum above a well-defined collar as in Metridium ; oral disk almost circular, mouth small, oval, lips with about twelve lobes, not prom- inent ; one or two, occasionally three siphonoglyphs. Tentacles tapering, slender, pointed, variable in number, most often 45-50 in perfect individuals. Color of column dark brown, tentacles and disk green. Many individuals with light longitudinal stripe of variable width on column (zone of regeneration after fission). Mesenteries unusu- ally variable in number and arrangement. Reproduction sexual and non-sexual ; latter the cause of irregularities in number and arrangement of tentacles and mesenteries. 2 It has since been found in San Diego Bay, Cal. 203 2O4 HARRY BEAL TORREV. it was uncovered about half each day by the falling of the tide, during which periods its tentacles were completely retracted, con- trary to the custom of such typical sand dwellers as Harenactis attenuata? and another species of Sagartia from San Pedro as yet undescribed. It may live permanently submerged, however, and thrives in aquaria. Though ordinarily attached to a solid substratum, it is occa- sionally found free on the sand. Its powers of locomotion are considerable. By means of multicellular amoeboid processes of the foot disk, readily seen with a hand lens at the edge of the disk, it is capable of creeping more than an inch in an hour. The polyps often leave the clam to which they are attached when placed in an aquarium, especially when they are on the lower valve. They may occasionally creep along the surface of the water, hanging from the surface film. The inverted position is not long retained, however, for 5. davisi has a marked tendency to assume as erect a posture as its situation will permit. An example of this tendency is provided in the case just mentioned, of the relatively greater haste of the polyps in leaving the lower than the upper valve of a clam lying on the aquarium floor. Moreover, the axes of polyps clinging to the vertical sides of the aquarium are either perpendicular to the sides or bent upward. They never bend downward if the polyps are submerged. The orientation of 5. davisi is, then, partly a result of geotro- pic stimulation. The same may be said of the locomotion of the species. There is a definite tendency of the polyps on the walls of the aquaria to collect near the surface, although the aquaria may be sealed jars completely filled with water, or furnished with green water plants evenly distributed (precautions against the possible influence of oxygen at the surface). The polyps on the floor of the aquarium, if it be horizontal, move about but little, and when they do, sporadically and without certainty of direc- tion. When by chance, however, they reach the angle made by the floor and a side of the aquarium and begin an ascent, there is never a retrograde movement, seldom a halt, until they draw near the surface. This locomotor geotropism is especially inter- 1 Torrey, 1901. HABITS AND REACTIONS OF SAGARTIA DAVISI. 205 esting from the fact that the major axis of the animal is not par- allel with the direction of locomotion, a peculiarity which dis- tinguishes it from the reactions of the majority of animals to directive stimuli. The major axis is the axis of geotropic orien- tation, but it can only be the axis of locomotion in swimming forms ' and those which lack a foot disk and creep on the column (e. g., Pcachia]. It is possible to reconcile these different cases if we think of the foot disk merely as a differentiated portion of the body wall. Edivardsia has no well defined foot, though its aboral end is rounded and adhesive. The hydroid, Coryuiorpha, again, has no foot such as is possessed by Hydra, its aboral end coming to a point ; yet the sides of this tapering extremity are adherent, and through their amoeboid cells the hydroid, orienting negatively to gravity, tends also to move vertically upward. It adheres in this case by a portion of what may truly be called its lateral wall ; in consequence of which the axes of locomotion and geotropic orien- tation coincide. 6". davisi is an extreme case in the other direction. Having a large and well defined foot, it can hardly be said to cling obviously by a portion of the lateral (i. e., column) wall. At the same time, when on a vertical surface, its axes of locomo- tion and orientation are as nearly parallel as the differentiation of foot and column will allow. This, however, is not equivalent to saying that the direction of locomotion in response to a directive stimulus is determined by the orientation of the major axis of the polyp, for the elements of the foot may be directly affected by gravity. Loeb ('91, p. 70) has said that Cerianthus and Actinia equina went from smooth glass to a mussel shell or piece of ulva more readily than in the reverse direction in his experiments. This indicates a certain "contact irritability," which seems to be pos- sessed also by .$. davisi, as the latter moves about more freely on smooth glass than on rough surfaces. The reaction to the contact stimulus, however, is not so strong as the orienting re- 1 Besides the pelagic species which Andres describes among the Minyadidce, all non-adherent, there is an interesting polyp abundant in the harbor of Honolulu which, I am told by my friend Mr. Loye H. Miller, leads both a sedentary and a free exis- tence. It appears to have no pedal float, sustaining itself by means of rhythmic move- ments of the tentacles which send it along foot foremost at a fair rate of speed. 2C>6 HARRY BEAL TORREY. action in response to the stimulus of gravity, so that as a result- ant of the opposing responses, the polyp leaves the shell for the glass as stated above. Light does not appear to stimulate 5. davisi in any way. The polyps neither bend nor move toward the light when it comes from but one side of the aquarium, in all degrees up to the in- tensity of bright daylight. Neither do flashes of sunlight falling upon polyps in a darkened aquarium produce any mus- cular responses. 5". davisi differs in this respect from Ccrian- thns mcmbranaceus and Edwardsia lucifuga, according to Nagel ('94, p. 545). The responses of anemones to mechanical and chemical stim- uli have been investigated already by Pollock ('82), Loeb ('91 and '95), Nagel ('92, '94^, '94^) and Parker ('96). With most of the conclusions of these investigators my own observations accord. I must differ with some, adding also a few facts which to my knowledge, have not been published heretofore. Two quite distinct reactions, usually follow the stimulation of a tentacle of 5. davisi by means of a slight touch with a needle or glass rod. The first is a bend at and toward the point of stimulation, whether the latter be near tip or base, on right side or left, above or below, and appears to be due to the response of the muscles involved to a direct stimulus. The second is a con- traction of the whole tentacle, with a simultaneous bending of the tentacle toward the mouth. Evidently all the longitudinal mus- cles of the tentacle not previously active are indirectly excited to produce this reaction, those on the inner (upper) side between base and point stimulated contracting more strongly than the outer (lower) muscles. This unequal contraction is probably to be explained by the greater strength of the inner muscles, which play the greater part in the chief work of the tentacles — carrying food to the mouth. The hydroid Corymorpha shows this inequal- ity still more strikingly ; the first reaction of S. davisi is en- tirely wanting and the outer muscles are in use only when the tentacles are slowly returning to their expanded condition after a contraction. But I have been unable to demonstrate histolog- ically any difference in size between outer and inner muscles, in either animal. HABITS AND REACTIONS OF SAGARTIA DAVISI. 2O/ The result of the second reaction is varied. Often the tentacle merely waves stiffly inward. At other times it may arch so that its point is directed toward the mouth. On the whole, however, its movements are less definitely adaptive than those which Parker describes for Mctridimn. The second reaction does not always follow the first. The general contraction does not appear to be induced by contact alone. If the tentacle be touched lightly and for but an instant, only the first reaction occurs. If, however, the stimulating ob- ject rest against the tentacle sufficiently long to allow the latter to adhere to it, the second reaction immediately follows. Whether this results from the adhesion itself, or the duration of the stimu- lus, or a tension in the muscles due to the resistance of the stimulating object, I am unable fully to decide. Such small ob- jects are capable of producing this reaction that the third possi- bility seems to be excluded. Whichever of the other two be the efficient stimulus, it produces a strong contraction of the muscles directly affected. This strong contraction probably serves as a direct stimulus for contiguous muscles, the contrac- tion of these for others, and so on, until all are involved. In no case did the evidence enforce the assumption of the presence of nerves, in the tentacular responses. So far the movements of but a single tentacle have been con- sidered, without relation to the others. And it should be said here that tentacles cut from the polyp behave in all essential respects as they do under normal circumstances. Often the stimulus applied to one tentacle is sufficient, unless care be used, to induce contractions in several. It may be that only a few tentacles on each side of the one touched will react ; with a stronger stimulus the entire set of tentacles may contract with vigor. There is no more evidence, however, that this correla- tion of parts is attained by the aid of nervous tissue than there was in the case of the single tentacles. Communication from one tentacle to the next is largely through the oral disk. The proper degree of contraction of a tentacle induces a con- traction in neighboring muscles in the oral disk, and pos- sibly in contiguous tentacles directly. The vigor of the stim- ulus, if it be local, appears to determine the extent of the 2O8 HARRY BEAL TORREY. response, which spreads by the direct effect of the tension of one muscle on those near it. The usual response of a tentacle stimulated indirectly in the manner just described is a waving or arching toward the mouth, with or without vigorous shortening of the whole tentacle. Occa- sionally the response is quite opposite to this, the stimulation of one tentacle producing an outward waving of neighboring tenta- cles. The anomaly is only apparent, not real, for as a matter of fact the muscles of the neighboring tentacles are not involved at all in the latter case. The tentacles neither shorten nor bend. They move outward stiffly, owing to a local contraction of muscles in the oral disk or the capitulum. If the stimulus applied to a tentacle be sufficiently strong, all of the tentacles may shorten simultaneously, may even be entirely withdrawn into the body by the contraction of mesenterial muscles and hidden by the contraction of the sphincter ; the column may shorten also, and the foot disk may change its shape. All of these movements seem to be induced by the direct passage of the stimulus from muscle to muscle without the aid of nerve tissue. The oral disk, between tentacles and mouth, is almost insensi- ble to mechanical stimuli. Stimuli applied to the column produce the inward movement of several or all tentacles, the outward movement of a few, or the contraction of column and foot disk, according to the strength of the stimulus. Stimulation of the foot disk, either at the edge or on the lower surface, produces local contraction of the foot and base of the column, and acontia are usually emitted near the point stimu- lated. The tentacles may contract also, but always as a whole, the same general reaction following stimulation at different points of the disk instead of a local reaction as in the cases of the foot, column and acontia. This inability of the tentacles to recognize the direction of the stimulus is also characteristic of the reaction of the tentacles of Coryuwrpha to stimulation of the column, and is due, I believe, to the opportunities for diffusion of the stimu- lation impulse owing to the distance of the reacting structures from the point at which the stimulus is applied. The entire surface of 5. davisi, with the possible exception of HABITS AND REACTIONS OF SAGARTIA DAVISI. 209 a small zone between mouth and tentacles, responds to a mechan- ical stimulation, the greatest irritability being manifested by the tentacles, the tactile organs par excellence. The latter exhibit a very definite adaptive reaction. The preliminary bend of an irri- tated tentacle at and toward the point stimulated makes it possible for the polyp in a sense to pursue its prey actively if, indeed, to but a limited extent. The great advantage of this reaction over the simple inward movement of the tentacle indirectly stimulated is obvious. The latter is also adaptive, however, since it is the most likely movement to clutch food organisms in a polyp whose tentacles are habitually outstretched. Supporting this idea is the fact that the tentacles of hydroids react only in this way, whether stimulated directly or indirectly. It is the simpler, more primi- tive reaction. A more efficient adaptive reaction, also indirectly induced, is the extrusion of acontia at the point stimulated, for purposes of defense. Though the reaction is always the same-, the acontia always move in the most desirable direction, which is not always the case with tentacles. The passive outward movement of tentacles due to the con- traction of muscles in the oral disk or capitulum is not directed by the position of the stimulating object. It may be toward the latter, but only when the stimulus happens to be applied at a point external to the tentacles, that is, at some point which is less likely to be stimulated by a food organism than points on their inner surface. Wlien a tentacle o'f an outer whorl is moved pas- sively as a result of the stimulation of a tentacle of an inner whorl, the movement is away from, not toward the tentacle stimulated. The reaction in this direction is of no obvious im- portance to the polyp in- this case, and seems to be of no more importance, in the sum of all cases, than a movement in any direction. It appears, therefore, to have no adaptive value whatever. By means of its varying sensitiveness to different chemical sub- stances and its ability to discriminate between mechanical and chemical stimuli, 5. davisi is enabled to make certain choices in its quest for food. This capacity, which it possesses in common with other anemones, has been described as olfactory by Romanes 2IO HARRY BEAL TORREY. and Nagel, and as gustatory by Jourdain. Such expressions, how- ever, are essentially psychological, and Loeb ('91) has justly insisted upon the substitution for them of some physiological expression, such as chemical irritability. This power of dis- crimination has been shown by Loeb to reside not only in the tentacles (Nagel, '92), but also in other regions. Actinia cqnina discriminated between crab's flesh and small rolls of paper as definitely after he had removed the tentacles by a transverse cut as before. Parker ('96) demonstrated later that Mctridiuin diantlins reacts in different ways to mechanical and chemical stimuli. Actinia cqnina, from Loeb's observations, is so definite in its choices that chemically inert paper pellets were never taken into the mouth. Parker found that Metridium would swallow pieces of white india rubber as well as flesh, though the former were sometimes disgorged before they had passed out of the oesophagus. Since he has shown that the cilia covering the lips of Mctndiiun and beating outward in the absence of chem- ical stimuli, reverse their dominant beat in response to the stim- ulation of meat juices, their behavior when stimulated by ap- parently chemically inert india rubber leaves a doubt as to whether or not they can be reversed by purely mechanical means. There is, however, no doubt of such a reaction in S. daiisi, as will be shown in the course of the following ac- count of my experiments. It may be well to begin with the effects of various chemicals. Cane sugar in solutions of various strengths produced no ap- preciable reactions in any part of the polyps on trial. Strong picric acid and 4 per cent, formalin caused the retraction of all the tentacles, indicating stimulation of body muscles. One half per cent, hydrochloric acid caused a general contraction of tentacles. From a knowledge of the behavior of Corymorpha, which, though unable to detect the presence of flesh until touched by it, yet reacts strongly to strong alcohol and acetic acid, I am led to suspect that these substances irritate the polyp in the same way that they irritate one's skin, through the tactile organs merely. Crab's muscle, bits of limpet and nnm lid worm were used as HABITS AND REACTIONS OF SAGARTIA DAVIS1. 2 I I stimulators with uniform results. Small amphipods were devoured with avidity. The response differed according as the stimulus was applied locally or generally. For my first experiment I placed a small piece of worm on several of the outstretched ten- tacles of a polyp. The tentacles immediately adhered, bending at and toward the point stimulated, as though responding to a purely mechanical stimulus, and then contracted, dragging the morsel to the mouth. For some seconds the tentacles not in contact with it remained motionless. Then, one or two at a time, they waved slowly inward and grasped the flesh, almost every tentacle finally becoming thus engaged. This experiment was tried many times with similar results. Apparently the tentacles not mechanically stimulated were irritated by substances in solu- tion diffusing out of the flesh, and the reaction was as definite as it would have been if induced by a mechanical stimulus. The movement was toward the stimulus. The possibility of an indirect stimulation of these tentacles through the oral disk from the tentacles touching the flesh was eliminated by holding a piece of worm flesh immediately above the mouth of another polyp. In a few seconds some of the tentacles began to twitch slightly, and a little later all began to wave slowly inward, toward the flesh, finally grasping it. A similar result followed numerous trials. Next, a bit of flesh was placed on the aquarium floor, near, but not in contact with the foot disk of another polyp. Would the tentacles bend in the direction of the flesh now, or toward the mouth? This experiment, repeated a number of times, did not give uniform results. In the majority of cases the tentacles waved toward the mouth, away from the flesh. In the rest they moved toward the flesh in the most definite and unmistakable manner. Not only that ; the column, in several cases, bent toward the morsel which was seized by the tentacles nearest it and dragged toward the mouth. There is no doubt here that the movements were in the direction of the stimulating object, and are thus comparable to the well-known movements of the manubria of various mendusae toward stimulated points on the subumbrella. The proboscis of Corymorpha reacts similarly, as will be shown in a forthcoming paper. 212 HARRY BEAL TORREY. Pollock ('82) observed this fact but was unable to reconcile his varying results. The reason for his failure was, I believe, that he failed to distinguish between general and local stimulation. When the meat juices of the annelid used previously were dis- charged gently over a polyp from a pipette, I observed that the tentacles always waved inward, without regard for the direction from which the juice was coming. This general chemical stimu- lation produced the same response from the tentacles that a me- chanical stimulation of the foot disk provoked. In both cases then, in which the tentacles waved inward and away from the flesh, the diffusion of soluble substances from the latter was probably so rapid that the tentacles were stimulated on all sides so nearly at the same time that no differential of stimulation beween opposite sides of the tentacles was established, the neces- sary condition of a directive reaction. But why the movement toward the mouth ? Because it is the primitive clutching move- ment already spoken of as most likely to capture food organisms, in a polyp whose tentacles are habitually outstretched. It is the simplest adaptation of the prehensile mechanism, common to hydroids as well. The responses of the tentacles to mechanical and chemical stimuli are essentially the same. The bend is toward the stimu- lus when the stimulation is local, toward the mouth when it is general, whether direct or indirect. If we turn now to the phenomena of swallowing, we shall see that the cilia of both lips and oesophagus may respond to mechani- cal as well as chemical stimulation by waving more strongly inward than outward. I early observed that not only were pieces of flesh occasionally rejected, but bits of shell and gravel were sometimes taken in. With the idea in mind that the size and shape of the object might affect the reaction, several substances, presumably chemically inert, were given to various polyps, in pieces varying in these respects. Pieces of very thin paper, from i mm. to 3 mm. square, when placed npon the tentacles, were cast off in half an hour. A piece of cork, about one fourth as large as the polyp, was likewise rejected. A much smaller piece, capable of being easily ingested, was taken into the gullet and retained for thirty minutes. A piece of paraffine of similar HABITS AND REACTIONS OF SAGARTIA DAVISI. 213 size was swallowed in three minutes, and a half-cube of heavy drawing paper, of about the same size, was also swallowed, though more slowly. Tiny bits of glass were frequently swal- lowed. I can say definitely that these objects were, not carried in by the beat of the cilia covering the siphonoglyphs and producing an insetting current, but by cilia covering the lips and oesophagus between the siphonoglyphs and producing a current which ordi- narily sets outward. It would seem, then, that chemically inert substances, if small enough to be taken easily into the mouth and thus brought into direct contact with the ciliated cells lining the oesophagus, are ingested under some conditions. Other ex- periments show that one of these conditions, probably the most important, is the degree of hunger of the polyp. Starving polyps were always more ready than well fed individuals to swallow chemically inert substances. Some explanation of this fact may be derived from the further fact that hungry polyps are in gen- eral unusually sensitive to both chemical and mechanical stimuli. Increased sensitiveness means increased effectiveness of a given stimulus ; this is equivalent to saying that the stimulus is more intense. 5". davisi, then, responds only to certain intensities of the same stimulus, so far as the ciliated cells of the lips and oesoph- agus are concerned. Under mechanical stimulation of a given intensity, the cilia do not reverse their beat ; an increase in the intensity or, if you will, effectiveness of the stimulation may produce this reversal. To chemical stimuli, or to mechanical which are above a certain degree of intensity (i. e., when the stimuli polyp is starving), the response is usually positive ; to a weakened mechanical stimulus there is less likelihood of any response. The positive response to mechanical stimuli is undoubtedly advantageous to the polyp. It is apparent that substances with even a very small food value must be of some importance to a starving polyp although they would not be desirable as food for a well nourished animal. For the latter they would come into the category of useless substances, which the ciliary cur- rents on (Esophagus, lips and tentacles are admirably adapted to remove. 214 HARRY BEAL TORREY. The disgorgement of non-nutritious bodies may now be briefly considered. All harmless non-nutritious bodies, and all food stuffs from which the nutrient juices have been taken during the process of digestion, are sooner or later cast out of the mouth. The cause of the ejection is to be found in the behavior, under varying stimulation, of the oesophageal cilia. The mesenterial filaments bordering the mesenteries, and the defensive filamentous acontia, are ciliated, but probably take no part in the process, for several reasons.1 First, the mesenterial filaments pursue excessively meandering courses along the edges of the mesenteries, and their cilia pro- duce many currents which are antagonistic instead of proceeding in one general direction. I have not been able to determine whether the cilia beat more strongly away from or toward the mouth. In all parts of each filament, however, they appear to beat in the same direction ; and this beat is not reversed by con- tact with meat or meat juices. Second, the cilia on the acontia beat always more strongly toward their free ends, and they too do not reverse their beat in the presence of meat juices. Since the acontia are attached by one end only, have a marked ten- dency to coil, and occupy without regularity of arrangement any position in the ccelenteron, they can hardly be concerned with the phenomena of disgorgement. It may be noted in passing, however, that when they are thrust through mouth or cinclides, their cilia, in carrying toward their tips whatever foreign particles may come in contact with them, are performing what must be in the long run an advantageous service. Finally, the oesophagus itself, ordinarily more than half the length of the column, reaches nearer to the foot disk when the polyp contracts as it does with food substances within it. The objects taken into the coelenteron never get far away from the lower edge of the oesophagus. Under the influence of the mesen- terial and acont'al cilia, they may, if small enough, rotate aim- lessly about during the period of digestion and absorption. In the absence of direct stimulation, the oesophageal cilia resume 1 The following facts concerning the behavior of the cilia on mesenterial filaments and acontia were obtained from Metri,/im>i, but I feel confident that the same results would have followed an investigation of S. dbr'/.r/ had the supply of material permitted. HABITS AND REACTION'S OF SAGARTIA DAVISI. 215 their dominant outward beat, and are able to carry away non- stimulating objects. At the end of the period of digestion and absorption, the ingested bodies have reached their minimum of stimulating power ; and now, no longer able to reverse the dominant beat of the cesophageal cilia, they are carried out by the latter just as soon as they come into their sphere of influence. Why chemically inert bodies, once swallowed, should be dis- gorged, may be explained, I believe, by assuming inability on the part of the cesophageal cilia to continue reversing their dom- inant beat in the presence of a persistent or frequently applied mechanical stimulus which was originally weakly positive. This is in entire harmony with Parker's demonstration that after repeated applications of a weak chemical stimulus to the lips of MctriJiuin, there comes a time when no positive reaction results. Peristaltic movements of the oesophagus may assist the cilia, but I have no evidence that they take more than a very subordi- nate part in the phenomena of swallowing or disgorgement. UNIVERSITY OF CALIFORNIA, January 1 1, 1904. 2l6 HARRY BEAL TORREV BIBLIOGRAPHY. Jourdain, E. '8g Les Sens chez les Animaux Inferieurs. Paris. Loeb, J. '91 Untersuchungen zur physiologischen Morphologic. I, Ueber Heteromor- phose. Wurzburg. '95 Zur Physiologic und Psychologic der Actinien. Arch. f. ges. Phys., L1V, P- 4I5- '02 Comparative Physiology of the Brain and Comparative Psychology. New York. Nagel, W. A. '92 Der Geschmackssinn der Actinien. Zool. Anz., XV, p. 334. '94a Experimentelle sinnesphysiologische Untersuchungen an Coelenteraten. Arch. f. ges. Phys., LVII, p. 495. '94b Vergleichend Physiologische und anatomische Untersuchungen ueber den Geruchs- und Geschmackssinn und ihre Organe. Bibl. Zool., Heft 18. Parker, G. H. '96 The Reactions of Metridium to Food and other Substances. Bull. Mus. Comp. Zool., XXIX, No. 2, p. 107. Pollock, W. H. '82 On Indications of the Sense of Smell in Actiniae, with an Addendum by George J. Romanes. Jour. Linn. Soc. Lond., Zool., XVI, p. 474. Torrey, H. B. *O2a Papers from the Harriman Alaska Expedition. XXX, Anemones, with Discussion of Yariation in Metridium. Proc. Wash. Ac. Sc. , IV, p. 373. 'o2b The Hydroida of the Pacific Coast of North America. Un. of Cal. Publ., Zool., I, p. I. VARIATION IN BEES. FRANK E. LUTZ. In the study of evolution, there is nothing more important than the investigation of variations, since the whole doctrine rests upon the premise that organisms do vary. There was a time when it was sufficient in such an investigation to take a series of specimens and from the general looks of things postulate theor- ies. But the world has become more critical now and demands that when a statement is made concerning some phenomenon, exact data accompany the statement. Hence, the statistical study of variation which attempts to exactly measure the varia- tions and correlations of different organs and to set them down in figures which "cannot lie." And here we cannot allow the other proverb which says that figures will prove anything, for figures truthfully handled can only prove the truth. But there is, on the other hand, great danger that, having collected a set of measurements, we make a show of accuracy that will lead us and others astray by reason of careless or insufficient analysis. Such work is most troublesome because of its seeming exactness and the difficulty of detecting errors. Messrs. Casteel and Phillips, in the December (1903) number of this BULLETIN, have taken up a very interesting and vitally im- portant problem. The comparative variability of the drone and worker bees hits, in a way, at the very root of the variation question. Accordingly, while we lament with the authors the smallness of their series, it seems well worth while to consider a few points about the paper. In the first place, we have to disagree with the statement that if the variability is "due to chance," it is "not in accordance with any law," for it is well known, and has been for years, that nothing is more bound by law or more expressible in mathe- matical formulae than "chance." However, we will heartily agree with them that the "true test of the relative variability" is the " descent in numbers of individuals " in the different classes as they are removed from the mean ; but we wronder greatly 217 218 F. E. LUTZ. why they did not apply this simple test. It is called the standard deviation and must be known to everyone who has ever done any statistical work. The phrases just quoted are taken from the discussion of the counts of the hooks on the hind wings. Let us therefore examine them by means of this confessedly bet- ter measure. We find that for the drones we have : Lo>. No. of Specimens. Standard Deviation. Probable Error. I. 50 2.1548 0.1453 II. IOO I-5435 0.0736 III. 100 1.7716 0.0845 IV. 100 1.6486 0.0786 V. 50 2.0988 0.1416 VI. 98 1-9377 0.0934 For the workers we have : Lot. No. of Specimens. I. II. III. 50 35° 100 Standard Deviation. 1.5223 I-5564 I-5523 Probable Error o. 1027 0.0397 0.0740 This gives an average standard deviation, or variability, for the drones of 1.8592 ±0.1028; and for the workers of 1.5437 ±0.0721. But we see that the difference between the averages for the two sexes is less than the difference between the two sets of drones from the same hive (I. and III.) ; and, considering the probable errors, neither is significant. If we omit the three small series because of their large probable errors, we see that the dif- ference between the variabilities of the two sexes is even smaller and clearly not significant. It is also unfortunate that the work should have been passed by both the authors and still two of the nine averages be wrong. The average for lot II. of the workers is 20.99, n°t 21.08 ; and that for lot VI. of the drones is either 22.65 or 22-75, according as we do or do not include the indi- vidual with 12 hooks, but it is surely not 22.42. This was prob- ably gotten by including this individual (although he was excluded by their argument above), and then using 100 as the total num- ber, but for some strange reason there are only 99 of the 100 said to have been studied which are listed. VARIATION IN BEES. 2IQ Passing over the grave error of lumping the different series of ratios (p. 27) because they seemed to be alike, when really their only claim to homogeneity is that they are of the same sex and all bees- - Italians, hybrids, " peculiar strains," ct <•?/., from cen- tral Ohio to eastern Pennsylvania being jumbled together — let us take up the first table (vein R) as we have done the last. We find that the standard deviation of lot I. of the workers is 1.5637- ± 0.1055, and for lot V. of the drones--" from the same hive " — is 2.25 17 d= o.i 5 19. There is here a difference of 0.6880. The probable error of this difference is d= o. 1 849. The standard deviation of lot I. of drones is 2.4023 d= 0.1620 and that of lot III., of the same sex, also from the same hive is, 2.9598 =t 0.1412. The difference here is 0.5575 ^ 0.2149. This is due to the ex- tremely small size of the series measured. Since the formula for the probable error of the standard deviation is 0.6745 (stand. dev./i/27z), we see how rapidly an increase of " n ''' the number of individuals measured --decreases the error of the result. But it is manifest that the differences between the two sexes, as shown by these data are of no significance ; for, far within the probable error of our work, we get as great a differ- ence between two lots of drones (one lot being twice as large as either of the two considered in the comparison of the different sexes) as we get between the two sexes. I have not taken the time to go over the rest of Messrs. Cas- teel and Phillip's work ; but, having reached the above results with the first and the last tables will leave them to go over the intervening ones. It is also probably unnecessary to remark that, even if it turns out that the greater variability of the drones can be established, their proofs of their theory to account for this difference seem rather unsatisfactory. HULL ZOOLOGICAL LABORATORY, UNIVERSITY OF CHICAGO. THE SEXUAL ELEMENTS OF THE GIANT SALA- MANDER, CRYPTOBRANCHUS ALLEGHENIENSIS. ALBERT M. REESE. In the spring and early summer of 1902, the author made strenuous efforts to obtain embryological material for investigating the development of the hellbender (CryptobraiiclinsAllcgJicniaisis}. These unsuccessful efforts have been described in an article en- titled "The Habits of the Giant Salamander." l In the fall of 1903, another effort was made to obtain the de- sired material, and a dozen or more live hellbenders were ob- tained as the result of a trip to the region of the Allegheny River, in western Pennsylvania. These animals were all about the same size, 45 cm. in length, and were sent by express from their native stream to Syracuse, N. Y. Upon opening the box, in which they were shipped, after its arrival in Syracuse, a number of eggs were found scattered through the grass that had been placed there to protect the animals dur- ing their trip. There seemed to be no difference in the colora- tion of the males and females, and the only way in which they could be distinguished was by the fact that, in the males, the lips of the cloaca were considerably swollen by the enlargement of an elongated mass of glandular tissue on each side. In handling one of the ripe females of this lot of hellbenders, the author was bitten on the thumb ; this was the only time in which any attempt to bite had been noticed, though many dozen animals had been handled at many different times. The bite was not at all serious, being merely a painful pinch which scarcely broke the skin. In removing one of the females from the box in which they had arrived, an egg, enclosed in its jelly- like envelope, was seen protruding from the cloaca. By gently pulling this extruded egg, it was found that a whole string of eggs could be drawn from the cloaca, without apparently injur- ing them in the least. 1 I\>f>itlar Scii'iice Monthly, May, 1903. 22O SEXUAL ELEMENTS OF THE GIANT SALAMANDER. 221 Each egg is a spherical yellow body, about 6 mm. in diameter, resembling somewhat the yolk of a miniature hen's egg. It is surrounded by a clear gelatinous envelope, which is arranged in two distinct layers (Fig. i). When removed from the gelatinous envelopes, as may easily be done by cutting through the latter with a pair of fine scissors, the egg is seen to be enclosed in a very thin and delicate vitel- line membrane which is easily torn in handling. The yolk, which is apparently evenly distributed throughout the egg, is made up of a compact mass of granules of various sizes (Fig. 2). 00 O 'f\J «0 c ooO or-^-'/ O^o >a°r^oQ ^^ti^s' FIG. 2. Fie. i. The egg is surrounded by a small amount of watery ma- terial (Fig. /i, 7C') which is, in turn, enclosed in a capsule of more dense jelly, the inner envelope (Fig. i, /. e.}. The inner envelope is continued as a solid, more or less tough cord of jelly (ir. e'.) from egg to egg, and binds them together in the continuous strings that have already been mentioned. The dis- tance between two adjacent eggs of the string is usually about four or five times the diameter of the egg, but the elasticity of the jelly will, of course, permit the eggs to be drawn much further apart. 222 A. M. REESE. The outline of the inner envelope is sharp and even, while that of the outer envelope (Fig. I, o. e.) is more or less irregular and uneven. The outer envelope is composed of such trans- parent jelly that it might easily be overlooked at the first glance. It forms a continuous layer over the entire mass of eggs. When the unfertilized eggs are left for some days in water, they become very much swollen, by the osmosis of water through the vitelline membrane, and may eventually burst. There was no apparent swelling of the gelatinous envelopes on coming in contact with water as is de- scribed in connection with some other amphibian eggs. Several dozen eggs were obtained from one average-sized female, about two dozen being^drawn, without appar- ent injury, from the cloaca, while the rest were obtained only after killing the animal and opening the body cavity. All the eggs obtained in the latter way were found to be contained in the right oviduct, the ova of the left ovary being nearly all in a very immature condition. Whether or not this was a normal condition, indicating perhaps, a very prolonged breeding season, it was not possible to say. The spermatozoa were obtained as a milky fluid from the living males by the usual process of stripping, though considerable pressure had, in most cases, to be exerted. They were immediately examined under the higher powers of the microscope, but no motion could be detected, though it would naturally be expected that spermatozoa obtained in this way would show the usual activity of mature spermatozoa. An attempt was made to artificially fertilize the eggs by put- ting them into a dish of water into which a great number of spermatozoa had been stirred, but the attempt was entirely un- successful. FIG. 3. SEXUAL ELEMENTS OF THE GIANT SALAMANDER. 223 No structures resembling spermatophores were discovered, and there was nothing that would seem to give any indication of the method by which the act of fertilization was accomplished. A single spermatozoon, as seen under a magnification of about 1,300 diameters, is shown in Fig. 3. Fairly good preparations were easily made by drying them rapidly on the slide, and staining in haematoxylin and eosin. The nucleus, //, is very much elongated, so that it makes up almost one third of the entire length of the spermatozoon. It is capped, at its anterior end, by a sharp, gradually-tapering apical body, a, which is plainly differentiated from the nucleus proper by the fact that it does not take up the stain to any great ex- tent. No structural details in the nucleus or apical body can be discerned with the magnification used, nor is any middle-piece distinguishable. The tail, which is comparatively stout, consists of a central supporting fiber, s. f., which takes up the stain slightly, surrounded by a transparent envelope, e, which does not stain at all. The envelope is usually considerably wrinkled and twisted, probably by the rough method of fixation. ZOOLOGICAL LABORATORY, SYRACUSE UNIVERSITY. THE RHYTHM OF IMMUNITY AND SUSCEPTIBILITY OF FERTILIZED SEA-URCHIN EGGS TO ETHER, TO HC1, AND TO SOME SALTS. E. G. Sl'AULDING. INTRODUCTION. The experiments described in this paper were undertaken during the summer of 1902 at the Marine Biological Laboratory upon the suggestion of Dr. A. P. Mathevvs ; their publication has been delayed because of the pressure of other work and of the desire to, if possible, get beyond their mere description to their meaning. This end is believed to have been attained in connec- tion with the working out of a synthesis of the artificial parthen- ogenetic methods, the detailed results of which attempt appear in a preceding paper.1 The effectiveness of all these methods and so the special physico-chemical result of normal fertilization and the nature of cleavage processes can, it is believed, be explained from a unitary standpoint, viz.: if it is considered that in the process of cleavage an average decrease in surface tension takes place as a result of the equilibrating of a potential difference between os- motic pressure and surface tension, accompanied by such elec- trolytic changes as cause the constricted form. That this average decrease in surface tension takes place is a necessary inference from the change from the approximately spherical to the con- stricted form of the egg at cleavage, for this means an increase in surface. It carries with it, therefore, the decrease in that po- tential, osmotic, which opposes surface tension in direction. The preceding cause in these events is the creation of a potential dif- ference by first increasing the osmotic pressure, which is done artificially by each of the parthenogenetic methods. Accor- dingly with the equalization of this difference, caused, c. g., by a splitting up of colloidal particles or molecules, there is in the case of eggs of marine forms an absorption of water. Both of these 1 Spaulding, E. G. " The special physics of segmentation as shown by the syn- thesis, from the standpoint of universally valid dynamic principles, of all the artificial parthenogenetic methods." BIOLOGICAL BULLETIN, February, 1904. 224 IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 22$ last events either alone or together may constitute what is termed liquefaction. The results of the experiments given below can, it is believed, be interpreted in agreement with this view of segmentation. They were undertaken primarily as an extension of experi- mental work which had already been done on the action of vari- ous chemical compounds on protoplasmic bodies, but were limited to the study of such action on the eggs of Arbacia at successive periods after fertilization. The existence of a rhythm of immunity and susceptibility has been shown already by Lyon in studying the effect of KCN and of lack of oxygen upon the fertilized eggs and embryos of the same form,1 and it has been found also that many eggs do not segment at all in the absence of oxygen, notably Arbacia2 and Ctenolabrus? Lyon also found this summer that Arbacia eggs required more oxygen during precleavage and gave off more CO,, during cleavage than at other times. 5 From these results it may be inferred in analogy to a large number of instances well known in chemistry that at least the ultimate effect of oxygen on the processes conditioning cleavage is the causing of analytic chemical changes ; /. c., fermentation, one might say, occurs and CO2 is given off, as Lyon found.. Previous to this, however, synthetic processes may take place which in turn as certain preferments become active 4 give rise to. molecular splitting. The result of such analytic change is that increase in osmotic pressure and therefore the creation of that potential difference between it and the surface tension which we have found to be necessary and in the equalization of which both potentials decrease, water is absorbed, and the egg cleaves. The hypothesis to be deduced from this and which might serve as a guide in our experimentation is that any method either (i) of preventing this necessary preliminary increase in osmotic pressure, or of compensating it after it has been created, or (2) of increasing it beyond a certain point, will tend to do away with 1 Lyon, E. P., American Journal of Physiology, VII., i. 2 Lyon, loc. cit. 3Loeb, ]., Archiv fitr die gesammte Physiologie, 1895, LXII. 4 Cf. Hofmeister, " Chemische Organisation der Zelle." 5 Personal communication. 226 E. G. SPAULDING. the event of cleavage. To the first two possibilities correspond the effect respectively of lack of oxygen and the use of strongly hypertonic solutions on the fertilized egg ; to the second method our own results with ether, HC1, etc. From this hypothesis it can also be reasonably inferred that the nearer to the point of termination of the preparatory process that either method is used, so much the less will its effect in general be, and this supposition is again confirmed by experimental results. Lyon l found that the effect of KCN on the fertilized Arbacia egg, taking, c. g., various strengths of a titrated solution mixed with sea water, was the indication of " successive stages of relatively high and low resistance in each cleavage." Putting the eggs into the solution at successive five-minute periods after fertilization and allowing them to remain perhaps one hour, then washing and removing to sea water, he found that "there is a stage about ten or fifteen minutes after fertilization when the egg is especially susceptible to KNC." " Again soon offer the first cleavage comes a second stage of small resistance ; a third fol- lows the second division." " The resistance of the egg to KNC increases up to a maximum up to the time of separation into the two cells." " The effect of KNC is the same as lack of oxygen." In interpretation of these results Lyon says that the processes dependent upon oxygen seem to begin about 10—15 minutes after fertilization, for if they are inhibited the egg does not seg- ment and they recur at each segmentation. To identify them with the morphological processes of the splitting and separation of the chromosomes or with the dissolution of the nuclear mem- brane seems to him to be impossible, for these occur too late to be directly affected. Wilson and Matthews,2 he says, mention how- ever two processes which occur sufficiently near to the suscep- tible stage to be worthy of consideration in this respect. One is the growth and division of the sperm aster, the other the growth of the nucleus. From the part which in order to explain the constricted form of cleavage3 must be attributed to, as played by each of these processes, the supposition that they are affected by a lack of oxygen, by KCN, etc., receives confirmatory evidence. 1 Lo;. cit. '2 Journal of Morphology, 1895, X., p. 319. ;! Lillie, R. S., BIOLOGICAL BULLETIN, IV., March, 1903; and Am. Jour, of /v'r.*/'i'/(;;'T, VIII., 4, Jan. 1903. IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 22/ But furthermore, whatever the morphological elements may be, it must also be admitted that in the processes leading up to and culminating in cleavage, we are dealing with chemical and elec- trolytic and consequently also with osmotic phenomena, coex- isting with those of surface tension. That these first two which condition the other two are, however, not uniform, but, rather, are varying, /. c., rhythmical, during that period must be ad- mitted to explain the observed rhythm in morphological changes. The experiments herein described serve the purpose then of testing the above-mentioned hypothesis of the existence of a liquefaction during the event of cleavage, and of a rhythm of in- creasing immunity up to and of marked susceptibility during that time. To this end use was made of ethyl ether, HC1, KC1, NaCl and sodium citrate solutions. THEORIES OF THE NATURE OF THEIR ACTION. From the position that has been taken in this and a previous paper that, inasmuch as in protoplasm we are dealing with col- loidal (probably also electrolytic) particles in solution, we there- fore in segmentation necessarily have to do ultimately with the relations of two kinds of energy, osmotic and surface, and that the cleavage process itself depends upon the existence of an un- compensated potential difference between these, from this it fol- lows that this potential difference might be caused in eitlier of two ways, viz., at the same time that either one is kept constant, by changing the other, /. c., either increasing the osmotic or decreas- ing the tension factor, the former being identical with the energy of the particles in solution, the latter with that of the solvent. The theories also which we find advanced in order to explain the nature of stimulation seems to us to be in complete agreement with this view. For example, we find the statement that " stimu- lation consists in the precipitation, /. c., gelation, of colloidal particles and is due in the case of positively charged particles to the negative ions, i. c., to the charge;1 the inhibition of this stimulation, /. c., what in some cases is termed poisoning, to the positive ions"; for negative particles the converse would hold 1 Mathews, A. P., "The Nature of Nerve Stimulation, etc.," Science, March 28, 1902. * 228 E. G. SPAULDING. true. In any case the osmotic pressure would necessarily be af- fected. Both the stimulating and poisoning effect have further- more been correlated with the valency.1 In the case of a com- pound of anion and kation, both of which are monovalent, like NaCl which does and KC1 which does not stimulate easily and when in both therefore the charges might seem to offset each other, the stimulating effect, c. g., on the nerve has nevertheless been said to be due to the " overbalancing " of the kation by the anion, and conversely for the inhibitory effect. This of course is not real explanation unless the difference in effect can be cor- related with a difference in some such quality as velocity of dif- fusion or solution tension, and Mathews has this summer shown that the poisoning qualities of the metals and non-metals as well are in fact a function of this latter. Some ground for this " overbalancing" effect seems to be furnished by the fact that in the case in which c. g., a divalent anion is combined with a monovalent kation (2) a greater stimulating effect is observed. Thus KC1 does not stimulate the nerve at its osmotic pressure, K.,SO4 does occasionally, K3 citrate stimulates in solutions of a gram molecule to 22,000 c.c. H.,O. But even here the number of opposing charges is the same. The kations therefore differ in some way other than in their mere number of charges. This must also hold true of the anions because of the increasing stimulating effect on the nerve of NaCl, NaBr, Nal and NaFl. The suggestion has been made that the difference in effect when the charges are the same in number is due to a difference in the translatory path of the charge around the atom ; but as a cause for this latter difference must in turn be assigned the admission of an ultimate difference in the atoms themselves would seem to be necessitated. While the salts therefore seem to affect the colloidal particles directly, the known inhibitory action of the anaesthetics would accordingly have to be identified with a direct effect on the sol- vent and so only indirectly on the solute. Thus it may be con- sidered that the anaesthetics as being better solvents in most cases than water have the same effect on colloidal particles as do like charges, which repel ; therefore they increase the osmotic 1 Loeb, Afchiv filr die gesch. Physiologic, Bd. 88. IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 2 29 pressure. It is believed that on this basis the varying effect of ether on the eggs of Arbacia at successive periods after fertiliza- tion can be explained. EXPERIMENTAL. i. The Effect of Ethyl Ether. The general methods of experimentation may be outlined as follows : First, a slightly supersaturated solution of ether in sea water was prepared, kept tightly corked, and when used a por- tion was drawn from the bottom, thus ensuring a saturated solu- tion. The eggs were fertilized in the usual way, good lots from among a number being selected. At successive periods these eggs were transferred to staining jars containing 50 c.c. of the solution used. The strength of the solution actually used was accurately controlled by starting with twice that strength and then diluting exactly one half, in part with the sea water neces- sary for transferring purposes. The eggs were allowed to stand in these covered jars for the length of time selected ; the solution was then carefully drawn off; the eggs were thoroughly washed twice with sea water, which was again added, and given time to develop. In important experiments the lots were each observed twice. All the experiments were conducted at the room tem- perature, about 20° C. The practical problem presented was to get such a strength of solution and time of exposure that of the eggs transferred at the various periods after fertilization some would be stopped in their development, others not. For each value of the one factor there would probably be a corresponding value of the other, the prod- uct being a constant. An abstract record of what were essenti- ally preliminary experiments is given on the next page. From these results it seemed that the correct relation between the two variables, time and solution strength, had been found ; accordingly in the next experiment a one sixty-fourth saturated ether solution was used for 25', which gave very satisfactory results. These are tabulated and plotted on page 008. In the plotting of the curve of these results the abscissae repre- sent the times of transferral after fertilization, the ordinates the per cent, of swimmers found by as accurate observation as possible. 230 E. G. SPAULDING. Exper. Strength of Solution. Period Time of Observation. Control. After Fertilization. Exposure. I. Saturated. Every 15'. 8 lots. I hour. All dead in the Well July 24. stage of treatment, developed III. Xand/^sat- Just before and Various. All dead. Well July 26. sol. by dilut- after each cleavage. developed ing with sea water (same lot of eggs). IV., A. Jjc sat. sol.; At " critical " pe- 30' All dead. Control July 29. one lot of riods, z. e. , just be- good, eggs for A, B, fore and after each regular. and C. cleavage, as below in B. IV., B. 3^2 sat sol. 15 minutes. (Lyon's 45' 50% unsegmen- Lot (i). ist critical point. ) ted, but pigmented and swollen, some in 4 and S cell stage ; 2O% swim- ming. (2) 50', ist. cleavage 30' So% dead,swol-" « just beginning. len, pigmented, 20% swimming. Segmen- (3) I hr. 10', toward 30' 87% dead, irreg. tation go- end of cleavage. segment. , some ing on. in 16 cell stage 13% swimming. (4) 2 hrs., during 2d. 30' All stopped in 4 Control cleavage. cell stage. good. (5) 2 hrs. 40', after 2d. 3C/ All dead. cleavage. IV., C. ^L sat. sol. I5/, Lyon's crit- 45' 8 hrs. afterward Control (i) ical point. all had segmented, good. 8, 16, 32 cell stage; 25 % swimming. (2) 50', cleavage just 30' (Like C i.) beginning. (3) 70', toward end of 30' Nearly all swim- cleavage. ming. (4) 2 hrs., during 2d. 30' 2-32 cell stages cleavage. present ; decom- posed and pig- mented ; J~ swim- ming. (5) 2 hrs. 4O/, after 30' | swimming. 2d. cleavage. IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 23 I Exper. V. July 30. Solution 1/64 Sat. Time After Fertiliza- tion. Lot i 2' " 2 V " 3 12' " 4 17' " 5 22' " 6 . 21' " 7 32/ " 8 37/ " 9 42' " 10 47' " ii 5*' " 12 57' "13 62' ' H 67' " 15 72' " 16 n' " 17 82' "18 87' " 19 92' " 20 97^ " 21 102' " 22 IO7' "23 112' "24 117' Exposed to Solution. Observation. Control. 25' 20% swimming. • 20 1 4 2O 4 4 50 < ( 62 ^ 85 4 i 88 4( 90 4£ IO% in 2-cell stage, 44' after fertilization. 95 4 t 0 4 4 20% segmented. all dead, pigmented, and swollen. 20% 87 5% swimming. 90 i 4 90% f\ f 4 I « < t I 95 90 ft 90 (C 90 i « 95% 90 4 4 95 4 ( 95 4 t 33% in 4- cell stage. 55 C 4 7 5 2 ( 6 30 + 232 E. G. SPAULDING. The character of this rather remarkable curve is obvious. Up to within twelve minutes after fertilization the resistance remains the same, but from this point on it gradually rises up to either just before or the beginning of the first cleavage ; during the earl}' part of cleavage it falls to zero, with a sharp rise afterwards and a fall at the second segmentation. The more important question however is to get at its meaning. To get at this we take, corresponding to the general rise in im- munity up to the time that cleavage is beginning, the greater demand for oxygen, established by Lyon in his work this summer. This might mean in view of the fact that either at least just pre- ceding or for sonic time before cleavage an increase in osmotic pressure must take place, as we have shown, either one of two things to account for this, viz., either that the oxidation process is at first synthetic and subsequently determines analytic events ; or that it is analytic from the start. Also to be correlated with this is the known effect of ether as a better solvent than is water. This is identical with its causing a greater degree of solution and consequently an increased osmotic pressure. The inhibiting effect of the ether on the eggs at the critical period in the above curve may be ascribed then, we believe, to its augmentation of the normal predominence in osmotic pressure at that time, i. e., to its increase of that difference of potential in the direction of pressure-tension necessary for cleavage. Accordingly in the equalization of this augmented potential difference the eggs would be expected to increase in size more than usual in their attempt to divide, and this is confirmed by the observed sivollen appear- ance, even when as in some cases division takes place once and then stops. This increasing immunity up to the maximum can be explained then in two possible ways. If synthetic as simple oxidation proc- esses precede the analytic then during that period there is some- thing to oppose the dissolving effect of the ether ; but since this opposition would seem to exist equally all through the precleavage period, the rise in immunity would be hard to account for in this way. On the other hand if analytic processes take place from the start as a result of the use of oxygen then the longer before cleavage that the exposure to ether is made the greater should be IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 233 its effect in augmenting the normally occurring increase in the pressure, and the point of greatest susceptibility would be at such points and also at that of the normal maximum pressure, viz., just before or during cleavage. This explanation therefore accords best with the sharp rise in the curve, and is supported also by the evidence from the parthenogenetic methods for Arbada, in which the pressure is first increased, sometime before the cleavage, which takes place after the return from the hypertonic solution to the sea water. The characteristics of the above curve were confirmed in general by Experiment IV., B and C, already presented, and more especially by three subsequent experiments, as can be seen from the following records : EXPERIMENT V]I. August 22, 1/64 sat. sol. Time of exposure, 1/2 hr. Lot. Period After Fertil- ization. Observation. Control. I 3' 66% unsegmented. 2 19' 90% living. 3 3i' 90% " 4 56' 95% dead, many in 2-cell stage, pigmented. Middle point of seg- swollen. mentation. 5 63' 25% swimming. 6 79' 25% 7 96' All dead in 4-cell stage. During second cleavage. 8 112' it t 4 * < « < c Experiments P//7. and IX., August 23 and 29. Solution, one sixty-fourth saturated ether, one half hour exposure. Lot i. one half hour after fertilization all living. Lot 2, during- see- o o & mentation, all dead in 2-cell stage. EXPERIMENTS WITH HC1. Preliminary and theoretical. According to the views that we have previously discussed the hydrogen ion in the case of the nerve is held to inhibit the stimulating action of the chlorine ion, "overbalancing'1 this more than do either K, Li, NH4 or Na. This may be due to the greater velocity of diffusion of H, which is 325, that of Cl 70.2 at 25° C.1 On the other hand it has been supposed that the H ion brings about the parthenogenetic devel- 1 Ostwald. 234 E- G- SPAULDING. opment in Asterias. These two seemingly contradictory effects cannot, however, be so in reality and the difficulty may be done away with if it is borne in mind that the effect depends as much on the character (electronic) of the colloid as on the agent. For on positively charged particles the H ion would have a repelling, /. c., dissolving ; on negatively charged, the opposite effect. If all the protoplasm of the Arbacia egg was uniformly posi- tive just prior to or during segmentation it accordingly might be deduced that H ions would have the same effect in increasing the osmotic pressure as does ether, and so of inhibiting development. Lillie,1 however, has shown that at cleavage the cytoplasm is markedly electropositive, the nucleus negative. Accordingly at that time he holds that the periphery repels the free kations within the egg and the nucleus the anions, so that the kations then predominate at the center, the anions at the periphery. As like charges repel each other, this is made to account for the con- stricted form of the egg at cleavage. In agreement with this view the effectiveness of the H ions, in parthenogenetic methods, in the environment might be considered to be due to their induc- tion of a predominance of negative charges at the surface and this in turn of positive charges at the astral centers. If this be so, however, then other kations ought to have the same effect ; but they do not. This indicates a specific action by the H ion, which it might have in accordance with its high diffusion velocity. It alone might therefore be considered to penetrate the egg mem- brane because of its and the latter's definite chemical make-up ; yet there remain difficulties even here in explaining why it should do this, since, if, at least before cleavage, the membrane and cyto- plasm are themselves positive they would tend to repel the H ions rather than to attract them. Only provided the surface were negative from the start could the attraction be explained. If, however, the egg, when jus f about to divide, were put into such a medium of H ions, it is reasonable to suppose that since the surface at least then is negative these might be attracted ; but again it is difficult to understand how they can go further, since the cytoplasm is even yet positive. However, if they succeed 1 Lillie, R. S., Am. Jour, of Physiology^ VIII., IV., and BIOI OGICAL BULLETIN, IV., 4. IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 235 in acting on the cytoplasm the effect would be a repulsion of its particles and an increase in pressure, the same as that of ether. Accordingly it would a priori be probable that the period of least immunity to HC1 would coincide with that of the greatest nor- mal pressure, viz., just before cleavage. EXPERIMENTAL. Two preliminary experiments with seventeen different strengths showed that the proper strength of solution, for an exposure of one half hour, was between a and --i- normal HC1 soluion. Accordingly a ^ ^ n solution was next tried with the following results : EXPERIMENT IV. August 3, 1/450 n HC1 solution (by diluting with sea water); three series, 25/> 3O/ an0' 6f 70' 7siot.,Rd. 88, 1901. 2 Loeb, Am. Jour, of Physiology, III., VIII. 3 Loeb, Am. Jour, of Physiology, III., IX. 4 Am. /'itr. of Physiology, VI., VI. 238 E. G. SPAULDING. more, as an illustration of the lack of a consistent theory here, are the views of Loeb, that the antitoxic as well as toxic effect is a function of specific kations, and of others that where the kations are toxic the anions are antitoxic. For instance, in 100 c.c. f « NaCl + 8 c.c. -^n CaSO4 or Ca(NO3)2 70 per cent, of Fnn- ditlns eggs develop, while if Na.,SO., be substituted they do not. Ca is therefore considered to be antitoxic to Na. A1.,C1.{ and Cr0(SO4)3 also have the same inhibitory effect on Na, but in smaller quantities. Loeb therefore concludes that the toxic and antitoxic effect of the ions is a function of their valence, but that only kations are poisonous. The necessity for such " balanced" solutions also holds good according to Loeb for muscle and for the contractions of Gonionemus ; " margin and center must con- tain three ions, Na, K and Ca."1 The opposite view is that in the instance of, c. g.y NaCl poison- ing but KC1 not, the difference in effect is due to a greater over- balancing by the Cl ion in one case than in the other.2 The effect is due in any case to both the ions. Both may be either toxic or antitoxic according as the colloid is like or unlike in charge and the normal event is one of gelation or of liquefaction. Accordingly if the normal progress of cleavage in Arbacia de- mands liquefaction, /. e., increased pressure and absorption of water, any salt either preventing this by unlike charges or aug- menting it by like beyond a certain point may be assumed to interfere with or inhibit division. And from the data at hand we may expect NaCl to do this to a less degree than KC1. Experiment IV., 7 pts., |H KC1 sol. to 2 of sea water, the other conditions as in III., gave exactly tJic same results, and is tabulated on the next page. Examination of this record makes manifest a decrease in im- munity beginning I 5 minutes before cleavage, much as with HC1, but reaching its maximum during segmentation. The same ex- planation of this effect that was made for HC1 and ether holds good, we believe, here ; but the thirty-minute fall obtained with HC1 is not present in these series, which indicates a different spe- cific chemical effect of H and of K ions. Two subsequent KC1 experiments confirmed these results. 1 Am. Jour, of Physiology., III., VII. 2Mathe\vs, Science, March 28, 1902, and May 8, 1903. IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 239 EXPERIMENTS WITH KC1. Solution. After Fertilization. Exposure. Observation. Control. Exper. I., Aug. 16. J;/. Lots I. -III. Lot IV. At critical periods before cleavage. During cleavage, 66' after fertili- I hour. I " 9°-95 % swim- ming. 75% swimming. Good. zation. Exper. II., Aug. 17. Lots I.-1V. fan KC1. Before cleavage. I hour. All dead in 2 cells. Good. Lot. V. During cleavage. Dead. Exper. III., Aug. 19. 8 pts. \n KC1 -(- 2 of sea H2O in t r a n s f er- every 5' I hour. ring. Lot I. 5' 80% swimming. So % swim- "II.-VIII. (ic/-4o') So ming. " IX. " X. " XL " XII. • 45' 5°' 55' 60' 70 60 5° 25 Remainder dead and disinte- Segmentation began here, 60' after. grated. " XIII. and XIV. 65'-75' 5-10% swim- ming, remain- der pigmented and disinte- grated. Experiment /., August 29, with NaCl, 15 pts. n to i of sea water, exposure one hour, at chosen critical periods. Observa- tion showed a slight rise in immunity from 70 to 95 per cent, up to the beginning of segmentation, then a fall to 70 per cent, during that process, but as opportunity for further experimentation was lacking this result is hardly conclusive, though by itself it is con- firmatory of the supposed greater immunity to NaCl than to KC1. Experiment /., with the Na citrate, August 13 ; 3 series, viz., 2 c.c. of seven-fourths ;/ Na citrate to 300, 400, 500 c.c. respec- tively of sea water, diluted one half at transferral. 15' periods, I hour exposure. Parallel observations showed that the eggs so treated were ahead of control both in beginning to segment 240 E. G. SPAULDING. and in the number segmented at all cleavages. No period of susceptibility was indicated. This acceleration effect might according to the views above discussed be ascribed to the so- called "overbalancing effect" of the trivalent radical, but the experiment was not repeated because of lack of time. SUMMARY AND CONCLUSIONS. 1. There is a pronounced rise in iunn unity of fertilized sea- urchin eggs to ether up to eithery/^Y before or the beginning of segmentation ; the exact point is impossible to determine owing to the unevenness of the cleavage. A sharp decrease then occurs, followed by a sharp rise toward the end of the cleavage. A repe- tition of this occurs at the second segmentation. 2. Similar changes are found resulting from the use of HC1, KC1, and NaCl with the difference that the fall in immunity comes somewhat earlier with KC1 than with HC1 and with this than with ether. All of these differ therefore from Lyon's inter- pretation of the susceptible points with KNC as occurring "after division," nor is a 15' period found, although with HC1 there is a fall in immunity at 30' after fertilization. 3. The marked decrease in immunity " at cleavage" caused by the four agents employed seems to be explainable on the basis that all, in one way or another, augment beyond a certain point the increase in osmotic pressure normally necessary for cleavage. The results obtained seem therefore to be confirma- tory of the position presented by the author elsewhere * that cleavage is due to the equalization (Ausgleichung und Umfor- mung) of an uncompensated potential difference between osmotic pressure and surface tension, accompanied by electronic phe- nomena which cause constriction. The author wishes to acknowledge his appreciation of the many opportunities offered him by the Laboratory and of the kind advice and suggestions of Dr. Mathews. COLLEGE OF THE CITY OF NEW YORK, DEPARTMENT OF PHILOSOPHY, January, 1904. 1 UIOLOCICAL IJri.i.ETiN, February, 1904. BUDDING TENTACLES OF GONIONEMUS. GEORGE T. HARGITT. While looking over specimens of Gonioncnnts for class work, and being somewhat on the lookout for any cases of variation, etc., my attention was arrested by an unusual appearance of a tentacle. Near the distal end was present a small knob which at first glance appeared simply as a protuberance, apparently without any very definite form or structure. On further and more careful examination it seemed to me to warrant a careful study. It was somewhat similar to some of the conditions found by Hargitt l in his work on the variation of FIG. i. FIG. 2. this form. The conditions referred to are the presence of bifur- cated tentacles. He found a number of tentacles which had double, or in one case triple tips. In all these cases the extra tip or bud seemed to arise either from the suctorial pad or else immediately proximal to it. Fig. I represents a rather typical example of bifid tentacle. The specimen is one I found in C. 111 Variation among Hydromedusce," BIOL. BULL., Vol. II., No. 5, 1901. 241 242 GEORGE T. HARGITT. W. Hargitt's collection, but which he did not use in his paper on variation. The extra tip is seen to be much shorter than the main tip. Each tip is supplied with one of the suctorial pads, but the shorter one arises from a point considerably proximal to the pad on the main tentacle and directly from the tissue of the tentacle. No sign of injury is present. The appearance of this tentacle suggests the probable result of further growth of the bud shown in Fig. 3, except of course the lack of the pad at the base of the bud. Fig. 2, showing a trifid tentacle, is taken from Hargitt's paper on variation (pp. cit.}. It shows two branches arising from near the end of the main tentacle which seems to be degenerate as mentioned later. The buds here do not seem to arise from the suctorial pad of the main tentacle, which is not shown, but each bud is supplied with a pad near its tip. The knob on the tentacle under consideration, however, had more the appearance of a bud than a bifurcation. This was due chiefly to its small size which rather suggested that it was a very early stage in the formation of an extra tip to the tentacle. The bud arose from a definite base which presented almost exactly the same external appearance as the normal suctorial pad. This similarity consisted not only in the smooth appearance, due to the absence of the ectodermal ridges found on the other parts of the tentacle, but also in its concave form, and the further presence of a bend or " knee " in the tentacle at this point ; all of which are characteristic of the normal suctorial pad (Figs. 3-5). The bud arose from a depression in the base (Fig. 4) due to the cup-like shape of the pad already mentioned. No external sign of injury was found either in this pad or in the surrounding tissue. The pad was of course not functional as an adhesive organ, another functional one being present nearer the distal end of the tentacle (Figs. 3 and 5). Whether this new pad formed after the beginning of the development of the bud from the old pad, whose functional activity would thus be destroyed ; or whether a second pad formed first, and a bud began to develop from the old one (which would not then be necessary) simply as a result of the capacity for regeneration, or rather duplication of parts, inherent in the tentacles, is an ex- tremely interesting question. Of course no direct answer can be BUDDING TENTACLES OF GONIONEMUS. 243 made without a considerable body of facts before us, coming from actual observations on this particular point ; facts which it would be very difficult if not impossible to obtain. It seems to me, however, that we can suggest a probable answer from con- ditions observed in other cases, which bear more or less directly on this point. A comparison of -Figs, i and 2 with some of those in Hargitt's paper show that buds do not always arise from pads. Indeed of the six figures of bifid and trifid tentacles shown in that paper and in this, only two show the bud as arising from the pad. This alone would show quite conclusively that there is no necessary or regular connection between the bucl FIG. 3. Fn:. 4. and the suctorial pad. This is further emphasized by the fact that F found nine tentacles having each three pads, apparently well developed and functional, and yet there was not the least sign of buds arising from any of the pads. These facts all point directly toward an unusual predisposition to a duplication of organs, and this perhaps offers the most satisfactory explanation of the budding and bifurcation of tentacles. C5 The bud showed a constriction near its middle region, but did not present externally any signs of annulation, or rather of the ectodermal ridges present on the old tentacle. However, nemato- cysts were present in abundance. The general appearance of the 244 GEORGE T. HARGITT. bud is similar to that of the old tentacle, with the exception noted above, and is undoubtedly of the same structure. Hargitt (op. cit., p. 244) in referring to bifid and trifid tenta- cles suggested that they might have orginataed is the result of some injury to the distal end of the tentacle. This seemed to be especially indicated by the one trifid tentacle found. In this case there seemed to be a degeneration or atrophy of the median branch, which was probably the end of the original tentacle. From the sides of this tentacle two branches arose opposite each other which were considerably longer than the median tip (cf. Fig. 2). He says concerning the cause of this : " The degenerating middle tip would very naturally suggest the probability that an injury might have been the predisposing cause of the secondary tips ; on the other hand, it must not be overlooked that in each of the other specimens with double tips no such cause seems at all evident." It was with the thought of trying to determine whether there was any sign of injury which might have influenced the forma- tion of a bud in that region, as well as to determine the histo- genic changes involved in its formation, that I was led to under- take a careful study of this budding tentacle. The tentacle was stained in toto with borax carmine. Sections were cut transversely across the tentacle, thus making the sec- tions of the bud longitudinal. Fig. 6 represents a section of the entire tentacle showing the bud in its general relations. The entoderm of the bud is seen to be directly continuous with the entoderm of the tentacle. The bud is solid with the exception of a cavity at the distal end and there is no connection between this cavity and the cavity of the tentacle. It will be noticed, however, that the cells are arranged more or less definitely in two rows with the dividing line quite distinctly marked in the proximal region, as though in further growth these would pull apart and thus connect the distal cavity of the bud with the cavity of the tentacle. On either side of the bud are masses of the rather dense tissue which makes up the suc- torial or adhesive pad already mentioned. This tissue resembles very much muscular tissue rather than glandular tissue, suggest- ing that the pad acts by virtue of its muscularity, rather than by BUDDING TENTACLES OF GONIONEMUS. 245 means of a secreted adhesive substance as suggested by Perkins.1 At the edges of this pad is present the collar-like expansion of the ectoderm which is characteristic of this structure normally. Indeed, the shape, size and contents of these cells, as well as their method of staining and general appearance, is almost exactly the same as in the normal pad. Thus there seems little doubt that there was originally present here a functional suctorial pad. So FIG. 6. far as we can tell from the sections the pad might have formed after the bud began its development, though this is very unlikely, since in such case the function would be quite limited. Indeed the pad would lose its power of functioning normally if the bud increased in size to any extent. The ectoderm of the bud is thrown into the folds or ridges, which are characteristic of the normal tenta- cles, and nematocysts are limited to these ridges (cf. figures). 1 " The Development of Gonionema Murbachii,'1'' Proc. Acad. Nat. 6V/., Phila- delphia, p. 764, 1902. 246 GEORGE T. HARGITT. Figs. 7, 8, 9 show only the bud and the tissues immediately adjoining. The same general features already noted are also seen here. In Fig. 7 the muscular pad on each side of the base of the bud is nearer the same size than in Fig. 6. The relation of the^entoderm of the bud and tentacle is shown in about the same j way, but the arrangement of the bud entoderm into two rows is not so distinctly marked. In Fig. 8 this arrangement of ento- dermfis scarcely indicated, the cells being more or less massed together and not showing any apparent regularity. The charac- ter of the tissue of the muscular pad is shown better as are also the ectodermal ridges. The cavity at the distal end of the bud FIG. 7. is larger than shown in the other figures. In Figs. 8 and 9 the muscular pad is shown on only one side of the base of the bud, showing that the bud is not completely surrounded by it, a feature also indicated in Figs. 3 and 5, where the pad is seen to have a notch or sinus on one side. Fig. 9 represents a section cut one side of the long axis of the bud, so that the entoderm of the bud and tentacle is not continuous. It will have been noticed that in all the figures the bud is solid with the exception of a cavity at the distal end. Perkins (of. cit., p. 785) referring to the development of the normal tentacle states BUDDING TENTACLES OF GONIONEMUS. 247 that it is at first solid, but that later " the cavity of the circular canal is drawn into it." "The entodermal cells, arranged radi- ally about the central axis, thicken until they are forced away from the center and a tubular cavity is left." This process, he states, begins at the proximal end and the cavity is gradually " carried out along the axis of the tentacle toward the tip." In this bud there seems to be present the cavity at the distal end so FIG. 8. that the method just mentioned does not apply, at least not wholly. In Fig. 6 the cells are evidently arranging themselves in rows next the supporting layer, with their edges meeting near the center, suggesting a drawing away from the axis and a forma- tion of a cavity (as Perkins suggests) connecting the cavity of the tentacle with the cavity already formed in the bud. Fig. 9, however, would seem to indicate a somewhat modified process. The cell outlines are not distinct, so that their arrangement cannot be definitely determined, but in the central part of the core are a 248 GEORGE T. HARGITT. number of irregular cavities. This suggests the possibility of these cavities enlarging and running together, the cells at the same time taking up a regular position next the supporting layer, and thus the cavity of the bud being formed. In neither case, however, would the process necessarily begin at the proximal end. Furthermore, it is not quite certain just how the cavity at the distal end of the bud forms, or why it should form so early and not involve the proximal portion of the bud. In regard to the early method of formation of the bud little can be suggested since it has developed beyond the initial stage. It can be said, however, that not the slightest trace of injury was found, which might be a predisposing cause. Alb. Lang l from Fir,. 9. his work on budding in Hydra, Endcndnmn and Plumularia tried to show that the bud originated from the ectoderm entirely, that the previous view that both layers were active could not be main- tained ; ectoderm cells migrated through the supporting layer and formed the bud entoderm, the old entoderm being absorbed. Seeliger2 and Braem,3 however, working on Hydra, Eudendrium, Plumularia, Obelia and Sertularclla claimed that Lang's results were entirely incorrect and the conclusions drawn from them 1 Zeitschr. f. wiss. ZooL, Bd. 54, 1892, pp. 365-385. * Zeilsckr. f. wiss. ZooL, Bd. 58, 1894, pp. 152-188. 3 Biol. Centralbl., Bd. 14, 1894, pp. 140-161. BUDDING TENTACLES OF GONIONEMUS. 249 not warranted. They found dividing cells in the entoderm as well as in the ectoderm, a condition which Lang did not find. They found no trace of ectoderm cells migrating into the ento- derm even in the earliest stages, of the two layers running into each other, of the entoderm being pushed aside and absorbed. Many others also have questioned Lang's claims and maintain that results obtained in many species of hydroids, as well as in other forms of animal life, confirm the old view. My own work on hydroids l confirm these results of Seeliger, Braem and others. In working on the regeneration of Tnbnlaria crocca, T. tcnella, T. larynx, Eudendrium rainosnin and Pcnnaria tiarclla particular attention was paid to structures which form either wholly or in part by budding. Sections made through these buds in all stages showed none of the features claimed by Lang, such as migrating entoderm cells, dissolution of the supporting layer with the ac- companying disappearance of sharp contrast between the two layers, and absorption of the entoderm. Often, usually indeed, the entoderm seemed to be the layer most active in this process. Mitotic division was observed in both ectoderm and entoderm though not abundant, reasons for which are discussed in the above-mentioned paper. Amitotic division was also more or less prevalent in both layers. Perkins (op. cit., p. 784) referring to the formation of the tentacle in Gonioncunts says the three layers, ectoderm, entoderm and supporting layer " are pushed out somewhat in the growth of the tentacle, the region of greatest activity being the endodermal layer, where the core of the tentacle is formed by a rapid outgrowth of the cells of the body wall ac- companied by multiplication of these same cells." He further states that there is not even an initial thickening of the ectoderm in the region where the tentacle is to appear. In regard to budding in the larval form likewise, he states that the ectoderm and ento- derm cells divide, the entoderm pushes out gradually, the ecto- derm growing so regularly as to cover it with a layer of constant thickness. Thus it has been conclusively proved that budding in many hydroids, as well as in the formation of the tentacle and in other buds of Gonioiicimis, involves both layers, and cells in both layers increase rapidly by division. This would entirely dis- ^ Arch. f. Ent-mech., Bd., XVII., Heft I, 1903, pp. 64-91. 250 GEORGE T. H ARC ITT. credit the universality of Lang's claims, even if they held true for a few forms. Since this is the method of the formation of the tentacles, and of buds in other regions of Gonionemus, we may safely assume that .probably the same process would be active in the formation of the bud from the tentacle, or at least to a great extent. Greater support is given this assumption by the fact of the presence of mitotically dividing nuclei found in the entoderm of the bud. They were not definitely determined in the ectoderm though doubtless present. I am glad to acknowledge my indebtedness to my father, C. W. Hargitt, for permission to reproduce Fig. 2, which appeared in his paper on " Variation among Hydromedusae," and also for allowing me to examine his collections of Gonionemus. ZOOLOGICAL LABORATORY, SYRACUSE UNIVERSITY, January 18, 1904. Vol. VI. May, 1904. No. 6 BIOLOGICAL BULLETIN A CRUSTACEAN-EATING ANT (LEPTOGENYS ELON- GATA BUCKLEY). WILLIAM MORTON WHEELER. There are few more profitable fields for the comparative study of instinct than the larger genera of the social Hymenoptera. This is especially true of the larger genera of ants, such as Cauipo- notns, Formica, Myniiccocystns, Lcptotliora.v, Plicidolc, Atta and Crematogaster. To these genera, each of which embraces species presenting a considerable range of ethological peculiarities while differing but little in morphological characters, we must also add Leptogenys, with its subgenus Lobopelta, a rather large tropico- politan congeries of species belonging to the primitive Ponerine subfamily.1 Only a single member of this genus, Lcptogcnys (Lobopcltd] clongata Buckley, is known to occur north of the Rio Grande River. It is not uncommon in the semiarid regions of Central Texas (Travis and Comal counties) and has been taken even as far north as Colorado and the District of Columbia. Frequent observations during the past three years have enabled me to confirm and extend my former account of the habits of this very interesting ant.2 I am now able to state positively that the peculiar apterous females, indistinguishable from the workers except for the shorter and more rounded petiolar node and the more voluminous gaster, are the only females produced in the colonies of L. clongata. Each colony contains only a single one of these females and no other is tolerated in the nest. Even the young virgin females leave the formicary very soon after hatching and acquiring their 1 Lobopclta differs from Leptogenys sensu stricto in having broader mandibles which, when closed, leave little or no space between their inner borders and the anterior margin of the clypeus. 2" A Study of Some Texan Ponerinx," Bioi.. BULL., Vol. II., No. i, Oct., 1900. 251 252 \V. M. WHEELER. red adult coloration. There can, of course, be no true nuptial flight, since the females are wingless. Most conclusive evidence in regard of the nature of these females has been furnished by my former pupil, Miss Margaret Holliday,1 who found them to possess not only well-developed ovaries but a typical receptac- ulum seminis. It is interesting to note that the slight morpho- logical differences separating these females from the workers are still further diminished by Miss Holliday's discovery that the latter have as many ovarian tubules as the former and may oc- casionally possess a receptaculum. That the numerous tropical species of Leptogenys agree with the Texan species in having very ergatoid females, is indicated, first by the fact that no winged Leptogenys females have been seen, though many species of the genus have been known for years, and secondly by Wroughton's observations on the Indian Leptogenys diminnta Smith, recorded by Forel : ~ "At my re- quest Mr. Wroughton has excavated an enormous formicary of L. diuiinuta to a considerable depth, but has looked in vain for a female among the many thousands of workers. All he could find was a worker whose abdomen was conspicuously distended with the ovaries. This worker differed in absolutely no particular from the others, and there is nothing very extraordinary even about its abdomen. This result would seem to confirm Emery's opinion."3 In my paper on the Texan Ponerinae I failed to furnish con- clusive proof of the identity of the males of L. clongata, as up to that time I had not taken this sex in the formicaries. More recently I have repeatedly seen the males in the natural nests and have bred them from larvae and cocoons in captivity. They are of a rich yellow color, retaining throughout life the tint exhibited by the workers and females only during their callow stages. Even when quite mature the males are seized by the workers, when- 1 •' A Study of Some Ergatogynic Ants," Zool. Jahrb. Abth.f. Syst., I5d. XIX., Heft 4, 1903, pp. 295-297. 2 " Les Formicidcs de 1'Empire des Indes et de Ceylan," Part VII., Journ. l!<>»i- bay Nat. Hist. Soc., Vol. XIII., p. 312. 3 Emery advanced the opinion that in the genus Leptogenys the function of the females may have been usurped by the workers. This is not strictly true, at least in L. elongata, since the petiole of the female is clearly different from that of the worker, as it is in the winged females of many other species of Ponerinse. A CRUSTACEAN-EATING ANT. 253 ever the nest is disturbed, and carried away like the very slender larvae and cocoons, with their bodies tucked away between the legs of the workers. The males leave the nests at night, like the peculiar males of Eciton --another genus in which the females are apterous — and are often lured into the houses by the elec- tric lights during, the late spring and early summer months, especially during the latter part of May and early June. It would be extremely interesting to learn something of the mating habits of these highly heliotactic males and wingless females. Do the males, during the breeding season, seek out and enter strange nests of their own species in order to fecundate the virgin females? This seems improbable when we stop to con- sider that male ants are so very stupid that they are unable to find their way back to their parental nest when once they have strayed away from it. Are the wingless females fecundated by the males of the same colony, /. c., by the offspring of the same mother ? This is possible but improbable, since this would be a flagrant case of inbreeding. It seems more likely that the vir- gin females leave the parental nest and wander about as pedes- trians, till they are found and fecundated by the winged males, as in the case of the 'Mutillidae. The same problems and an- swers seem to be suggested by the large winged males and dichthadiiform females of Eciton and Dorylns. My former paper contained no account of the feeding habits of L. elongata in a state of nature. In my artificial nests the insects and their larvae were fed on termites. I have since found that these ants, under natural conditions, feed very largely, if not exclusively on the common wood-slaters (Onis- cns and Arjiuidil/uiiiini) which abound under stones and logs in the shady places where the formicaries are excavated. I have repeatedly seen workers of L. elongata returning to their nests, carrying dead slaters in their mandibles. The earth sur- rounding the entrances to the nests is invariably white with innumerable bleaching limbs and segments of the crustaceans, showing that great numbers of these animals must be habitually destroyed by the ants. Their long, toothless mandibles resemble scissors and seem to be admirably adapted for cutting through the intersegmental membranes of their prey and exposing the 254 w- M- WHEELER. edible parts. L. elongata is, to my knowledge, the only ant known to feed on crustaceans as a regular diet. Other ants are either insectivorous, granivorous, mycetophagous, or collectors of the saccharine exudations or juices of insects and plants. The little that is known concerning the habits of other species of Lcptogenys would indicate that the North American form is peculiar in the character of its food. Wroughton has studied the habits of two of the Indian species of Lobopclta, L. dis- tinguenda Emery and L. cliiucnsis Mayr.1 Concerning the for- mer he writes as follows : "This species is fairly common from Poona westwards to the Ghats. The idea of a disciplined army has been fairly developed in this genus. L. distingnenda may sometimes, it is true, be found loafing about singly, but these individuals are probably only scouts ; ordinarily, she is only met, in the early morning or late in the afternoon, travelling in an un- broken column four to six or eight abreast, straight, or rather by the easiest road, to the scene of operations. This is usually a colony of white ants whose galleries have been broken open by the hoof of a passing beast, or some similar accident. Arrived at destination, each worker seizes her termite prey, tucks it under her thorax in the orthodox ponerine fashion, and the column then returns (but marching ' at ease ' and much less regularly than on the outward journey) to the nest. I have never succeeded in finding a nest ; on one occasion I tracked a column for more than fifty paces, only to lose it in a patch of prickly pear. I do not think that L. distingnenda, any more than any other ant, ever has the inspiration to open a termite gallery for herself; on the occasion mentioned above, the column passed close to several, and even over one colony of white ants before reaching its destination ; I believe, however, I saw a worker break open a piece of tunnel, into which a termite had retreated, but cannot be sure, and the practice certainly was not general. Nor are the termites followed into the galleries, partly, perhaps, because the passage is too small for a Lobopclta, but equally, I imagine, because such a measure would be very like ' drawing ' a badger ' only more so.' Mr. Aitken tells me he has seen ' hun- dreds going into a hole in the ground and emerging with white ants,' but this is very different from entering a termite gallery." 1 "Our Ants," Journ. Bombay Nat. ffisf. Soc., 1892, p. 56-58. A CRUSTACEAN-EATING ANT. 255 Concerning L. cliinciisis Wroughton makes the following statements : " This species is even commoner than the last. Distinguenda would seem to be a denizen of forests, while Chi- ncnsis prefers more open and inhabited country. I have only once seen Cliinensis on the warpath, and then the objective, a large worm, in several pieces, had been reached, and the column was on its way home. The column I must say was more a mob than a disciplined army, but this may have been due to the fact that the normal irregularity of the homeward march was en- hanced by the size and shape of the booty, which did not admit '' ( Lobopclta) donga fa Buckley, male, female and worker. of being carried ' according to the regulations.' On the other hand, I have often, during the early part of the rains, witnessed a migration (or was it a colonization, in no case was a female, even apterous, present ?) when the discipline and regularity of the column left nothing to be desired. My experience seems to show that Cliinensis prefers a formation in fours, at any rate when carrying her own larvae and pupae. Mr. Aitken lias fur- nished me with the following most interesting note on Chiucnsis. ' There is a populous community of this ant, in a hole, in the 256 W. M. WHEELER. foundations of my house, at Goa. From the nest there is a well marked " road," crossing a broad gravel path, and then ramifying all over the tennis ground. They issue after sunset, and march along one of the main branches, or break up into parties and take different routes. When they come to a place where the termites have thrown up new earthworks, and are busy eating the dead grass underneath, they collect in dense masses, waiting for an opportunity of breaking in, which they very likely find when the termites attempt to extend their works on any side. Then the slaughter begins. Sometimes the poor termites are killed far faster than they can be carried off; and on one occasion, as late as 7 a. m., I saw the ground still heaped with slain, and an unbroken stream of ants, fifty-six yards long, carrying them away. Each ant had two or three in her jaws. If these ants cross the grounds of a community of ' harvesters ' (? Holcomyrmex) after the latter are up in the morning, they have to flee in their turn. A Lobopclta, when once a worker major has laid hold of her by the leg, appears to be perfectly helpless, she can neither kill her enemy nor shake her off. Sometimes another Lobopclta will come to her assistance, and, after vainly trying to tear off the aggressor, will pick up her comrade and carry her and her enemy off together.' Apparently some of the American species of Leptogenys also prey on termites: The nest of a single colony of L. Wheel cri Forel which I observed at Cuernavaca, Mexico, was almost embedded in a Eutenncs nest, and I have no doubt that the ants were in the habit of using their neighbors as a convenient larder. The only other Leptogenys of which I find the habits recorded is Lobopclta diuiinnta Smith var. bismarckensis Forel from the Bismarck Archipelago. Dahl 1 compares this species with the amazon ant (Polyergns ritfcsccns of Europe and North America), on account of the sickle-shaped, toothless mandibles. He says : " Although I have drawn a comparison between Leptogenys. bismarckensis, and Polyergns rufcscens, I must clearly emphasize the fact that I never found the nest of the former species and 1 " Das Leben der Ameisen im Bismarck- Archipel, nach eigenen Beobachtungen vergleichend dargestellt," Berlin, R. Friedlander und Sohn, 1901, p. 52. A CRUSTACEAN- EATING ANT. 257 could not, therefore, observe whether the work of the colony is carried on by slaves. What firmly convinced me, nevertheless, that this species is a slave-holding ant, was the following : The mandibles are long and sickle-shaped, almost as much so as in our German slave-holders, and little adapted for working, as they have no masticatory border. They could of course, be of use in killing termites, as Forel supposes to be the case in this genus, but the place were I found the species under consider- ation was far removed from all termite nests. It was on the sea-shore between blocks of coral, where I saw about fifty indi- viduals in a troop, as it were, on the march. It was evident that the troop had left the nest for the purpose of perpetrating a robbery in common. That this robbery, after what has been said, was for the purpose of obtaining slaves, seems probable." I venture to maintain that Dahl is mistaken in supposing that L. bisuiarckcnsis is a dulotic ant, as a perusal of the above quoted passages from Wroughton's work will suffice to show. It is also evident from the observations of Wroughton and Aitken that the Indian species of Lcptogcnys differ widely in habit from L. elongata. The colonies of the latter species are very small, rarely containing more than a hundred individuals, whereas the colonies of the Indian species appear to contain thousands of workers. Moreover, the workers of L. elongata leave the nest singly and hunt about timidly for their phlegmatic and defenceless prey, whereas the Indian species hunt in well organized files somewhat after the manner of the driver ants and ants of visita- tion (Dorvlns and Ecitoii). And such diversity of instinct is exhibited not only in the same genus but within the confines of the same subgenus (Lobopelta). In conclusion I give the synonymy and a description of all three phases of the North American Lcptogenys. LEPTOGEXYS (LOBOPELTA) ELONGATA (Buckley) Wheeler. Ponera elongata Buckley, Proc. Ent. Soc. Phila., Vol. VI., 1866-67, p. 172, £. ? Ponera Icxana Buckley, ibid., p. 170, §. Lobopclta septentrionalis Mayr., Verhandl. k. k. zool. bot. Hes. Wien, Bel 36, 1886, pp. 438, 439- §• Leptogenvs septcntrinnalis Emery, Zool. Jahrb. Abth. f. Syst., Bel. 8, 1894, p. 268, '4 . 258 W. M. WHEELER. Leptogenys (Lobopeltd) elongata (Buckley) Wheeler, Biol. Bull., Vol. II., No. i, Oct., 1900, p. 2, 7, Fig. 4. 5! ?c?; Trans. Tex. Acad. Sci., Vol. IV., Pt. II., No. 2, 1902, p. 9 WORKER. --Length 5-6.5 mm. Head slender, excluding the mandibles, longer than broad, somewhat broader in front than behind. Eyes moderate, flattened, situated a little in front of the middle of the sides of the head. Mandibles slender, nearly two thirds as long as the head, gradually increasing in breadth as far as their apical third, forming a sharp, toothless blade and thence narrowing more suddenly to the acute, curved tip. Clypeus prolonged forward in the middle to a rather acute point and with a prominent median keel ; lateral emarginations distinct but not very deep. Frontal furrow extending back a little beyond a line connecting the posterior orbits of the two eyes. Antennae slender ; scapes extending about one third their length beyond the posterior corner of the head. First and third funicular joints subequal, decidedly shorter than the second joint ; joints 4-1 1 subequal, shorter than joints I and 3. Thorax elongate, its dorsal surface horizontal, meso- and epinotal regions laterally compressed ; depression behind the small meso- notum short but rather deep. Basal surface of epinotum twice as long as the sloping declivity, which is somewhat flattened and transversely margi- nate below and behind. Petiole in profile as high as the thorax, somewhat higher behind than in front and nearly as long as high ; anterior, dorsal and posterior surfaces flattened ; the dorsal and posterior meeting at a somewhat sharper angle than the anterior and dorsal surfaces. There is a distinct tooth at the anterior, ventral border of the petiole. Seen from above this segment is pyriform, twice as broad behind as in front, with flattened sides and rounded dorsal surface. Gaster slender, somewhat deeper than the petiole, distinctly constricted between the first and second segments. Sting prominent. Legs long and slender ; claws with long pectination. Mandibles smooth and shining, the former with a few scattered punc- tures and faint traces of striation ; the latter thin and submembranaceous along its anterior border and obliquely rugose on the sides. Head, thorax and petiole subopaque, rather densely and uniformly covered with shallow punctures. Region between the eyes and clypeus longitudinally rugose. Gaster shining, very sparsely and finely punctate. Body and appendages clothed with delicate, grayish yellow pubescence ; the head, thorax and abdomen also with sparse, grayish yellow hairs, which are long and projecting on the clypeus and terminal segments of the gaster. Deep red ; edges of mandibles and thoracic sutures somewhat black- ened. Antennae and legs a little paler than the trunk ; tip of gaster, sting, anterior border of the clypeus and the spines of the tibiae, yellow. FEMALE. --Length 6.5-8 mm. Apterous and decidedly ergatoid in form, indistinguishable from the A CRUSTACEAN-EATING ANT. 259 worker in the head and thorax, even lacking ocelli and with eyes no larger than those of the worker. The petiole is proportionally shorter and higher, so that when seen from above, it is little if any longer than broad behind, shaped like an eqilateral triangle with rounded angles. In some females the upper surface of the segment in profile is flattened like that of the worker, but in most cases it is more convex and in this respect some- what like the petiole of the male. Gaster conspicuously larger, both broader and higher than that of the worker. Sting like that of the worker. MALE. — Length 5.5-6.5 mm. Head, including the very prominent, somewhat reniform eyes, distinctly broader than long ; cheeks and postocular regions very short, ocelli large and protruding. Mandibles small, hardly meeting with their tips, slightly geniculate, broadest at the base and suddenly tapering to a slender point. Clypeus less produced, less pointed in front and with a blunter keel than in the worker. Antenna; long and filiform, 13-jointed ; joints 3-13 long and cylindrical, subequal ; scape very short, hardly a third as long as the third and succeeding joints ; second joint very small, somewhat narrower than the scape and hardly longer than broad. Thorax rather robust, with prominent Mayrian furrows on the mesonotum ; epinotum long and slop- ing. Petiole small, hardly half as high as the first gastric segment, about as high as long, in profile rounded above, with sloping anterior and pos- terior surfaces ; seen from above it is oblong, a little longer than broad, hardly wider behind than in front. Gaster with well developed constric- tion between the first and second segments and large, cultriform, exserted genital appendages, which are fully half as long as the remainder of the gaster. Legs long and slender. Smooth and shining, except the antennae, which are opaque. Surface of thorax indistinctly and finely punctate. Body covered with yellowish hairs, which are long and prominent on the gaster, shorter on the head and thorax, and still shorter and denser, and more like pubescence, on the appendages. Body and legs light yellow throughout. First and second antennal joints yellow, remaining joints brown. Wings grayish hyaline with brownish veins and stigma. Type Locality. - - Austin, Texas. Otlicr Localities.- -"District of Columbia (Pergande, Mayr) ; Colorado (Cresson, Emery) ; New Braunfels and Belton, Texas (Wheeler). THE MARCHING OF THE LARVA OF THE MAIA MOTH, HEMILEUCA MAIA. WM. S. MARSHALL. In the autumn of 1901 while collecting along the marshy shore of Lake Wingra, near Madison, I noticed many specimens of the maia- or buck-moth, HcinUcnca uiaia, flying low over the marsh. Both males and females were present, and many of the latter, having settled, were laying their eggs on the grass. These were placed in a somewhat irregular set of spirals closely packed together so that when they hardened, the grass could often be pulled away leaving the eggs stuck together and forming a short tube. The process of oviposition and the arrangement of the eggs has been described by Riley ' and copied by Packard.2 Without having any definite plans in view I collected a great many of the eggs most of which I put in a cold place, but a few I left in an open bottle in my room. One morning I noticed on the neck of this bottle a black mass which was found to be a group of young caterpillars ; they had evidently hatched but a short time before. Later in the morning I again looked at the eggs and found that more had hatched ; all io the first bunch, having in the meantime left the bottle, were marching in a line on the table. Again, later in the day other groups were seen, and in nearly every instance, each group had formed a line marching in a regular procession and following the leader which- ever way he turned. I placed some large sheets of paper on the table ; upon these the different groups were soon marching, and could be much more easily seen than when upon the darker table. I now, with a pencil, knocked the leader away from one line and was surprised to see the next in the line, now the leader, stop when he reached the place occupied by the first leader prior to his removal. Here he stopped and raising himself upon his prolegs moved the anterior part of his body to and fro as if he 1 Riley, C. V., "Fifth Missouri Report," p. 128. ^Packard, A. S., " Insects Injurious to Forest and Shade Trees," Washington, 1890. 260 THE MARCHING OF THE LARVA OF THE MAIA MOTH. 26 1 were trying to scent the leader. He soon discontinued this and resumed his natural position again, appearing, however, for some time, very restless. While this had been going on, the rest of the caterpillars had crowded up to the front one ; they appeared for some time very restless, but finally settled in a close bunch, in which position they all remained : one here and one there would often become restless for a few minutes, but end by set- tling again in its former position in the bunch. I now marked the original leader (he had been kept away from the others all of this time) by putting a little white paint on his back and then picking him up on a small piece of paper dropped him back at the edge of the group. The leader, in the meantime, had been walking around evidently seeking the other caterpillars, and when he returned to the bunch, began to walk restlessly around near its edge. In a few minutes he started off away from the others and these began to follow him, moving in a regular procession. Different masses of the eggs were now brought into my room, a few each week, and when the caterpillars hatched, a few ex- periments were carried on to see if the removal of the leader always affected the followers in the same way, and if a new cater- pillar would not assume the leadership and be followed by the rest. The results I have thought it best to write out briefly and not to arrange them in a tabulated form. The first set contains those in which the old leader, upon being returned to the bunch, resumed command, and the second lot, those experiments in which a new caterpillar became the leader. There then follow a few experiments differing from those contained in the first two lots. When the number of caterpillars forming the line was counted, this is given ; but in some cases, however, the number which formed the line was not noted. In nearly every instance the removal of the leader brought about at first the same result. When I removed him I would draw a line on the paper marking a place where his head was before removal. When the second caterpillar in the line reached this point, he always stopped, rarely crossing the line, and when the bunch was formed, it was always back of the line. I. Sixty-four in line. 8: 1 5, leader removed ; 8:30, all bunched ; 8:31, leader returned; 8:35, all restless; 9:00, leader started, and at 9:08, all were in line and moving. 262 W. S. MARSHALL. 2. Six in line. 8:45, leader removed, others soon bunched ; 8:5 I, leader returned ; 8:57, old leader started ; others following. 3. 8:47, leader removed; 8:50, all bunched; 8:51, leader re- turned ; he passed to back of bunch and started away, others following; 8:43, all in line. 4. Ten in line. 8:31, leader removed and all bunched one inch back of line ; 8:36, leader returned ; 8:55, leader starts and others follow. 5. 8:57, leader removed and put back at once, seventh in the line ; all bunched ; 4:06, P. M., leader started, others following. 6. 8:16, leader removed; 8:26, leader returned; 8:31, leader started, others following. 7. 8:21, leader removed; a caterpillar, the next in line, goes on half an inch and here they all bunch ; 8:30, leader returned ; 8:42, leader starts and others follow. 8. 2:32, leader removed ; 2:47, leader returned to bunch ; 3:55, leader started, others following. 9. One hundred and fifty in line. 10:22, leader removed ; 10:40, bunched and leader returned; 11:25, leader started and others followed. The following are experiments where a new leader started : 1. Forty in line. Leader removed ; 9:10, bunched; 9:11, a caterpillar at back of bunch starts and others follow. 2. Twenty-one in line. 9:01, leader removed ; 9:08, bunched ; 9:10, one near front starts; 9:21, all in line following new leader. 3. Eighteen in line. 8:50, leader removed ; in a minute the next caterpillar turned and started; 8:55, all in line and follow- ing new leader. 4. Thirty-six in line. 8:42, leader removed and the next cat- erpillar assumes leadership ; 8:47, new leader removed and orig- inal one put back in middle of line ; all bunch ; and at 4:08, P. M., were still quiet. 5. Eighteen in line. 8:30, leader removed; 8:37, bunched; one went a short distance over the line but returned ; leader re- turned ; 8:46, new leader started and others follow. 6. Eleven in line. 8:38, leader removed; 8:41, next cater- pillar returned along the line two inches and then came back ; others bunched ; 8:45, start with new leader. THE MARCHING OF THE LARVA OF THE MAIA MOTH. 263 7. Ten in line. 10:34, leader removed ; line kept on ; 10:39, second leader removed; march continued; 10:40, third leader removed; 10:42, bunched; 10:48, started with the original leader, who had been returned, at head. 8. 8:13, leader removed; 8:22, leader returned; 8:56, new leader started, others following. 9. 2:56, leader removed; 3:05, leader returned; 4:05, new leader started and others follow. 10. Forty-six in line. 9:24, leader removed ; 9:33, all bunched ; leader returned ; 10:05, new leader starts and others follow. 11. One-hundred and twenty in line. 8:41, leader removed; when the entire line had bunched the leader was returned ; 11:45, new leader starts and others follow. 12. One-hundred and fifty in line. 8:43, leader removed; 9:25, mostly bunched and leader returned; 10:10, old leader started, rest following ; they went more than an inch and then returned to bunch again ; 2:30, P. M., a new leader starts and others follow. The following experiments were made by removing the leaders of two lines and then returning them each to the other's bunch : A. Six in line. 2:02, leader removed ; 2:06, bunched ; leader of A' placed near head ; 2:08, leader of A' starts and others follow. A' . Thirty-two in line. 2:02, leader removed ; 2:07, leader of A placed in bunch ; 2:1 1, one, not the leader, started but re- turned ; 2:35, leader of A starts and others follow. B. Thirty-two in line. 1:56, leader removed, when others bunched leader of B' placed with them; 2:18, leader from/?' starts and others follow. B' . Thirty-five in line. 1:56 leader removed and leader of B placed in bunch ; 2:03, leader of B starts and others follow. C. 1.22, leader removed ; 1:27, leader of O placed in bunch ; 3.20, line starts with entirely new leader. 0 . 1:22, leader removed ; 1:27, leader of C placed in bunch. Called away and unable to follow this to end. The following experiments I give separate from the others : 1. 8:24, leader removed ; 8:33, leader returned and at once started out, the other caterpillars remaining in bunch ; 8:38, 264 W. S. MARSHALL. leader returned again ; 8:42, he starts out again but none follow ; in two minutes he returns to the bunch himself and starts a third time, this time some follow buttsoon return to bunch and leader goes off by himself. 2. Eighteen in line. 8:32, leader removed ; 8:38, bunched ; leader returned and they remained in bunch all morning. 3. Thirteen in line. Same thing happened as in number two. In one lot, thirty-six in line, the leader had been marked and the line allowed to go on. The leader reached the last cater- pillar in the line so that a circle was formed. They all kept moving, the leader finally reaching the place where he was when the circle was first formed ; he then went half way around the circle and started off in another direction the others following. From the above, it will be seen that the removal of the leader affects the whole line, but that he is not necessary for the further progression of the caterpillars. I have been unable to find refer- ences to the procession-caterpillar, but notice that the caterpillars of Satnrnia iol march when young the same as the Maia-moth. Dubois2 notes that the procession-caterpillar spins a thread which the others in the line follow ; the young larvae of Hemilcnca do the same, the thread being seen with a hand lens back of the line. In nearly all of the long lines, which the larvae form, there is very apt to be at least one break where there is an inch or more between the nearest caterpillars, or such a break can be made by stopping one of the larvae until the preceding ones have gone ahead for some distance : at such a place the thread also can be seen. When a break occurs, it does not in any way affect the movements of the line, the caterpillars following along the regular path. Wishing to see how much the caterpillars depended upon this thread to enable them to follow in the exact path of the leader, I removed the thread a number of times when the distance between two neighboring caterpillars was great enough, and found that the course was not in the least altered. The caterpillars, upon reaching the end of the broken thread, generally kept straight on as if nothing had been done, failing to show a dependence 1 nickersoii, Mary C., " Moths and Butterflies," 1901. 2 Dubois, .-/;///. Soc. Linn. Lyon., XLXL, 1900, p. 125. THE MARCHING OF THE LARVA OF THE MAIA MOTH. 265 upon the thread alone in following the path of those ahead. I next removed the thread, and then dipping a finger in water, rubbed it rapidly a number of times across the path and then wiped the place dry. When the first caterpillar reached this spot, he halted, and for three minutes remained at the same place, raising the anterior part of the body in the air acting the same as if the leader had been removed. At the end of this time he started forward following, as near as I could judge, the original path. The following few experiments should have been made with the food plant of the caterpillar, but this being unobtainable at the time of the year when the caterpillars were hatching in my room, the leaf of the geranium, Pelargonium, which was easily obtained and possessed quite an odor, was used. 1 . A small piece of the leaf was placed 5 mm. away from a small group which had been quiet for two or three hours ; the caterpillars became at once restless and in two minutes, three had moved over and touched the leaf. 2. A small piece of the leaf was placed 5 mm. away from the leading caterpillars in a line ; they became at once restless and " broke rank " ; in four minutes two (not the leader) had reached the leaf. 3. A piece of leaf was placed 15 mm. from a group, nothing happened ; the leaf was moved to 10 mm. and left for ten min- utes, nothing occured ; moved to distance of 5 mm. from cater- pillars and all still remained quiet. I now moved the leaf to 3 mm. away, one immediately came out, touched the leaf and returned to its original position, in thirty seconds another came out, touched the leaf and returned. 4. A piece of leaf was placed 5 mm. from group, two came out, touched it and returned. ZOOLOGICAL LABORATORY, UNIVERSITY OF WISCONSIN, Madison, February, 1904. FORM-REGULATION IN CERIANTHUS, IV. THE ROLE OF WATER-PRESSURE IN REGENERATION. C. M. CHILD. INTRODUCTORY. The regeneration of the marginal tentacles in cylindrical pieces was described in the first paper of this series (Child, '03^). The tentacles do not arise from the new tissue closing the end, nor from the cut surface, but from the old body-wall itself, which first becomes thinner and loses its muscular layer in the region where the tentacles are to appear and then gives rise to small buds, corresponding in number and position to the intermesen- terial chambers. The fact that the new marginal tentacles arise some distance away from the cut surface by a local transformation of the already differentiated body-wall is of considerable importance. There must be some adequate ground for the localization of this pecu- liar process of transformation in a region of the body-wall which apparently differed in no way from adjacent parts before the cut was made. Very early in my study of regeneration in Ccriantlins it became evident that the rapidity of regeneration was more or less closely connected with the degree of distension of the piece. My obser- vations along this line were made chiefly upon C. solitarius. In cases where closure of the ends and consequently distension by water was possible regeneration was much more rapid than in pieces where communication between the enteron and the exterior - other then the mouth and the aboral pore --existed. Observa- tion of this apparent relation between water-pressure and regen- eration led to experiment and it was soon possible to demonstrate that a very close relation between water-pressure in the enteron and regeneration existed. I am aware, of course, that Loeb ('91, '02) regards certain phases of this relation in CcriantJins as osmotic in nature. The results of his experiments and his inter- pretation will be discussed at another time : it need only be 266 FORM REGULATION IN CERIANTHUS. 26/ said here that Loeb failed to understand the real conditions, otherwise he would scarcely have attempted to apply the osmotic hypothesis to this case. The evidence bearing upon the relation between regeneration and internal water-pressure is varied in character. In the re- maining portion of the present paper and in following papers the various lines of evidence will be discussed. First, however, it is necessary to call attention to certain features of the water-pres- sure and circulation in the enteron of normal animals. Except where statement to the contrary is made all observations and experiments were made upon C. solitarius. WATER-PRESSURE AND CIRCULATION IN THE ENTERON. Under ordinary conditions both the body-wall of Ceriantlnis and the tentacles are subjected to a considerable degree of ten- sion in consequence of internal water-pressure, the enteric cavity being distended with water. When contraction occurs this water issues through the aboral pore, at least in large part, though some passes out through the stomodaeum. After loss of water from the enteron the body and tentacles are shorter and smaller, and, if the loss was great, are more or less completely collapsed. This condition continues until the enteron again begins to fill with water. As it fills, the body gradually resumes the original form exactly as does a rubber balloon when inflated with air. This distension of body and tentacles by water under pressure is an indispensable condition of the characteristic form of body and tentacles. The animal is incapable of extending to its full length or attaining its full diameter in any other way than through the medium of the internal water-pressure. Extension of the tenta- cles and the turgid condition often designated as erection is just as completely the result of the enteric water-pressure. Turgor of individual cells has absolutely nothing to do with the condi- tion, as is evident to all who have examined with any care Ccri- anthns or other actinians. Loeb's belief ('91) that osmosis plays a part here is wholly without foundation, except so far as water may diffuse through the body-wall into the enteron. We may regard CcriantJins as a sac filled with water under pressure. Under certain conditions the walls of the sac may de- 268 C. M. CHILD. crease in size either locally or at all points, the result being in the first case increase of pressure and either stretching of other parts of the sac or loss of water through the pore or mouth, and in the second case always loss of water, commonly through the pore, and reduction in size of the whole body. While this sac has the power of contracting and forcing water out through certain openings, it has not the power of expanding actively and so drawing water in. Since water under pressure is normally present in the enteron, and since after collapse due to contraction the pressure is soon reestablished, means must exist by which water can be forced into the enteron against pressure. In my first paper (Child '03^) mention was made of the fact that regenerating pieces closed at both ends by their membranes of new tissue become distended with water in the course of a few days. It was suggested that in such cases water diffuses through the body-wall in consequence of the accumulation within the enteron of certain soluble products of metabolism. It is also possible that water may be secreted into the enteron together with these substances. To what degree the distension in normal animals is caused in this manner cannot be definitely ascertained, but it is certain that the rapid accumula- tion of water in the enteron of the normal animal after this has lost all or nearly all of its contents in consequence of violent con- traction cannot be due to diffusion or secretion. In such cases the animal frequently regains its usual degree of distension within fifteen or twenty minutes, whereas distension by diffusion in re- generating pieces requires several days. The few observations which have been made upon the sipho- noglyphes in actinians indicate that the function of these struc- tures is the production of currents through the stomodaeum. Hickson ('83) found that in certain Alcyonaria, which possesses only one siphonoglyphe, the current was inward or downward along the siphonoglyphe and outward on the other portions of the stomodseal surface. It is probable further, according to Hickson, that the inward or downward current is continued along the free edges of the mesenteries for a greater or less dis- tance below the siphonoglyphe, while on other mesenterial mar- gins the cilia beat in the reverse direction, like those of the gen- eral stomodaeal surface outside the siphonoglyphe. FORM REGULATION IN CERIANTHUS. 269 FIG. i. According to this view there' is an inward and an outward current in the stomodaeum and for a greater or less distance aboral to it in the central region of the enteron between the mar- gins of the mesenteries. According to my own observations con- ditions are very similar in Ccriantlnts. Only one siphonoglyphe is present (Fig. i) and along this the current is directed in- ward, though whether the cilia may ever re- verse this movement has not determined. The fact that small particles placed on the disc, if not ingested by muscular action, are gradually carried in a peripheral direction indicates that here the cilia beat so as to cause an outward current. The direction of the current on the lower parts of the oesophagus outside of the siphonoglyphe has not been determined, but is probably the same as upon the disc and upper portions. In Fig. 2 the opposing stomodaeal currents are indicated by arrows, the inward current along the siphonoglyphe by the longer arrow on the left and the outer currents by two shorter arrows on the right. The natural conclusion is that the accumulation of water in the enteron is in some way the result- ant of the stomodaeal currents, but the manner in which this result is attained requires consideration. When the enteron is distended with water under pressure the walls of the oesophagus must be brought into contact since pressure is exerted upon them from all sides by the water in the enteron. The radiating grooves corresponding to the lines of attachment of the mesenteries, which are visible upon the disc of Ccrianthns, extend the whole length of the stomodaeum and probably it is along these that the chief outward currents pass. As the walls of the oesophagus are pressed more more closely together these grooves are more and more nearly obliterated. Probably when internal pressure is high the out- ward currents are very slight or absent. The siphonoglyphe, however, is a relatively powerful organ, which produces an in- FIG. 2. 2/O C. M. CHILD. ward current. This groove is probably always open whether the body is distended or not. According to the laws of fluid pres- sure the force of the inward current need be only slight since the pressure it exerts is transferred to every unit of surface and in all directions in the enteron, /. e., the principle by which en- teric pressure is maintained is that of the hydraulic press ; a slight pressure exerted over a very small surface is transferred to a large body of confined fluid and so multiplied many times. If these suggestions are correct the passage of water in and out of the body and the process of distension and collapse are accomplished somewhat as follows : When the body is contracted it is probable that the oesophagus is more or less widely open ; observation of living animals or sections of contracted specimens (Fig. i) support this view. This condition of the oesophagus is probably brought about by contraction of the mesenteries which possess a slightly developed transverse musculature. In the contracted state then there is free passage of water in and out. When the muscles relax the cesophageal walls are again brought into more or less close contact, this result being, perhaps, brought about in part by the elasticity of the cesophageal walls or by muscular contraction, and in part by the circulatory currents in the intermesenterial chambers on all sides of the oesophagus (see Figs. I and 2). The approximation of the oesophageal walls decreases the outward currents, while the inward current continues, since the siphonoglyphe remains open. Thus, more water enters than passes out, and the result is of course gradual distension of the body. As the internal pressure increases the walls of the oesophagus are more and more closely appressed and thus the outward currents are decreased still further while the inward current continues undiminished. The increase in pressure and distension will continue until the motive power ex- erted by the cilia of the siphonoglyphe is just sufficient to balance the internal pressure and prevent an outward movement of water, and to replace any loss through outward currents, if such still continue, as perhaps they do to a slight extent. So long as the various conditions remain unchanged distension continues. Let us now suppose that a sudden stimulus leads to contrac- tion of the body-muscles. In the first stages of this contraction FORM REGULATION IN CERIANTHUS. 2/1 the internal pressure must be greatly increased and probably the cesophageal walls are more closely appressed than before, since the muscles which separate them are weak. At this stage then little or no water escapes through the oesophagus. When the pressure reaches a certain point the aboral pore is forced open and rapid ejection of water occurs with considerable force. Thus the internal pressure is relieved, and now, as further contraction occurs, separation of the cesophageal walls takes place, and the remaining water passes out of the oesophagus, sometimes carry- ing with it mesenterial filaments in case of violent contraction. The point to which I desire to call especial attention is that when contraction begins the internal pressure closes the oesophagus all the more tightly, and it is not until this pressure is relieved by escape of water through the aboral pore that opening of the oesophagus can take place. Though few observations have been made on other species of actinians, I am inclined to believe that distension and collapse may be accomplished in much the same manner in other members of the group, the cinclides or other openings taking the place of the aboral pore. It is probable, however, that in many forms the muscles which separate the cesophageal walls are more powerful than in Ccriatitluis and so are able to bring about separation in spite of the internal pres- sure. It is perhaps needless to state that the ejection of mesen- terial filaments through the cinclides or mouth is purely passive, the filaments being merely carried out with the water. One other point may be mentioned in this connection : if the preceding observations and suggestions are correct it follows that the form of the oesophagus usually observed in transverse sec- tions, viz., an oval with grooves at one or both ends (Fig. i) is not what might be called the natural form, but a form resulting from contraction. Practically all fixed specimens of actinians are more or less contracted. It is extremely probable that when the animal is distended in the normal manner the cesopha- geal walls are always closely appressed except in the region of the siphonoglyphe. Dilation occurs of course in the taking of food, but it probably does not extend over the whole length of the oesophagus at one time and so does not cause any great loss of water. That the oesophagus is widely open in contracted 272 C. M. CHILD. actinozoa is very evident from the frequent forcing out through it of mesenterial filaments ; that it cannot be widely open during distension of the body by water is equally evident. Doubtless many differences in detail occur in different members of the group, but it is probable that the suggestions given here for Ccri- anthns vill apply more or less closely to a large number of forms. The presence of two siphonoglyphes must condition certain mod- ifications in these processes. The statement is made in several text-books that the siphonoglyphes serve to keep the water in cir- culation when the animal is contracted. According to the view given above, these organs serve rather to permit circulation when the body is distended. In addition to the inward and outward currents in the stomo- dseal region the water in the intermesenterial chambers and in the tentacles connected with them is in constant circulation, impelled by the movement of cilia. I have not been able to determine the details of this circulation in Ccrianthns, but have observed it in several transparent members of the group. In general it may be said that the current passes orally along the body-wall, periphe- rally along the aboral face of the tentacle, back again along its oral face, centrally beneath the disc, probably into and out of the labial tentacles, to the stomodseum, and aborally along the sto- modfeum. Whether cilia on the lateral surfaces of the mesenteries aid in forcing the water aborally I clo not know. In the forms observed, at any rate, the aboral current continued from the aboral end of the stomodaeum to the aboral end of the body. In all of the intermesenterial chambers are these circulatory cur- rents. In Ccriantlnts itself they are difficult to demonstrate with certainty, but there is little doubt that the current in the 'oral di- rection along the body-wall exists, and this being present, we may confidently assert that the return current is also present. These currents in the intermesenterial chambers and tentacles may be designated for convenience as the circulatory currents in distinc- tion from the inward and outward currents of the stomodaeal region. They are indicated diagrammatically by arrows in Fig. 2. How far the circulation of water in the central regions of the body is complicated by the complex arrangement of the mesen- FORM REGULATION IN CERIANTHUS. 2/3 terial margins and mesenterial filaments cannot be determined. Moreover, these are merely matters of detail and do not affect the general outlines of the process. Since the position of the organs is subject to considerable variation the direction of local currents produced by their cilia must vary likewise. This discussion of the internal circulation and pressure has been somewhat detailed, since I believe that the general internal pressure and perhaps also local pressure resulting from the impact of currents against the body-wall are important factors in form-regulation and without doubt also in development. INTERNAL PRESSURE, GROWTH AND FORM-REGULATION-- A PRELIMINARY SURVEY. It is necessary before proceeding to the account of my experi- ments to indicate briefly how growth and form-regulation may be affected by the internal water-pressure. It would be difficult otherwise in many cases to show the bearing of the experiments without extended explanation. It is conceivable that the presence of water in the enteron may affect the body-wall in two ways : first, by general pressure, the same in all parts of the body, which subjects the body-wall to tension ; second, by local pressure resulting from the impact of definitely directed currents upon some portion of the body-wall which interrupts their course, thus subjecting that part of the body-wall to a localized tension in addition to the general tension. The experiments to be described demonstrate clearly that regulation in Ccriantlnts is dependent in certain important respects upon the tension resulting from internal water-pressure. The tissues react to this tension by growth. In the absence of the tension the typical form of the animal does not appear. Up to this point the evidence afforded by the experiments can scarcely be doubted or refuted. An essential feature in form-regulation in Ceriantlnis, as well as in other forms, is localized growth which is especially notice- able in the formation of new tentacles. The problem of the cause of localized growth is one of the greatest importance in morphogenesis. According to one view the basis of morphological form is inherent in the organism, 274 C. M. CHILD. either in the structure of the protoplasm or as a governing prin- ciple distinct from physical and chemical factors to which the latter are subordinated. On the other hand, it is possible, at least theoretically, to regard organic form as the resultant of the complex of physical and chemical conditions internal and external which affect the protoplasm. According to this view, organic form is not strictly speaking inherent in the living substance ; but results indirectly from its activities and its environment. The experiments to be described demonstrate, I believe, that in the absence of internal water-pressure the living substance of Ccriantliits is absolutely incapable of producing the typical form of this animal. It is not merely that the parts are formed and remain collapsed in the absence of water-pressure ; either they are not formed or their form is atypical. The appearance of the tentacles is delayed or is inhibited ; the growth of the disc and the oesophagus does not occur when the internal pressure is reduced below a certain point. Thus it is possible in this case to demonstrate a general relation between internal water-pressure and growth. The question as to whether any relation between localized growth and localized internal pressure exists must not be con- fused with the preceding question as to a general relation be- tween the two phenomena. It is possible for instance to sup- pose that the region where new tentacles shall appear is deter- mined in some unknown manner, but that the internal pressure is a factor in causing their growth after their position has been determined. On the other hand, it is possible to conceive that the position of these structures is determined by local pressure due to currents in the enteron (the " circulatory currents"). It cannot be claimed for my experiments thus far that they decide which of these two possibilities is correct. Certain of the data, however, appear to me to indicate that the local pressure due to currents may play a certain role in determining the position of the marginal tentacles. Concerning the labial tentacles there is as yet no definite evidence and this fact must constitute a weak point in the evidence, at least until further experiments can be performed. Time need be taken here only for a brief consideration of the possibility of a direct relation between local internal pressure and FORM REGULATION IN CERIANTHUS. 2/5 the determination of localized structures at certain points. In each intermesenterial chamber the circulatory current passing orally along the inner surface of the body-v/all (Fig. 2) must strike against any part of the wall which is folded or rolled over in such a manner as to form an obstacle in its course. When a transverse cut in the body-wall of CcriantJms is made the cut edges roll inward in such a manner that the current passing orally in the chambers must strike the inner surface of the in- rolled portion. In this condition that portion of each intermes- enterial chamber just beneath the inrolled cut margin forms a blind sac into which water is continually being forced by the current. The cilia which in the normal animal provide for a return current have been in large part removed from this region. Local pressure upon the body- wall in this region must result. Now the marginal tentacles always make their appearance in exactly this region, a region which previously formed a part of the lateral body-wall, and which, since tentacle-regeneration is possible at any level except the extreme aboral region, cannot have possessed any special qualifications for tentacle formation. The question is, what causes tentacles to appear in this particular region of a piece, no matter what part of the body the original piece represents. Doubtless very few will regard the assump- tion of an " entelechy " (Driesch) or of "dominants" (Reinke) as an answer to this and similar questions, although it may possess the merit of many other unwarranted assumptions, viz., that of being an easy way out of difficulty. If we call in " hered- ity " to answer the question we still have no real answer, though it is evident that tentacles of characteristic form and structure would not appear if CcriantJins were not concerned. Are we not justified in seeking for definite, intelligible causes or condi- tions of one kind or another for phenomena of this nature ? Much work of recent years has given an affirmative answer to this question. In many cases undoubtedly these causes or con- ditions are internal, /. e., in the protoplasm, but it is by no means impossible or improbable that they are external in many other cases. Certain of my experimental results seem to me not only to admit the possibility of local pressure as a determinative factor 2/6 C. M. CHILD. in morphogenesis but to afford little basis for other interpreta- tions. I desire to give the data in descriptive form, but shall endeavor to point out their bearing in connection with this hy- pothesis. It should be borne in mind, however, that while the existence of a general relation between internal water-pressure and growth is demonstrated, my data upon the other problem are insufficient in my own opinion to establish a conclusion with- out doubt. At present, though the evidence in regard to the marginal tentacles is very strong, the impossibility of giving any special evidence concerning the labial tentacles constitutes a ser- ious defect. The regeneration of labial tentacles is certainly delayed or inhibited by reduced water-pressure, but I have not been able thus far to demonstrate that localized pressure upon the body-wall occurs in the region where they appear. This failure is due at least in part to the fact that my attention was concentrated chiefly on the marginal tentacles during my experi- ments. I hope that a future opportunity for renewed experi- mentation may render it possible to attain more definite conclu- sions on this point. Attention may be called to the possibility that the two sets of tentacles possessing different functions and appearing as they do at different times and under different con- ditions may perhaps be determined by very different factors. General similarity ©f form and structure is not necessarily indic- ative of similar conditions of origin. Even if it should be demon- strated that the marginal tentacles arise in response to the stimu- lus of localized pressure it would by no means necessarily follow that the labial tentacles are similarly produced. THE CLOSURE AND DISTENSION OF PIECES. The closure by new tissue of the two ends of cylindrical pieces has been described in the first paper of this series (Child, 'c>3) at the aboral end of a distal piece, and also one at the oral end (Fig. I, C) of a proximal piece. The results of this series of experiments are given in Table I. to IV. The first column gives the total number of individual stems operated upon ; the second column shows the time that elapsed between the removal of the hydranth and the cutting of the stem through the middle ; and in the following columns the results two, three, and four days after the operation are indicated. Hy. signifies the regeneration of a complete hy- dranth ; t. a. indicates the formation of tentacle anlagen only ; while 0 is used to indicate that no regeneration had taken place when the observations were made. The letters A, B, C and D refer to surfaces thus marked in Fig. I ; and the numbers in parentheses show the number of cases in which similar results were obtained. TABLE I. Number of Individuals. Interval Between Result in Cuttings. Two Days. Result in Three Days. Result in Four Days. (A...ky. (6) !A \A •t same. same. (£... 0 (6) B U 6 2 hours. ( C / *• "• ( ^ (3) (3) \C' I/"'. (4) •\t.a. (2) ( C hv. (6) (In: (2) (£>... O (6) \D t.a. (2) {D...\t.a. (I) 0 (4) (0 (3) Owing, probably, to differences in the temperature of the water in which they live, regeneration in Tubularia crocea is much slower than in Tubnlaria uicscnibryantlieuiuui. In the former species a new hydranth rarely develops until two days after the removal of the old hydranth, while in the latter species a new hydranth frequently regenerates in the course of twenty-four hours. In all of the experiments in this series, as shown in the above table, a polyp formed on the oral end, A, of the anterior REGENERATION IN TUBULARIA CROCEA. 289 piece, AB, before one developed on the oral end, C, of the prox- imal piece, CD. This result might, of course, be expected as the oral end, A, closed two hours before the end, C. In no case did a polyp develop at the aboral end, D, of the proximal piece as soon as one formed at the oral end, C. A start of only two hours, however, is sufficient, in most cases, to cause the for- mation of a hydranth at D before one develops at the aboral end, B, of the distal piece, AB. The results obtained when the intervals between the cuttings were four, six and eight hours are given in Tables II. to IV. TABLE II. Number of Individuals. Interval between Cuttings Result in Two Days Result in Three Days. Result in Four Days. (A \hy-W •\0 (4) [*•"{£. (A ( A ... hv. ( 10) (A ,7. (2) \B... 0(io) \S ... 0 (10) I " — \ 0 (8) 10 4 hours. (t.a. (i) \c--\0 (g) (hy. (2) (hy. (7) (C.... \t.a. (0 JC—U*. (3! (£>... O (10) 10 (3) | Mr. (i) \D /'-"-co "i° (9) •\0 (9) TABLE III. Number of Individuals. Interval between Cuttings. Result in Result in Two Days. Three Days. Result in Four Days. (hy. (3) J //,.. (6) (//..J /.«. (3) 1" "U«. (2) (A... hy. (8) ('0 (2) ( ^... o (8) \B... 0 (8) 8 6 hours. (£- ° (8) rr f/'r. (5) (C.... hy. (8) (C {hv. (I) | ' (t.a.(3) J (7) (^ | t.a. (4) \D"\0 (4) |Z>... (9 (8) \^ (4) TABLE IV. Number of Interval Between Result in Result in Result in Individuals. Cuttings. Two Days. Three Days. Four Days. (hy. (6) (A...hy. (12) ./ ...hy. (12) (A...\t.a.(4) \ 12 8 hours. (0 (3) [JB...O (12) /;...<_> (12) 1^... O (12) 1M/M2J C....hy (12) (C.. O (12) J (.'• • V'1^ J l 1 (hv. (2) f /'.''• (5) U... 0 (12) ^-]/.«.(3 lO (7) '"to (7) 2QO HELEN DEAN KING. There is a great similarity in the results of these experiments. In all cases a hydranth formed on the oral end, A, of the an- terior piece before one developed at any other cut surface, and the rate of development of a polyp from the aboral surface, D, was more rapid than that from the aboral surface, B, even in those cases in which D had only two hours start over B. In no case, however, did a polyp form at D before one developed at C, even in the experiments in which D closed eight hours before C. As was the case in the experiments made by Morgan, an interval of eight hours between the two cuttings has, apparently, no more effect on the regeneration than has an interval of only two hours. The formation of a polyp at the oral end, C, of the proximal piece, CD, does not prevent the early development of a polyp at the aboral end of the same piece, and in some cases there is only a few hours interval between the formation of the two hydranths. The earlier development of a hydranth at A, however, seems to check the formation of a hydranth at the aboral end, -B, of the distal piece for some time, as in no case did a hydranth develop at B until five days after the experiment began. This difference in the rate of development at D and at B cannot be due to a difference in the lengths of the pieces, because, in making the ex- periments, the stems in all cases were cut as nearly as possible through the middle and any difference in the lengths of the anterior and of the proximal pieces would be too slight to have any ap- preciable influence on the result. The earlier closing of the aboral end, D, of the proximal piece, CD, evidently counter- balances to some extent the influence of the oral end, as sug- gested by Morgan. As a result, the development of a polyp at D is hastened somewhat, although in no case is a hydranth formed here before or as soon as one develops at the oral end of the piece. The effect of the earlier closing of the aboral end of long pieces of the stem, in both Titbularia mesembryanthemum and in Tubularia crocca, is to hasten the development of the aboral sur- face. The influences that bring about this result are apparently not as strong in the latter species as in Tubularia mesembryan- themum where the aboral development may be hastened so much that polyps develop simultaneously at both ends of the piece. REGENERATION IN TUBULARIA CROCEA. 29 1 This seeming difference between the two species may possibly be due to the fact that Tubitlaria crocca, which lives in cold water, regenerates very slowly and, therefore, comparatively slight dif- ferences in the rate of regeneration at the oral and aboral ends of the stem can be readily noted. Tubitlaria incscinbrvantlicinnni, on the other hand, lives in much warmer water and its regeneration takes place so quickly that it is difficult to detect slight differences in the rate of development of the polyps at the cut oral and aboral surfaces. In a variation of the above experiment, a piece of silk thread was tied tightly around the stem about 2 mm. below thehydranth, and another piece was tied about 30-40 mm. below the first. Both ends of a long piece of stem were, therefore, closed at prac- tically the same time in such a way that no regeneration was possible from either end of the piece. After the ends had been tied, the stem was cut transversely through the middle as in Fig. I, B, C, in order to ascertain whether subsequent regeneration from the cut surfaces, B, and C, would be hastened in comparison with the rate of regeneration from similar surfaces of pieces of stem of the same length, cut at the same time, but not closed artificially at one end. The control pieces of stem were kept in the same dishes with those used in the experiment, and both sets, therefore, were under the same external conditions. Eight long pieces of stem were used in this experiment. Two days after the operation, tentacle anlagen had appeared at the cut ends of all of the sixteen pieces, but they were not as well developed on the aboral end, B, of the anterior piece as they were on the oral surface, C, of the posterior piece. At this time there was no indication of any regeneration at the aboral sur- face of the anterior piece in the control set of stems, although in some cases complete hydranths, in other tentacle anlagen, were present on the oral end of the proximal pieces. On the third day after the operation, polyps were found on the oral end, C, of all of the proximal pieces, both in the control and in the tied stems. The development from the aboral surface, B, of the an- terior pieces, however, did not keep pace with that at the oral end, C, of the proximal pieces, as at this time only two of the pieces of stem tied at one end had produced hydranths at the aboral sur- 292 HELEN DEAN KING. face, B, the rest had, as yet, developed only tentacle anlagen ; in the control stems, no development from the aboral surface, B, had taken place in any case. It is seen from the above experiments, that the development of a hydranth at the oral end of a piece of the stem of Tubit- laria crocca is not hastened by artificially closing the aboral end. Tying the oral end of a distal piece of the stem, however, hastens the development of the aboral end of the piece as compared with the development that takes place from the aboral surface of a piece of stem of similar length that is not closed at the oral end, as Driesch has shown. This result also agrees with that obtained by Loeb (7) in experiments in which he stuck the oral end of pieces of the stem of Tulntlaria mesembryanthemum in fine sand, leaving the other end freely surrounded by water. He found that " Durch Hemmung der Polypenbildung am oralen Ende kann man also die Polypenbildung am aboralen Ende besch- leunigen." In a third set of ten experiments, hydranth bearing stems about 30 mm. in length were removed from the colony and kept until a polyp formed on the cut aboral end. The time required for the development of these aboral polyps varied from three to five days in different cases. After all of the pieces had developed hydranths at the aboral end, each stem was cut transversely through the middle as in Fig. 2, B, C. The object of these experiments was to ascertain whether the presence of a hydranth at the aboral end, D, of the proximal piece, CD, would alter the polarity of the piece and thus prevent or retard the development of a hydranth at the oral end, C. The stems were cut through the middle region on June 19. Not until June 22 were there any indications of a development of a hydranth at the oral sur- face, C, and then only faint traces of tentacle anlagen were found in three stems. At this time, no development had taken place from the aboral surface, B, of any of the anterior pieces, AB. For control experiments, pieces of stem about 30 mm. in length were cut through the middle as in Fig. I, BC, at the same time that the transverse cuts were made across the stems bearing a hydranth at each end, and on June 22, well developed hydranths were present at the oral end of all of the proximal pieces. On REGENERATION IN TUBULARIA CROCEA. 293 June 23, three of the ten proximal pieces of stem bearing aboral hydranths had also developed hydranths at the oral end ; while the oral end of two other pieces of stem showed well developed tentacle anlagen which developed into hydranths on the following day. No further changes took place in any of the pieces although they were kept for some ten days longer. It is seen from the above experiments, that the presence of a hydranth at the aboral end of a piece of the stem of Tubularia delays, but does not prevent, the development of a hydranth at the oral end of the piece. This result can- not be due simply to the fact that the proximal end of the piece was closed by the presence of the aboral polyp, because, in the previous set of experi- ments, it was shown that closing the aboral end of a piece of stem by tying does not delay the development of a polyp at the oral end. It seems prob- able that the polarity of the piece was changed, for a time at least, by the presence of a hydranth at its aboral end and, therefore, the influences for hydranth formation at the freshly cut oral surface were not strong enough to bring about the development of a polyp for some days. II. EXPERIMENTS ON BRANCHING STEMS. The following series of experiments were made in order to as- certain whether the development of a hydranth on the oral end of a stem will influence the rate of development of a hydranth on the distal end of a long or of a short piece of a branch, and also to determine what conditions are necessary in order that the formation of a hydranth at the one place will prevent the forma- tion of a hvdranth at the other. FIG. 2. 294 HELEN DEAN KING. Series i .- - On June 24, twenty experiments were made in which a branch" was cut off about I mm. from its origin in the stern, and then the anterior end of each stem was removed by a transverse cut leaving a piece from 10-20 mm. above the place of union with the branch (Fig. 3). In one case, a hydranth developed on the cut end of the branch two days after the operation, and at the same time a polyp also formed on the oral end of the main stem which in this instance was 20 mm. in length above the origin of the branch. In all other individuals at this time tentacle anlagen had formed at the oral end of the stem, but there was no indication of the devel- opment of a hydranth at the cut end of any of the branches. On June 27 hydranths were found at the oral end of all of the stems and also on the distal end of four branches ; all the remaining branches had well developed tentacle anlagen excepting one which showed no signs of regeneration during the course of the week that the hydroids were kept. In this set of experiments, therefore, with the exception of the one case noted, regeneration of a hydranth took place at the distal end of the long stem before a polyp formed at the oral end of the short branch. The results of this set of experiments might possibly be con- sidered to be due to the fact that the longer piece exerted some kind of an influence over the shorter piece that would tend to alter the polarity of the shorter piece and thus retard develop- ment from its cut oral surface. That a larger piece of a hydrozoa can influence the polarity of a smaller piece is shown unques- tionable in grafting experiments that I made on Hydra viridis (King, 6) in which the larger component of the graft either absorbed the smaller component or formed a permanent union with it. In the latter case, the polarity of the smaller piece was completely reversed, if necessary, in order that a structure might regenerate on its cut surface that would produce a normal polyp. Another factor that might, possibly, cause a delay in the development of a hydranth from the cut surface of the shorter piece is the length of the piece. Morgan has shown that in REGENERATION IN TUBULARIA CROCEA. 295 FIG. 4. branching stems a short piece of a branch or of a stem regener- ates as does an isolated short piece, /. e., the region of the ten- tacle anlagen is reduced and the rate of development of a hydranth is much slower than that of a hydranth from the cut end of a long piece of stem. Both of these factors may help to bring about the delay in the development of a hydranth from the oral end of the shorter piece in all of these experiments with branching stems in which there is a marked difference in the length of the stem and of the branch. Series 2.-- Seventeen branching stems from different colonies were cut so that the anterior portion of the stem above the place of union with the branch was only about i mm. in length, while the length of the branch varied in different cases from 8—25 mm. (Fig. 4). Two days after the opera- tion, hydranths had developed at the cut ends of thirteen branches, and well developed tentacle anlagen were found at the oral ends of the other four branches. No hydranths were found at this time at the oral end of any of the stems, and tentacle anlagen were only faintly defined in some few cases. On the next day, hydranths had developed at the distal end of all of the branches, but only five stems bore hydranths at the oral end. In this set of experiments the development of a hydranth took place more rapidly from the cut end of the lone branch than from the oral end of o the main stem. Series j. - - Twelve experiments were made in which the anterior end of the stem and the distal end of the branch were cut off so that the length of the branch and of the stem above the place of union was practically the same, varying in different cases FIG. 5. 296 HELEN DEAN KING. from 8-20 mm. (Fig. 5). Two days after the operation, there were well developed hydranths on the ends of three branches and only tentacle anlagen on the oral ends of the correspond- ing stems. In two cases hydranths had developed on the end of the branch and also on the cut surface of the stem ; while in the other cases only tentacle analgen were found, and they were equally well-developed on the branch and on the oral end of the stem. During the following two days, hydranths formed on all of the cut ends, sometimes the hydranth developed on the end of the stem before it did on the branch, and some- times the hydranth appeared first on the branch. As a general result of this series of experiments it can be stated that hydranths develop at about the same rate when both the branch and the anterior portion of the stem are approximately the same length. Series 4.-- In the previous set of experiments both the branch and the anterior portion of the stem were of considerable length and both developed at about the same rate. In order to see if similar results would be obtained if the pieces were very short, fifteen experiments were made in which the branch and the stem were cut off about I mm. above their point of union (Fig. 6). When the hydroids were examined three clays after the operation, hydranths were found on the oral end of the stem and not on the cut end of the branch in five cases ; while in four hydroids, polyps had developed on the branch and not on the oral end of the stem ; in the remaining six cases, hydranths were present at the distal end of both branch and stem. In those cases in which one or the other cut sur- face failed to develop a hydranth, the ccenosarc appeared to be entirely with drawn from this part. When these stems were ex- amined under the microscope, the streaming of granules in the interior cavity was visible only in the proximal part of the main stem and in the part of the stem or branch that had regenerated. In the cases in which hydranths formed at both cut surfaces, the streaming of granules was found in all parts of the stem and also in the branch. In this set of experiments, regeneration seemed to take place REGENERATION IN TUBULARIA CROCEA. 297 with equal rapidity from the cut oral surface of the branch and of the stem, as was the case in the experiments described under series 3. If the pieces above the place of union of the branch and stem are of approximately the same length, no matter how long or how short they may be, the influences for hydranth for- mation appear to be alike in both, and one piece has, seemingly, no effect on the other. Where the ccenosarc is withdrawn entirely from one part of the hydroid, as was noted in some few cases in the last set of experiments, no regeneration of the piece is possible. In experimenting on Tubnlaria crocea, Morgan (9) found that if he cut off both the main stem and the branch a little above the place of union, the results varied considerably in different cases. In some instances he obtained the regeneration of a hydranth on the branch and not on the oral end of the stem ; in other cases a polyp formed only at the oral end of the main stem ; and in still other individuals regeneration took place from the cut surfaces of both branch and stem. It seems probable that the lack of uniformity in these results can be attributed to a difference in the relative lengths of the branch and of anterior portion of the stem above the point of insertion of the branch. It is, of course, impossible to cut the branch and stem at absolutely equal distances from the place of their union, and in those cases in which regeneration from one cut surface took place before it did from the other, there may have been just enough difference in the lengths of the pieces to bring about the earlier regeneration of a hydranth on the cut oral surface of the longer piece. Scries •>'. — In twenty-eight cases the stem was cut off transversely just above the origin of the branch as shown in Fig. 7. The oral end of the branch was then removed leaving a piece, from 3 to 5 mm. in length, still attached to the stem. The object of these experiments was to see whether the regeneration of a hydranth at the oral end of the stem could be entirely pre- vented by this means. Two days after the operation, hydranths had developed at the cut end of the branch in nine of the hy- 298 HELEN DEAN KING. droids and well-developed tentacle anlagen were present on the other branches ; there was no indication of a regeneration at the oral end of any of the stems. The next day all of the branches bore hydranths, and in but one case had any regeneration taken place at the oral end of the stem. In this instance, the stem extended about o. 5 mm. above the place of insertion of the branch and a considerable amount of red pigment had collected at its extreme oral end. In the course of forty-eight hours more a polyp formed on the oral end of this stem but no regeneration took place at the oral end of any of the other stems, although they were kept for over a week. Series 6. — Sixteen experiments were made in which the branches were cut off very close to their origin on the stem. The oral end of the stem was then removed leaving a piece about 5 mm. in length above the origin of the branch, in order to see whether the formation of a hydranth at the oral end of the stem would prevent or merely delay the formation of a polyp at the place where the branch was removed. In all cases the wound in the side of the stem healed over very quickly and, although the hydroids were kept alive for a number of days, no regeneration of any kind took place at the point of injury. Series 7. - - In ten cases the entire branch was removed from the stem, but the old hydranth at the distal end of the stem was not cut off. The result was the same as in the previous set ot experiments, as the cut surface was very soon covered over and no subsequent regeneration took place from it. Series 8. - - In sixteen cases where long pieces of stem bore from two to four branches, the anterior end of the stem and the apical end of each branch were removed by transverse cuts leav- ing the lengths of the branches approximately the same as that of the stem above the origin of the most anterior branch. The experiments were made to see if there is any difference in the relative rate of regeneration of the anterior branches and of the proximal ones. There was no uniformity whatever in the results of this set of experiments. In some cases a hydranth regenerated on a posterior branch before it did on the oral end of the main stem ; and in other cases all of the branches produced hydranths at the same time that one developed at the oral end of the stem. REGENERATION IN TUBULARIA CROCEA. 299 III. THE REGENERATION OE SHORT PIECES OF THE STEM OF TUBULARIA. It was first noted by Bickford (i), and later confirmed by Driesch (2) and by Morgan, that small pieces of the stem of Tubularia about I mm. in length are capable of regenerating. In a recent paper, Hargitt (5) states that he was unable to obtain any regeneration from pieces of the stem of Tubularia crocea and of Tubularia teueUa that were as much as 3-4 mm. in length. This result was probably due to the poor condition of the stems when the experiments were made. Small pieces of the stem of some other hydroids, do not appear to possess as great a power of regeneration as Tubularia, for Cast and Godlewski (4) have found that pieces of the stem of Pcnnaria cavolinii about i mm. in length never produce hydranths and pieces 2 mm. in length regenerate hydranths but rarely. Bickford's experiments on small pieces of the stem of Tubularia tenella show that, in this species, regenerative processes are not restricted to any special region of the stem, and also that such short pieces tend to form one complete hydranth rather than to produce double abnormal structures. In experimenting on Tubularia mesembryanthemum, Driesch (2) found that of 82 short pieces of stem, 5 formed a single proboscis, 26 formed a double proboscis, and the remaining 5 i pieces produced hydranths. These results agree with those of the earlier experiments of Bickford. In a later paper, Driesch (3) states that at the oral end of the stem one seldom gets a whole hydranth, but usually a single or a double proboscis ; from the middle zone hydranths usually develop ; while from the aboral end of the stem, these structures are rarely produced. This difference in the kind of regeneration from the various parts of the stem Driesch attributes to the situation of the small piece in the onVinal individual and to the different distribution of the O hydranth-forming pigment in the coenosarc of the different parts. Since Morgan and also Stevens (i 1-12) have proven the fallacy of the hypothesis of "red formative stuff" in Tubularia, this portion of Driesch's explanation is, of course, no longer tenable. Morgan (S-io) has made an extended series of experiments with small pieces of the stem of both Tubularia imsembryanthemum !OO HELEN DEAN KING. and of Tnbularia crocea. He finds, as did Driesch, that pieces about I mm. in length from the region immediately behind the old hydranth usually die, even when longer than pieces from a more proximal region that regenerate. When this distal region does regenerate, it produces a greater number of single proboscides than of other forms, a result that might be expected as this part of the stem ordinarily goes into the proboscis of the new hydranth when a long piece of stem is regenerating. In another set of experiments, Morgan cut pieces of the stem •of Tubnlaria mesembryanthemum into a series of small pieces about i mm. in length in order to observe the behavior of con- secutive pieces from one stem and to compare the results with those obtained from similar pieces cut from other stems. His tables do not show any very definite results although there seems to be a certain similarity in the behavior of pieces of the same stem, and the incomplete structures are found most frequently at the distal end of the stem. At the suggestion of Professor Morgan, I repeated his experi- ments, using Tnbularia ci-occa, in order to furnish more data from which definite conclusions could be drawn. In making the ex- periments the old hydranths were removed and the distal part of the stem was cut into consecutive pieces about I mm. in length. The pieces were than laid in rows on the bottom of flat dishes filled with fresh sea water. For the sake of brevity the follow- ing abbreviations are used in the tables given : hy. = complete hydranth without any stalk ; hy. + stalk = hydranth with a short stalk that has been formed by a withdrawal of the coeno- sarc from the perisarc ; hy. -f stem = hydranth with a stem attached to the perisarc ; pb. = single proboscis ; d. pb = double proboscis ; reprod. = reproductive organs. The results tabulated are from observations made three to four days after the operation. • TABLE V. Number of Kind of Structure Number of Kind of Structure Piece. Regenerated. Piece. Regenerated. I pb. with no tentacles. 6 pb. 2 d. pb. 7 d. pb. 3 pb. 8 hy. + stem. 4 d. pb. -)- reprod. 9 d. pb. 5 pb. 10 d. pb. REGENERATION IN TUBULARIA CROCEA. TABLE VI. 301 Number of Piece. Kind of Structure Regenerated. Number of Piece. Kind of Structure Regenerated. I pb. 6 dead. 2 d. pb. 7 pb. 3 d. pb. 8 pb. 4 dead. 9 dead. S d. pb. 10 dead. TABLE VII. Number of Kind of Structure Number of Kind of Structure Piece. Regenerated. Piece. Regenerated. I d. pb. 6 d. pb. 2 pb. 7 d. pb. -)- reprod. 3 pb. 8 pb. 4 Pb. 9 by. + stem. 5 hy. 10 pb. TABLE VIII. Number of Kind of Structure Number of Kind of Structure Piece. Regenerated. Piece. Regenerated. I dead. 6 pb. 2 d. pb. 7 Pb. 3 d. pb. 8 hy. ~ stalk. 4 pb. 9 pb. 5 pb. -U reprod. 10 d. pb. TABLE IX. Number of Piece. Kind of Structure Regenerated. Number of Piece. Kind of Structure Regenerated. I dead. 6 pb. -j- reprod. 2 pb. 7 pb. 3 pb. 8 hy. -•- stem. 4 hy. -|- stem. 9 pb. 5 pb. 10 dead. TABLE X. Number of Piece. Kind of Structure Regenerated. Number of Piece. Kind of Structure Regenerated. I pb. 6 pb. 2 d. pb. 7 hy. -|- stem. 3 d. pb. 8 pb. 4 d. pb. -:- reprod. 9 hy. 5 pb 10 dead. 3- 3 hy. 7 pb. 4 dead. 8 dead. TABLE XIII. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 dead. 2 dead. D pb. -j- reprod. 3 d. pb. 7 pb. 4 dead. 8 Pb; TABLE XIV. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I hy. 5 dead. 2 Pb- 6 dead. T. Pb. 7 pb. 4 pb. 8 dead. 304 HELEN DEAN KING. TABLE XV. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 pb. 2 pb. 6 dead. 3 pb. f reprod. 7 dead. 4 pb. 8 dead. TABLE XVI. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 pb. 2 pb. 6 pb. 3 pb. 7 pb. 4 hy. -)- stem. 8 dead. TABLE XVII. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 pb. 2 pb. -j- reprod. 6 dead. 3 pb. 7 dead. 4 pb. -f- reprod. 8 dead. TABLE XVIII. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I dead. 5 dead. 2 pb. 6 hy. -f stalk. 3 pb. 7 pb. 4 pb. 8 dead. TABLE XIX. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 dead. 2 pb. -)- reprod. 6 dead. 3 hy. + stalk. 7 hy.+ stem. 4 pb. 8 hy.-(- stem. TABLE XX. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 pb. 2 pb. 6 pb. 3 hy. -f- stem. 7 pb. 4 dead. 8 dead. REGENERATION IN TUBULARIA CROCEA. 305 TABLE XXI. Number of Piece. Structure Produced. Number of Piece. Structure Produced I pb. 5 dead. 2 pb. -f- reprod. 6 pb. 3 pb. 7 pb. 4 hy. -(- stem. 8 dead. TABLE XXII. Number of Piece. Structure Produced. Number of Piece. Structure Produced. I pb. 5 pb. 2 dead. 6 hy. + stalk. 3 pb. -f- reprod. 7 pb. 4 pb. 8 dead. The results of these experiments confirm those obtained by Morgan in every respect, as double structures were produced very rarely, only one being obtained in the entire series of experiments. There seemed to be no distinctive individual dif- ferences in the pieces of stem as regards the structures produced. In only one case (Table XIX.) were as many as three hydranths produced, while in the tables given by Morgan for Tubularia mesembryanthcimini, whole series of pieces from the same stem produced hydranths. If a comparison is made between the results shown in Tables XI. to XXII. and those shown in Tables V. to X., the most noticeable difference is that a very much greater number of double structures were produced when short pieces of the stem were lying on their sides during the process of regeneration. In both sets of experiments single proboscides were the structures most frequently produced, and very little individual difference could be detected in the stems regarding the kind of structure that they would tend to produce. The development of small pieces of the stem of Tiilndana standing on one end is considerably slower than that of similar pieces lying on one side. In the latter case, development takes place in about two days ; while in the former case the various structures never appear under three days, and usually not under four or five days. Many pieces, usually those nearer the proxi- 306 HELEN DEAN KING. mal end of the stem, die when the pieces stand on one end. This result may be due to the fact that the conditions under which regeneration takes place are not as favorable when the end of a small piece of stem is closed by contact with some foreign sub- stance, as when the piece lies on one side and the cut ends are allowed to close in a normal manner. BRYN MAWR COLLEGE, Bryn Mawr, Pa., March 23, 1904. LITERATURE. 1. Bickford, Elizabeth. '94. Notes on Regeneration and Heteromorphosis of Tubularian Hydroids. Journ. of Morph., Vol. IX. 2. Driesch, Hans. '97. Studien iiber das Regulationsvermogen der Organismen, I. Von dem Regu- lativen Wachsthums- und Differenenzirungsfahigkeiten der Tubularia. Arch. Entwickelungsmech., Bd. V. 3. Driesch, Hans. '99. Studien iiber das Regulationsvermogen der Organismen, II. Quantitative Regulationen bei der Reparation der Tubularia. Arch. Entvvickelungsmech., Bd. IX. 4. Gast, R., und Godlewski, E. '03. Die Regulatonserscheinungen bei Pennaria cavolinii. Arch. Entwickelungs- mech., Bd. XVI. 5. Hargitt, G. T. '03. Regeneration in Hydromedusre. Arch. Entwickelungsmech., Bd. XVII. 6. King, H. D. '03. Further Studies on Regeneration in Hydra viridis. Arch. Entwickelungs- mech., Bd. XVI. 7. Loeb, J. '92. Untersuchungen zur Physiologischen Morphologic der Thiere, II. Organ- bildung und Wachsthum. Wurzburg. 8. Morgan, T. H. '01. Regeneration in Tubularia. Arch. Entwickelungsmech., Bd. XI. 9. Morgan, T. H. '02. Further Experiments on the Regeneration of Tubularia. Arch. Entwickel- ungsmech., Bd. VIII. 10. Morgan, T. H. '03. Some Factors in the Regeneration of Tubularia. Arch. Entwickelungs- mech., Bd. XVI. - ii. Stevens, N. M. '01. Regeneration in Tubularia mesembryanthemum, I. Arch. Entwickelungs- mech., Bd. XIII. 12 Stevens, N. M. '02. Regeneration in Tubularia mesembryanthemum, II. Arch. Entwickelungs- mech., Bd. XV. RESEARCH SEMINAR OF THE MARINE BIOLOGI- CAL LABORATORY FOR THE YEAR 1903. The Research Seminar, begun in 1903, was devoted chiefly to informal reports and discussion of work carried on at the Labora- tory. It found abundant support and was continued for some weeks after the regular courses ended. The range of subjects reported upon contrasts strikingly with that of earlier years, when embryology was the main field of productive research at the Laboratory. The following is a list of the subjects presented, with brief sum- maries : July ij. The Anatomy and Embryology of Pecten tenuicost- atus. By GILMAN A. DREW. Drawings illustrating the structure and development of this form were exhibited and points of interest were discussed. Much of the discussion concerned the nervous system, the circulatory system, the formation of organs, and the excessive development of the posterior portion of this animal. The visceral ganglia, which supply nerves to the greater part of the body, are very complex, and the different structural regions are very well marked. The pallial nerves have taken on gang- lionic structure probably because of the man}' tentacles and eyes on the mantle margin that they supply. The cerebral and pedal ganglia are small, corresponding to the reduced condition of the anterior portion of the body. Octocystic canals opening on the surface of the body are present. The circulatory system comprises a definite system of blood channels, but they are probably not lined with endothelium, so that there is no sharp distinction between blood and lymph. July 75. The Origin and Relationships of the Rock Pigeons as Revealed in their Color-Patterns. By C. O. WHITMAN. The wild rock pigeons present two very distinct color-patterns - (i) the chequered type, and (2) the barred type. 30? 308 RESEARCH SEMINAR. Two black wing-bars on a gray ground have always been held to be the more primitive pattern, and birds of this pattern are supposed to represent the typical Columba Uina. The form with black chequers evenly distributed over the wing and back, although once named C. affinis, as a distinct species, was regarded by Darwin as a variety derived from the two-barred rock, and his opinion has stood undisputed. It appears from a comparative study of many species of wild pigeons, and from a study of the variations in domestic species, that the relationship is just the reverse : C. affinis is the original rock dove, and C. livia is the derived type. Domestic pigeons have come from both sources. Columba affinis, however, is not the most primitive form among the wild pigeons. It was derived from a more ancient type, best preserved in the turtle doves (Turtur orientalis and Turtur tiirtnr}. In the turtle dove type each feather has a dark center and light edge. The turtle dove pattern is at the same time a general avian pattern. The turtle type and the rock type coexist in some forms (e. g., Pliaps chalcoptera). The two-barred pattern of Colnmba livia was reached in the simplest way by an even reduction of the dark pigment, which would result, at one stage, in leaving remnant spots on the long coverts and the secondaries. The process of reduction has run in one direction in many, if not all pigeons, and present species have reached different stages, varying all the way from a uniformly spotted condition to four, three, two, one or part of one bar, or no bars. It appears to be impossible to reverse the direction of evolu- tion, and to advance from one or two bars to a complete or even a partial chequered state. The white-winged pigeon (Melopelia leucopterd] has no black spots or bars on the wing in the adult plumage, but unmistakable evidence of their former existence is seen in structural imprints still left on many of the coverts, and in vestigial traces of spots in a few of the Juvenal feathers. RESEARCH SEMINAR. 309 July 17. Effects of Light-rays on an Ant. By ADELE M. FlELDE. The species used for the experiment was Stcnainina fnkmui, a myrmicid ant. Five queens and about two hundred workers were kept for ten months in each of five artificial nests, one nest roofed with transparent glass, one with opaque glass, one with glass transmitting only red and green rays, and two with glass transmitting only rays of shorter wave-length than blue. These nests were exposed to like temperature, and the ants were fed on the same foods. In all the nests the young passed safely through the egg, the larval andk the pupal stage, and reached active life, proving that the cause of the usual hasty withdrawal of the inert young from daylight does not lie in any injury by any of the rays of the spectrum. The ants are blind to all rays of light other than the ultra-vio- let, and they avoid the ultra-violet rays in proportion to the in- tensity of the illumination. They often congregated with their young in the spots where the illumination from the red and green rays was most intense. Orange glass which excludes most of the ultra-violet rays may be used for roofing their artificial nests, and then the ants may be studied with the certainty that they be- have as if they were in darkness. At the end of ten months marked ants from each of the five nests were introduced into each of the other four nests, where they were amicably received, and it was thus determined that exposure to any ray of light in the spectrum does not affect the odor whereby the ants recognize one another. July 20. Further Experiments in the Embryology of the Chick. By F. R. LILLIE. First, three cases in which the cerebral hemisphere of the right side was destroyed at an early stage, and which resulted, after farther growth of the embryo for four or five days, in displace- ment of the wing rudiments of the opposite side. This displace- ment was the same in all cases and was never found in normal embryos. The conclusion was that there is some trophic relation between the higher brain centers of one side and the embryonic tissues, especially muscular tissues, of the opposite side; this be- 3IO RESEARCH SEMINAR. ing expressed in the case under consideration by the abnormal growth of the wing. The sections of these embryos show that the parts of the brain posterior to the cerebral hemispheres on the defective side were less developed than on the uninjured side, owing to the absence of descending tracts from the cerebrum of that side. This defect disappeared in the region of the medulla, from which it may be concluded either that the tracts had de- scended no farther, or that approximately half had decussated. The trophic effect, if any, would be indirect, through the inter- mediation of the motor neurons of the chord. Subject still under investigation. A second series of experiments was made involving destruction of the hind end of the embryo, showing, first, that there is no re- generation, and second, that the uninjured embryonic parts de- veloped precisely as they would have done in a normal embryo. Thus, if but a single leg-somite remained, a rudiment of the leg was formed in a developing embryo ; but if all the leg-somites were destroyed no rudiment appeared. Absence of the allantois was noted in such embryos. Other modifications of internal organs were described. 22. Variation and Selection in Saturnid Lepidoptera. By HENRY EDWARD CRAMPTON. An account was given of a study upon variation and. its rela- tion to elimination in PJnlosaniia cyntkia, as well as of additional studies which have been prosecuted during the past five years upon the same, and other problems in the case of Cynthia and othersaturnid moths. The first points determined were : (i) That when normally formed pupae which died before metamorphosis, were compared sex by sex with pupae which lived through the metamorphosis, the two groups were markedly different in several characters, as regards the typical condition as well as the variability with reference to such typical condition. Thus pupal elimination and variation were shown to be related. (2) A com- parison of the pupne which metamorphosed perfectly with those which formed more or less imperfect moths revealed a second period of elimination which again proceeded hand in hand with variation. Thus "secular" and "periodic" selection, so-called RESEARCH SEMINAR. 31 I by Pearson, were proven for Cynthia of this year (1898-1899). Annual series of the same species exhibited the same phenomena. Three lots of Cccropia have been likewise examined, in much greater detail (28 characters), and these also show both kinds of selection. An enquiry into the occurrence of reproductive selection grew naturally out of the foregoing. Two annual series of Cynthia (more than two hundred matings) and two annual series of Cc- cropia (130 and 125 matings respectively) have afforded the basis for the following conclusions : Pupal characters of mating males, or females, when compared with similar characters of the non- mating males, or females, prove to be different in type and re- stricted in variability. Thus a third period of selection is shown to be correlated with variability. In discussing the nature of the selective processes, reasons were given for regarding the correlations between the several characters as the real bases for the action of eliminating processes, and not the individual characters themselves. Further studies, now in progress, were briefly described. These deal with the phenomena of correlation between characters of the individual in its successive larval, pupal and imaginal con- ditions ; with inheritance in pure breeds, and in mixed breeds ; and with the correlation of certain physiological reactions with structural characters. July 2j. White Feathers. By R. M. STRONG. The few references in the literature of feather coloration to the causes of white in feathers state that the whiteness is due to the presence of air in the feather substance. This is misleading, for the larger proportion of the white effect is produced by the bar- bules which do not have air-spaces of significant size. The bar- bules are white for the same reason that powdered ice or glass and other transparent substances in a fine state of division appears white. July 2+. Nervous Regulation of the Heart of Venus Merce- naria. By R. A. BUDINGTON. The details of the nervous control of the heart of vertebrates have been wrorked out in more and more minuteness since the time when 312 RESEARCH SEMINAR. attention was first called to the matter by the Weber brothers in 1846. A similar relation of the nervous system and heart has been shown to hold among the invertebrates also, notably through the work of Conant and Clark on arthropods, of Ransom, Straub, and Yung on mollusca. The last-named worker has described inhibition of the heart of lamellibranchs on stimulation of the visceral ganglion, but no graphic records have been published illustrating experimental investigations of this kind. In this seminar experimental work on the heart of l^enns mer- ccnaria was described, and kymograph tracings exhibited, which seem to warrant the following conclusions : 1. Normal rate and character of the heart-beat varies widely with different individuals. 2. Partially exhausted hearts show extreme irregularities. 3. Electrical stimulation of the visceral ganglion causes arrest of the heart. 4. Stimulation of the cerebral ganglion produces no effect. 5. Stimulation of nerves passing from the visceral ganglion to the heart gives results comparable in every way to those obtained from stimulation of the vasrus in the vertebrates, viz : lone after- <_> o effects of strong stimulation, and typical escape from weak stimulation. 6. No evidence of acceleration was ever present. Definitely localized cardiac organs are found in no group of ani- mals generally considered lower than the lamellibranch molluscs. It would therefore seem that, notwithstanding one or two doubt- ful exceptions, cardiac muscle, wherever found, does not perform its functions entirely independent of inhibitory influences of the central nervous system. July 2-j. The Relation Between the Solution Tension and Physi- ological Action of the Elements. By ALBERT P. MATHEWS. As already pointed out by Loeb and the author, there is a relationship between the valence of an ion and its physiological action. For the motor nerve anions with three charges are some- what more than three times as powerful stimulants as anions with one charge. There are, however, for other tissues wide diver- gencies from this rule, particularly for the cathions. For ex- RESEARCH SEMINAR. 313 ample, mercury and copper are enormously more powerful than the bivalent alkaline earth metals ; and hydrogen with one charge is more active than other univalent metals. The author sug- gested that this divergence from the theoiy probably meant that it was the motion of the valence or charge which ultimately determined its action and that the velocities or orbits differed for the charges attached to different elements. Dr. Stieglitz sug- gested that the affinity of the atom for its charge might vary and account for these discrepancies. It occurred to the writer that this affinity might be represented by the solution tension of the element. The more easily the element is separated from a solu- tion of its ions, the less is its affinity for the charge it carries as an ion. I have accordingly compared the poisonous action of the metals and negative elements with their relative solution ten- sion as given by Nernst, using Fundulus eggs as test objects. The least concentration of the solution of the chlorides of the different metals which would just prevent the formation of an embryo was determined. The result showed a marked inverse ratio between the solution tension and poisonous action. Potassium with its great solution tension is almost the least poisonous ; silver with a very low tension is the most poisonous. The other metals arranged them- selves in proper order, except that ferric iron, zinc and cadmium were more poisonous than their position in the list of metals would indicate. This discrepancy is probably due to the fact that the solution tension is but a poor measure of affinity between the charge and the element. The anions arrange themselves in order, those parting with their negative charges most easily being most poisonous. The correspondence is so close as to indicate clearly that the affinity between the electric charge and the atom is a powerful factor in determining physiological action, and that the less strongly the charge is held, the more powerful is the action of any ion. Experiments were also tried demonstrating that the action of any cathion is modified by the action of the anion ; and that of any anion is modified by the action of the cathion. There is hence an antagonistic action between anions and cathions and the RESEARCH SEMINAR. physiological action of any salt is equal to the sum of the actions of the two ions. In the toxic and antitoxic action of salts as described by Loeb, both the anion and the cathion are of importance. There is no. simple relationship of antitoxic action between monovalent and bivalent cathions. July 31. The Cranial Nerves of Squalus acanthias. By O. S. STRONG. The object of the work is to confirm, using serial sections, the partial analysis of the cranial nerves into their components ac- complished by means of gross dissection by previous investigators, and to extend that analysis further. The components are found to be the same and to have the same general typical arrangement as in other vertebrate types. The trigeminus is purely " general cutaneous." The facialis has four roots : two lateral line roots (R. ophthalmicus superficialis, buccalis, mandibularis externus, etc.) distributed to the canal and ampullary organs of the head, one communis root to the mouth (R. palatinus, mandibularis internus, etc.) and one motor root. The post-auditory roots are the lateral line root, including also a small separate root to one or two canal organs via the glossopharyngeus, a series of com- munis roots and a series of motor roots. The communis roots are distributed into the pre- and post-branchial and visceral branches, innervating the branchial cavities, pharynx and viscera. July 31. A Case of Almost Complete Absence of the Left Cerebellar Hemisphere in the Brain of a Child Three Years and Four Months Old. By O. S. STRONG. The external appearances have been reported elsewhere (Jouni. Couip. Ncnr., Vol. XL, No. i). The sections show the follow- ing principal abnormalities : absence, usually complete or nearly so, of the right olive and the cerebello-olivary fibers connected with it, of the left restiform body, of the transverse pontine fibers to the left cerebellar hemisphere, of the right nucleus pontis and of the left superior peduncle. The \efc.formatio rcticidaris con- tained more longitudinal fibers than the right. The left lem- niscus showed some degeneration and the left corpus quadri- gcinimnn anterior was much smaller than the right. RESEARCH SEMINAR. 3 i 5 July 29. Hybrids from Wild Species of Pigeons, Crossed inter se and with Domestic Races. By C. O. WHITMAN. The species thus far employed with some success in crossing- are the following : FERAL SPECIES : 1. Oriental turtle (Tnrtnr oricntalis). 2. European turtle (Tnrtnr turtnr*). 3. Chinese turtle (Spilopclia1 chinensis}. 4. Surat turtle (S. tigrina). 5. Blond ring dove (Strcptopdicr risoria). 6. White ring dove (S. alba). 7. Oriental ring dove (S. torqnata). 8. Red ring dove (S. /in mil is}. 9. Passenger pigeon (Ectopistes migratorins). 10. Mourning dove (Zenaidnra carolinensis). 1 1. White- winged pigeon (Melopelia Icncoptcrd). 12. Wood pigeon (Columba palumbus). 13. Guinea pigeon (Columba guinea). DOMESTIC RACES : 14. Homer (Columba tabcllarid). 15. Fantail (Columba laticandd). 1 6. Tumbler (Columba gyrans). 17. Archangel (Coliunba illyrica). 1 8. Mondain (Colinnba adinistd). 19. Chequered rock (Columba affinis domesticd). 20. Owl-rock hybrid (C. lurbata x C. livia). The more important hybrids and their parent species were exhibited, and the importance of known ancestry, for definite re- sults, was clearly demonstrated. With a few exceptions, the hybrids were remarkably close intermediates. Reciprocal crosses gave like hybrids. Several series of fertile hybrids have been obtained. The most remarkable case was a male hybrid between a male chequered rock pigeon (C. affinis domestica) and a female oriental 1 Sundev, Math. Nat. Av. disp. Tent., p. 100, 1872. The species 1-8 are usually included in the genus Turtiir. The Streptopelias and Spilopelias are both sufficiently distinct for generic rank, and it is convenient to deal with them ae genera. 2 Bp.-Comsp. Av., II., p. 63, 1854. 316 RESEARCH SEMINAR. turtle dove (Turtur orientalis). The offspring of this hybrid, mated with the domestic pigeon, exhibited segregation ; but neither in the first nor the second generation were there " domi- nants " and " recessives " in the Mendelian sense. No support was found for the so-called " principle of pure germ-cells " in hybrids. Segregation is probably never com- plete ; and in some cases at least the intermediate character seems . to be permanent. August j. On the Presence of Specific Coagulins in Animal Tissues. By LEO LOEB. It has been known before that extracts of many animal tissues have an accelerating effect upon the coagulation of the blood. It can, moreover, be shown that there exist in animal tissues spe- cific substances which act specifically upon the blood of animals in which they are found, or upon the blood of related species. I have previously found these specific substances in mammals, birds, reptiles and amphibia. Further investigations show that they exist also in invertebrates. The muscle of the lobster acts more strongly upon the blood plasma of the lobster than upon the blood-plasma of the blue crab ; the muscle of the blue crab acts more strongly upon the blood-plasma of the blue-crab than upon the blood-plasma of the lobster. Pieces of muscle of other animals than arthropods, which have so far been tried, are with- out effect upon the blood-plasma of the lobster. For vertebrate blood there exist non-specific or less specific substances like peptone. Similar substances can also be found in certain animal tissues. The specificity of blood-coagula of verte- brates could not be demonstrated among vertebrates, although they become specific if used with invertebrate blood upon which they do not act at all. Non-specific or less specific substances exist also in inverte- brate tissues, as for instance in the ova of Arbacia and other animals. They are able to cause a coagulation of the blood of invertebrates which are not closely related. The origin of these substances, which are peculiarly useful, will probably have to be explained by a process of auto-immuniza- tion. This explanation is suggested by the great similarity be- RESEARCH SEMINAR. 317 tween the specific coagulins and substances derived by artifi- cial immunization. August j. The Retinal Nerve-endings in the Eye of Pecten. By IDA H. HYDE. Although the more important methods of staining were tested, none proved so satisfactory as the methylin blue method of Bethe, which was used in a somewhat modified form. The chief facts regarding the nerve -structure of the eye are as follows : There are two kinds of nerve-tissue. The first is an efferent, or, possibly, a trophic nerve-system, the fibers of which form the side-branch of the optic nerve, and are continuous throughout the peculiar twine-cells to their terminations on the supporting as well as on the nerve-cells in the eye. These fibers penetrate the eye on one side and form a single layer of large unique structures called by the author "twine-cells," as they have the appearance of fibers much coiled in the form of balls of twine. This layer of twine-cells lies between the lens and the retinal disc and on the disc. From these cells fibers extend to other twine-cells, to the tapetum and argentia, as well as to the supporting cells of the retina and as far as the rods. The fibers are very fine, have a beaded appearance, and are hi every respect different from the sensory nerve-fibers, which form the second kind of nerve-tissue. These sensory nerve-fibers arise as modified nerve-cells, and form the rods which connect with long bipolar cells by means of small dendritic processes. The bipolar cells join the large mar- ginal ganglia by means of nerve-endings in the ganglion. The marginal ganglia form a border of large ganglia cells around the retinal disc, the axones of the ganglia passing in the form of a cup-shaped case over the eye to form the optic nerve. The sensory nerve fibers and cells are surrounded by a hyaline sheath, which is absent in the efferent fibers. August j. The Static Function in Gonionemus. By Louis M-u REACH. The movements of the medusa Gonionemus would indicate that it has a definite sense of equilibrium, and some preliminary exper- im jnts confirmed this view. To localize this sense experiments 318 RESEARCH SEMINAR. were made to change the center of gravity by weights, etc. These were followed by the removal of different organs, the tentacles, tfie manubrium and the gonads, but without producing any marked disorientation. It seemed, then, that the otocyst organs to which this function is usually ascribed were thus shown to be active. At first, solution of the otoliths was attempted with various acids ; but it was found that all acids tried, if strong enough to dissolve the otoliths, also killed the animals. Then the vesicles were punctured, putting the otocysts out of function, but in the light of all the experiments there was no definite disorientation, except when the velum was severely mutilated. While this was not a proof that the otocyst organs do not function as the principal organs of equilibrium, the latter observation suggested that equi- libration in this medusa is, to a large extent, to be ascribed to " muscular sensation." Further experiments were finally made, cutting away the margin of the bell, including the bases of the tentacles and the otocyst organs. Intermediate portions of the margin were left until healing had taken place ; then the remain- ing portions of the margin were cut away and the a*nimal tested. Although the medusa was more or less imperfect from the opera- tion it was not seriously disoriented but moved in definite direc- tions, including swimming to the surface of a shallow dish of sea water and turning over, also lying with the opening of the bell turned up. From all the experiments it seems to follow that the otocyst organs serve very little in the equilibrium of the medusae, and that the muscular sensation is probably the principal factor. August 7. Nestling and Juvenile Plumages of Sterna hirundo and S. dougalli. By LYNDS JONES. The ventral downs of the nestling plumage are wholly white, except the throat, where the tips are dusky-black. All dorsal downs have a dusky-black base, then a tawny area, then a dusky-black area, and many a tawny tip. In the nestling hii- undo these dusky tips are arranged in mottled pattern, but in dougalli in stripes. Both patterns are protective, dougalli nest- ing mostly among grasses, hirundo originally on the beach among pebbles and seaweed. RESEARCH SEMINAR. 319 The juvenile plumages of the two species present the same general pattern, but differ in detail. In both the ventral plum- age is essentially pure white, doitgalli showing a faint rosy tint. All dorsal feathers are pure white at the base, then, except the remiges and rectrices, which are nearly like the adult feathers, a pearl-gray area, followed by a dusky area, and all feathers with a tawny tip. The inner tertiaries and their lower coverts, and the lower rows of scapulars, have an added area of tawny and of dusky colors. Thus the tawny and dusky areas of the nestling downs are reproduced in the juvenile feather, with the addition, at the inner half of the juvenile feather, of an area of pearl-gray and a white base. The pearl-gray area corresponds in color to the color of the adult feather. In hirundo these markings produce a barred effect, except on the head, which is white at the base of the bill, gradually dark- ening to black on the crown and occiput. In dougalli the outer tawny and dusky areas are parallel to the border of the feather, in the more strongly marked feathers, for fully a third of the length of the feather, and therefore present a more mottled pat- tern. Some feathers are even distinctly barred with dusky color about their outer third. The essential pattern of the two plumages is, therefore, a defi- nite barring of each feather on the dorsal surface, and a lack of any marking on the ventral surface. The greatly modified rem- iges and rectrices closely resemble the adult remiges and rec- trices, thus requiring no transition stage, but the rest of the dor- sal plumage undergoes a distinct transition from the nestling to the adult, the intermediate, or juvenile plumage, resembling the nestling plumage for its outer half, at least, and the adult plum- age for its basal half. August 7. The Internal Factors of Regeneration in Alpheus. By CHARLES T. BRUES. Alphcus is a small decapod crustacean in which one of the chelae is larger than the corresponding one on the other side. When one of the larger chelae is cut off at the base, it has been shown by Przibram, and also by Wilson, that its stump regener- ates a chela of the small type, while the originally small one is 32O RESEARCH SEMINAR. remodeled into a large one. Section of the nerve at the base of the small chela inhibits this reversal, wholly or partially. With regard to the internal factors which induce this reversal it seems at first sight that the nervous system must be the con- trolling one. Histological examination shows the following facts : (i) Nerves of the right and left sides are not morpholog- ically different except that they branch differently in the two types of claw. (2) During regeneration or remodeling changes occur in their definitive places, i. e,, growth does not occur at one end only. (3) The nervous shock of amputation may produce a weakness of the cut side, and also add to the nutriment of the other side of the ganglion, thus inducing reversal. (4) The nerve evi- dently carries the stimulus to grow, but may be only passive. (5) The cutting of the nerve in the claw may cause a general disease of that organ, such as might be caused by injuring the musculature, and thus prevent perfect reversal. It appears, therefore, that we must not attribute to the nervous system as much importance in the remodeling as would seem at first sight necessary when it is found that section of the nerve inhibits reversal. August 12. The Development of the Vascular System of Ce- ratodus. By WM. E. KELLICOTT. The vascular system of the adult Ccratodns shows resem- blances to both the elasmobranchs and the amphibia. It was thought that the investigation of its development might throw some light upon the significance of this curious combination. A few of the points of interest which appeared in the course of the study are the following : The heart develops similarly to that of the frog or Urodelc, from a pair of folds of the splanchnopleure, the somatopleure forming the pericardium. The branchial arteries also develop similarly to those of the amphibia. They are four in number and early form continuous passages from the ventral to the dorsal aorta. Later they divide into the afferent and efferent vessels and finally each efferent di- vides, forming the two efferent branchials characteristic of the adult. The vessels of the hyoid arch are well developed early, RESEARCH SEMINAR. 321 but later disappear. The vessels of the mandibular arch are only slightly indicated. The veins are arranged symmetrically during the early stages. The arrangement of the cardinal veins is typical. A well-marked subintestinal vessel is present very early ; later it disappears in part, and in part becomes connected with the hepatic-portal vein. The vena cava is for a time independent of the cardinal system ; later the anterior part of the right posterior cardinal disappears, while the vena cava connects with its posterior section. The pulmonary vein appears very late. Many of the features of the development of the vascular sys- tem are closely similar to those of the Urodela. Some, though not all, of the elasmobranch resemblances appear quite late in the process of development and are preceded by conditions which are typically amphibian. There are, however, typical elasmobranch characters present from the first. August 12. The Metamerism of the Nervous System in Areni- cola cristata. By C. P. LOMMEN. The arrangement of the nervous system of Arenicola cristata is definitely related to the annuli forming its somites. A pair of nerves from the ventral cord proceed dorsally between the longi- tudinal and circular muscles along each of the grooves that sepa- rate the annuli. These nerves give off numerous tiny branches, and do not unite dorsally to form rings. In the setigerous annuli there are two additional nerves which are imbedded in the circu- lar muscles, one on each side of the neuropodium. The posterior one divides into two branches, one passing into the gill and the other into the seta-sac. Anteriorly the number of annuli in the somites is gradually reduced from five to two. From the outside of each connective a series of eight nerves is given off, all of which are presumably homologous to the nerves from the cord. This homology has been shown with certainty only in the case of three. Of the remaining five, one innervates the otocyst, the position and muscular connection of which are suggestive of homology with a seta-sac. Fifteen nerves from the inside of the connectives give off some branches to the body-wall and then bend back into the wall of the pharynx, innervating its ventral 322 RESEARCH SEMINAR. and lateral sides, and passing then into the oesophagus. The dorsal wall of the pharynx is supplied by a plexus of nerve-trunks from the brain and the nearest portions of the connectives. The plexuses on the two sides of the body are seldom alike. From the posterior lobes of the brain several nerves pass to the nuchal organ. In the caudal region, the number of annuli in each somite, and with them the number of nerves, may vary from three in the anterior part to nine or ten in the posterior part. Augtist 14. The Organization and Orientation of the Ascidian Egg. By E. G. CONKLIN. During this summer I have studied the development of three species of solitary ascidians, viz. Styela {Cynthia) partita, Molgula Manhattensis and dona intcstinalis, with the purpose of finding out how much of organization can be recognized in the unseg- mented eggs of these animals. In the living eggs of all these ascidians, but particularly in the first mentioned, one can recognize the substance of the ectoderm, the endoderm and at least a portion of the mesoderm before the first cleavage occurs. The spermatozoon enters at the vegetative pole in an area of cytoplasm free from yolk and in Styela a dense mass of orange pigment aggregates at this spot and slowly spreads over the vegetative hemisphere. Subsequently it withdraws to one side of this hemisphere, thus forming an orange crescent which lies just below the equator on the posterior side of the egg. The vegetative pole then becomes slate-gray in color and the animal pole a light gray. The study of the subsequent development shows that all the axes of the future animal are now established and that the slate-gray substance forms endoderm, the light gray ectoderm and the orange crescent the muscular system of the tadpole. In Ciona and Molgula the crescent is present as in Styela, but is nearly colorless, while the substance of the ecto- derm and of the endoderm may also be recognized in the unseg- mented egg. An incidental result of this work is to prove beyond question than Van Beneden and Julin were right in their orientation of the ascidian egg and that the polar bodies are found at the ectodermal and not at the endodermal pole, as Castle has maintained. RESEARCH SEMINAR. 323 August ij. Rhythms of Susceptibility and of CO , Production in Cleavage. By E. P. LYON. Two years ago the author found that the Arbacia egg about fifteen minutes alter fertilization is very susceptible to lack of oxygen or to KNC. At other times during the first cleavage it is more resistant. This rhythm of susceptibility and resistance recurs in each succeeding cleavage period. Further investigation shows that cold acts like lack of oxygen. If the eggs are kept at about o° C. for a number of hours, those which are placed on ice about fifteen minutes after fertilization are much injured, and may wholly fail to develop, while little harm is done to eggs which have passed the critical stage before being cooled. This rhythm recurs in successive cleavages. Heating the eggs to 33°~38° C. for a few minutes reveals the opposite rhythm. They are most susceptible at the time of cleavage and are little injured ten or fifteen minutes after fertiliza- tion or at corresponding stages in the following cleavages. The CO2 output of a mass of eggs is greatest at the time of cleavage. At the time when oxygen is most needed, apparently little CO2 is produced. This shows that the oxygen, in all prob- ability, is needed for synthetic processes and that the CO2 is pro- duced by splitting and not by oxidation. The rhythm of CO., production can be demonstrated also in the second cleavage. August ij. The Voices of Pigeons. I. The Voice of the Ring Dove (Turtur risorius). By WALLACE CRAIG (demon- strations with the doves). Nearly five hundred species of wild pigeons are known, and, so far as observation goes, each species has a perfectly distinct and constant set of notes. These voices have had a common origin, and the problem is to discover this and trace the derivation of homologous elements. The work consists of two parts, a study of the voice in each species, and a comparison of different species and their hybrids. The former may be further divided into : (i) A description of the different notes, the attitudes which accompany each, and their whole significance in the life of the bird ; (2) the development of the voice in the young ; (3) a history of the seasonal changes in voice and behavior in the adult bird. The first and second of these subdivisions were reported upon in this seminar, and the 324 RESEARCH SEMINAR. main facts presented were as follows : The adult ring dove has only three principal calls, but these have a number of modifica- tions, which, together with many expressive movements, afford a considerable variety of expression. All these modes of behavior seem to be strictly inherited. They develop in the young bird very gradually, and in a definite order which is probably also the order of their development in the race. August 2+. Some Reactions of Mnemiopsis Leydyi (A. Ag.). By GEORGE WILLIAM HUNTER, Jr. Mnemiopsis orients itself with reference to gravity, being nega- tively or positively geotropic under differing conditions. It has two characteristic resting positions, one at the bottom with the aboral pole upward and one at the surface of the water with the oral pole upward. In strong and moderate intensities of light it may be first negatively and later positively photopathic ; to very weak inten- sity of light it may be positively photopathic. Some evidences of phototaxis are found under strong light, the aboral end being directed toward the light. The animal reacts toward a moderately strong constant cur- rent (one half to three volts) by turning the aboral pole toward the anode and moving to the cathode. A weaker current may cause orientation without movement toward the cathode. The action of the "make" and "break" upon muscles and cilia depends upon the position of the electrodes and the strength of the current. Mnemiopsis is relatively more resistant to decrease than to increase in the temperature of the water. Responses to electrical stimulation under conditions of greater heat than normal show decrease in reaction time up to about 29° C., then rapid increase in reaction time. Responses to electrical stimulation under condi- tions of decrease from normal temperature show little change in reaction time to about 15° C., then a slow increase in reaction time. August 24. The Reaction Time of Gonionemus Murbachii to Electric and Photic Stimuli. By ROBERT MEARNS YERKES. This experimental study of the time relations of the neural processes of Gonionemus indicates : (i) A reaction time to elec- RESEARCH SEMINAR. 325 trical stimuli of from .6" to 2.0" according to the intensity of the stimulus and the position of the organism ; (2) a shorter reaction time when the radial canal regions are stimulated than when the inter-radial regions receive the stimulus ; (3) rapid fatigue with repetition of the stimulus ; (4) a reaction time to photic stimuli of i" to 10" dependent upon conditions; (5) a much quicker reaction to light when the organism is resting subumbrellar surface uppermost (exposed to the light) than when the exumbrellar surface receives the stimulus ; (6) a specialization of organs for the reception of photic stimuli ; (7) the existence of highly irritable and conductile tissues (nervous system) ; (8) that excised organs rapidly lose their irritability ; (9) that varia- bility of reaction time for comparative work should be expressed in terms of percentage of the reaction time as well as in absolute terms. This relative variability may be called the variation coefficient. August 2j. The Establishment of "Association by Con- tiguity" in Hermit Crabs, Eupagurus longicarpus. By E. G. SPAULDING. Crabs are naturally positively heliotropic. They were taught to react against this by feeding them in a darkened portion of aquarium. Coefficient of daily improvement in eight days = 6.00. The darkening screen was removed each time after feeding. After seven days the crabs go behind the screen, when inserted, although no food is there. This proves the existence (a) of asso- ciation between two different stimuli and (£) that when one of these, the screen, is presented, the usual effect of the other is internally reproduced. This may be interpreted as conscious memory. August 24. The Resin Gnat and Three Parasites : A Study Made in July and August Under the Direction of Mr. Chas. T. Brues. By L. ECKEL. This study of Diplosis resinicola brought to notice three para- sites, among a number of additional facts about this insect, whose larval stage is well known to be passed within the lumps of resin that exude from various species of pine, and which pupates within the resin : i. Polygnotus pinicola, a proctotrupid which has been reported as parasitic upon Diplosis pini-inopis. It destroys many of the 326 RESEARCH SEMINAR. larvas of Diplosis rcsinicola in July, twenty transforming in one Diplosis larval skin. 2. Two Clialcis flies ; one a species of Syntasis, described now for the first time as Syntasis diplosidis, the second as yet unde- termined. They destroy many pupae of Diplosis rcsijiicola in early August. August 26. On the Artificial Creation of Mixed Nests of Ants. By ADELE M. FIELDE. Natural mixed nests of ants have been described, but such nests are always of ants belonging to the same subfamily, and not more than two species of ants ever inhabit the same nest. There are two ways in which an artificial mixed nest may be created ; one is by depriving all its residents of the sense of smell by removing the funicles of the antennas ; the other is by accustoming all the resident ants from their earliest hours to the odor of each kind of ant that is to occupy the artificial nest. If a nest not larger than a watch-glass be made, and one or more ants from each selected colony be sequestered in this nest, within twelve hours from the moment of hatching, these ants will each touch all the others with the antennas and will thus become ac- customed to and unafraid of the odor of species unlike their own. An ant reared in isolation will not affiliate with any whose odor differs from its own. Its criterion of correct ant-odor having been formed within three days after hatching, it continues hostile through life to all ants whose odor disagrees with its standard. But by the process of ant-education herein indicated, any ant may be induced to live peacefully with those of a different genus, or even of another subfamily. Several artificial nests were shown in which ants of four genera, or of all the three subfamilies, were living together, and in which young ants of onegenus were snuggling the queen of another genus. Were the ants in the artificial nests set free, their unlike re- quirements relating to temperature, humidity and food would soon separate them ; but it is improbable that these individual ants would ever fight with one another on subsequently meeting, although any of them would fight with ants of other colonies than those in which their early companions originated. MBL/WHOI LIBRARY UH 17JG