cphephal os uf Poin te ‘en sr of iS tw ot Seat K, Fc i Yay Behe e aty eaeerate rasdie tw . ae oeh Ge Haraperrens a hts aves te pop setada feta la tas) * } Haas Ppp Teeny Ree aa RD, 4 HE be i , ett. 7 % Bi Meg Ree aT 34s 4 Le Mirae ae f oe * preter rats ie: ahias Kee, ¥. iy rg Tele Foe apy : iigrere “1 aE ey pia ay Pattee Tega 7eae rttyeg 15F 2 i 1% wi) ys m1 y { 5 ay ey: Pi Neh ms pL ty) ay THE JOURNAL OF EXPERIMENTAL ZOOLOGY ' EDITED BY WILLIAM K. BROOKS FRANK R. LILLIE Johns Hopkins University University of Chicago WILLIAM B. CASTLE JACQQES LOEB Harvard University University of California EDWIN G. CONKLIN THOMAS H. MORGAN University of Pennsylvania Columbia University CHARLES B. DAVENPORT GEORGE H. PARKER Carnegie Institution Harvard University HERBERT S. JENNINGS CHARLES O. WHITMAN ; University of Pennsylvania University of Chicago EDMUND B. WILSON, Columbia University AND ROSS G. HARRISON Johns Hopkins University Manacine EpiItTorR . VOLUME I THE JOURNAL OF EXPERIMENTAL ZOOLOGY BALTIMORE 1905 A § CONTENTS. No. 1.—April, 1905. CHARLES ZELENY, Compensatory Regulation. With 29 figures..............:seeeeee eee | Amos W. PETERS, Phasphorescence in Cheniophores.:. .:.2'9-<. <.'6 4 << « ae eels sig woes ee o's 103 IsaBEL McCRACKEN, A Study of the Inheritance of Dichromatism in Lina Lapponica. With leplate ands hiplires'in the text,“ eeneu tn sian lerghee asaiaeh see eine 117 C. B. DavENPoRT, : Hivolubion- without: MAitation.. ii aie. so waeatete aig ddirp oie eaten oo oa enka oc 137 No. 2.—May, 1905. -= Epwin G. ConkELIN, Mosaic Development in Ascidian Eggs. With 82 figures................ 145 C. W. Haun, Dimorphism and Regeneration in Metridium. With 2 figures............ 225 Cuar.es G. RoceErs, The Effect of Various Salts upon the Survival of the Invertebrate Heart. NAGI PLUG ss to-scenc yy eles hob enehaiars Sacre eolaflsaretis 4 aut evolve caLietate 237 C. M. Cuip, Studies on Regulation. WII. Further Experiments on Form Regulation Bin Leptoplana. With 34 figures... . 0.0.0... secs e eee sees tee nese 253 ¥ NaonwE YarTsu, The Formation of Centrosomes in Enucleated Egg-Fragments. With 8 RUINS Se gee g tee Sn cts Se shattewise sans soels Sim Rca wa shepeteieks 287 No. 3.—August, 1905. N. M. Stevens, A Study of the Germ Cells of Aphis Rosze and Aphis Ginothere. With 4 POLICES Scio ie he oe Sistas nao etc Og aie oe cee) Saha’ - iw htne 6 ate ey en 313 N. M. Stevens Anp A. M. Borne, Regeneration in Polychoerus Caudatus. Part I. Observations on Living Material. “By IN. M: Stevens; With 21 figures. ..... 0.0.5.0. cosh wee 335 Part II. Histology. By A. M. Boring. With 2 plates and 1 figure in the COG) hee cant ROR Met RTE Bs eee oney sy a /ope Pm epspatvelans, wahdie se tate,« eccge sues, is oe Me 340 CHARLES ZELENY, The Relation of the Degree of Injury to the Rate of Regeneration. With CPTI S Peon tan care them tor lar Ome, Coie eee ae ONE oes tel ae Se 347 Epmunp B. WILson, Studies on Chromosomes. I. The Behavior of the Idiochromosomes in Eleni bers.- SWiHbL. 7 MIS UEES sp si ose oe ans. sno oe) want ceaye es oeiwin mets ag exe 371 G. H. Parker, The Movements of the Swimming Plates in Ctenophores, with Reference to the Theories of Ciliary Metachronism. With 2 figures................ 407 Henry Epwarp CRAMPTON, On a General Theory of Adaptation and Selection....................4- 425 WarrREN Harmon LEwIs, Experimental Studies on the Development of the Eye in Amphibia. II. On MINE Ones. VILA es MALES MEM cette x. crqictaccltin jon cis: 4< «baa wipe Sqciantnts eee 431 No. 4.—November, 1905. H. S. JENNINGS, Modifiability in Behavior. JI. Behavior of Sea Anemones............... 447 H. 8. JENNINGS, The Method of Regulation in Behavior and in Other Fields.............. 473 T. H. Morean, “Polarity ’’ Considered as a Phenomenon of Gradation of Materials....... 495 Epmunp B. Witson, Studies on Chromosomes. II. The Paired Microchromosomes, Idiochro- mosomes and Heterotropic Chromosomes in Hemiptera............... 507 Cuas. W. Harairt, Variations Among Scyphomeduse. With 1 plate and 17 figures in the GEG oc Sear coh meu tens staccato ee ea ore Maen ele gc ered yet ca eh altstiate tone aaa vas (Ra 547 LoraNDE Loss WoopRrvrr, An Experimental Study on the Life-History of Hypotrichous Infusoria. With 3 plates and 12 figures in the text... . 2.0.0.2... se eeeseneencces 585 COMPENSAVORY, REGULATION. BY CHARLES ZELENY. Generalalatrocuct arse vseve steerer ict cke nveices oecieraye/s!alel is toutlotne viele slots eis neiars 5 4 kPais 4 deh ae ee 2 Delia n.c0 ny do dobpsacadead AO sid HAbd oo NOD gaUSn OU DL Oe od Out Hood ot Obra e Sap EHO Eeae Hu.ceaae se 5 leeaihres Zeatletsrotathe: © ompoundsledhes reser ee ese nee sei ae ceils etes 5 II. The Rate of Regeneration of the Arms in the Brittle-star, Ophioglypha lacertosa.... 7 Me Mn trod uctiomer -\. ce crocs cite ticle ite ee ieee asatiots tinea cine: 2e cen Moree 7 Den Method iste ccc esses cee Sie Nee he. eile RAMEE eee bod: {en eee 8 Goi] DEES ire hee eh eae Sec aie eRe ere OOM OP oS GO Er a ois ek EE DCRR Pate co Skahics 14 APY AUDI CUSSION dope.) ol 2 s10) 6 102 Ge tect oe FAAS A acto ie tol Aa PS a MORN ae SREB ohn Bich MER Ne 16 Le TP her@ percula ofiSenpulidsii os 22-0 5 estas sure ce snes cvstte Tee eRe Seley a letenaters eRe ae 18 Toy COMParatlyevATIAtOMUY, s-po.cd.< Giattiss- lafosmsoeeheyal Ss dye pees ielibe emoreau! ware Ate ee 19 Tew Mhevpentis, Hy droid ese 4. Salstai.cne Ps ialt s1efeleletwiet co aoe hae ere etaa ee ae 19 2. Otherieneralohserpulidsiac. saa -)ta see ts dene re ee etree aes So Tees 3- Distribution of the Opercula between Right and Left Siders ce Aaa 32 4. Exceptional Degrees of Development and One Case of a Supernumerary @percultir jad asfe pak sass heroes eet Pe EL hice ie ican e Se 37 2. Development of Opercula— Tepe Ontorenetic Developments. sie stsrae ee restate is ie eee eee tence 38 Gsdintrod Uctiomy. 5). sis stat = ayes cana Gente atc Ser iets serine ees 38 b5.: Historical Review «stu st < ccrins scree tae tee ice Saeee sei de seleto eels 39 cw Observations s\.( jnece saisa/ cca ae eee eee une om OO eee aie Pee icrsaste 41 dy Summanyiol Data:and Discussion) cere teers oo acide fie si 2: Repeneratory..Development:4......2020qoh1 Seen ote oes foe deca taeise Dy ASA a lntroductionie crassa sree eae reer. debovecnss oboe doupecosnacage: 54 b. Unoperated Condition of the Opercula in H. dianthus................ 55 é-, OperationsionPunctionaly@ penelope sad srs< is =e) oi. ciate os foiale 55 a.) @perations on) Rudimentary; © perculumasn sare. Or 65 iy Wagoeimaeiahs Gre (CikaioyIN/cooccoa baddoaeebehu SmoeneOnnaabaIbds ooD bs 67 ope xh. 'B. Wilson and’to Prof: Tae Morgan, for inspiration and aid in carrying out the work, and to the members of the staffs at the three laboratories where it was done, for their uniform kindness in giving every convenience in the course of the investigation. The data will be considered in five sections as follows: I. In the first section (p. 5) the experiments on the leaflets of the compound leaf will be briefly referred to as constituting a case of regulation of a system in which there is no regeneration of the removed part. The readjustment 1 is here confined to the unin- jured portions and the assumption of an interaction between the members of the leaf constitutes an important factor in the explana- tion of the changes that take place. II. Inthe second section (p. 7) the rate of regeneration of the arms of the brittle-star, Ophioglypha, is taken up end studied with special reference to the influence which parts of the animal away from the injured surface have upon the nature of the regeneration at that surface. HI. The third section (p. 18) consists of experiments on the opercula of the Serpulids made with a view to the analysis of the factors involved in the control of the asymmetry of these animals. Observations on the comparative anatomy of the opercula and on their ontogenetic development made with special reference to the problem of compensatory regulation are included. IV. The fourth section (p. 77) contains observations on the regulation of the rate of differentiation in the regeneration of the opercula of the Serpulid, Apomatus ampullifera. ‘Uhe influence of the removal of the posterior part of the body upon the opercula istaken up. ‘The experiments are put into a section separate from the main one on the opercula of Serpulids because they are not directly concerned with the interaction of the two lateral sides of Compensatory Regulation. 5 the body, 7. e., with the factors controlling the asymmetry of the opercula, but rather with the influence of the presence or absence of the posterior regions of the body upon the process as a whole. V. The fijth section (p. 81) gives an account of experiments on the regeneration of the checks of the Decapod Crustaceans, Gelaannus. and Alpheus, with reference, jirst, to the problem of control of the asymmetry of the chelz in the male Gelasimus and in the male and female Alpheus; second, with reference to the general problem of the influence of parts away from an injured surface upon the character of the regeneration at that surface; and, third, with reference to the influence of the character of the opera- tion upon the moult period. Finally, all the data are brought together in a general discussion of the facts of compensatory regulation andtheir relation to the point of view which considers the organism as a system of mutually interacting parts (p. 96). DaTa. I. THE LEAFLETS OF THE COMPOUND LEAF. ‘The simplest instance of the application of the method employed in the present paper is furnished by the experiments on the com- pound leaf of the palmate type, as described in my paper on “The Dimensional Relations of the Members of Compound Leaves.” It will not be necessary to go into the details of that paper but a sample result may be of sale) because the case there described ished pure instance of change in the uninjured organs without regeneration of the injured one. ‘The main point of the experi- ments may be briefly illustrated by the following quotation from the introduction as given there: The individual members of the compound leaf as well as of other parts of the plant respond to stimuli in a definite way. Each member is, however, limited in its reaction by its mechanical and organic relations to the other parts of the leaf. This limitation is mutual and as a result of it we get an equilibrium of forces which results in a configuration more or less definite for each species. As a further conse- quence each member must respond not as a unit but as part of a system. If now we have a system of this kind with a definite configuration due to the mutual inter- action of its members and we remove one of the component parts, we must get a disturbance of the equilibrium leading to changes in the relations of the remaining 6 . Charles Zeleny. parts limited only by the extent to which the rigidity of the skeletal structures may counteract such a tendency. We may in this manner get at the forces which are active in correlation at the time of and subsequent to the operation. The main difficulty with the method must consist in the reaction to the stimulus of the injury itself, a factor which does not enter into the normal relations of the parts. A sample result will illustrate the method in a more concrete manner. When an asymmetrically placed leaflet of a five-leaved form (white lupine or Virginia Creeper) is removed at the earliest possible stage, the remaining four leaflets take up positions Fic. 1. Virginia Creeper, Parthenocissus quinquefolia. Diagram of changes in position and length of leaflets of compound leaf after removal of one of their number (<4). Unbroken lines—Original positions. Dotted lines—Resultant positions. Barred lines—Removed leaflet. P—Petiole. Arrow—Direction of movement. A, B, C, D, E—Original positions. 41, By, C;, E;—Resultant positions. which tend to approach those of the units of a symmetrical jour-leaved system, the chief change of position being confined to the two leaflets which were asymmetrically placed after the opera- tion. In the case of the Virginia Sa: the resultant — of position was +5°.9 for leaflet A, +12°.2 for leaflet B, +24°.4 for leaflet C, and —3°.2 for leaflet E. (See Fig. 1.) Likewise in a three-leaved system (the red clover) after removal of an asym- Compensatory Regulation. 7 metrically placed leaflet the resulting two- leaved system tends to be symmetrical with respect to the petiole. This shows very definitely that the norma] symmetrical palmate leaf has a definite configuration as a result of the interaction of its units and that the manner of this interaction may be revealed by the removal of a unit, an operation which brings about a new mode of interaction leading to a new resultant state of equilibrium. Likewise after such an operation, which is performed at an early stage before the leaflets have fully unfolded, the remaining leaflets do not attain their full normal size, the average decrease for the leaflets of the three species being 6.8 per cent of the normal length. These changes in position and length for the Virginia Creeper are shown in the accompanying figure. (Fig. 1.) In the following sections the experiments have to deal with more complicated cases because there is a regeneration of a new organ or organs in place of the old, and the changes in the system are the result not only of the interactions of ‘ie: uninjured parts but also of these upon the regenerating part and in turn of the latter upon the former. Il. THE RATE OF REGENERATION OF THE ARMS IN THE BRITTLE- STAR, OPHIOGLYPHA LACERTOSA.' 1. Introduction. In this section we have a case of evident influence of organs situated away jrom a regenerating surface upon the character of the regeneration at that surface. The influences exerted by the regenerating organ or organs upon the character of the uninjured organs, or the changes produced among the uninjured organs inemeelves by the new interactions resulting from the new con- ditions, were not studied because the material was evidently unsuitable for that purpose. The experiments to be described are, therefore, concerned entirely with the rate of regeneration of the arms as influenced by the number of arms removed. Inci- dentally the variation of the rate of regeneration of the arms with the size (1. e., age [ t]) of the animal must be considered. The principal results of this section were given in a preliminary paper already mentioned. (Zeleny, ’o3b.) | 8 Charles Zeleny. The experiments were performed at the Naples Zodlogical Station during the winter of 1902-03. [he common five-armed brittle-star, Ophioglypha lacertosa, was used and five series of experiments, with one, two, three, four and five arms removed, respectively, were kept for 46 days after the operation and no food was supplied to them during the whole period. The resulting data show that the rate of regeneration of the arms varies on the one hand with the size of the animal and on the other with the number of removed arms. Medium-sized individuals show the maximum rate of regeneration and there is a pronounced decrease both for smaller and for larger ones. ‘The second corre- lation and the one that concerns us especially in the present paper gives an increase in the rate of regeneration of an arm as we pass fom the cases with a smaller to those with a greater number of removed arms. ‘(he series with all five arms missing is excepted in the statement because the animals in this lot in every instance died or showed evidences of decay before the completion of the experiment. 2. Method. Forty-five perfect specimens were divided into five equal groups of nine each, care being taken to distribute them in such a way as to make the sets approximately equivalent as regards size of individuals. ‘he operations consisted in the removal of one or more arms by a transverse cut at the disk level. In the first series one arm was removed, in the second two contiguous arms, in the third three contiguous arms, in the fourth four, and in the fifth five arms. [he animals were kept in ten “battery” jars, two for each series and were not fed during the whole pertod of the experiment. Measurements of the lengths of the regenerating arms were taken 22, 33 and 46 days after the operation. As “stated above, the specimens of the series where all five arms were removed did not retain their vitality for a sufficient length of time to allow of com- plete comparison with the others, and they will therefore not be included in the main comparison, though the data concerning them are given in Table I. The results are given in the accompanying table (Table [), which shows for each of the five series the disk diameters of the specimens and the lengths of the regenerating arm or arms 22, 33 and 46 days after the operation. Where more than one arm Compensatory Regulation. TaBLe I. Series I—One Arm Cut Off. Series II—Two Arms Cut Off. Specimen Bis ay || 1B | 46 Berens Spee Disks 225133 \tva6 | eens No. Diam.} days | days | days | No. | Diam. days days | days | I 4.8 Bh | fr Ap ee a) | I 6-0) | —— | = | | dead (22) 2, Gee, gsi set ||) U0 2 | 6.5] 55) -45 0 | 3 6.6 | 1.4 | 2.4 | 3-0 | 3 N27 a 5L.9) | 1.95/20" 4 TO) |) 226) ||) 2-0) |) 2.0 | 4 [LORS 0 tS TG .65| } | | | 5 ites | eon 3eo) | -Aso 5 ere Oi eet Olt ——ilideat(22)) 6 13).2 2 | 2501-3 04 | 6 Teese Cates | ok 7 | 13-5 | — | — | — | dead (22) 7 TAO} Tes | 2203.30 Se) ges | T-04, — | — dead (3) 8 Ego" 85) 2.6 | zon) 9 letig-8; || <0.) 20 0" 9 LOeayieeeOn |) ZN ine-Ou| Series I1I—Three Arms Cut Off. Series [V—Four Arms Cut Off. Specimen Eigs 22,1) 33)) ||) 46) | eae Specimen} Disk | 22 | 33 | 46 Rens No. | Diam. days | days days He SIE Diam. days | days | days I Bab — | — | dead (22) | I [o heQr iP OR iATCOn lt rubs 2 6.5 | .6| 1.05). — | dead (46) | 2) || 6204) (7.00), 0765) 0.9 3 90 |= | — | — | dead (22) | 3 | 8.2 | 2.45) 4.6 | 6.2 4 | 11.0 | 1.15 12 75\| 93.25) 4 | 11.3 | 1.85) 3.65) 5.3 | 5 12-5 1.35 2-35) 3-2, 5 Walzeege WezioU Gd a ees Greil aes) 2.254. 261) 6.65 6 r208)| 16) 2.0 || 14.05 Fer, ata Sale orl 3 oa | 74.90) fe 12.8 |) ©.g5) 2-9) e855 8 | Ley igte || Teepe eid 8 Vu ttefeede) SitsG ils eel Ae fad lls Lay 9 | A@sO) |) Sey) SGI) dicted 9 meet || Texte) | teay iis) Stay Serres V—Five Arms Cut Off. Specimen | Disk (a 46 | Pssnardes No. Diam. days _ days | days I Goal: 268s at oe _ dying (46) 2 6.5 | PONE | .o | dying (46) 3 7.0| .6| .46| .o | dying (46) 4 aren | 2.26 2.86) 2.66 5 tog — | — > — | — | dead (22) EXPLANATION OF Taste I. The data for the experiments on the regeneration of the arms in Ophioglypha lacertosa after their removal by a cut level with the circumference of the disk. The measurements are in millimeters. In each of the sub-tables the second column gives the disk diameters, the third column the average lengths of the regenerating arms 22 days after the operation, the fourth column the lengths after 33 days and the fifth column after 46 days. In the four last tables (7. e., in all except Series I) the lengths as given are the averages of all the regenerating arms of the individuals. The numbers under ‘‘Remarks”’ represent the time of death (in days) of specimens which did not outlive the experiment. IO Charles Zeleny. was removed, 1. ¢., in all except the first series, this length repre- sents the average length of all the regenerating: arms of the individ- ual. In some specimens there is a decrease in the length of the regenerated arm from one period to another. ‘This is due to a gradual disintegration and breaking off of the regenerating arm, Wp visy ale atsye io al7( salty aly 2A) oT . lees T t Ti r 22 AL ~_— 4 ———}- days. \y Ne See 7, a eee oe Ns A ' SSS z4 : ies a ve | | | | | | | 33 A Z - = oe days. 5 : “EF Well | 7 Pasar gy ia | | 3 wa | Pies | 5 3 7 | += ‘et + all i : Hi = 4 at Se BE 2 = ion SeStiG asi =i i —o =i Bere ek | ; | | | Za g 1 + oe = | = } ~ - BE Vf \ a = 0 | S | 5 9 | Je Ht Is | 4 =e is & ere | | eee fave [ | | = 74 ecole + + —t =: Al = ft tt — + + = aM a 64 } iE 5 aia 7 ute i i 4 / | rs \ “nN 5 1 UN es ae wa Baile ie eel <(-toae i | gan | ice 1 Me = =f eae ++ 4 al 4 + oe ays. / | rf uv Sut + s! iyi + — = i = = 7) . if sr | a 4 AEA | | lies | ! if 0 d 4 5 6 7 8 9) TOT 2 Zs 1S les low Ges als hoe 20 Disk diameters in millimeters. Fie. 2. Brittle-star, Ophioglypha lacertosa. Comparison of lengths of regenerated arms in Series IV (four arms removed), dotted lines, with Series I (one arm removed), unbroken lines. Upper curves, 22 days; middle curves, 33 days; and lower curves, 46 days after operation. The lengths of the regenerated arms are distinctly greater in Series IV than in Series I. Compensatory Regulation. ial it process which begins at the distal end and is usually accom- panied by a loss of vitality with the approach of general bodily decay. In Series V, where all five arms were removed, none of the specimens retained its full vitality for the whole 46 days, though one did so for 33 days. A glance at the table (Table I) is sufficient to show both of the main points brought out in the general statement of the results. In the first place the rate of regeneration of the arms is greatest in medium-sized individuals, decreasing for both the smaller and the larger ones and second, the rate of regeneration is dependent on the number of arms removed. Excepting Series V, where all the arms are removed, there is an evident increase in the rate of regen- eration of the arms as we pass from the cases with the fewer to the cases with the greater number of removed arms. As both these factors enter into the individual measurements the proper relations can best be represented graphically by means of “curves.” Such “curves” are shown in Figs. 2 and 3 and the relative rates of regeneration of the arms are also represented for two typical individuals in Fig. 4, which gives two specimens, one from Series I (one arm removed) and the other from Series IV (four arms removed). In each of the individuals in Fig. 4 the condition of the arms 46 days after the operation is represented. Each of the four removed arms of the individual from Series 1V has evidently regenerated more rapidly than the single removed arm of the individual from Series I. In Fig. 2, Series I and IV, with respectively one and four arms removed, are compared. ‘The individual cases are shown by dots. The abscissz give the sizes of the animals as represented by the disk diameters in millimeters. The ordinates give the lengths of the regenerating arms also in millimeters. In the series where more than one arm was operated on the regeneration length as given is an average of all the regenerating arms of the individual. The individual cases of each series are connected by lines so as to give a basis for comparison of the two groups. ‘The unbroken lines represent the lengths of the members of Series | and the dotted lines those of the members of Series [V. The upper two curves give the measurements as taken 22 days after the operation, the middle pair those at 33 days and the lower pair those at 46 days. In each group the curve showing the rate of regeneration of the arms in the series with four arms removed is well above the 12 Charles Zeleny. one with only one arm removed. Fig. 2 likewise shows the increase in the rate of regeneration of the arms as we pass from the individuals with a smaller disk diameter to those with a medium disk diameter (about 12 mm.) which show the maximum rate. Then there is a decrease in rate of regeneration until indi- i 8 9 10 1G pale wiley alee aS MG ae ak ake) Lengths of regenerated Arms in millimeters. 7 8 9 10 sll ale wales Se alias aU alr > shy 2) Disk diameters in millimeters. Fic. 3. Brittle-star, Ophioglypha lacertosa.° Comparison of lengths of regenerated arms in Series III and IV combined (three and four arms removed), dotted lines, with Series I and II combined (one and two arms removed), unbroken lines. Upper curves, 22 days; middle curves, 33 days; lower curves, 46 days after operation. The regenerated arm lengths are greater in Series IIT and IV than in Series I and IT. Compensatory Regulation. 13 viduals of 19 or 20 mm. are reached when the regeneration length for 46 days arrives at a second minimum. A similar relation between rate of regeneration and disk diameter is shown in Fics 3 In Fig. 3 all the data for the Series I, II, [1 and IV are included. It was not, however, possible to put in the individual measure- ments without confusing the general effect of the curves. “There- fore Series I and II are combined in one curve (the unbroken Fic. 4. Brittle-star, Ophioglypha lacertosa. Left figure—Typical specimen with one regenerating arm. Right figure—Typical specimen with four regenerating arms. Both 46 days after operation. In left specimen two unoperated arms are shortened for lack of space in figure ( X 1/5). line) and Series III and IV in another (the dotted line). As the individual disk diameters are not exactly equivalent in the different series, it was found convenient in taking the averages for the com- bination curves to use arbitrarily disk diameters equal to whole millimeters as the points for comparison. The values for such disk diameters are obtained from the separate curves constructed from the individual measurements for each series according to the method shown in Fig. 2. The average curve for Series I and LV in Fig. 2 would be one equidistant between them. ‘The combina- tion curves for Series I and II and Series III and IV, as given in Fig. 3, are constructed on this basis. “The unbroken line gives 14 Charles Zeleny. the average of the former group (one and two arms removed) and the broken line the average of the latter group (three and four arms removed). 3: Data. The curves show very distinctly the correlation between the rate of regeneration on the one hand and the size of the animal and the number of removed arms on the other. 1. [aking up first the size correlation and using Fig. 3 as our basis of comparison, since it contains all the cases except those of Series V and therefore gives a more uniform and complete curve, we find that starting with the smaller individuals as we advance toward the larger ones there is a general increase up to a maximum at a diameter of 12 to 15 mm. This is most striking in the two later measurements, taken 33 days and 46 days after the operation. Thus in the 33-day measurement for Series I and II (Fig. 3), the regenerated length increases from 1.07 mm. for a disk diameter of 7mm. to a maximum of 2.37 mm. for a disk diameter of 14 mm., and then goes down to .21 mm. for a 1g 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 18 mm. The medium-sized individuals thus have the maximum rate of regeneration.! 2. More striking still is the very constant difference between the regenerated lengths for Series I and those for Series IV in Fig. 2, and between the lengths for the combination of Series | and II and those for the combination of Series III and IV in Fig. 3. This shows a very decided advantage in favor of the animals with the greater number of removed arms. ‘The difference 1s evident in the upper curves of Fig. 3 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 12 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 and II, and of 3.45 mm. for Series 1 Dr. Hans Przibram has called my‘attention to the fact that the specific rate of regeneration of the arms, 7. e., the amount of regeneration per unit of disk diameter as obtained from my data, does not show this increase from the smallest up to the medium-sized indivjduals, but gives a fairly constant figure up to 12 or14 mm. ‘The higher diameters then decline rapidly toward a minimum. Compensatory Regulation. 15 III and 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 and IJ has its maximum regeneration) we get 2.37 mm. for Series I and II and 2.77 mm. for Series III and IV, an advantage of .4 mm. or 17 per cent in favor of Series [I] and IV. Ina similar manner in the curves obtained from the 46-day measurements we get at a 12 mm. disk diameter a regenerated length of 2.46 mm. for Series I and II and 5.42 mm. for Series III and IV, and at a 15 mm. diameter 3.14 mm. for Series I and II and 3.72 mm. for Series III and IV, which represents an advantage for the group with the greater number of removed arms of respectively 2.96 mm. (=120 per cent) and .58 mm. ( = 18 per cent) for the two points named. The difference between Series I and Series IV as represented in Fig. 2 is still greater. “Lhe regenerated lengths are on the whole at least twice as great in Series [V where four arms were removed as in Series I where only one arm was removed. ‘Thus at a disk diameter of 8 mm. the regenerated length in Series I is 1.05 mm. and the average regenerated length in Series IV is 2.3 mm., an increase of 1.25 mm. or II1g per cent. For the same diameter at 33 days the respective values are 1.65 mm. and 4.4 mm., an increase of 2.75 mm. or 167 per cent. At 46 days the correspond- ing values are 2.0 mm. and 5.85 mm., an increase of 3.85 mm. or 192 per cent. Likewise at a 12 mm. disk diameter for 22 days the values are .Q mm. and 2.1 mm., an advantage of 1.2 mm. or 133 per cent. At 33 days the values for a 12 mm. disk diameter are 2.3 mm. and 4.2 mm., an advantage of 1.9 mm. or 83 per cent, and at 46 days the corresponding values are 3.05 mm. ae 6.5 mm., an advantage of 3.45 mm. or I13 per cent. We may sum up the 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 (7. ¢., age Lem of the animal and the rate of regeneration of its arms. T he mas 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. fie ereater the number of removed arms (excepting the case where all are removed) the greater 1s the rate of regeneration of each arm. 16 Charles Zeleny. A+) Diseussvon: 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 regen- erative energy greater in this case, but the energy expressed in each arm is greater than the total energy when only one arm 1s removed. Expressing this in mathematical form, if E, represents the regenerative 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, aad En represents the energy exhibited in regeneration when more than one arm is removed, 7 being the Aunnber of absent arms, then not only is En > E, but also © > E, or Ex > n E, Therefore, when we remove 7 arms we increase the total regenera- tive energy by more than 7 times the amount exhibited 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- Stak with arms 4, .65-C, D andl, im which apo psee amen, represent the respective lengths these arms will attain after a definite period of regeneration, supposing that one alone 1s 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 a,>a,, b,>b,, c4>¢,, d,>d,. Now in the first case we cannot assume that the stimulus of removal and the resultant reaction of regeneration are purely local and concern only the tissues in the crmmediate 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 (r,) to each of the original pe regenera- tion lengths to get the new regeneration length, CLUS a quer ‘Then Pei cee ay ee +¢e,+d,+R, hese Re, (ir) represents the total response of the organism as a whole which must be added to the local effects of the operation stimulus. If, on the other hand, we consider the influence of the organism as a Compensatory Regulation. L7 whole on the regeneration of its arms as one of retardation, we must take the values a,, b,, c, and d, as representing most nearly the original local stimulus effect. “Then without changing the values of r, or R, we may rearrange the formulz, making a, = a,— r, etc., and a,+6b,+c,+d,=a,+6,+¢,+d,—R,. The regeneration of the arms of Ophioglypha thus offers us a very good example of the influence of conditions away from an injured surface upon the regeneration at that surface. The result may be stated in two ways and each mode of statement may be made to lead to a separate mode of interpretation. We may say that the rate of regeneration increases with an increase in the number of removed arms. With this statement as a starting point it is natural to assume that, in the cases where more than one arm is removed, the stimulus of the additional operations or of the additional regenerating organs exerts an accelerating influence upon the regenerating tissues at any one such ihre: Another mode of statement is the following: The increase in the number of removed arms is necessarily accompanied by a decrease in the number of uninjured arms present, and the rate of regeneration of a removed arm therefore increases as the number of uninjured arms still remaining decreases. If the uninjured arms exert a retarding influence upon the regenerating tissue at an injured surface we can understand why a removal of additional arms may bring about an increase in rate of regenera- tion of each. The discussion of this interpretation involves the whole problem of nutrition and perhaps the whole general problem of form regulation as well. It will be best to reserve further dis- cussion until we have examined the other experiments to be described in the following pages. 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 as determined by the direct action of the operation. It seems, on the other hand, to be bound up with intricate reactions affect- ing the whole character of the activities and organization of the animal. ba 4) Charles Zeleny. Hil. THE OPERCULA OF SERPULIDS. We have now considered a case (section one) in which there is a readjustment in the uninjured portions of a system as a result of their mutual interaction. This interaction is not complicated by the addition of a regenerating organ. The result is a new system in equilibnum, based on the resultant of the interactions of the uninjured parts. In a second section (section two) a case was considered in which it was possible to study the effect of the presence or absence of uninjured portions of the animal upon the rate of regeneration of the removed ones. The reaction is here more complicated than in the first case, because there may be here an action of other regenerating surfaces upon any particular wounded surface as a as the action of the uninjured organs themselves. . In the present section (section three) a case will be considered in which there are two organs, dissimilar in size, situated on mor- phologically similar opposite sides of the median line. An extremely close interaction is found to exist between these two organs so that any disturbance im one is refiected in changes in the other. This close interrelation between the opercula of the Serpulids, the organs in question, gives a good basis for the study of such interactions as were outlined in the general introduction. The opercula of the Serpulids furthermore furnish exceptionally good material for this study of compensatory regulation because of the various degrees of asymmetry present in the different species. In the following account it will be necessary to go into paths not in the line of the main discussion, but such a course cannot be avoided in a study of the factors controlling the regulation of the opercula. In the adult Serpulids of the genus Hydroides we have an asymmetrical stable system with the functional operculum on the right side and the rudimentary operculum on the left or vice versa. The nature of this case will first be taken up. Then the opercula of other members of the family will be described. This will be followed in turn by a description of the ontogenetic development, the regeneratory development, some speculations as to the prob- able phylogenetic development, a discussion on the comparison of regeneratory, ontogenetic and probable phylogenetic develop- Compensatory Regulation. ie) ment and finally by a discussion of the facts of compensatory regulation as here exhibited. In a separate section (section four) a special series of experi- ments on the regulation of the rate of differentiation of the oper- cula in the Serpulid, Apomatus ampullifera, will be treated. ae Comparative Anatomy. 1. The Genus Hydroides. The opercula and branchie of the genus Hydroides will serve as the type in our description of the anatomy of these structures throughout the family. The branchiz are brought in only incidentally as our main purpose is to get the details of the struc- ture of the opercula to serve as a basis for the regeneration and regulation experiments to be described later. Unless otherwise stated the description applies to H. dianthus. H. uncinata and H. pectinata, living in the Mediterranean at Naples, were also used in the experiments and the differences will be pointed out at the close of the special description. H. dianthus is found on the Atlantic coast of North America living attached to stones, mollusk shells and other hard materials, from low water mark to a depth of several fathoms. The worm lives in an irregularly twisted calcareous tube which is attached by its side to the supporting surface. [he tube increases in size from the posterior end anteriorly and is continually being built up at the anterior end by additions from the special fale eee glands. The body of the animal is very distinctly divided into the thorax and the abdomen. The first-named region is marked by the presence of the broad, flat fold of the thoracic membrane, which is continued at both the anterior-ventral and the posterior- ventral ends as a projecting membrane. At the anterior end this membrane forms a collar which, except for a slight break on the dorsal side, completely surrounds the head end. (See Fig. 5a.) Upon the anterior surface thus enclosed by the collar are isc the two semicircular rows of branchiz, one on each side of the mouth, which apparently serve on the one hand as organs of respiration and on the other through their cilia as agents for the creation of a current of water carrying food particles to the mouth. The two rows of branchiz are placed on slight ridge-like eleva- tions, the branchial ridges. These are not strictly semicircular 20 Charles Zeleny. in shape but the ends of each are curved inward. ‘This shape evidently has some connection with the proper collection of the food particles carried downward by the cilia. (See Fig. 58, c.) The number of the branchiz increases with the age of the animal and in fully grown individuals there are about fourteen on each VW Y \\ \ \ WS \\ NIN Fi@. 5. Hydroides dianthus. A—Dorsal view of right-handed specimen, showing relations of parts. Ends of branchiz and functional operculum not given (<6). B, C—Diagram of anterior surface of head of left-handed and right-handed specimens (6). D—Branchia viewed from inner surface (X25). F—Rudimentary operculum (30). 4—Functional operculum ( X 30). Compensatory Reguiation. 21 Each branchia consists of a long axis bearing two rows of secondary processes, the pinnules. (See Fig. 5p.) The axis is continued for a short distance beyond the region of the secondary processes as a slender tapering thread. ‘The pinnule rows slope inward so as to inclose a trough-like area, V-shaped in cross section and with the cavity of the trough pointing inward, 7. e., toward the mouth. ‘The surfaces bordering this area are ciliated and it is along them that the food-bearing currents are formed. Near the dorsal end of one of the branchial ridges, not in the line of the branchiz but dorsal to it, there is a stout, naked stalk of approximately the same length as a branchia but bearing at its distal end a funnel-shaped expansion. (See Fig. 5r.) The whole organ constitutes the functional operculum. ‘The edge of the expanded portion is marked by teeth-like serrations, the hollows between which are continued for some distance down the outside. From the center of the terminal circular area within this row of serrations there arises a group of secondary pro- cesses, arranged so as to form a cup-shaped figure. The ends of the processes are usually hooked and considerable foreign material often clings to them. The whole organ serves as a very efficient plug for the open end of the tube when the animal has retired within for protection. An examination of the place of attachment of the opercular stalk shows that it is located dorsal to the first branchia or sometimes nearly opposite the interval between the first and second branchiz. Near the base of the stalk there is a transverse suture varying in distinctness in different cases and which, as we shall see later, is a “breaking joint,” an important structure in the experiments. On the opposite side of the mid-dorsal line, and in a position corresponding in all respects with that of the large operculum, is a small organ consisting of a slender stalk with a slight terminal enlargement. (Fig. 5£.) It also shows a distinct line of demarca- tion between a darker colored more basal region and the lighter remainder of its body. ‘This small organ, the “ pseudopercule’’ of de St. Joseph is most commonly called the rudimentary oper- culum. A study of the relative positions of the opercula 1s interesting. An examination of 244 adult individuals of H. dianthus gave 139 or 57 per cent with the functional operculum on the right side and 105 or 43 per cent with it on the left. The distribution between 22 Charles Zeleny. right and left is thus fairly equal though there is a considerable advantage in favor of those with the functional operculum on the right side and the rudimentary on the left. Similarly in H. unci- nata out of 16 specimens ten had the functional operculum on the right side and six on the left, and in H. pectinata out of 41 speci- mens 21 were right handed and 20 left handed. An examination of the internal structure of the branchiz and opercula brings out a close agreement between the two in ana- tomical details. “Uheir morphological agreement has been espe- cially emphasized by Orley and Meyer. Orley (’84) compares the internal anatomy of the branchia and the functional operculum in Serpula. He makes no mention of the rudimentary operculum. According to him an operculum corresponds morphologically with a branchial stalk, all the pinnules of which have been col- lected at the end in one bundle. He describes the presence of an axial blood vessel in both branchial and opercular stalks. In the branchial stalk, however, he saw only one nerve trunk (the axial one) while in the opercular stalk two lateral ones were shown. Meyer (’88) showed the more complete similarity of the branchia and operculum in Eupomatus uncinata (= Hydroides uncinata), while at the same time pointing out the incompleteness of Orley’s observations and the error in his mode of homology. He describes three nerve trunks in both branchiz and operculum, although the middle one is very small in the opércular stalk and does not reach much more than halfway to the distal end. In the branchia the two lateral ones likewise are very insignificant. Meyer points out that this difference is probably due to the fact that the pinnules and ciliated groove are innervated from the niiddle nerve, so that this has a greater development in the branchiz where pinnules and ciliated groove are present than in the opercular stalk where they are absent. ‘The stalk of the operculum is thus made directly homologous with a branchial axis lacking its pinnules. A study of the internal structure of the branchiz and opercula of Hydroides dianthus brings out points which are in entire agree- ment with the conclusions of Meyer and which emphasize the close similarity of the branchiz and opercula. An interesting characteristic is further made out in the func- tional operculum. ‘There is a difference between the cells near the basal region and those in the middle and terminal regions of the stalk. Near the base of the stalk in the region below the Compensatory Regulation. 22 “breaking joint” the cells of the connective tissue have more of an embryonic character than elsewhere, having fewer and shorter processes and less intercellular material. ‘This distinction has already been noted by Orley (’84) in his description of the con- nective tissue of the opercular stalk in Serpula vermicularis. He says, speaking of this tissue, “Die Modificationen dieses Bindeyewebes sind nach den Ortsverhaltnissen sehr verschieden. Im innersten Theile des Stielcs wo dieser mit dem Kiemenlappen zusammen hangt, findet man kleine weniger verzweigte Zellen in der sehr sparlichen Intercellularsubstanz. Es abnen sebr der embryonaler Form. Etwas hoher trifft man bereits Zellen an, die sich durch Grosze und durch die Zahl ihrer Ausliufe auszeichen und eine gut entwickelte Intercellularsubstanz haben.”! The significance of the differences of the regions will be brought out in connection with the experiments described later in the paper (Pp. 55). he rudimentary operculum of H. dianthus has two well- defined regions. ‘The cells distal to the “breaking joint” are distinctly embryonic in form and general character. Those proximal to the breaking joint have among them well-developed supporting cells of the type found in the branchiz and functional operculum, though these cells are not as highly differentiated as in the latter organs. A comparison of the two other members of the genus Hydroides with H. dianthus brings out only slight differences in the charac- ter of the opercula and branchie. H. pectinata, however, has pectinate secondary processes as opposed to the unbranched ones of H. uncinata and H..dianthus. Any conclusion drawn from direct anatomical evidence must emphasize a very close resemblance in the internal structure as well as in the position of the opercula and branchiz. A similar conclusion as regards the morphological worth of the rudimentary operculum can be reached by a recognition of similarity in position on the one hand and the nearly equal appearance of right and left- handed individuals on the other. The functional operculum in Hydroides is therefore morpho- logically a branchia which has formed an expansion at its distal end and which has at the same time lost its respiratory pinnules. Mtalics mine. 2.4 Charles Zeleny. The increase in strength of the supporting axis is a necessary con- comitant of the other changes. The rudimentary operculum cannot be compared directly with a branchia because of its bud-like appearance and embryonic tissues. In position it, however, corresponds perfectly with the functional one and therefore with the branchiz as well. 2. Other Genera of Serpulids. An examination of the different groups of Serpulids brings out the fact that we have almost all gradations between forms with no opercular modification of the branchiz and forms with the single operculum possessing scarcely any trace of a branchial character. a. Group I. No Opercular Differentiation. Examples of Serpulids with no opercular modification of the branchie are Protula (Risso) and Protis (Ehlers). Each branchia possesses respiratory pinnules and tapers to a point at its distal end. ‘The branchiz resemble one another throughout both right and left circlets. “Che members of this group are able to retreat for a lon distance back into the tube, in this respect resembling the Sabellids which also have no opercula. (See Fig. 6.) b. Group II. Each Branchia with a Terminal Enlargement. In Salmacina Dysteri Huxl. there are eight branchiz, four on each side and each has a terminal club-shaped enlargement. ‘The branchial stalk or axis has from fifteen to twenty pairs of ciliated pinnules. ‘lhe two rows of pinnules are bordered on the outside by enlarged mucous cells which near the distal end spread out along the sides of the club. ‘This enlarged region bears no pinnules. The eight branchiz are similar in their characters. It is evident that when the animal retreats into its tube these enlarged ends must collectively serve as a stopper for the opening and thus barri- cade the end more effectively than those of Protula which bear no = 1 such enlargements (Fig. 68). c. Group III. Two Equal Opercula, Right and Left, on Ends of Branchie. Branchial Pinnules Present. Filograna implexa resembles Salmacina in having eight branchiz. ‘The dorsal one on each side is, however, terminated by a small, transparent, chitinous, spoon- shaped structure obliquely attached to the side of the tip of the axis of the branchia. ‘The other branchiz end ‘de St. ae however, seems unwilling to admit an opercular function for these structures. Compensatory Regulation. 25 in short blunt points. ‘The two opercula are equal in size and the stalks which bear them retain the pinnules and other branchial characters (Fig. 6c). When the animal has withdrawn into its tube the branchiz are twisted in spiral form and the two opercula are superimposed, the one upon the other. The more anterior Fic. 6. A—End of branchia of Protula. B—Club-shaped end of branchia of Salmacina (after de St. Joseph). C—One of the two opercula of Filograna (after de St. Joseph). D—Tip of non-operculate branchia of Apomatus ampullifera (17). £, F—Tip of rudimentary operculum (£) and functional operculum (F) of same (X17). G—Distal portion of functional operculum of Serpula vermicularis ( X 19). one closes the tube after the manner of forms with but one oper- culum. ‘The more posterior operculum, therefore, serves as a protection only in the cases where the barricade formed by the first is not effective. As compared with Salmacina, to which it is otherwise closely related, Filograna has two, more effective oper- 26 Charles Zeleny. cula’ instead of eight less effective club-shaped enlargements. The fact that when the animal is retracted the expanded portion of one operculum occupies a position in front of the other may be of importance in connection with a theory of the development of asymmetry in these organs in other members of the group. d. Group IV. One Functional Operculum and One Rudi- mentary Operculum. Both on ends of Branchiea. Pinnules present. Examples—Apomatus, Josephella. In Apomatus the next to the dorsal branchia on either the right or the left side is expanded at its end into a globular almost trans- parent operculum. ‘The chitinous shell of the sphere itself con- tains irregularly branched blood vessels, the green-colored blood of which makes them very conspicuous. The branchia in a corresponding position on the opposite side has a small ovoid enlargement with a very pronounced network of blood vessels containing distinctly pulsating green blood. Both these opercula are placed at the ends of stalks which retain all the branchial characters in an unchanged condition.' (Fig. 6p, £, F.) In a few cases the branchia occupying the place of the rudimen- tary operculum ended in a tapering point instead of an ovoid enlargement. ‘There are usually about twenty pairs of branchiz in the adult. Each of the two circlets breaks aff very readily along a definite breaking plane level with the anterior surface of the head. ‘The division plane is very pronounced and the break is clean cut and takes place so readily that it 1s very hard to remove the animal from its tube without causing it to throw off both of the branchial circlets. e. Group V. One Functional Operculum and One Rudimen- tary Operculum. Functional Operculum with Naked Stalk. Rudimentary O perculum Not on End of Long Stalk. Examples— Serpula, Crucigera, Hydroides. The description given above for Hydroides (p. 21) is sufficient as a general characterization of this type. ‘The functional oper- culum may be either on the right or on the left side, the rudimen- tary operculum in each case occupying the opposite position. ‘The opercula are not in the line of the branchia but occupy a position ‘According to de St. Joseph (94) the functional operculum appears on the left side and the rudimen- tary, his ‘‘pseudopercule,” on the right. He does not mention the possibility of the reverse arrangement. The specimens which I examined at Naples showed a preponderance of the right-handed condition. (See p. 32.) Compensatory Regulation. 27] dorsal to the first dorsal branchiz or to the interval between the first and second dorsal ones. Serpula differs from Hydroides in the entire absence of the secondary group of processes in the oper- culum (Fig. 6G). Crucigera has only four secondary processes and these are arranged in the form of a cross (de St. Joseph, ’94). Spirorbis Pagenstecheri. Ventral (slightly anterior) view showing branchie and operculum with its brood chamber containing embryos (40). ~ j. Group VI. One Operculum. No Rudimentary Operculum. ‘The members of this group have only one operculum. ‘There is no rudimentary operculum. Examples are Spirorbis, Pileolaria, Ditrupa, Filogranula (?), Pomatoceros, Vermilia. This group may be further subdivided according as the oper- culum has a position in the line with the branchiz (Ditrupa, fe 28 Charles Zeleny. Fic. 8 A—Ditrupa subulata, showing single naked stalked operculum in line with branchie of left side. Dorsal view. Note indication of basal suture (15). B, C—Side view and dorsal view of operculum of Pomatoceres triquetroides, showing highly modified terminal and lateral spines. Basal suture present (10). D—Operculum of Vermilia multivaricosa, showing curved stalk and absence of a basal suture. Dorsal view—Left-handed individual (8). Compensatory Regulation. 29 Spirorbis, Pileolaria, Filogranula [ f]) or dorsal to the line of the branchiz (Pomatoceros, Vermilia and others). In the latter case the operculum may further be either at one side of the median line (most species of Pomatoceros and Vermilia) or in the middle line itself (Pomatoceros elaphus, Haswell). In Ditrupa there is a large cup-shaped operculum with a naked stalk situated on the left side in line with the branchizw. It occu- pies a position on the median side of the most dorsal branchia of that side (Fig. 8a). I had only four specimens for examination. In all of these the operculum was on the left side, but whether this was merely a coincidence or not it is impossible to say. The tube _of Ditrupa subulata lies freely on the sea bottom usually at a con- siderable depth. Its substance is extremely hard and difficult to break. ‘The tube is slightly curved and resembles very much the shell of the mollusk Dentalium. The Spirorbis-like forms (Spirorbis, Pileolaria, etc.) have closely coiled tubes attached by the dorsal side to a flat surface. In some the tube is attached for its whole length but in others (some species of Pileolaria) the end may rise up from the level of the surrounding surface. ‘The direction of the coil of the tube is constant for any one species but varies in the different species. Thus dextral and sinstral species are distinguished according as the tube is coiled clockwise or counter clockwise. The dorsal side of the animal is next to the attached surface and the posterior end of the animal upon removal has a pronounced curve to the right or left according as the tube is dextral or sinstral. In the dextral species the operculum is on the right side and in the sinstral on the left so that in all cases the operculum is on the side next to the concave curve of the shell. “The number of branchiz varies in the different species from five to twelve, according to Caullery and Mesnil (’96). Of the two species examined by me Spirorbis Pagenstecheri had four branchiz on the left side and three plus the operculum on the right and Pileolaria sp. had five branchiz on the right and four plus the operculum on the left. In both cases the operculum occupies the position of the next to the dorsal branchia on its side, 7. e., the right side in Spirorbis and the left side in Pileolaria. In all members of the group the operculum is in line with the branchiz. It is as a rule much smaller than the opening of the tube so that the animal can retreat to a considerable distance within the tube. ‘There is no sign in either of the two a 30 Charles Zeleny. genera of any modification of the branchiz to compare with the rudimentary operculum of Apomatus or Hydroides. ‘The right or left position of the operculum is definitely correlated with the direction of curvature of the tube and as the latter 1s constant for any one species the former must be also. In Spuirorbis Pagenstecheri the operculum serves as a brood pouch and 1s of a trumpet shape. ‘The branchial pinnules are comparatively large and it is sometimes hard to tell whether a basal pinnule should or should not represent a branchial stalk. (Fig. 7.) Pileolaria sp. also uses its Sime as a brood pouch. Other species, how- ever (5. borealis, { or example, according to Alex. Agassiz, *66), keep the eggs in a string within the tube on the ventral side of the body. Next comes the group in which the single operculum does not occupy the line of the branchiz but is dorsal to it. First are the cases in which it is lateral. In Pomatoceros triquetroides the operculum is very large and stout. ‘There are two lateral processes beyond which comes an expansion ending in a cap of three spines. There is a very pronounced suture near the base. ‘The whole region below the two lateral processes is flattened dorso-ventrally. In the cases examined the operculum was always on the left side (Pig. 3B, Cc). In Vermilia multivaricosa the operculum occupies a position corresponding with that of P. triquetroides but it may be either on the left or on the right side. ‘The stalk of the operculum is approximately circular in cross section though possessing a corru- gated outer surface. It loops around from its point of attachment toward the median line. ‘The terminal region is thus brought nearer to the median line than is the proximal region. ‘The ter- minal portion is very large and heavy. ‘There is a basal globular portion upon which rests a heavy cone-shaped body (Fig. 8p). Haswell (85) describes a species of Pomatoceros (P. elaphus) with a large median operculum which 1s short and flattened dorso- ventrally. At the sides of the proximal portion are two wing-like lobes bearing small processes. “Terminally there are Aer, pro- cesses with Anitler: like branches. In another Serpulid (Vermilia czespitosa), according to Haswell, there is also a large operculum on a short stubby stalk (but not median judging by the figure, though there is no statement in the paper on this point). ‘This operculum is armed terminally with peculiar spines and serrated Compensatory Regulation. 31 processes and also has two lateral wings like those of Pomatoceros elaphus but not as well developed as the latter. E. Meyer (’88) concludes that these opercula have been formed by the union of two lateral ones, but the evidence as regards this point is by no means conclusive, since we have species where similar opercula are evidently lateral in position (P. triquetroides and V. multivaricosa, for example). g- Summary. The principal modifications of the opercula throughout the family of Serpulids have now been passed over briefly and the general characters may be summarized. In the jirst group are the forms with no opercular modification (Protula, Protis). In the second each branchia has a small club-shaped enlargement, the combination of the enlarged ends no doubt making a more or less effective barricade against invaders when the animal has retreated into its tube (Salmacina). In the third group (Filograna) the modification is confined to the most dorsal branchia on each side. All the others lack opercular differentia- tions. [he two dorsal ones mentioned also retain their branchial characters, but in addition each has an operculum of sufficient size to close up the opening of the tube. In the fourth group (Apomatus, Josephella) there is one functional and one rudi- mentary operculum, one on the end of each of the two next to the dorsal branchia. ‘The stalks supporting these opercula retain their branchial pinnules and other branchial characters. In the jijth group (Serpula, Crucigera, Hydroides) there are likewise one functional and one rudimentary operculum, the distribution between right-handed and left-handed forms being fairly equal in adults. The opercula, however, do not possess branchial pinnules though their internal anatomy and position indicate branchial characters. ‘The rudimentary operculum is not situ- ated on the end of a long stalk but is a small bud-shaped organ corresponding in position with the functional operculum. Judg- ing by their position the opercula seem to have moved down from the interval between the most dorsal and the next to the dorsal branchie on‘each side. Finally in the szxth group there is only one operculum and this retains but little of its branchial character. In some of the group, however (Ditrupa, Spirorbis, Pileolaria), it retains its position in the branchial circlet. In some cases it may be used as a brood pouch (Spirorbis, Pileolaria). In other forms (Pomatoceros, Vermilia) it is a considerable distance below 32 Charles Zeleny. the branchial region and is large and massive. In some of the species as P. triquetroides and V. multivaricosa it has a lateral position and in others, as P. elaphus, a median one. These six groups form a very complete morphological series which points strikingly toward the homology of the opercula and branchie in all the forms. 3. Distribution of the Opercula between Right and Left Sides. The data upon this point will be presented under this separate heading because of their special interest in connection with later discussions. ‘The first three of the groups of Serpulids mentioned above exhibit no asymmetry in their opercula and need not be considered here. “The three others will be discussed in turn: a. The fourth group have one functional and one rudimentary operculum. Both opercular stalks have branchial pinnules. (Examples—Apomatus, Josephella.) b. The fijth group have one functional and one rudimentary operculum, each with a naked stalk. Rudimentary operculum not on end of a long stalk. (Examples—Serpula, Crucigera, Hydroides.) c. The sixth group have only one operculum. No rudimen- tary operculum is present. (Examples—Ditrupa, Spirorbis, Pileolaria, Pomatoceros, Vermilia. ) (a.) Inthe fourth group thirteen specimens of Apomatus were examined and of these ten had the functional operculum on the right side and the rudimentary on the left. ‘The other three had hie reverse condition with the functional on the left and the rudi- mentary on the right. Taste II. F=Right F=Left Total Ne. Scie | Ree APOMIAES ampMllitera. vse tjecicxd evacuees 13 fe) 3 RETOOME ee arta set dtd Site, oie aye acter Ne ae Se — 77, 2g (0.) In the fifth group Hydisides eae H. uncinata, H. pectinata and one specimen of Serpula vermicularis were exam- Compensatory Regulation. Ba ined for the distribution of opercula. By far the most extensive observations are on Hydroides dianthus. Tas_Le III. A ydroides dianthus. Position of Opercula. F=Right. F=Left. Not like other Locality and Date. Total No. Re Tele) ee ee eee Woods Hole, Mass. ......... EG) 31 24 ae 1901/1 X/16-IX/19. Cold Spring Harbor, L.I.... 74 39 30 5t 1902/VII/5-6-7. Wold"Spung iMarbor, U.1:...| 10+ ?7t 69 51 net 1902/VIII/g to 20. 25 to iy Tin AGA erat sap a) betes Zee =258 139 105 =14 53-97% | 40-77% 5-47 EXPLANATION OF TABLE. F = Functional operculum; R = Rudimentary operculum. *These two specimens had a rudimentary operculum on each side. {These irregulars come under four heads: 1. One specimen with Right = operculum missing; Left = operculum between rudimentary and functional stage. 2. One specimen with Right = between rudi- mentary and functional; Left = rudimentary operculum. 3. Two specimens with Right = functional operculum; Left = two-thirds developed functional. 4. One specimen with Right = rudimentary; Left = one-half developed functional. {In this case the number of unclassified irregular cases was unfortunately not put down. My notes give only the indefinite statement ‘‘several abnormal and incomplete ones observed are not included in the present list.” Assuming that the percentage of such cases is the same as in the other two groups, where it is 5.4 per cent of the total number, we will not be far astray in making the unknown number equal to seven. The relative relation between right-handed and left-handed forms is expressed to better advantage if the irregular cases are not included. Removing the last column from the former table we get the relations expressed in the following one: An examination of this table shows that 57 per cent of the “nor- mal”’ cases have the functional operculum on the right and the rudimentary on the left, while 43 per cent have the opposite arrangement. ‘The striking agreement in the three sets of figures, 34 Charles Zeleny. one from Woods Hole and the other two from Cold Spring Har- bor, indicates the probability of some organic basis back of the fact. “This matter will come up again later in the discussion of the development in ontogeny and during regeneration. At the same time the considerable number of cases (fourteen [?] or 5.4 per Tape IV. Hydroides dianthus. Position of Opercula (not including irregular ones). F=Right F=Left Locality and Date | R=Left | RR Total Woods Hole, Mass., No. | 31 | 24 | 55 POOR) UX TO=1G,< . .h. Tejeeree Percents 5Or4 |. 2436 = Cold Spring Harbor, L. I., No. 39 30 69 TG02) VEI/ 5-6-7000. os wstoewion | Rerreenteleea5O.5 |) Ag 5s" ete Cold Spring Harbor, L. I., | No: 69 51 Nios (250) moo2/ VEU /9=20;4e 0a. eae | Per cent Fos) aa ok el ee No. ~ | 139 105 Dae MPotalhs ate. nee ee Per cent | 57 , 348 == Tasie V. Hydroides uncinata. Position of Opercula. ~Right | P= Locality and Date Bo Rights) Peon Total R=Left | R=Right t No: | fe) | 6 16 Naples, go2/X1/21 4. oye e ame eae [Percent A)? GResan nn Magy a5 -- cent of the whole number) which do not come under either of these groups will be taken up. Sixteen specimens of Hydroides: uncinata were examined at Naples. Of these ten, or 62.5 per cent, had the functional oper- culum on the right and the rudimentary on the left, and six, or 37-5 per cent, had the reciprocal arrangement; a considerable advantage in favor of the right-handed ones. ‘ Compensatory Regulation. | 35 Likewise 41 specimens of Hydroides pectinata were examined at Naples, and of these 21 or 51.2 per cent had the functional oper- culum on the right side and the rudimentary on the left while 48.8 per cent had the reciprocal arrangement, a slight advantage in favor of the right-handed ones. Tas_e VI. AH ydroides pectinata. Position of Opercula. Locality and Date R= Lek Re ight Naples, 1902/IX/27 No. 21 20 41 MOO a MUM OTe. Caste exe Per cent KIS! Hi anes -- | In Serpula vermicularis [ examined only one specimen and this had the functional operculum on the left side and the rudimentary on the right. Our general conclusion regarding the distribution of the oper- cula in the fifth group of Serpulids, those with the naked-stalked functional and small bud-like rudimentary is that the right and left-handed forms are nearly equal in number, but there is a slight advantage in favor of the right-handed ones. (c.) Inthe sixth group there is only one operculum. In Ditrupa subulata only four specimens were examined as regards this point. All four of these had the operculum on the left side.! In the dextrally coiled Spirorbis Pagenstecheri eleven specimens were examined and all had the operculum on the right side, while in the sinstrally coiled Pileolaria the one specimen examined had the operculum on the left. The observations of Caullery and Mesnil (’96) show that in the species with dextrally coiled tubes the operculum is always on the right side and in the sinstrally- coiled ones always on the left side. ‘de St. Joseph (98) gives a description of Ditrupa arietina, O. F. Miiller (= D. subulata, Desh.) in which he mentions the operculum as a structure of the left side in agreement with the present observa- tions. Also the left side according to him has one less branchia than the right, 7. e., there are 11 branchie (plus the operculum) on the left and 12 on the right. 36 Charles Zeleny. In Pomatoceros triquetroides 21 specimens were examined and all had the operculum on the left side. This makes a fairly strong probability in favor of the permanence of such a charac- teristic. As a further argument may be mentioned the statement of de St. Joseph (’94), who mentions the observation of Grube on 63 specimens of P. triqueter, L.( = P. triquetroides, D. Ch.) and of himself on many more than this number which showed the oper- culum in every case on the left side, so that we may be fairly certain that in this species the organ is a permanent structure of the left side. In Vermilia multivaricosa eleven specimens were examined. Six had the operculum on the right and five on the left side. In this case, therefore, there seems to be a fairly equal distribution between the two sides. The data for Group VI are collected in the following table: TaBLeE VII. aes Six. Position es O perculum. cae Bay of Naples. | Name and Date. | No. | Rowen | Operc. | Per cent Per cent | Right. Left. | Right. || Lee Ditmupaisubulatay. 5. 22th eee ee eer O | 4 | O | 100 1903/I/1o. | | Spirotbis Papenstechen! jaya: 5... 2 a0 II Tis ° | 100 | fe) 1903/I to V. | | | PALEOlATIA Sis ( f)\ 9. ert hier: 5.« 5 eas taels hata: O | I o | 100 1903/III/2. | Pomatoceras triquetrordes, 20. .:.5... 22 Oo 22 Oo 100 1903/I/25, 1V/3, IV/s. | Vermulia multiyaricosay. 25 .8b.4 0.5 II 6 Sal 55 45 1903/1/16, III/5. Discussion of the Evidence from Group VI. In Spirorbis Pagenstecheri and Pileolaria sp. the position of the operculum 1Former observations on the position of the operculum in this group are those of Caullery and Mesnil (’96), who state that dextrally coiled Spirorbis-like Serpulids always have the operculum on the right side (example—Spirorbis Pagenstecheri) while sinistrally coiled ones haye it on the left side (example— Pileolaria sp.[?]). de St. Joseph (’98) describes the operculum of Ditrupa subulata, Desh., as a struc- ture of the left side. de St. Joseph (94) for himself and also for Grube describes Pomatoceros trique- troides as always left-handed. Compensatory Regulation. ay bears a direct relation to the direction of the coil of the tube. Caullery and Mesnil (’96) have shown that the operculum of Spirorbis and its relatives is always located on the side next to the concave surface of the coil, 7. ¢., on the right hand side in dextral tubes and on the left-hand side in sinistral ones. Whether or not any such relation can be made out in other members of Group VI it is not possible to say. In Ditrupa there is a slight curvature of a definite form in the tube but the relation of the body within the tube has not been made out definitely enough to determine the significance of the constancy of position of the operculum. In Pomatoceros triquetroides there is a definitely fixed left-handed- ness though the tube is irregularly coiled, and in Vermilia multi- varicosa there is an almost equal distribution between the two sides, the tube being again irregularly coiled. A discussion of the factors controlling the determination of the position of the opercula must be left nel the ontogenetic and regeneratory development of these organs have been studied. 4. Exceptional Degrees of Development in General and One Case of a Supernumerary Operculum. The two opercula in Groups IV and V in the vast majority of cases consist of a fairly typical functional and a fairly typical rudimentary operculum. ‘There are, however, exceptions to this statement, as has already been indicated above. The main exceptions are due to the partial further development of what probably would otherwise correspond with the rudimentary oper- culum, either with or without the loss of the functional operculum. There thus arise either one functional operculum and one partly developed functional or a missing operculum and one partly developed one. In other individuals both opercula were found to be rudimentary or one rudimentary and one partly developed one were present. ‘The discussion of these cases must be reserved until we come to the experimental part of the paper. One case of a supernumerary operculum was found and this is interesting in connection with the problem of the regulation of the opercular development. ‘The accompanying figure (Fig. 9) gives the relations of the opercula. On the left-hand side there is a rudimentary operculum of the typical form in the usual position. On the right-hand side in a corresponding position is a typical 38 Charles Zeleny. functional operculum, but in addition to it there is an added rudi- mentary operculum with its point of attachment posterior to that of the functional. ‘This added rudimentary operculum agrees in all respects with the one on the opposite side, so that we have two rudimentary opercula and one functional one. The specimen indicates that the influence which determines the development of a functional or a rudimentary operculum is not always a bilater- ally differentiated one. A more complete discussion must be reserved until the experimental data have been given. 2. Development of Opercula. 1. Ontogenetic Development. a. Introduction. Ina paper, entitled “A Case of Compensa- tory Regulation in the Regeneration of Hydroides Dianthus,” I described some experiments showing that when the func- tional operculum of this Serpulid is removed the rudimentary operculum on the opposite side develops into a new functional oper- culum similar to the old one while in place of the old functional stalk a new rudi- mentary bud develops. In the discussion of this and similar experiments it was stated that a knowledge of the ontogenetic development Hydroides dianthus with three opercula, two rudi- of the OES = highly desir- mentary and one functional. Note that one of the able before we can be in a rudimentary opercula is attached near the base of the position to discuss the data functional one ( 15). intheirfull relations. With this object in view the writer undertook to raise the larva up to the stage where both opercula have attained their normal adult development. In attempting a provisional explanation of the compensatory regulation of the opercula it was stated in the above paper that there may be a restraining influence exerted by the fully developed Fic. 9. Compensatory Regulation. 39 operculum upon the rudimentary one which prevents the latter from attaining its full development. ‘The removal of the func- tional operculum removes the restraining influence and the rudi- mentary continues its development. The new functional oper- culum in turn restricts the new bud developing in place of the old functional operculum and holds it at the rudimentary stage. The plausibility of this explanation is increased if it is found that in the ontogenetic development one operculum develops before the other, and therefore can hold the latter in check in the manner before indicated. With this object in view the investigation of the ontogenetic development was undertaken. b. Historical Review. The first recorded observations on the development of the branchial apparatus in Serpulids which I have been able to find are those of Milne-Edwards (’45), on the development of the young Protula. He saw the larve attach themselves to solid objects at the bottom and sides of his dish. Here they secreted a cylindrical tube which at first was open at both ends and shorter than the length of the larva. At about the same time two lobes were differentiated at the anterior end of the larva, one on each side of the median line. At a slightly later period he thought he saw digitations of these lobes and he took them to be the beginnings of the branchiz. Pagenstecher (’63) gives an account of the development of the branchie and operculum in Spirorbis. He states that the first traces of the branchiz are exhibited in the form of three knobs upon each of the two head lobes. The operculum 1s not differentiated until a later time when there is formed “der Fortsatz welcher ihn tragen soll und der von den Tentakeln durch eine Runzelung oder seichte Kerbung der Oberflache ausgezeichnet war.” Judging by Pagenstecher’s figure there 1s very little difference at the above-mentioned stage between the so-called opercular outgrowth and the other branchie. ‘The figure gives two branchiz on one side and two plus the opercular outgrowth on the other. Fritz Miller (’64) noticed on the side of a glass vessel which he had on his study table a young Serpulid with three pairs of branchiw, and which he took to be a member of the Protula group because of the absence of an operculum. However, a short time later he noticed that one of the branchia had an opercular enlargement at its end though it still retained its branchial pinnules. Still later the branchial pinnules disappeared also and he had a Serpulid with the typical genus-Serpula- type of operculum, which had developed by a modification of a branchia. In the meantime a new pair of branchiz had been added to the oral crown making three ,branchiz plus one operculum on one side and four branchiz on the other. ‘This is he only recorded observation of the transformation of a branchia into an operculum. 40 Charles Zeleny. In 1866 Agassiz described the development of branchie and operculum in Spirorbis spirillum, Gould (not Lamarck), and made out an alternate appearance of the tentacles (branchia). ‘The first tentacle appears on the right, next comes the corresponding tentacle on the left and only later the rudiment of the odd opercular tentacle (on the right side).” The rudiment of the operculum, though at first somewhat resembling that of the tentacles, shows a difference from the start. Claparede and Mecznikow (69), on the contrary, make out a paired mode of forma- tion of the branchiz in other species of Spirorbis. | Willemoes-Suhm (’70) speaks again of an alternate mode in Spirorbis. Giard (’76b) raised the larve of Salmacina Dysteri. He found two lateral head lobes each of which soon showed a threefold division. These divisions elongated to form the first three pairs of branchiw. On each side there were two dorsal and one ventral branchia, the latter, however, dividing into two on the fifth day, so that there were then present eight branchial trunks, four on each side. ‘The first pinnule appeared on the eighth day on the upper third of the external dorsal branchia. This is the only notice of pinnule formation I have found. In Manayunkia, a fresh water Serpulid, Leidy (’83), describes the head lobes as showing the branchial digitations from the first trace of formation of the former. Salensky (’83) likewise states for Pileolaria sp. that four branchia and the operculum appear at the same time from a median dorsal plate. The opercular “anlage” is from the beginning three to four times as large as the branchial “‘anlagen.”” In Salensky’s words: “‘On voit d’apres cette description, que, chez Pileolaria la formation des branchies et de l’opercule s’opere en méme temps, et non comme Agassiz et Pagenstecher le montrent pour Spirorbis spirillum.” _ Meyer (88) describes the development of the branchia in Eupomatus (= Hy- droides). He makes out the appearance of the two head lobes from each of which the three processes representing the first three branchie sprout out. The develop- ment was not carried further than this. “This method of formation agrees also with that described by Roule (’85) for the larvze of Dasychone. From the foregoing notes it is evident that further observa- tions on the early development of the branchiz are necessary in order to clear up our ideas regarding the matter, and when we come to the opercular development we have practically nothing outside of the observations on the highly modified forms Spiror- bis and Pileolaria except the short note of Fritz Miller upon the change of a branchia into an operculum. Regarding the forma- tion of the rudimentary operculum there is nothing at all, and we therefore get very little aid in our study of the correlation between Compensatory Regulation. AI the two opercula at their first appearance and up to the time when they assume the final adult condition. The following observations on H. dianthus were made at the Cold Spring Harbor Biological Laboratory on Long Island, N. Y., during July, August and September, 1902. ‘The observations on the other species, H. uncinata and H. pectinata, were made at the Naples Zoological Station in the winter of 1g02—-03. ‘The obser- vations on the method of rearing the larve and on their general activities will be given in a separate short note. c. Observations. When the free-swimming larva is about nine days old its body is considerably elongated and shows external signs of segmentation. ‘The apical iui; is long and two very promi- nent. reddish eyespots are present. (Fig. toa.) As the move- ments of the animal become more and more sluggish just before its fixation to a solid object the apical cilium gradually grows smaller until it disappears entirely. At the same time three pairs of sete are formed, the first pair being especially long and promi- nent. (Fig. 108.) Fig. roc shows a larva, 17 days old, with the tube covering about one-half of the body. Here each of the two lateral head lobes already shows the division into three blunt pro- cesses, the forerunners of the branchiz. ‘These divisions of the head lobe appear very soon after the formation of the head lobe itself but the latter has a short separate existence before the tripartite subdivision appears. The two dorsal processes are more closely connected together than with the third and more ventral one. ‘The relation is more clearly made out in Fig. rop, where the mutual union of the two dorsal pairs is very evident. The two larve (Fig. toc and Fig. 10p) are of the same age (17 days) and come from the same dish. In Fig. 10o£ there are still the same three pairs of processes though the branchial character 1s more evident than before. This specimen is from the same dish as the others (16 days after fertilization). It is evident from these data that the rate of de- velopment of the different larve in a single dish varies within wide limits. At the stage represented in Fig. ror the inner sur- faces of the branchiz are covered with very active cilia. In order 1Biological Bulletin, °o5, vol. viii. 2The tube secreted by the animal is at first a very short cylinder which is quite transparent and covers only a small part of the larva. The ring at first is situated near the anterior end of the body just back of the eyes. 42 Charles Zeleny. to get at the method in which these primary processes branch later on, I have designated them arbitrarily by the Roman numerals I, I and III. I represents the most dorsal pair, II the middle pair and III the ventral pair, R or L being prefixed to represent right or left when there is any need for distinction between the two sides. II iI D Z I 2 vee II 3 LMI III ” Fic. 10. Hydroides dianthus. Larvae. Stages of transformation from free-swimming to sedentary life. Dorsal views. A—Free-swimming larva. Age, 9 days. Shows long apical cilium ( X208). B— Swimming but sluggish larva. Age, 9.days. Shows sete and shortened apical cilium (> 185). C—Attached larva (go). Age, 17 days. Three pairs of head lobes. Beginning of secretion of tube. D—Age, 17 days (go). E—Age, 16 days (85). F—Age,17 days. Second of original three pairs of branchize shows secondary branches ( 70). : Compensatory Regulation. 43 In Fig. tor, also 17 days after fertilization, we find a stage con- siderably more advanced in which each of the middle branchiz has already sent out three branches, which may be labeled according “to age, I]—1, Il—2 and II—3. Of these it will be seen that [I—1 very early takes on the character of one of the main branchial trunks, so that we thus geta stage with four pairs of branchiz. ° I1—2’and II—3 retain the characters of pin- nules of the Branchia II. Neither I nor III has any branches at this stage. Fig. 11A shows a later stage taken from the same dish at the same time (17 days). Here we _ still have no branches of I and III. Branchia II has five branches and the first branch of L-Il (L-II—1) a branchlet of its own (L-[]—1—1) just appearing as a very small knob. It is very evident that Branchia II is rapidly outgrowing I and III in strength. I } eae LE | Yu if LT1 LEE A fi : lk L/-£ 1 B LET Fie. 11. Hydroides dianthus. Dorsal views. d—Age, 17 days (X47). B—Age, 19 days (X 62). Fig. 118 (19 days old) gives a still older stage. Branchia Ion each side has not increased much in size and is hardly larger than 44 Charles Zeleny. the older branches of [I]. L-I[—1 has two branchlets ( =. pin- nules) and R-I]—1 has one branchlet. Branchia II on each side now shows the beginning of the sixth branchlet or pinnule. Branchia III also has not increased much in size. In Fig. 12 (23 days old) Branch IJ—1 with its six pinnules has very evidently taken its place as a prominent part of the branchial L[-r Fic. 12. Hydroides dianthus. Age, 23 days (X75). The first secondary branch of Branch II has assumed the character of an independent main branch with branchlets of its own. The original third pair of branches is not shown. apparatus. Branchia II has now added its eighth pinnule. Branchiz I and III are still unbranched and have increased com- paratively little in size. In Fig. 13 (23 days old), in which only the right side is repre- sented as both sides are essentially similar, Branchia I for the first time shows a trace of branching, seven little branchlets (pinnules) appearing simultaneously. Branchia I] has added one new branchlet making nine in all counting Branchia I[I—1 as the first Compensatory Regulation. 45 one. Branchia I]—1 has eight pinnules. The character of Branchia III was not made out. The operculum was seen for the first time six days later at the stage shown in Fig. 15. Fig. 14, taken five days later still (34 days ‘old), shows an earlier stage of the operculum. Here it appears as a rounded knob on the end of Branchia L-II. vf This branchia has at yn \\ this time nine branch- lets counting the one ({I—1) which has as- sumed the character of a main. branch. The eight represented as pinnules are []—2 to II—g9. The knob at the end of the Ley branchia is conical in > form with the base of the cone free and the apex attached to the stalk. The corres- ponding branchia on the right side (R-II) has likewise at this stage nine branchlets counting the independ- ent one (II—1)or eight dependent ones (pin- nules) II—2 to II—g without this, but there - 1s no modification at the tip of the matin stalk Hydroides dianthus. Age, 23 days. Dorsal view (75). assoccurs on the lett Original third pair is not shown. First pair shows beginnings side. In Fig. 15 the opercular character of the knob on L-II is very evident. ‘The cup of the operculum has a notched edge with eleven serrations and resembles in its character the Serpula type and not the Hydroides type. The eight dependent (II—2 to I1—g) and one independent branch are very prominently developed BiG: 13: of secondary branches. 46 Charles Zeleny. and evidently all of them retain their respiratory character. The corresponding branchia (R-II) on the right side has branchlets similar to those of the branchia (L-II) on the left side though it shows no opercular modifications. It is similar to the latter and different from all the other branchiz in one essential respect. While I, II—1 and III show buds of new developing pinnules its pinnules (7. ¢., the eight dependent ones II—2 to II—9) are all Fie. 14. Hydroides dianthus. Age, 34 days. The next to the dorsal branchia on each side. The left one shows the beginning of the knob of the functional operculum. The right one later drops off and the rudimentary operculum regenerates from the remaining stump. well grown, indicating, no doubt, that the organ has reached its limit of development as a branchia. With the beginning of the opercular differentiation Branchia I enters on a new period. It increases in size and new branchlets (pinnules) sprout out in rapid succession, the first ones appearing simultaneously. Thus in Fig. 15, I already has thirteen well formed pinnules with several buds crowded into a zone at the base of the terminal flament. Branchia IJ—1 has likewise been Compensatory Regulation. 47 increasing in size and now has 12 pinnules plus a thirteenth bud on each side ___ Branchia III is also branched but the character of its pinnules is not given in the figure in order that complication may be avoided. This pair of branchiz is directed away from the observer toward the ventral side of the animal. At all these stages the Branchie L-II and R-II both retain their flexible and branchia-like char- acter in all respects except for the ter- minal enlargement im -1f: In Fig. 16 the pin- nules of L-II have disappeared, leaving a cup-like opercu- lum with a serrated edge on the end of a long, slender, flexi- ble but bare stalk. The corresponding branchia on _ the other side (R-ID) has dropped off, leaving only a small round knob at the place of its former attach- ment. : The other Hydroides dianthus. Age, 28 days. Shows the three most dorsal branchia, te II—1 pairs of branchie. The ventral pair directed away from observer is and ne all are keep- not shown. The opercular knob of the left side is notched and its ing on with their ‘talk still retains the respiratory pinnules. The next to the dorsal branchia of the night side has eight pinnules but differs from other Fic. 15. branchi velop- = al dey elop branchie, except the opercular one, in the absence of new pinnule ment by continually ,.4. adding new pinnules to the old ones. We thus have three pairs of branchiz at this stage with a functional operculum of the Serpula type on the left side and a rudimentary operculum on the right side. No young Serpulids were observed that showed an exception to this order of appearance of.the opercula. Numerous observations were made 48 Charles Zeleny. upon specimens before a count was undertaken. In this count twenty individuals were noted. All without exception, the former as well as the latter, showed the functional operculum appearing on the left side. Fig. 174 shows the rudimentary operculum at a slightly later stage in which it has more definitely assumed its typical condition. ei ae \ Fic. 16. Hydroides dianthus. Age, 34 days. Dorsal view (38). On left side is functional operculum of Serpula type with naked stalk and cup with one row of serrations. On right side is rudimentary bud developed from the base of the cast-off second branchia of that side. The three pairs of typical branchie also are shown. The condition at this time resembles very closely that of the adult Serpula, as the functional operculum as shown in Fig. 16 is without doubt of the Serpula type. The further changes were not followed in H. dianthus, the species at Cold Spring Harbor, but were made out in the two Naples species, H. pectinata and H. uncinata. Compensatory Regulation. 49 Fic. 17. A—H. dianthus. Age, 34 days. Primary rudimentary operculum, right side (50). B—H. pectinata. Age, 45 days. Dorsal view. Functional operculum of Serpula type on left. Rudimentary operculum on right. Pinnules are shown in only one branchia, the other branchie being essentially similar to this one. C—Tube of H. pectinata. Age, 45 days (6). D—Hydroides uncinata. Age, 46 days. Ventral view. Shows Serpula type of functional operculum on left side and rudimentary operculum on right. E—Diagram of cross-section of a branchia of H. uncinata showing method of attachment of pinnules. /—H. uncinata. Age, 179 days or nearly six months. Original or Primary functional operculum as appearing after it has dropped off. 50 Charles Zeleny. Evidently the adult condition with an operculum situated on either the right or the left side and having two rows of processes at its distal end is not fully explained by the Cold Spring Harbor observations. Only three larve out of a great many sets of eggs started at Naples lived through the period of attachment. ‘Two of these were H. uncinata and the other H. pectinata. ‘These were first care- fully observed only after they had developed up to the stage corre- sponding to Fig. 16 of H. dianthus. In Fig. 17B 1s represented a specimen of H. pectinata with essen- tially the same characters as those of H. dianthus in Fig. 16. ‘The number of branchiz has, however, meantime increased and there are here besides the opercula five branchiz on the left side and four plus the bud of a fifth on the right side. ‘The functional operculum has the essential characters of the Serpula type (see above). ‘he new branchiz are being added on the ventral edge of each of the branchial ridges. Both opercula have moved down from the line of the branchiz and the gap left in the line by their absence 1s being closed up. ‘The character of the tube at this stage is shown in Fig. 17c. The tube was so extremely irregular in shape, largely because it was detached from the glass frequently in order to facilitate observation. Practically the same conditions are shown at this time in H. uncinata where there are on each side five branchiz plus the oper- culum. ‘The opercula here as before have the characters of the Serpula type. (Fig. 17D.) No further changes in the opercula were noticed for a long time. Finally, six months after the fertilization of the ova, the animals were again carefully observed, and it was noticed that the primary functional operculum (left side) had fallen off (Fig. 17F) and in its place a rudimentary one had developed, while the primary rudi- mentary operculum of the other (right) side had developed into a functional one. (Fig. 18a, B, c.) ‘The two specimens of H. uncinata retained their simple Serpula-like operculum longer than did H. pectinata. Returning to the-specimen of H. pectinata as it was found after the reversal of the opercula we find all the adult characters except the full number of branchia. The branchiz increase in number by additions along the ventral edge of each branchial ridge. In speci- mens at this stage there are beside the opercula seven branchiz Compensatory Regulation. 51 plus a very small bud on the left side and six branchiz plus a large bud on the right. ‘The functional operculum has a basal funnel- shaped cup with a serrated edge. From the upper flat end of this cup there projects a new secondary cup, the individual serrations of which reach nearly to the base and are strongly toothed. ie G Fic. 18. Hydroides pectinata. Age, 181 days or 6 months. A—Ventral (somewhat inclined) view, showing position of secondary functional and rudimentary opercula. Pinnules of branchie are not shown in the figure (16). B—Secondary functional operculum (adult Hydroides type with two rows of pro- cesses) (32). C—Rudimentary operculum ( X 32). _d. Summary of Data and Discussion. ‘The above results con- cerning the ontogenetic development of the opercula may be sum- marized as follows: At the stage with four pairs of branchiz the next to the dorsal one on each side stops its development when it has eight long slender pinnules. “The two branchiz of the third pair, counting from the dorsal side, arise originally as branches which resemble in all respects the ordinary pinnules, but instead of retaining their 52 Charles Zeleny. dependent condition increase in strength, develop secondary branches of their own and soon take their place as independent branchiz coequal with the three primary pairs. After reaching its limit of growth as a branchia the next to the dorsalmost branchia on the Jeft side starts a new differentiation at its end, developing a knob which rapidly increases in size and soon assumes the shape of an inverted cone. Along the edge of the upturned base notches appear so that the whole knob has the general character of the opercular cup of members of the genus Serpula. All this time, however, the stalk has retained its eight branchial filaments and the corresponding branchia on the other side has remained unchanged. ‘This stage corresponds in general with the adult of Filograna or rather with Apomatus except that only one operculum is present. ‘The branchial filaments of the stalk, however, soon disappear. Whether they drop off or are resorbed was not made out but the former supposition is the more probable one, as they were still very long a short time before they had entirely disappeared; or, in other words, no intermediate stages of resorption were seen. Almost coincidentally with the disap- pearance of these pinnules the next to the dorsal branchia of the right side drops off, the region of the break being near the base. From this broken stump a bud develops which in a few days has reached its limit of development for the time being. ‘This bud, which remains as the primary rudimentary operculum for several months, is a regenerated structure, a true case of physiological heteromor phosis. Furthermore, 1 it is restricted in its development by some forces acting from without its own substance. At this stage the opercula remain for a considerable time (several months) orien Fe further change, although at the same time the animal 1s increasing in size, is building up its tube and new branchiz are being added on the ventral edge of each of the branchial ridges. In its essential characters this stage is equivalent to that of adult members of the genus Serpula with the functional operculum on the left side. After this long period of no opercular change the primary func- tional operculum drops off, the stalk breaking near its base. Immediately the primary rudimentary operculum on the right side, no longer restricted by outside forces, starts its further devel- opment and becomes a functional opercular organ. However, it does not develop into an operculum of the simple type like the pri- Compensatory Regulation. 53 mary functional one. Instead it takes on the characters of the adult Hydroides operculum with two rows of serrations. Beside the inverted cone with serrations around its upper edge there is an additional circlet of pointed and often hooked processes, which constitute the most important character of the Hydroides group as distinguished from the Serpula group. At the same time the broken stump on the left side has started to develop a knob of embryonic tissue which grows only up to the stage represented by the rudimentary operculum of the adult and is in its turn restricted in its further development by some force most likely similar to the one which in the first place restricted the original primary rudi- mentary operculum. ‘There are, therefore, at this time a secondary functional operculum on the right side and a secondary rudi- mentary operculum on the left side. These have the essential characters of the opercula of adult specimens of Hydroides. However, one point of difference is evident in specimens taken at random from the sea. It is found that approximately the same number have the operculum on the left side as on the right though there is uniformly a slight advantage in favor of the right-handed ones (57 per cent right-handed to 43 per cent left-handed in H. dianthus), while all the larvae appeared first as left-handed ones and later by reversal changed to right-handed ones. How does this change occur? Either we must suppose that the similarity in character of all the larve was accidental or that the reversal takes place in nature during the life of the individual more than the one time described for the young animal. The first supposition seems improbable because the larvae came from a great many different individuals, and moreover the order of appearance was found to be the same in the two Naples species (H. uncinata and H. pectinata). We are, therefore, forced to assume a further reversal as taking place in nature. This reversal may be a purely physiological one, induced by the normal activities of the animal, like the first reversal already described, or may be induced by some injury to the functional operculum of the character which was found to cause such a reversal in my experiments. (See below, p. 55 7.) However, unfortunately, there is no experimental evidence to show that physiological reversal takes place in nature after the first time already described. As is mentioned elsewhere (p. 65) in this paper the experiments undertaken to determine whether worms 54 Charles Zeleny. kept in dishes in the laboratory exhibited physiological reversal were negative, although the time was in all cases too short to constitute a good test. “There was no change in any case unless the functional operculum was injured. However, the very near equality between right and left-handed individuals seems to pre- clude the possibility of all reversal being due to injury of the func- tional operculum. And we have beside the analogous case of physiological reversal in the young animals as has been empha- sized before. 2. Regeneratory Development. a. Introduction. The nature of the opercular modifications among the Serpulids has now been outlined in the discussion of the comparative anatomy and their orzgin within the individual life history has been traced in the genus Hydroides. ‘There now remains an attempt at an experimental analysis of the factors involved in the development and maintenance of the adult charac- ters. Partly because of the imperfection of the method and partly because it is not desirable to dissect the data too minutely while presenting them, the latter will be given in the descriptive portion of this section as parts of individual experiments without perfect regard to logical development of the analysis of the factors involved. ‘The latter will be attempted more fully in the general discussions to follow the descriptive portions. In a former paper a preliminary report of some of my experi- mental results on compensatory regulation in the regeneration of Hydroides dianthus was given. Since that time a more detailed series of experiments has been undertaken along the same lines and the work has been extended to other species of the family. Two distinct problems have come up. In the first place is the study of the factors involved in the compensatory regulation of the opercula which, as stated above, is the main object of the present paper. In the second place a comparison of the regen- eratory development of the opercula with the ontogenetic and with the probable phylogenetic development brings up an ex- tremely interesting qecuecion with important bearings on the recapitulation theory. The great majority of the experiments were performed on lek demehus and this form will be discussed first. “Then will follow the other members of Group V, namely, H. uncinata, Compensatory Regulation. 55 H. pectinata and Serpula vermicularis, then a member of Group IV, Apomatus ampullifera, and finally the members of Group VI, Ditrupa subulata, Spirorbis Pagenstecheri, Pomatoceros tri- quetroides and Vermilia multivaricosa. The adult condition of the opercula which has already been described in detail in the anatomical portion of the paper will be again briefly noted, as it must serve as the basis of our experiments. [The experiments will then be described in turn, and finally the results will be discussed. b. Unoperated Condition of the O percula in H ydroides Dianthus. The character of the adult opercula has already been given on p- 21 fff. and is also shown in Fig. 5, F. A repetition of these data is therefore unnecessary. In all the experiments about to be described the animal was first removed from its tube and placed in a dish of sea-water, the desired operation was performed under a dissecting microscope and the animal was kept in its individual dish either with running or standing water, in the latter case the water being changed once or twice a day as required. The running water was not found as favorable as the standing because of the collection of a fine deposit on the animals notwithstanding the greatest care exercised. ‘The dishes with standing water were found very favorable if provided with a glass cover to keep out the dust. ‘The observations were in most cases made on the living animals. c. Operations on arenowal Operculum. The results may best be arranged around a description of the effect of a cross cut through the stalk three-fourths of the distance from the base to the beginning of the terminal expansion. The stump of the functional stalk remains attached to the animal for two or three days as a rule or even longer in some cases. It then breaks off from the body, separating by a clean division at ae basal suture or “breaking joint’’ described above (pp. 21-23). the distal end of the small stump still remaining attached to i animal a small bud now appears and gradually increases in size until it reaches the dimensions and character of the former rudi- mentary operculum of the opposite side. At this point it stops and proceeds no further. In order to understand the result it is neces- sary to look away from the immediate vicinity of the operated organ and to note the change going on in a corresponding position on the other side of the animal. Even before the attached stump of the 56 Charles Zeleny. operated functional operculum has fallen off the rudimentary operculum has started to enlarge and processes appear at its distal end. Gradually the structure increases in size and assumes Hirth Pd I Ca ee: b c d Fic. 19. Hydroides dianthus. A, a-f—Stages in the development of a rudimentary operculum from the stump of an old functional one (X54). B, a-e—Stages in the regeneration of a rudimentary operculum after a cut at the region indicated by the double-headed arrow (X54). C, a-d—Stages in the trans- formation of the old rudimentary operculum into the new functional after removal of the old functional operculum. The final condition of the functional operculum is shown in Fig. 5r. Compensatory Regulation. Fa) more and more the character of the former functional operculum. After 15 to 20 days the development is complete and we have a complete reversal of the opercula. ‘The former functional oper- culum is now the rudimentary and the former rudimentary has become the functional. ‘The resulting arrangement is the exact reciprocal of the former one. The case just briefly outlined will now be taken up in more detail. Before the stalk of the functional operculum 1s cut it is almost impossible to pull it off from the animal. ‘The basal suture resists breaking as well as the solid material of the stalk. The hardest kind of a pull that can be given with a pairof forceps is not suth- cient to dislodge the organ. Inside of a few days after the operation, how- ever, the opercular stalk as we have seen comes off of its own accord, so that great changes must be assumed to have taken place in the region. The time at which the stalk drops off varies greatly. In one case it had not come off 5 days after the operation and in another after 6 days it was still attached. Very soon after the stalk has dropped off a bud appears at the top of the stump and this stead- ily increases in size until the typical form of a rudimentary operculum 1S days after transverse section of stalk reached. (Fig. IQ A, a- f.) It seems (26). Exceptional case. Differentia- very probable that the breaking off of tion at the distal end of a functional the remanent of the stalk is induced stalk which had been cut two-thirds of by histological changes in the suture | aint ine ieee region which are aes ponethey na : beginning of the dev clopment of the new bud. Evidence in favor of this view will be given later in another place. Except in one case no differentiation of tissues took place at the cut end of the functional stalk. In this case there was an expan- sion on each of the two sides of the stalk near its tip. ‘The stalk did not drop off for 5 days after the operation. ‘The changes are represented in Fig. 20. Fic. 20. Opercula of Hydroides dianthus, five the distance from the base to the termi- 58 Charles Zeleny. The changes taking place on the other side of the body are very evident 3 or 4 days after the operation, often before the rema- nent of the functional stalk has dropped off. ‘This fact that the rudimentary operculum begins to develop even before the func- tional has dropped off seems to argue against the mere retarding influence of the organ asa mechanical weight, though of course the weight is considerably diminished by the removal of the part of the operculum distal to the cut. ‘The stages through which the rudimentary operculum passes in changing into a functional one are given in the accompanying figures. (Fig. 19c, a-d.) The essential points to be emphasized in this development are: first, the fact that throughout there 1s no sign of the appearance of spectal bran- chial characters, and, second, that the secondary processes of the operculum appear before the primary ones. ‘The regeneratory development, therefore, differs widely from the ontogenetic or probable phylogenetic one as regards these points. A series of operations was performed on the functional opercu- lum to determine the amount of injury necessary to bring about reversal. ‘The cases overlap slightly but it is found that in general the cutting off of the distal circlet of processes does not induce reversal while cuts through the main enlarged portion of the oper- culum bring about a reversal of the opercula. In one case the stalk of the injured operculum remained attached though the rudi- mentary operculum in the meantime had reached a stage equal to three-fourths of the normal functional development. It may be concluded that a removal of the secondary circlet of processes of the junctional operculum does not as a rule cause reversal, while a similar injury below this point to the main portion of the cup or to the stalk of the operculum always brings about such a result. d. Operations on Rudimentary Operculum. When the rudi- mentary operculum alone is removed there is no effect upon the functional operculum and a new rudimentary develops in place of the old one which had been cut off. The cut end rounds off, a bud-like mass of new tissue appears there, and the whole, both old and new tissue together, gradually assumes the shape of the old rudimentary operculum, The greater part of the change from the beginning is, however, accomplished by the growth of new tissue and only very little by the change in form of the old. No further development takes place. The result is the same no matter what the level of the cut may be. One of the levels at Compensatory Regulation. 59 which cuts were made in my experiments is shown in the accom- panying figure. In none of them did the functional operculum changt its character or did any structure other than a rudimen- tary operculum develop in place of the old rudimentary. (Fig. LOB, a—C:) e. Operations on Both Oper- cula. When both opercula were cut off it was found that while in some cases there was a reversal, in others two functional opercula were developed, one on each side, while in still others characteristics differing from either of these two combinations were formed. An attempt was made to find the cause of the difference in results and with this object in view cuts were made at different levels. The difference of level in the cuts in the rudimentary operculum could not be easily controlled, but since in the former cases where only the rudimentary oper- culum was removed there was no specific influence either on the opposite functional operculum or on the new regenerating one, it is supposed that these differences of level have here also no influence upon the character of the result. In every case care was taken to bring the cut well down toward the base of the rudimentary operculum. ‘The different levels iG. 21. Hydroides dianthus (X 30). Operations made on both opercula simultaneously. I, I, II, IV represent the regions of functional operculum in which the cuts of the four groups of experiments were made. The shaded portion of the rudi- mentary operculum between the two dotted lines includes all the cuts on this side. on the functional operculum are indicated in Fig. 21. A summary of the results is made in lable VIII. If we neglect the differences in the levels of the cuts in the rudi- mentary operculum and divide those of the functional one into 60 Charles Zeleny. four groups we get a very interesting correlation between the regions and the corresponding results of the operation. In this way we get four fairly well marked groups. Group I consists of accurately located cases near the distal end of the stalk where it expands into the cup. Group II has the earlier cases located from description alone, i in most cases stating that the “functional stalk was cut near its middle.” Group III has accurately determined cases located about one-fifth of the length of the opercular stalk from the basal suture. Finally, Group IV contains the cuts made just distal to the basal suture. (Fig. 21.) TasLe VIII. Hydroides dianthus. Both Opercula Removed. Bias Pa se F, = F. fs hey ease Ri = Rz Ro =e Ri = Fe i ine Ry = Groups lisewewccr: Oy 3 fo) fe) fe) Group WW Se ae: I I 3 I I Gseoivy oy bl bye emcees fo) 4 fe) fo) GrODP JING secel st ohce fo) fo) o] fo) F\= Original functional operculum. R= Resultant rudimentary operculum. Ri= Original rudimentary operculum. S = Functional stalk remains attached. F.= Resultant functional operculum. r’, r’= Small undifferentiated buds of new tissue. In Group I the three valid cases (see Table VIII) all showed a reversal of the opercula, the old functional becoming the new rudi- mentary and the old rudimentary becoming the new foperional: In Group II where the region of the cut was not so accurately located the results are scattering. Seven of the cases give results valid for our purpose. Of these three developed two functional opercula, one showed a reversal similar to that of Group I, one showed the development of a new functional in place of the old and a new rudimentary in place of the old rudimentary, in one the functional stalk did not become detached and the old rudimentary developed into a functional, and in still another case there were rudimentary buds on both sides, neither of which reached a stage beyond a rounded knob and were, therefore, not developed even up to the rudimentary stage proper. Compensatory Regulation. 61 In Group III, consisting of accurately determined levels, four of the five cases showing valid results developed two functional oper- cula, the fifth one showed a reversal of the opercula. In Group IV, also consisting of accurately determined levels, five cases showed a clear result sie all of them had a reversal of the opercula. * The result is a peculiar one in that the most distal and the most proximal groups agree in giving rise to a reversal of the opercula, while the intermediate two groups give rise in a majority of the cases to two functional opercula. An attempt at an explanation is hazardous and can be little more thana guess. Such a provisional attempt may, however, be made, for by so doing some light may be thrown on the regulation of the “normal”’ condition in the animal. An examination of the whole number of cases where both opercula are cut off shows that in all but two the rudimentary operculum after regeneration did not stop at the rudimentary stage but kept on developing until it reached the functional stage. ‘The difference in the results 1s then due to differences in the regeneratory development of the old func- tional operculum. What factor or factors hold it in the rudimen- tary stage in some cases, while in others it is allowed to develop into a full-sized functional organ? ‘Iwo factors are to be con- sidered: first, the influence of the position of the cut upon the initial stages of change in the embryonic tissue in the neighbor- hood of the basal suture and, second, the possibility of a retard- ing influence emanating from the new functional operculum which is rapidly developing on the opposite side from the stump of the old rudimentary. First Factor. It has been shown above (p. 58), in the series of experiments with an uninjured rudimentary operculum, that injury to the extreme distal portion of the functional operculum does not lead to the dropping off of the injured organ or to the development of the opposite rudimentary operculum into a func- tional. Further, from the same series of experiments it is seen that the development of the opposite rudimentary operculum 1s more easily induced by a terminal injury to the old functional than is the dropping off of the old functional stalk. ‘The latter point is well illustrated by several cases in which the injured stalk remained attached to the animal while the opposite rudimentary had already developed into a full sized new functional. From these data we 62 Charles Zeleny. may assume that the distal cuts do not affect the embryonic tissue at the basal suture as quickly as do the more proximal cuts and for this reason the tissue will have had only a small start when the opposite rudimentary already has a very considerable one. The latter may then restrict the further development of the bud after the old stalk has fallen off. In Group IV, on the other hand, the cut 1s so near the embryonic tissue of the suture itself that it may directly injure the cells which are to give rise to the mechanism of a cleaving plane or.else leave such short leverage in the distal por- tion of the stalk as to compel the growing tissue to do all the work in pushing off the useless portion and thus to retard its growth. It is possible also that in some of the cases in Group IV the short por- tion distal to the suture is not cast off, but that the tissues are re-formed and thus give rise to the growing bud. However, no observations were made on this last point and it 1s merely put down as a possibility in view of some of the later experiments on Pomatoceros (p- 70). In the above paragraph an attempt has been made to show how the embryonic tissues at the basal suture in Group I and in Group IV may develop up to the stage of a rudimentary operculum less rapidly than those of Groups II and III. Second Factor. Admitting this greater development of the bud of the old functional side in Groups II and III than in Groups I and IV, and further assuming the uniform development of the bud of the old rudimentary side in all four groups, we are led to the consideration that if one of the opercula when well developed com- pels the other to stay in a rudimentary stage such a cause may act in Groups I and IV and not in Groups II and III. Therefore, in the former cases, the result of the simultaneous operation on both opercula 1s a reversal of. the original condition, while in the latter it ts the production of two “functional” opercula. }. Body Cut in Two. Regeneration of Opercula at the Anterior End. When the body is cut in two in the thoracic region two opercula and groups of branchiz are regenerated on the two sides of the median line but the opercula instead of being differen- tiated into a large one and a small one are both of the large func- tional type. We must assume in this case that since both had an equal start in development the retarding influence of the one upon the other which occurs in other cases did not occur here. The newly developed opercula were in some cases exact dupli- Compensatory Regulation. 63 cates, the one of the other, but in others one operculum was considerably larger than the other, though both showed the true ce = 9 bi functional”’ characters. “[he extremes of the different cases are given in the accompanying figure. (Fig. 22.) The resultant opercula usually differed from the normal func- tional one in being shorter and stubbier than the latter. It is to be noted that two fully developed opercula of the kind indicated can A ea at \ i , | AN f | \ \ / y| \ W WN. iY Uy \\ N y \ | N ‘ | / V I | | \ y \ vA porn Ks YY) Y\ V\ AA yf} ly Wy) PUR avy} Fic. 22. Hydroides dianthus. Opercula as regenerated at the anterior end of the posterior piece after trans- verse section in the thoracic region (> 17). Left, case with equal opercula. Right, case with unequal opercula. be of little value in closing up the opening of the tube as each one stands in the way of the other. The animals in the experiments were, however, not kept in their tubes so that the actions under such circumstances were not observed. It seems that the anterior missing segments were not regenerated in any case. Whether such regeneration would be possible under favorable conditions cannot be said: In my specimens bacteria and infusoria developed on the tender regenerating tissues and the growth was retarded and finally stopped. The main point as regards the opercula made out in the group of experiments where the body was cut in two in the thoracic region is this: When the opercular buds have an equal start in development both develop into functional opercula. 64 Charles Zeleny. The relation of the developing opercula and branchiz of each side to the nerve cord of that side is very interesting. This is most noticeable in the regeneration of these organs from a cut near the posterior end of the thorax where the nerve cords are widely separated. In the Serpulidz it will be remembered the nerve cords do not come together ventrally as in most Annelids but remain widely separated, forming two latero-ventral trunks. “The principal blood vessels, however, do not have this arrangement. The branchial circlets, each with its operculum, regenerating from a cut near the posterior end of the thorax, are always widely sepa- rated and seem to be located in intimate relation with the nerve trunks of the corresponding sides. ‘This fact agrees very, well with the data as made out by Morgan (’02) for the regeneration of the head of the earthworm which showed that the regenerating head always develops in connection with the anterior cut end of the nerve cord. A similar relation has been made out for other forms. A further discussion of this and other cases of nervous control in regeneration 1s reserved for a future time. The results of a transverse cut in the abdominal region were in every case negative as far as the posterior piece is concerned. Its anterior cut parce in every case healed over and no regeneration took place. ‘The piece lived for a considerable time hae did not show any signs of regenerating tissue. In this connection two other groups of experiments may be described. The first concerns the regeneration and regulation following the /ongitudinal dorso-ventral division of the body into equal right and left parts. In this group a dorso-ventral longitudinal cut divided the body into approximately equal right and ‘jeft halves. Fourteen speci- mens were operated on in this way and of these several showed traces of the regeneration of knob-like elevations near the anterior end of the cut surface. Two of these showed especially clear structures which correspond very well with young regenerating branchial circlets from the anterior end of a posterior piece after transverse section of the thorax. It 1s probable that the new structures may be located at a cut end of a nerve cord. In one of the cases the new circlet in question was a considerable dis- tance behind the old branchial circlet. In no case did the animal live long enough to allow of a full development of the new organs. Compensatory Regulation. 65 The effect of the cut upon the old organs is as follows: The rudimentary operculum in several cases showed a slight develop- ment though only one advanced to the stage with both rows of opercular processes present. Usually the rudimentary operculum remained unchanged or a slight development of the secondary processes took place. No changes were observed under similar circumstances in the functional operculum. The second group of experiments concerns the regeneration from a posterior cut surface of a half (right or left) Serpulid. ‘Two of the cases of regeneration at the posterior cut surface after longitudinal dorso-ventral section of the whole organism showed very clearly the character of the new tail bud regenerating there. After transverse section of the whole body in the abdominal region the new tail bud is always very evidently double. In the present experiments, however, where only half of the animal was used the regenerating bud always showed a single tail knob. As the two components of the ventral nerve-cord in Serpulids and Sabellids are widely separated this singleness of structure may be correlated with the presence of only one of these nerve cords at the posterior end when the animal is cut in two longitudinally before the cross cut 1s made. g. . Progressive Changes in Opercula. Progressive changes in the opercula of adult specimens were not observed. Several groups of specimens were kept under observation for varying periods of time but in no case was evidence of such a change noted. It must, however, be stated that the periods were all rela- tively short, not over a month at most. The indirect arguments in favor of the occurrence of such changes as furnished by speci- mens in nature with intermediate stages of reversal, etc., are given elsewhere. (p. 33.) b. Experiments on Group V. (See p. 26 for definition.) A series of experiments on H. uncinata was undertaken with the object of determining the relative capacity for regeneration at different levels in the body. ‘The most posterior region showing regeneration of branchial and opercular structures was an anterior cut surface located between the next to the last (sixth) and the last (seventh) thoracic segments. Several posterior pieces back of this point lived for a sufficient length of time to allow of regenera- tion if it were to occur at all, but all these healed up at the cut surface and showed no regeneration of head structures. We 66 Charles Zeleny. D 4 Fic. 23. A, B—Hydroides uncinata. Regenerating branchie and opercula at anterior cut surface after trans- verse section between fourth and fifth thoracic segments (93). 4—13 days after operation. B— 14 days after operation. C—H.uncinata. Regenerating branchie and opercula at anterior cut surface after transverse section between first and second thoracic segments, 18 days after operation ( X 60). D—Dorsal view of Apomatus ampullifera showing functional and rudimentary opercula ( X 5). Pinnules not represented. (See also Fig. 6 p, £, F). E, F, G—Apomatus ampullifera. Regenerating branchie and opercula at anterior cut surface after transverse section between the third and fourth thoracic segments, 23 days after operation (X 60). E—Dorsal view of both branchial groups. #—Left group as viewed from right side. G—Right group as viewed from left side. - Compensatory Reguiation. 67 must, therefore, for lack of positive evidence to the contrary decide that in H. uncinata the power to regenerate head struc- tures is found only in the thoracic region and that anterior surfaces of the posterior pieces after transverse section through the abdomen do not possess this power. The manner in which the regeneration takes place is extremely interesting when compared with the development of the same structures in ontogeny. It was found that in each of the cases at the earliest stages three branchial buds and one opercular bud were present on each side. (Fig. 23a, B, c.) This corresponds with the number present in the ontogenetic development of Hydroides at the first appearance of the opercula. A similar relation holds for the regeneration of the branchial circlets of Apomatus after a transverse cut in the thoracic region. A series of experiments was performed on H. pectinata to deter- mine whether the cutting of the animal in two by a transverse cut through the second and third segments of the thorax would cause any changes in the opercula remaining at the anterior end. In this series the opercula were not disturbed. ‘The result was not completely satisfactory because most of the specimens died at an early stage but it was found that the cut did not cause the opercula to change. A severe bodily injury, therefore, need not cause a reversal. Another series was undertaken in the hope of finding the influence of sectioning of the thorax upon the differentiation of the opercula after the functional stalk had been cut at its middle. It was found that the separation of the region of the body back of the fourth thoracic segment from the rest does not retard the changes of reversal in the opercula which usually take place after a section of the functional stalk at its middle. A single specimen of Serpula vermicularis was operated on. Both opercula were cut off, the functional one at its middle. ‘The result was a teversal of the former condition. “The animal was kept in its dish unobserved for about three months and was then found to have reversed back again to its original condition. 1. Experiments on Group IV. Apomatus ampullifera. “lwo characteristics of the branchiz and opercula of Apomatus need to be taken into account before going on with a description of the experiments. (See also description, p. 26.) 68 Charles Zeleny. In the first place there are two opercula, one a large spherical body and the other a very small terminal enlargement, each at the end of the branchial stalk occupying the next to the dorsal position in its branchial circlet. ‘This stalk is in each case a typical branchia except for the opercular enlargement and apparently carries on its full respiratory as well as its opercular function. (Fig. 23D.) In the second place each branchial semicirclet taken as a whole breaks off very readily along a definite line at its base so that all the branchiz including the opercular one come off together. ‘Thus a very slight irritation is sufficient to cause the animal to throw off the whole branchial apparatus, including the opercula. The fission plane is in a very definite region at the base of the branchial circlet and after coming off the whole branchial crown holds together in one piece because of the union of the branchiz near their bases. The right and left branchial circlets act independ- ently in the matter since it often happens that only one 1s cast off. Usually, however, both are thrown off. Such an operation as the removal of the animal from its tube usually brings about this autotomy of the branchiz. Out of 42 specimens removed from their tubes on November 6, 1902, thirty lost both branchial circlets, 6 lost one of the circlets and only 6 retained the whole branchial crown. For this reason it was not possible to repeat the ordinary operculum reversal experiments on Apomatus as after such an operation the branchial circlet was cast off. After the casting off of the branchial crowns in these cases a regeneration of two functional opercula usually followed, though one was often larger than the other. Probably correlated with the differentiation of a “breaking joint” at the base of the circlet is the fact that the regeneration of the branchial crown does not show only three branchiz plus the operculum at the first differen- tiation as in Hydroides but at once brings out several branchiz on each side. One of these may show the opercular differentiation from the start, while in other cases it develops first as a branchia and only later shows the opercular character. The regeneration at the anterior end of a posterior piece after transverse section through the thoracic region showed a less highly differentiated character of the new organ at the start than when the regeneration took place from the ‘ “breaking joint. . Aeteat number of operations were made, but the animal proved very Compensatory Regulation. 69 sensitive to the injury and nearly all the individuals died very early. However, the beginning of the process of regeneration was observed in a few cases. In one of these which had been cut through the third thoracic segment there was, 23 days after the operation, a distinct indication of the young branchial circlets at the anterior end of the posterior piece in the form of three branchial knobs on the left side and four on the right. (Fig. 23E, F,G.) Of the latter four the ventral one was very small and the next to the dorsal one evidently larger than the others and showing thus early its opercular anes The regeneratin tissue appears first as an undifferentiated mound. When dif- ferentiation does occur it takes on the form of three or four knobs in each mound, corresponding evidently with those of Hydroides dianthus after a similar section and reminding one strongly of the first branchial differentiation in the young of the Larter species where, as we have seen, each branchial circlet z appears first as three processes, one of which divides at its base, forming four in all. The early differentiation of the opercular knob after a thoracic cut in Apomatus as in Hydroides, however, brings in a point of dif- ference as compared with the ontogenetic development. }. Experiments on Group VI. In four specimens of Ditrupa subulata the functional stalk was cut intwo just below the terminal cup. [he embryonic tissues at the base of the stalk increase in bulk and bulge out, showing an oblique suture. ‘The stalk drops off a few days after the operation and a new operculum develops from its stump. Evidently the increase in the embryonic tissues serves as a mechanical stimulus for the dropping off of the old stalk. See Fig. 24a, B. All the specimens of Spirorbis Pagenstecher1 in which the operculum was removed died. ‘There is, however, evidence that regeneration of the operculum takes place. A considerable number of the specimens just removed from their tubes showed stages of growth of the operculum from a small bud to a large full-sized operculum. Whether the process is a direct physiolog- ical one or is due to injury cannot of course be definitely stated. A periodic replacement may be connected with a possible periodic injury during the breaking out of the embryos from the brood pouch, though evidence is also lacking as to the length of life of the animals. Several experiments on the regeneration of the opercula in 70 Charles Zeleny. Pomatoceros triquetroides were started. It will be remembered that the operculum has a distinct basal suture. In about half of the specimens which were removed from their tubes it was found that the oper- culum had_ been thrown off at this basal suture, the distal portion of the operculum remain- ing in the tube. ry The plane of the ; fracture is not a straight one but the middle is pointed forward so as to give in a dorsal view the form of an inverted A. Figs. SB, Cs 2ACh Ds Ee Three series of op- erations were Car- ried out. In the first the operculum was cut in two es distal to the basal suture. Here it Fic. 24. seems that the part A—Ditrupa subulata. Stalk, two days after operation, showing pro- of the operculum jection of new tissue at one side of basal portion below breaking joint (X20). B—Regenerating operculum of D. subulata, 9 days after above the suture operation (X 20). did not drop off but C, D, E—Pomatoceros triquetroides. Stages in regeneration of new the regeneration operculum from breaking joint level (X10). C—Operculum just took place by a pulled off. D—z3 days after operation. E—8 days after operation. growth from the F—Vermilia multivaricosa. Stalk of operculum two days after removal of cup (X16). Note projecting knob of new tissue at side of ee surface. The stalk. first sign of this regeneration was a swelling of the terminal region from which three knobs developed, which evidently became the terminal processes of the new oper- culum. The evidence for this change and for the later changes in general is not complete as the later stages were not followed out. Compensatory Regulation. 71 The interesting general point is that regeneration takes place from the cut surface without a breaking off at the basal suture. When the operculum was pulled off at the time of removal of the animal from its tube the break always took place at the A-shaped suture and the regeneration then naturally followed from this level. In a third set of experiments the animal was cut in two in the thoracic region. All such specimens, however, died before the appearance of regeneratory changes. The operculum was removed in five specimens of Ver- milia multivaricosa. In no case was there any regeneration of the organ. In one individual the cut was made through the narrow portion of the stalk just below the terminal cup. In this case (Fig. 24F), two days after the operation, there was a protruding knob on the median side of the stalk which may represent the beginning of an opercular regeneration, such as that shown in the case of Ditrupa. (Fig. 24.) In the other four specimens the cut was through the cup portion of the operculum. In all of these there was no regeneration, though three of them lived more than eleven days after the operation. k. Discussion of the Data. It has been seen that the char- acter of the regeneratory process varies according to the loca- tion of the cut. When the regeneration takes place from the breaking joint of the operculum (Hydroides, etc.) or of the branchial circlet (Apomatus) the regeneration is highly special- ized and the stages do not follow the ontogenetic ones very closely. When, however, the regeneration 1s from a thoracic cut, where the branchial and opercular tissues are not as highly special- ized with respect to the mechanism of regeneration, the organs pass through a stage which may very well be compared with a corre- sponding stage in the ontogeny of Hydroides. However, even here the regeneratory development does not follow the other closely because the operculum Is very evidently differentiated as such from the start in regeneration though not 1n ontogeny. Our general conclusion may, therefore, be that when there is no definite mechanism for the autotomy of a region of the body the regenerating tissue may in its various stages resemble ontogenetic stages quite closely, but where a definite mechanism is present the resemblances are much less close, the development being hastened in the latter as compared with the former and both being hastened, 72 Charles ZLeleny. though in different degrees, as compared with the ontogenetic development. The discussion of the regulation of the process may be referred to the general discussion of compensatory regulation in the group of Serpulids, as given on p. 76. 3. Probable Phylogenetic Development. The opercula and branchiz of the family Serpulidz furnish as good a case of a morphological series as can be found within the animal kingdom. ‘There are all gradations between species with no modification of the branchiz up to those with a degree of © opercular modification so great thatno branchial characters can be made out inthe organ. Furthermore, in the ontogeny of the one form studied (Hydroides), in which there is a high degree of modifi- cation, each of the two opercula passes through a stage in which it 1s to all appearances a functional branchia. The paleontological evidence, however, is fragmentary. Our only knowledge is obtained from the calcareous tubes and it is not always possible to decide whether the animal inhabitant was or was not operculate. Tubes evidently belonging to the genus Spirorbis are, however, found as low down as the upper Silurian. The morphological and ontogenetic evidence leads us to the probable conclusion that the ancestors of the present day opercu- late Serpulids were non-operculate forms and that the opercula arose inthe course of phylogeny by the development of enlarge- ments upon the branchiz which served to close the opening of the tube in which the animal lived. Some speculations as to the origin of the asymmetry of the opercula in the Serpulids may be permissible if it is recognized that the course of the probable phylogeny can at present be no more than guessed at. ‘The existence of a morphological series running From forms with no opercular modification of the branchiz (Protula) through forms with a terminal enlargement at the end of each branchia (Salmacina), others with two equal opercular knobs one on each side of the median line attached to stalks still retaining respiratory pinnules (Filograna), to still others with a large operculum on one side and a small one on the other (Hydroides, etc.) or with one operculum and that lateral in posi- tion (Ditrupa, etc.) indicates that the early differentiations of the Compensatory Regulation. Ge: operculum were symmetrically arranged with respect to the median line. However, the ontogeny of Hydroides shows an asymmetry from the very first appearance of the opercular modification. Furthermore, the fact that this earliest development always occurs on the left side indicates some correlation between the character of the tube and the position of the organ. In Hydroides, how- ever, there is an irregularity in the coiling of the tube from the very start, so that we get no evidence here of such a relation. An examination of several Serpulids brings out the following relation between the adult position of the functional operculum and the character of the coils of the tube. A tabulation of the result is given below: TasBLe IN. Genus Funct. Operculum Tube SPILORDIS pin 4 e ayeraw ts ony Always right... . 0dc.0s tt Dextral coil. Pileohanars xs pistot.saster 3 ARRAYS NCEE a tay as 40). Operation: Autotomy of both branchial circlets at breaking joint and removal of body posterior to second thoracic segment. Note pronounced opercular differentiation on both sides. these limitations it is evident that we have a definite acceleration of the rate of differentiation of the opercula. Two probable factors may be Oe as concerned in the bringing on of the acceleration. The shock of the transverse division of the body may lead to eh an increase in rapidity of differentiation. 2. The small size of the piece itself may directly influence the process and bring about such a differentiation. This action may result because of the difference in the interactions of the organs in the one case as compared with the other. Compensatory Regulation. 81 V. REGULATION OF THE RATE OF GROWTH AND NATURE OF DIF- FERENTIATION DURING REGENERATION OF THE CHELAE OF GELASIMUS AND ALPHEUS. 1. Introduction. The general problem to be taken up in the experiments on the chelz of the two Decapod Crustaceans mentioned corresponds with that already given for the Serpulids. ‘The interactions of the two chele naturally constitutes the principal point of study. Likewise the influence of the removal of one or both chelz upon the rate of moulting of the animals will be discussed and some further incidental points will be touched. In Gelasimus pugilator the two chele are of nearly the same size and character in the female but differ widely in the male. In Alpheus dentipes the chelz differ both in size and character in both male and female. 2. Gelasimus Pugilator. In Gelasimus the male has one of the two chele enormously developed. ‘This large chela is nearly equally distributed between right and left sides in a group of individuals taken at random. In the female the two chelz are small and equal in size. ‘Che animals readily autotomize their legs if a needle is inserted between two of the joints distal to the “breaking joint”? so as to touch the nerve. In the following experiments the animals were made to throw off their chela in the way mentioned. ‘They were kept in glass dishes with just sufficient water to keep them moist and fed with bits of the horse-mussel, Mytilus. Under these conditions they lived very well, though unfortunately the growth of the new legs was extremely slow and the experiments could not be com- pleted as satisfactorily as was wished. 1. Experiments on Males. a. Large Chela Alone Removed. ‘The first object of the work was to determine whether reversal of the character of that of Hydroides takes place in these forms. ‘The large chela was autoto- mized in the manner already indicated. In the great majority of the cases the animals lived through the 62 days after the opera- tion, but in only a few did a moult take place so that the results are 82 Charles Zeleny. not entirely satisfactory. “[wenty specimens, ten with the large chela on the right side, and ten with it on the left, were treated in this way. Five specimens had moulted at the end of 62 days after the operation when the experiment was closed. ‘The first one moulted 54 days after the operation and in this the regen- erated chela ( = former large one) was as yet smaller than ihe other (= former small one). The old small one had no pro- nounced change as a result of the moult. In the four other specimens, one of which moulted 59 and the other three 62 days after the operation, the new regenerated chela was in each case larger than the opposite old one, though it had not as yet attained the full size and characteristics of the typical large one. It may be safely concluded from the above observation that no reversal of the chele in the sense of the reversal of opercula in Hydroides takes place in the males of Gelasimus after removal of the large chela, for it seems evident that in the first case men- tioned, where the regenerated chela was as yet smaller than the old small one of the opposite side, it had not yet reached its full growth. In further support of this view is the fact that no pro- nounced change in the old chela was noticed. b. Small Chela Alone Removed. ‘The following results were obtained when the small chela alone was removed: Only four of the ten specimens moulted before the end of the 62 days, con- stituting the limit of the experiments. In all of these the newly regenerated chela were much smaller than the opposite large ones ao approached i in character the ordinary small chele. ‘The four specimens mentioned moulted, respectively, 48, 61, 61 and 62 days after the operation. ‘Therefore, here also there is no reversal of the chelz. c. Both Chele Removed. In this set of experiments both chelaee were autotomized. ‘Ten specimens were kept for 62 days and eighteen for 42 days. Seven of the former moulted and showed the characters of the regenerated chela. In each of the seven a large chela was regenerated in place of the former large one and a small chela in place of the former small one. ‘There was no reversal. The chelz after the first moult did not of course as yet have the full size of the old ones but the difference in size was very evident. In one of the seven cases mentioned here as having moulted so as to show the characters of the regenerated ‘chelz one specimen Compensatory Regulation. 83 showed an abnormality in that the smaller regenerated chela had two pinchers at its end. ‘This case will be described in a separate note at another time. d. Two normal male specimens kept in glass dishes for 62 days did not moult or show any changes. They were fed on fragments of Mytilus 1 in the same manner as the others. 2. Experiments on Females. a. Removal of One Chela (right or left). Two of the six specimens moulted before the completion of the experiment. In one of these the regenerated chela was a trifle smaller than the opposite one. In the other the two chelz, the old and the regener- ated one, were nearly equal in size after the moult. One of these specimens moulted 56 days, the other 62 days after the operation. b. Removal of Both Chele. Only three specimens were oper- ated on in this way. All had moulted within 46 days after the operation. ‘The new animals regenerated two new and equal chelz. ‘The regenerated structures, therefore, repeat the char- acter of the removed appendages. 3. The Rate of Regeneration and of Moulting After the Opera- tion. (Male and Female.) The data show very plainly that the moulting takes place sooner in the cases where both of the chelz are removed than in the cases where only one or none are removed. In fact all three mem- bers of the female set with both chela removed moulted before any of those with only one chela removed had done so. It does not seem possible that the matter of accident can come in here as there are too many cases both as regards this point and as regards other related ones. A general comparison in both males and females of the cases in which both chelz were removed with those in which only one or none were removed bring out an interesting result. 1. Time of moulting. The specimens with both chele removed moulted sooner than those with only one chela removed. De = Both chele (Cu+ Sn) removed. In the upper data the upper line is drawn to fit the first two groups of cases (one chela alone removed) and the lower line to fit the last group (both chele removed). Compensatory Regulation. 87 “the reversal in some cases at least does not take place or is incom- plete.” Brues (Wilson, ’03, p. 210) adds the interesting fact that in A. heterochelis the nerves supplying the two chelz and the ganglionic centers from which they proceed do not differ percep- tibly in size. 2 whesWata. The experiments about to be described in the present section of the paper were performed at the Naples Zoological Station in the winter of 1902-03. They confirm the general facts of reversal of the chelz as given above. Their main object, however, was the dis ina een first, of the effect of the removal of one or both chelz upon the rate of moulting of the animal, and, second, of the influence of the presence or absence of the opposite chela upon the rate of regeneration of a chela. a. The Influence of the Removal of One or Both Chele upon the Rate of Moulting of the Animal. ‘Three sets of specimens were operated on. In one set (Sm) the snapping chela alone was removed. Ina second (Cw) the cutting chela alone was removed. Inathird (Sn + Cu) both snapping and cutting chela were removed. The animals were kept in isolated dishes for 59 days after the operation and were fed either every day or ev ery other day on small pieces of fresh fish meat. Without taking into account the cases where the legs were accidentally autotomized a second time during the experiment, or in which other disturbances occurred, we have the following relation between the time of moulting and the post-spinous thoracic length of the animals without reference to the character of the operation: On the codrdinate paper (Fig. 28, p. 86) the abscissz represent the thoracic lengths in millimeters and the vertical columns (ordinates) the days after the operation when moulting occurred. ‘The animals were killed 59 days after the operation. The data from the first set (Cu) are represented by the symbol ©, those of the second set ($7) by the symbol © and of the third set (Sn + Cu) by x. It will be seen that in general the moulting interval increases with the size of the animal as repre- sented by the thoracic length. On pp. 88 and 89 the data are put in Tables X, XI and XII, each of the sets being placed by itself. ‘The interval of time in days between the first moult and the second moult is put down in a separate column. Upon averaging this interval in the three 88 Charles Zeleny. sets separately it is found that the interval decreases from 29.6 days jor the Cu set and 28.7 days for the Sn set to 22.9 days for the Sn + Cu set. This result is represented on codrdinate paper on p. 86, Fig. 28. When two chele are removed there is, therefore, a shortening of the period between the first two moults as compared with the cases where only one chela is removed. It will be seen that there is only a slight difference between the moult period in the two single chela cases. ‘This result agrees perfectly with that obtained for the time of appearance of the first moult in Gelasimus, where TasB_eE X. Alpheus dentipes. Time of Moulting. Cutting Chela (R or L) Removed. | Thoracic | Date of | Ist 2d 3d Interval Cat. No. | | | | Length. | | Operation. moult. moult. moult. Ist—2d. or cle ae ! | 1903s 562 6.6 1/8 | 5 2A Pal! oe ta 19 565 8.5 UAE 2 | 29 52 27 569 8.0 MiSteed | Pe OF esl) oe Me 39+ 573 8.5 Igy ath 22.9) 48 | =— |) 636 576 6.5 I/9 | TO |) a pee 2 579 Seem et Ce el ie: Gm eRe eA fe 582 | | I/9 | 200 | oe ee Oe Ave 29.6 it was found (p. 83, ffi.) that the specimens with both chelz removed in every case except one moulted before those with only one chela removed. The greater disturbance of the normal condition of the animal here again causes greater activity as regards the moulting period. Starting with the sipallest disturbance ada going upward we have a series ranging, respectively, through (1) cutting chela removed, through (2). snapping chela remov ed, to (3) both chelz removed. Correspondingly, the interval between the first and second moult decreases from 29.6 days, through 28.7 days to 22.9 days for the cases named. ; Compensatory Regulation. 89 TasLeE XI. Alpheus dentipes Time of Moulting. Snapping Chela (R or L) Removed. (Sige Thoracic Date of Ist 2d | 3d Interval Se lt Wenceh..| Operation. moult. moult. moult. Ist—2d. 561 8.7 1/7 24. 56 a 32 563 7-9 1/8 22 56 = 34 575 8.5 I/9 24, 55 oe 31 578 | 5.0 I/9 19 45 = 26 Som 850 I/9 16 45 oo 29 617 6.0 I/7 20 42 — 22 619 II.4 17 lh db 49 | — 23 620 ea 1/7 18 51 | = 33 Av. = 28.7 TaBLeE XII. Alpheus dentipes. Time of Moulting. Both Chele Removed. Thoracic | Dateof | 1st | 2d 3d 4th Interval Cat. No. 2 . Length. | Operation. moult. | moult. | moult. | moult. | Ist—2d. 1903 506) + 6.2 | E78 17 37 | — | 20 567 8.7 pen n/s 23 50 —- — 27 St |W etora IG 2) Tae ae AMR a 27 574 8.2 Howe) 23 50 a ae 577 5.5 I/9 2016 | AS Nc rh eee 25 580 4.9 I/9 20 £5 eet 25 583 5.0 I/9 20 OM (Mee So ke 584 Get I/9 pie Mi) ei ~- = 4 \|-" 20 585 a7 eileen Ei te 45 = 17 618 8.0 Wis, 4 | ozo: | Oe ae 16 Summary OF Tapes X, XI, XII. Comparison of Interval Between First and Second Moults. Cu Removed = 29.6 days. (Av. of 7 cases.) Sn Removed = 28.7 days. (Av. of 8 cases.) Cu+Sn Removed = 22.9 days. (Av. of Io cases.) go Charles Zeleny. Original cutting chela length in millimeters. 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 3 ° 5 a E ee wn 5 3 A E fi aq ~_ oo =] a) 3 S a 1) oo 5 = =] ° 3 z o S o ivi) ) 6S Ba re) EB ° i] o i) 3.0 4.0 5.0 6.0 7/40) 8.0 9.0 10.0 Original cutting chela length in millimeters. Fic. 29. Alpheus dentipes. Lengths of regenerating cutting chelz at the end of the first and second moults. Ordinates = Regenerated cutting chela lengths in mm. Abscisse = Original cutting chela lengths. ©O = Cutting chkela (Cu) alone removed. © = Snapping chela (Sn) alone removed. > = Both chele (Cu-+ Sn) removed. The broken lines fit the last group (both chele removed) and the unbroken lines fit the first two groups (one chela alone removed). i Compensatory Regulation. gI b. The Influence of the Presence or Absence of the Opposite Chela upon the Rate of Regeneration of a Removed Chela. A comparison was made of the regenerated lengths of the cutting chela in cases where only one chela was removed with cases where both chele were thrown off. When the cutting chela alone was autotomized a new cutting chela was regenerated in its place. When the snapping chela alone was removed there was a reversal and the old cutting chela was differentiated into the new large snapping chela, while in place of the removed snapping eels. a ty pical cutting chela was developed. In the second case this new cutting shel is the one taken in our measurements. In a third case Theeh chelz were thrown off, a new cutting chela regenerating in place of the old cutting chela and a new snapping chela in place of the old snap- ping one. ‘The lengths are taken from the moulted casts of the animal, the original length being taken from the cast of the first moult, the first moult condition from the cast of the second moult, etc. The final measurements are taken from the alcoholic speci- mens of the animals killed 57 days in each case after the operation. The lengths measured in the data about to be described are the greatest lengths of the cutting chela, 7, ¢., the distances from the tip of the pincher process of the fourth podomere to the farthest corner of the base. (Fig. 27.) The chelz or their casts were in every case drawn carefully to scale by the aid of a camera lucida and the measurements are taken from these drawings. Out. of 29 specimens kept for 59 days only 12 can serve for first moult data and 13 for second moult data. ‘The others are not valid because of the death or escape of the animal or the accidental secondary autotomy of one or both of its appendages. The relation of the regenerated chela lengths to the original lengths is shown on coordinate paper (Fig. 29) for both the first and the second moult. “The number of individual cases is small, but it is evident that the regenerated lengths of the cutting chela in the cases where both chelz were removed have a distinct advan- tage over the others. ‘This is especially clear for the first moult. The relation comes out very clearly when we take the ratio between the regenerated cutting chela length and the original length in that specimen as our basis for comparison, for we see that the regenerated length increases as we go from small original lengths to large original lengths. As the cases given on the coor- Q2 Charles Zeleny. dinate paper (p. 90, Fig. 29) show, the correlation is not a perfect one, 7. é., it is positive but equal to slightly less than one. ‘The results of such a comparison are given for both the first and the second moults in Tables XITI-XVI. TasLe XIII. Alpheus dentipes. Length of Regenerated Cutting Chela. Cutting Chela Removed. Cat. | Original | Reg. Cu. Lg. Regen. Cu. Reg. Cu. Lg. Reg. Lg. ee 100 jee Oo No. | Cu.Lg. | 1st moult. Orig. Cu. | .2dmoult. Orig. Lg. SUSU we as 80 tl 54.3 )/ groe hy” | jase 570)! 19.8 | 6:3 64.3 77 78.6 7 Ss | ae | 59-3 or | 74-3 Tasie XIV. Alpheus dentipes. Length of Regenerated Cutting Chela. Snapping Chela Removed. Cat. Original | Reg. Cu. Lg.| Regen. Cu. Reg. Cu. Lg.) Reg. Lg. | = = Xi1C —=—_= X 100 No. | Cu. Lg. | Ist moult. | Orig. Cu. 2d moult. | Orig. Lg. 561 Kamses | O27, iden! V7 87.8 563 Gul | 6.0 65.9 6.9 75ne Dias wos || 6.3 64.9 6.7 69.1 0 Ol ales een Naan 57-6 4.0 67.8 — | == — 66.1 == 75.1 The tables show that Cu + Sn has a very distinct advantage over Cu alone or Sn alone. ‘This advantage amounts to 19.1 per cent for the first moult and 16.5 per cent for the second moult. Just after the first moult the cutting chela regenerated from the breaking joint surface of what was “formerly the snapping claw has a ae advantage over the cutting chela regenerated from a removed cutting chela but this advantage 1s nearly overcome at the time of the senna moult. Compensatory Regulation. 93 TasLeE XV. Alpheus dentipes. Length of Regenerated Cutting Chela. Both Chele Removed. ce ia Reg. ars Rese PE al ee ae Se aeD sie oO. u. g- Ist mou Gs Orig. Leg. zy mou 1&5 | Orig. Ug: REG)! 5-7 3-7 64.9 eee ee la. 567 7.5 6.4 85.3 | 6.9 92.0 574 709 =| 57 72:2 6.4 | 81.0 Silat esos 3-9 68.4 | he 78.9 580 eae2) a — 3-4 | 106.2 583 4.6 Zia) 7823 | 4.0 | 86.9 584 520 329 78.0 | 4.5 go.o = = = 74.5 oe 87.0 | TasBL_e XVI. Summary of Tables XIII, XIV and XV. First Moult. | Second Moult. Original data. igecuetered Lg. oe a Cu 59.3 74.3 Original Lg. | Sn 66.1 Text Cu + Sn 74.5 87.0 Comparisons. | (Gas= Sn)i— Cul earse2 +12.7 Absolute difference ....... | (Cu + Sn) — Sn + 8.4 +11.9 { Sn — Cu + 6.8 + .8 Enea ee +25.6 +17.1 Cu | Per cent of increase ...... | (Cu + Sn) — Sn] +12.7 aaSe8 Sn | Sn — Cu —--- +11.5 + 1.1 Cu Q4 Charles Zeleny. The general result is clear. The regenerated cutting chela is larger in the case where two chelz are regenerating than where one alone is to be replaced. ‘This result is emphasized by the fact that the time between the first two moults is'shorter in the specimens with two chelaz removed than in those with only one PONE. 1( See p./07;) 3. Discussion. The significance of the data*may be emphasized by bringing them out in two ways, one of which lays special stress upon the fact of removal of a certain organ or organs and the necessity of a certain amount of regulation in restoring the normal form, and the other of which emphasizes rather the interactions of the two chelz as parts of a system normally stable at the condition with a large snapping chela on one side and a small cutting chela on the other. The first point of view in which the total necessary amount of regulation is compared with the rate of regeneration of a part of the whole will first be taken up. ‘The three series of experiments may be compared in the following way: 1. With the cutting chela alone gone the animal has merely to accomplish the regeneration of this organ in order to regain its normal condition. 2. With the snapping chela alone gone the animal has not only to regenerate a cutting chela in place of the old snapping one but also to differentiate the tissue of the old cutting chela into the new snapping chela. 3. With both chelz gone the animal has not only to regenerate a new cutting chela in place of the old one but also to regenerate a new snapping chela. Taking the ratios given in Table XVI, p. 93, we see that in the first case where the least work is to be accomplished, at the end of the first moult the cutting chela has regained but 59.3 per cent of its original size, while in the second case it has reached 66.1 per cent, and in the third case 74.5 per cent of its original size. At the end of the second moult likewise we have, respectively, 74.3 75-1 and 87.0 per cent for the three cases in question. Therefore, the amount. of actual regeneration accomplished in the cutting chela is greater the greater the amount of other work of a similar character to be accomplished at the same time. Compensatory Regulation. 95 But if we consider the matter from the second point of view, namely, of the influence of the presence of another similar and opposite organ upon the regenerating tissue, the apparent anomaly of the case is cleared up to a great extent. For it is seen that in the first case (Cu alone removed) we have an uninjured opposite large snapping chela to retard the growth of the regenerating cutting chela. In the second case (Sn alone removed) we have as the retarding agent at the beginning merely the smaller Cu chela which, however, gradually undergoes the changes in size and character leading up to the large snapping chela. Finally, in the third case (Cu + Sn removed) we have at the beginning no retarding agent, though gradually the snapping chela develops from this point. Expressing this in concise form we have the following relation referring to the retarding agent as indicated by the size and com- plexity of differentiation of the chela situated on the side opposite to the developing cutting chela: The snapping chela in the group where the cutting chela alone is removed is greater than the cutting chela developing into a snap- ping chela, as in the group where the snapping chela alone is removed and this in turn is greater than the retarding agent where both chela are removed, and which amounts to zero at the beginning with a gradual development up to a snapping chela condition. And referring to the corresponding regenerated lengths of the cutting chela we have: The amount of regeneration of the cutting chela in the first group (Cu alone removed) is less than the amount of regeneration of the cutting chela in the second group (Sz alone removed), and this in turn is less than the amount of regeneration of the cutting chela in the third group where both hela are removed. In graphic form this may be represented as follows: Snapping chela [as in Cu group] > Cutting chela (— snapping chela) [as in Sn group] > Zero (— snapping chela) [as in (Cu + Sn) group]. Correspondingly, Amount of regeneration of cutting chela in Cu group < Amount of regeneration of cutting chela in Sn group < Amount of regeneration of cutting chela in Cu + Sn group. ae 96 Charles Zeleny. Evidently the differences between the retarding influences of the three members are greatest near the beginning of the experiments, 1. é., immediately after the operation, and gradually decrease as we go away from this point. Correspondingly, the results show a greater comparative difference in regenerated material at the end of the first moult than at the end of the second moult. This result agrees very well with the experiments on the fiddler- crab Gelasimus (p. 81) and on the brittle-star Ophioglypha, (P- 7): s moulting involves not only an increase in bulk of the animal but also a complicated degree of differentiation of materials before it can be accomplished, we may likewise compare the acceleration of moulting in Alpheus and Gelasimus with the acceleration of the rate of differentiation of the opercula in Apomatus when the pos- terior region of the body is also removed as compared with the cases where this region is uninjured. GENERAL DIscussION. The following discussion does not serve as a summary of the data of the preceding sections. For this the reader must be referred to the summaries of the individual sections which are complete entities in themselves. It is the writer’s purpose in the general discussion to show the manner in which the various data, at first sight seeming to have little in common, can be brought under a common point of view. In the introduction it was stated that the standpoint of the present paper would be the consideration of the organism as a system made up of mutually interacting parts, the relations of which were to be studied by noting the disturbances produced as a result of the removal of one or more of the parts. In the paper on the dimensional relations of the members of compound leaves the relations of the parts of a system were studied in which the removal of one member was not followed by its regeneration but resulted in changes in size and position of the remainder. ‘The chief reactions were the following: In the five- leaved forms in which an asymmetrically placed leaflet was removed the other four leaflets tended to rotate to a position such that the new system was a symmetrical four-leaved one. Like- Compensatory Regulation. 97 wise in the three-leaved system after removal of one of the asym- metrically placed leaflets the two remaining leaflets tended to take up a position so as to form a symmetrical two-leaved system. From the position reactions it is evident that the parts of the normal compound leaf are exerting a continual influence upon each other which when resolved into its resultants gives rise to a configuration very definite for a given species. “The removal of one of the parts changes the whole system of reactions, and we have a tendency toward the formation of a new stable symmetrical system, with one less leaflet than the original number, the completeness of the new symmetry being only limited by the rigidity of the leaflets. In Ophioglypha we have a radial system in which the removed arms are regenerated. ‘The experiments on the rate of regenera- tion bring out the presence of an unsuspected interaction between the arms which must naturally be correlated with some interac- tion present in the perfect, unmutilated animal. ‘The data of the experiments show that (leaving out of consideration the cases where all five arms are removed and which cannot be used because of the early death of the animals) the rate of regeneration of an arm is greater the greater the number of other arms removed at the same time. ‘This indicates an interesting interaction of the arms upon each other for the presence of unremoved arms seems to retard the rate at which the removed ones are regenerated, for it is not probable that the increase in rate in the one case is due entirely to the increase in stimulus to regeneration produced by the added injuries. The two members of a pair of appendages 1 in bilateral animals have been shown by the present experiments to have a profound influence upon each other. Inthose Serpulids, for example, which have one large functional and one small rudimentary operculum it has been shown that either organ originally has the potentiality of developing into a functional operculum, which is to be developed in this way, depending upon the matter of an early start. When one side gets a start over the other the development of the latter is restricted to a rudimentary stage, while the former develops to a full functional size. Also, when the functional operculum is removed its restricting influence being removed at the same time, the rudimentary operculum immediately develops into a functional one, which in turn restricts the developing new bud of the other side. When both develop at 98 . Charles Zeleny. the same time, as from the anterior cut surface of the thorax, two functional opercula are formed. Similarly, in the Decapod Crustaceans the two chelz have a profound influence upon each other. In Alpheus there are two chela, one a larger “ snapping” chela and the other a smaller “cutting” chela. The snapping chela seems to hold the cutting chela in check, for as soon as the former is thrown off the cutting chela changes over to the snapping chela by a qualitative and quantitative change combined. ‘The new organ regenerating in place of the old snapping chela comes into a system no longer relatively like the old, so that the inter- action of parts forces it into a different niche in the new order of things. ‘The reversal, here as in the Serpulids, is easily under- stood in the way mentioned, if we consider the systems as asym- metrical interacting systems such that the removal of one part can lead only to the development of a certain definite structure. The removal of the organ (functional operculum or snapping chela) brings about an instability in the system which because of the reactions between the parts tends to assume the condition of a new stable system. ‘This new system reacts now in a different way on the regenerating organ, causing it to develop into a different structure. The readjustment in the old material and in the regenerating material is further complicated by the fact that both processes go on at the same time, the final outcome being the resultant of both. In Alpheus as also in Gelasimus we have an interesting relation between the two chelz, in that when both are removed the rate of regeneration is greater in each than when one alone is removed. Evidently this comes under the Ophioglypha relation that the presence of an unremoved organ retards the rate of regeneration of a removed one. Likewise if we consider the cutting chela of Alpheus as a stage in the development of the snapping chela (Wilson, ’03) and the rudimentary operculum as a stage in the development of the functional, we can say that the presence of the larger organ retards the differentiation of the smaller one. This comes into relation with the series of experiments on the rate of differentiation of the regenerating opercula in Apomatus, in which it was found that the absence of the posterior region of the body back of the second or fourth thoracic segment accelerates the rate of differentiation of the regenerating opercula. Compensatory Regulation. 99 From the point of view of the retarding influence exerted by one organ upon another the data that have been brought out in the present paper may be collected in the following concise form: 1. Opbhioglypha. Arms a, }, c, d, e. a1 = Rate of regeneration of an arm when it alone is cut off. a2 = Rate of regeneration of an arm when two arms are cut off, etc. Ta, = Retardation as result of influence of remaining arms on ai, etc. Then as = 04 + Ta, = G3 az = G2 + ra, = a1 + Tay arms remaining = none, e, dande, c,dande, b,c,dande. Inthe above as > as > Grae ida > 4a atidun COME wiiage. | Ve, 1 Bane RG Ta, ays 2. HH ydroides. Opercula, OFgori and ORrorr. OFr ort = Functional operculum, right or left. Or:x ork = Rudimentary operculum, left or right. Taking for the sake of simplicity the opercula as Org and Orxz we have: O peration. Result. Explanation on “ Retardation” Theory. (1) Both off. (thoracic cut) Orr; + Or. No retardation of one by the other. Therefore both reach full develop- ment. (2) Functional off. = Orr+Orz. Reversal of opercula. Release of normal retardation of old rudimen- tary and the presence of a new retardation influence upon the new rudimentary. .*. a reversal. (3) Rudimentary off. = OFg + Ort Full action of old retardation influence upon the new bud and therefore no change. 3. Gelasimus. Chelae Cr, Ci. Asymmetry in J only. In & one chela is considerably larger than the other, but the condition is fixed and cannot be reversed as can the opercula of Hydroides. In 9 the two chelz are equal. Cr + CL, as compared with Cr alone or Ci alone, shows an acceleration of the time of moult and has each chela bud larger than the single bud of the latter. 4. Alpheus. Chele Cr, Cir. Asymmetry in both Gand 9. The basal ganglia controlling the chelz are similar on the two sides (Brues). (1.) Reversal takes place as in Hydroides. 100 Charles Zeleny. (2.) Relations of time of moult and size of regenerated structure similar to those of Gelasimus. 5. Apomatus. Serpulid. Opercula OFgort and ORrorr. (See Fig. 23D.) Operation. Removal of two branchial circlets + opercula at basal suture. Uniform in all experiments. (1) In one set of experiments body of animal is kept entire. (2) In other set of experiments body of animal back of second or fourth thoracic segment is removed. Result: Rate of differentiation of new _— Rate of differentiation of new opercula opercula in (1) -=> in (@). because Retarding influence of whole _ Retarding influence of anterior two or body (1) ae four thoracic segments (2). The superficiality of the attempted explanation of the data is very apparent, but it is from this point of view that the analysis of the problems must be attacked. ‘That the factors which it is attempted to isolate are real factors in normal development is shown by the physiological reversal of the opercula which takes place in the ontogeny of Hydroides, a process in all ways similar to the reversal after artificial section of the stalk of the functional operculum. From such a point of view the present analysis may be of value as affording a basis for further experimentation on the isolation of the factors involved in regulation. Hull Zodlogical Laboratory, University of Chicago, May 17, 1904. Compensatory Regulation. IOI LEIP ERATURE Crib iD: Acassiz, ALEX., ’66.—On the Young Stages of a Few Annelids. Ann. Lyc. N. H. N. Y., vol. vii, pp. 303-343. Bruges; C. T., ’03.—Reference in Wilson, E. B.,.’03. ’04.—Biol. Bulletin, vol. vi, No. 6, May, 1904, p. 3109. CaAuLLERY ET MEsnIL, ’97.—Etudes sur la morphologie comparée et la phylogénie des species chez les Spirorbes. Bull. Scient., France, Belg., t. 30, pp- 185-233. *97.—Sur les Spirorbis; asymétrie de ces Annelides et enchainment phylo- génique du genre. Compt. Rend., t. 124, pp. 48-5o. CLAPAREDE AND MEcznikow, '69.—Beitrage zur Erkenntnis der Entwicklungs- geschichte der Chetopoden. Zeit. wiss. Zool., Bd. 19, pp. 163-205. Grarp, A., ’76.—Note sur l’embryogénie de la Salmacina Dysteri Huxley. Compt. Rend., t. 82, pp. 233-235 et 285-288. GruBeE, Ep., ’40.—Actinien, Echinodermen und Wiirme des Adriatischen u. Mittelm. Konigsb., p. 64. Grusg, Ep., ’62.—Mittheilungen tiber die Serpulen mit besonderer Beriicksichti- gungihrer Deckel. Breslau. Sitzung der Schlesischen Gesellschaft fiir vaterlandische Kultur am 19 Juni, ’61. °78.—Annulata Semperiana. Mem. de l’Acad. des Sc. de St. Petersbourg. P- 279. Haswe Lt, W. A., ’85.—The Marine Annelids of the Order Serpulea. Some Observations on their Anatomy, with the Characteristics of the Australian Species. Proceedings of the Linnean Soc. of N. S. Wales, vol. 1x, part 3. Lerpy, J., ’83—Manayunkia speciosa. Proceed. Acad. Nat. Sc. Philadelphia, 1883, pp. 204-212. Meyer, Ep., ’88.—Studien iiber den Korperbau der Anneliden. IV. Die Koérperform der Serpulaceen und Hermellen. Mitth. aus der Zool. Stat. zu Neapel, viii, Bd. Mitne-Epwarps, M., ’45.—Observations sur le développement des Annélides. Annales des Sc. Nat. Troisiéme Serie. Tome troisiéme. Morean, T. H.,’02.—Experimental Studies of the Internal Factors of Regeneration in the Earthworm. Archiv. f. Entwicklungsmech. d. Organismen. xiv, Bd. 3 u. 4 Heft. ’04.—Notes on Regeneration. Biol. Bull., vol. vi, No. 4. V. Trans- positional or Compensatory Regeneration of the Chelz in some Crustacea. 102 Charles Zeleny. MU ter, Fritz, ’64.—Fiir Darwin, pp. 76-77. Ortey, L., ’84.—Die Kiemen der Serpulaceen und ihre morphologische Bedeu- tung. Mitth. aus der Zool. Station zu Neapel, 5 Bd. s. 197. PAGENSTECHER, A., °63.—Untersuchungen uber niedere Seethiere aus Cette. VII. Entwicklungsgeschichte und Brutpflege von Spirorbis spiril- lum. Zeit. wiss. Zool., 12 Bd., pp. 486-495. PrzipraM, H., ’o1.—Experimentelle Studien tber Regeneration. Archiv. f. Entwicklungsmech. d. Organismen, Bd. 11. ’o2.—Experimentelle Studien tber Regeneration. Zweite Mittheilung. Crustaceen. Archiv. f. Entwicklungsmech. der Organismen, Bd. 13, Heft 4. Route, L., °85.—Notes embryogéniques. Esquisses du développement de la Dasychone lucullana, D. Ch. Revue Sc. N. Montpellier (3), tome 4, p. 463-470. SALENSKY, W., °83.—Etudes sur le développement des Annélides. Premiére partie. No.3. Pileolaria. Arch. Biol., tome 4, p. 143. DE St. JosEpH, X.,’94.—Les Annélides Polychetes des Cotes de Dinard. Serpu- liens. Annales des Sc. Nat. Zool., vii serie, t. xvii. *98—Les Annélides Polychetes des Cotes de France. Ann. des Sc. Naturelles. Zool., viii serie, t. v, p. 209. WILLEMOES-SuHM, R. v., ’71.—Biologische Beobachtungen tiber niedere Meeres- thiere. Zeit. wiss. Zool., 21 Bd., pp. 380-396. Witson, E. B., ’03.—Notes on the Reversal of Asymmetry in the Regeneration of the Chel in Alpheus heterochelis. Biol. Bulletin, vol. iv, 1902-03. ZELENY, C., ’02.—A Case of Compensatory Regulation in the Regeneration of Hydroides dianthus. Archiv. fiir Entwickelungsmechanik der Organismen., xiii, Bd. 4 Heft. ’03.—The Dimensional Relations of the Members of Compound Leaves. Bull. of the N. Y. Botanical Garden, vol. iii, No. 9. ’03.—A Study of the Rate of Regeneration of the Arms in the Brittle-star, Ophioglypha lacertosa. Biol. Bull., vol. vi, No. 1. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Direcror.—No. 163. PHOSPHORESCENCE IN CTENOPHORES. BY AMOS W. PETERS. I. INTRODUCTION. The problems discussed in this paper are the localization of the power of phosphorescence 1 in mature and in young ctenophores, and the influence of certain factors, such as mechanical stimula- tion, light, and heat, upon the ability of these animals to phos- phoresce. The species upon which I have worked is the common summer ctenophore, Mnemiuopsis leidyi A. Agassiz. “These animals were to be found at Wood’s Hole, Mass., abundantly during August, 1902 and 1903. The phenomenon of phosphorescence which they exhibited in their native sea-water when mechanically agitated after dark was sug ggestive of laboratory experiments. In my experiments I found it necessary to use a dark chamber, which I constructed from a simple pine ‘box heavy ily covered first with paper and then with several layers of black cloth. The dark box was placed upon a table before the experimenter and its open front was provided with overhanging cloth, sufficient to include his head and shoulders. ‘This arrangement permitted both the observation of phosphorescence and the free use of the experi- menter’s hands for agitating the ctenophores, etc. “This appara- tus was not quite as eticient as a dark room, yet it was adequate for the work that was attempted in it. As observation of the animals required the continuous attention of the experimenter in the dark-box and as light must be excluded, the time was read and recorded by an assistant upon signals from the experimenter. This procedure also favored the adjustment of the experimenter’s eye to the conditions of observation after the change from daylight to darkness. “The time here recorded was read to tenths of a minute, and differences so small as this are nowhere of conse- 104 Amos W. Peters. quence in the following work. ‘The abundance ot the material made it possible to select animals of the same large size for most of the experiments. Lots of from four to eight were placed in glass or porcelain dishes containing about one liter of sea-water brought in with the animals. In these dishes most of the tests for phosphorescence were made. Several methods of mechanical stimulation, to be used in testing the animals for phosphorescence, were tried and compared. ‘The most efhcient of these was stirring the ctenophores by means of a glass rod. Simple contact with the rod frequently succeeded in bringing forth the response of phosphorescence when jarring, shaking, etc., failed. ‘The adult animals being of sufficient size and weight, the contact of the glass rod with them was easily perceptible through the skin and muscles of the experimenter in the dark. ‘This method of stimulation was uniformly adopted as a standard in this work, being also used for small parts of animals, embryos, and eggs. Unless a statement to the contrary is made, a fresh, previously unused lot of Se opeorest was used in each test. Strict uniformity of conditions and the constant presence of control animals excluded from the observations here recorded, it is hoped, errors arising from insufhcient adjustment of the eye as well as from other sources. That these experiments could profitably be repeated and extended with a much greater degree of refinement, is a point the writer desires to emphasize. He wishes to express here his indebtedness to Dr. G. H. Parker, of Harvard University, for critical advice and suggestion, and for the revision of the manuscript. He is also under obligation to the Humboldt Fund of the Museum of Comparative Zoology at Harvard College for financial assistance. Furthermore, his thanks are due to the authorities of the United States Fish Com- mission for the use of its laboratory at Wood’s Hole, Mass., during the summers of 1g02 and 1903. II. LOCALIZATION OF PHOSPHORESCENCE. 1. In Mature Animals. As is well known, ctenophores brought into the laboratory disintegrate quite readily. “The dead substance of such animals was frequently tested both in the dark-box and in the dark-room. Phosphorescence in Ctenophores. 105 In no case was any phosphorescence detected in the dead matter originating from ctenophores. It was observed that after rough weather many ctenophores were mutilated but nevertheless phosphorescent. Even separated portions of the animal show this reaction both in the sea and in the laboratory. Such pieces examined under the magnifier always showed movements of the paddle plates and frequently muscular contraction. In short, the pieces of the animal were found to be alive. All the observations made gave the result that only the living ctenophores or living parts of them phosphoresce. When either whole ctenophores of small size, or, much better, excised parts from various regions of the animal were examined under the magnifier in the dark, phosphorescence seems to be present only along the rows of paddle plates. When the paddle plates were numerous upon the excised piece, adjacent parts were often so illuminated as to’ make this determination uncertain. But when portions of the jelly entirely free from paddle plates were examined no phosphorescence was seen. Such jelly was alive, for when the same preparation was examined in the daylight muscular contraction could be seen in it. In the course of these experiments no phosphorescence could be obtained from jelly free from paddle plates. The smallest piece from which phosphorescence was obtained consisted of four connected paddle plates with, of course, some jelly adhering. Even single excised paddle plates were observed to live for many hours or a day, as judged by their motion, and yet all efforts to get phosphorescence from single excised paddle plates were unsuccessful. The excised auricles showed, under the magnifier, cilia but no paddle plates. No feet fber scence was obtained from them. The sense organ with adjacent parts was excised in a piece about two centimeters long and one centimeter broad. Under the magnifier no paddle plates were seen, but muscular contraction was evident. No phosphorescence could be obtained from such a piece. The previously described experiments with excised rows of paddle plates, or parts of them, are sufficient to show that phos- phorescence does not depend upon correlation of the part with the sense organ. Whether cut in two transversely, or longitudinally in such a manner as to leave the sense organ wholly in one part, 106 Amos W. Peters. the result was the same. In both cases the piece without the sense organ, as well as that with it, was phosphorescent. If the whole animal had been made phosphorescent in the dark- box before the operation, both pieces retained phosphorescence; if the whole animal was originally non-phosphorescent, the pieces acquired this property in the dark-box. Numerous tests were made to determine whether after trans- verse or longitudinal division the piece retaining the sense organ acquired phosphorescence sooner or later than the other piece. A normal animal, as a check, was subjected to the same test at the same time. The results seemed to follow the law of chance. Sometimes the piece with the sense organ phosphoresced more quickly than the other, sometimes more slowly. The results were hence negative and warrant the statement that the sense organ is not a controlling center for phosphorescence. It was now clear that phosphorescence was localized somewhere in or near the paddle plates, and that the reaction-chain from stimulation to response consists very probably of an anatomically short and entirely local series of elements, 7. ¢., there is no distant central station for the reception, modification, or dispatch of impulses. Although it was shown that phosphorescence bears a local relation to the paddle plates the question was still open whether any necessary relation existed. The attempt was therefore made to ascertain by experiment whether all movement of the paddle plates are accompanied with phosphorescence. A glass evaporating dish eight inches in diameter and three inahesed in depth was filled with sea-water to within half an inch of the top. At night a single medium-sized and strongly phosphorescent ctenophore was placed in the dish in the dark-room. “The whole was left undisturbed for some time to insure the absence of currents originating from external me- chanical disturbance of the dish. At intervals the dark-room was sufficiently illuminated to enable the observer to note the position of the animal in the dish. During the dark periods the attention of the experimenter was directed upon the dish for the purpose of observing phosphorescence, if any occurred. ‘The result was that though the ctenophore was almost constantly changing position, sometimes to the extent of half the diameter of the dish, yet it showed no phosphorescence during the great majority of the dark intervals. Evidently during such intervals the paddle plates are Phos phorescence in Ctenophores. 107 in motion and yet without being accompanied by phosphorescence. When in a dark period phosphorescence was seen, the light was immediately turned on, and it was observed that the ctenophore was adjacent to the side of the dish and had probably struck it in the course of locomotion. A slight mechanical stimulus, such as touching the animal with a glass rod, jarring the dish, or the table upon which it was placed, easily elicited the response of phos- phorescence, both before and after the experiment described above. It was clear that the animal was capable of phosphores- cence during all the periods of locomotion, but the necessary mechanical stimulus was absent except when the ctenophore came into contact with the side of the dish. 2. In Embryos. Further observations were directed toward finding how far back in the ontogeny of the animal phosphorescence could be traced. ‘The eggs were obtained as follows: On August 6, some ctenophores were brought into the laboratory and placed in glass evaporating dishes each containing about two liters of the sea- water brought in with them. ‘[wo animals were placed in each dish. The water was changed once or twice, only such being used as was brought directly from the sea. On the morning of August 7, a layer of eggs in various stages of development was found. upon the bottom of each dish. By withdrawing the sea- water above them and replacing it with fresh sea-water about twice a day, they were reared to fully formed young ctenophores. In no instance were eggs observed to be deposited in the day time. When a lot of eggs had developed to the stage in which the four sets of paddle plates first appear, phosphorescence could be demonstrated. If at night the embryos were stirred with a glass. | rod, or the dish containing them was jarred, numerous phos- phorescent specks would appear momentarily. [he experiment did not easily succeed in the day time, even if the eggs were kept in the dark-room. Perhaps the same rhythm in the intensity of phosphorescence belongs to them as tothe adults. In the latter \ it was observed (1903) that phosphorescence was more intense | and more easily excited at night than during: the daytime, even when the animals were kept continuously in the dark-room. Furthermore, the phosphorescence of these embryos could not be indefinitely repeated, but was exhausted after a few flashes. In 108 Amos W. Peters. this respect also they resemble the adults, except that exhaustion is much more quickly produced. Experiments were made to test for phosphorescence before the formation of the paddle plates. A single gastrula was isolated at night in a watch glass. It was still contained in the egg- capsule and showed ciliary movement but no paddle plates were as yet developed. It was placed in the dark-room and, to make the conditions as favorable for the reaction as possible, it was allowed to remain undisturbed for half an hour, when the watch glass was suddenly jarred and a flash resulted. Another flash could not be obtained until after a period of rest. Experiments were next made to test for phosphorescence in ae segmentation stages and in the egg. It had been several times observed during these studies that embryos from animals that had been kept in ie dark-room during the previous day were further advanced when examined the next morning than the embryos from animals kept in diffuse daylight during the previous day. Both lots originated from the same collection and were parallel in conditions except with regard to light. In this experiment the influence of light upon the time of egg laying was also tested. August II, 1903, 2 p.m. Collected Mnemiopsis. Distributed them into lots A and B, each consisting of several dishes. A was kept in the dark-room. 8B remained in diffuse daylight, later in artificial light, electricity and gas. FoVp. im.) « Noeres Ea 510, (11a: Ess present in lot A of the dark-room. No eggs in lot B. A number of eggs were immediately isolated in a solid watch glass and tested by stirring with a glass rod and by jarring, at intervals, in the dark-room. They were examined before and after the series of tests and were found to consist of one-cell stages. No phosphorescence could be detected in these undivided eggs August 12, 12.20 a. m. Cleavage stages from lot A were iso- lated in a solid watch glass. Esanieatin before and after the tests showed that no led (moving) embryo was as yet formed. Stages from one to thirty-two cells were present. After an undis- aanbed period in the dark-room stirring with a glass rod elicited phosphorescent flashes, but probably not from all the embryos. 12.45 a.m. No eggs in lot B. Phosphorescence in Ctenophores. 109 1.20 a.m. Many embryos in lot A were becoming gastrule. Also many undeveloped (dead ’) one-cell stages were still present in lot A. 1.30a.m. No eggs in lot B. 2.20 a.m. Some eggs in lot B. Some of these were isolated in solid watch glasses, examined before and after testing for phosphorescence and found to be in one-cell stages. 2.40 a. m. No phosphorescence was detected in the one-cell stage isolated above from lot B. An interesting result of this experiment is the difference of about three hours in the time of the laying of the eggs between lots A and B; lot A having been in the dark longer, deposited eggs sooner. Ina subsequent experiment it was observed that animals kept in the dark from 9 a. m. had not yet deposited eggs at 10.30 p- m., although eggs were present the next morning. Hence the ieeast a fe eges does not seem to occur after simply a giv en number of hours of darkness. “The indications favor the view that the deposition of eggs takes place in accordance with the daily rhythm of light and darkness, deposition occurring in the dark period, and being capable of retardation by light. Alte INFLUENCE OF CERTAIN FACTORS ON PHOSPHORESCENCE. ifs A gitation and Light. In determining what factors influence phosphorescence it has been found convenient to deal with agitation and light together. Preliminary tests showed that ctenophores removed to the dark- box at once from their native sea-water, where they had been exposed to direct sunlight, were not immediately phosphorescent. However, they became so after remaining in the dark for some time. Similar observations were first made on Beroé by Allman (62) and subsequently by Panceri (’72). “The above fact was the starting point for a series of experiments in which both light and agitation were factors. Experiment r. Lots A and B having been exposed to direct sunlight for about one hour, were both placed in the dark-box at the same time. ‘The ctenophores in A were then continually agitated with a glass rod, while B was left undisturbed except for momentary tests made at intervals. A phosphoresced first in 2.5 minutes; B in 3.0 minutes. I1O Amos W. Peters. The result shows that direct sunlight prevents the occurrence of phosphorescence and that mechanical stimulation accelerates it. Experiment 2. ‘Two phosphorescent lots, A and B, were exposed to direct sunlight for three minutes. ‘They were then both placed in the dark-box at the same time. ‘They were both found to be non-phosphorescent. A was then continuously agitated and B was left undisturbed except for tests, as above described. A phosphoresced first in 2.5 minutes; B in 3.0 min- utes. After permitting the phosphorescence to develop for a minute or two, A was exposed to direct sunlight for two minutes while B remained in diffuse daylight. A was continually agitated in the dark-box as above described. A phosphoresced first in I minute; B continued to phosphoresce. After some minutes both were exposed to. diffuse daylight and then tested as follows: A was agitated in the dark-box, while B remained undisturbed. A first phosphoresced in 1 minute; B in 2 minutes. The result indicates that exposure to direct sunlight not only prevents phosphorescence, as found in the preceding experiment, but also overcomes a previously acquired power to phosphoresce. Furthermore mechanical stimulation, as before, accelerates the aL aka of phosphorescence. Experiment 3. It was observed that Mnemiopsis was some- times phosphorescent and sometimes not so after standing for a time in the diffuse daylight of the laboratory. The object of this experiment was to test the power of diffuse daylight, of the inten- sity then prevailing in the laboratory, to inhibit or permit phos- phorescence, as well as to test further the influence of mechanical stimulation. ‘he ctenophores used had been exposed to diffuse daylight. A was agitated in the dark-box, but B, in the same box, was undisturbed except for tests. Both A and B were then again exposed to diffuse daylight. A was then put in the dark- box and agitated; B was undisturbed except for tests. A phos- phoresced first in 1.7 minutes; B in 2.5 minutes. The results show that diffuse daylight can check phosphores- cence and, as before, mechanical stimulation can accelerate its appearance. Experiment 4. In this experiment ctenophores in the dark-box were continuously agitated with a glass rod to determine whether Phos phorescence in Ctenophores. TTI the phosphorescent condition could be removed by excessive mechanical agitation. Reduction of intensity had frequently been observed after long continued agitation. 2.22 p.m. Strong phosphorescence. 2.42 p.m. Phosphorescence appears only in slight gleams, but these persist upon stimulation with the glass rod. The result indicates that sufficiently long-continued agitation reduces the intensity of phosphorescence, but does not entirely inhibit it. Experiment 5. ‘he object of this experiment was to determine whether the rate at which the ability to phosphoresce is acquired, varies with the intensity of the light. Lots A and B each with six ctenophores, were exposed to direct sunlight for five minutes. The temperature of the sea-water before the exposure was 21°.5 @; alter it, 22°.5 C., Then A was kept im the dark-box until phosphorescent, being tested at intervals (7. e., not continuously agitated). During the same time B was exposed to diffuse day- light and at intervals it was placed in the dark-box for a momen- tary test. B did not phosphoresce during the whole experiment (19.5 minutes). A phosphoresced first after three minutes in the dark-box, and though kept in diffuse daylight, it retained its phosphorescence over five minutes, after which it lost its phos- phorescence so long as it remained in the light. The result indicates that the ability to phosphoresce is acquired more quickly in darkness than in diffuse daylight and also that phosphorescence has a proportionate relation, in a negative sense, to the intensity of the light. Experiment 6. Preceding experiments have shown that: (1) darkness is at least one necessary condition for phosphorescence; (2) darkness alone does not result in phosphorescence; and (3) mechanical agitation can call forth and accelerate this phenom- enon in the dark. This comparison suggested the question, Can agitation alone produce phosphorescence? To make this deter- mination a lot of ctenophores were poured repeatedly from one dish to another in diffuse daylight and were tested at intervals in the dark-box. ‘The agitation including the tests was continued for a period of ten minutes. No phosphorescence whatever could be detected. The inability of agitation to produce this phenom- enon was frequently observed. Pre: Amos W. Peters. This result shows that a non- phosphorescent ctenophore is not made phosphorescent by mechanical agitation alone. Further- more, comparison of all preceding experiments shows that dark- ness accompanied by mechanical stimulation is at least one com- bination of conditions which is able to produce phosphorescence, but its two factors acting singly cannot produce this result. Other stimuli capable of eliciting phosphorescence may, of course, exist. Ds T em perature. Experiment 1. This experiment was made to determine the effects of physiological extremes of temperature. It was per- formed in a dark room. A pailful of fresh ctenophores standing there at a temperature of 21°.5 C. emitted, when jarred, enough light to illuminate the room to a considerable degree. From this supply four animals (lot A) were removed to the ice bath and four others (lot B) to the warm-water bath. ‘The respective cooling and warming of the two lots was done simultaneously. The ice bath consisted simply of a basin containing broken ice, in which the vessel containing lot A was partly immersed. Neither ice nor fresh water (from melting ice) came into contact with the animals. They were gradually cooled in their original sea-water. The other lot of animals (B) were warmed in sea-water by placing the vessel containing them over sufficiently warmed water. ‘The tests for phosphorescence were made at intervals by stroking the ctenophores as usual with a glass rod. The temperatures were taken with the bulb of the thermometer in contact with the surface of the animal. Since the phosphorescent parts, the paddle plates, are superficial, the temperatures given apply to these parts. The interior of the jelly might have been at a different temperature. Under these conditions the following record was obtained: Lot A at 21°.5 C. was strongly phosphorescent; seven minutes later at 12°.5 C. no phosphorescence could be observed. A was then removed to the warm-water bath whereupon the animals became, after some time, phosphorescent. Hence the previous cessation of phosphorescence was not due to death. Lot..B at:21°.5: C. was also strongly phosphorescent; five min- utes later at 37° C. no phosphorescence was observable. Phos phorescence in Ctenophores. 1 8 Lot B was then removed to the ice bath whereupon the animal became, after some time, phosphorescent. Hence the previous cessation of phosphorescence was not due to death. Experiment 2. The aim and methods of this experiment were the same as in ee io Lot A at 21°.5 C. was strongly phosphorescent; after 6.5 min- utes cooling it was much diminished, and after 13.5 minutes (9° C.) there was no phosphorescence. Lot A was then placed on the warm water-bath and in 13 minutes became again phos- phorescent. Lot B at 21°.5 C. was strongly phosphorescent; after seven minutes warming the phosphorescence was much diminished, and after ten minutes (38° C.) there was none. At this temperature the animals had completely disintegrated. Experiment 3. he aim and methods of this experiment were the same as in Experiments I and 2. Lot A, strongly phosphorescent at 21°.5 C., was cooled in ten minutes to 9°.5 C. and became non-phosphorescent. Lot B, strongly phosphorescent at 21°.5 C., was cooled in 12.5 minutes to 119.5 C. and became non-phosphorescent. Another series of experiments was made to determine the effects of variations of a few degrees only from the normal. Such varia- tions, of from one to foie degrees above and below the normal (21°.5 C.), showed, in all the trials but one, a diminution of phos- phorescence as compared with a control. In other words, phos- phorescence in the dark-box appeared sooner in the animals at normal temperature than at any other temperature. It would not have been surprising to find an optimum point slightly differ- ent from the normal temperature. “The experiments made upon this subject are not regarded as conclusive. The general result of this work upon temperature may be stated as follows: The phenomenon of phosphorescence in the ctenophores here investigated occurred during a range of temperature extending from about 9° C. to 37° C., with an optimum at or near 21°.5 C., which was the temperature of their native sea-water. The inten- sity of phosphorescence diminishes as physiological extremes of temperature are approached. 114 Amos W. Peters. IV. DISCUSSION OF RESULTS. The preceding experiments demonstrate that the power of phosphorescence is located in the mature animal solely in the region of the paddle plates. I am not aware of direct evidence for a more precise localization than that just given. Allman (62, pp. 518-519) and Chun (’80, p. 195) attributed this phenom- enon in Beroé to the germinal cells lying in the walls of the gastro- vascular tubes. ‘The supposed fatty, phosphorescent substance of Panceri according to Chun (’80, p. 195) does not exist. Phosphorescence was observed by A. Agassiz (’74, p. 371) in embryos. The experiments described in this paper also show that this property belongs to protoplasm that has but little organic differ- entiation, viz: that of the earlier stages of segmentation. When we inquire what service in the economy of the animal is rendered in the process of phosphorescence we find it difficult to give a satisfactory reply. I have never been able to obtain phosphor- escence in mature ctenophores without the motor activity of the paddle plates, but not every movement of these is accompanied by phosphorescence. Darkness and mechanical agitation are the two selective stimuli whose joint presence results in phosphores- cence. This important fact, taken in connection with the localiza- tion of the reaction, the acceleration of its appearance by mechan- ical agitation, and its complete inhibition by extremes of tempera- ture, lead to a probable conclusion regarding its nature. The phosphorescence of Mnemiopsis is a metabolic reaction which is dependent upon the formation of a substance in darkness, the katabolism of which takes place upon mechanical stimulation and becomes observable as the energy of light. “Che amount of substance so accumulated may be exhausted by continued mechan- ical stimulation in darkness or may be consumed as produced. When the animal is brought into the light the substance is no longer produced or, if so, it undergoes katabolic transformation rapidly, or the energy is given out in some other form than light. That the phosphorescent substance cannot accumulate in the light is shown by the fact that ctenophores removed from bright daylight or sunlight to darkness are not immediately phosphores- cent. ‘This is the case whether they have been previously agitated or have remained undisturbed. Phosphorescence in Ctenophores. 115 SUMMARY. 1. [The dead matter originating from ctenophores is not phosphorescent, 7. e., only living ctenophores or parts of them phosphoresce. 2. Phosphorescence appears along the rows of paddle plates and no phosphorescence was obtained from jelly free from paddle plates. 3. The smallest piece from which phosphorescence was obtained consisted of four connected paddle plates. 4. Movement of the paddle plates is not always accompanied by phosphorescence. 5. No phosphorescence was obtained from the excised auricles having cilia but no paddle plates. 6. ‘The sense-organ is not phosphorescent. 7. Phosphorescence does not depend upon correlation of the part with the sense organ. The sensory-motor circuits for phos- phorescence are local in character. 8. No phosphorescence could be obtained from the eggs of Mnemuopsis before segmentation. g. he early cleavage stages (without cilia) are phosphores- cent. 10. Gastrulz (ciliated) are phosphorescent, as are also all stages in which paddle plates are present. 11. [he phosphorescence of embryos is easily exhausted. 12. he deposition of eggs can be retarded by light. 13. Direct sunlight prevents the appearance of phosphores- cence, but in darkness, the power to phosphoresce upon stimula- tion, is acquired. 14. Direct sunlight inhibits a previously acquired power to phosphoresce. Diffuse daylight of sufhcient intensity has the same effect. 15. Phosphorescence has a proportionate relation, in a nega- tive sense, to the intensity of light. 16. Mechanical stimulation accelerates the appearance of phosphorescence in darkness. 17. Non-phosphorescent ctenophores do not become phos- phorescent by mechanical agitation alone. 18. Long-continued mechanical stimulation reduces the inten- sity of phosphorescence but does not easily inhibit the phenomenon entirely. 116 Amos W. Peters. 19. Darkness accompanied by mechanical stimulation is at least one combination of conditions which produces phosphores- cence, but these two factors acting singly cannot produce this result. 20. [he phenomenon of phosphorescence was observed at temperatures ranging from about 9° C. to 37° C., with an optimum at or near 21°.5 C., the temperature of the sea-water. 21. The intensity of phosphorescence diminishes as physiologi- cal extremes of temperature are approached. | 22. [he phosphorescence of Mnemuopsis is probably a meta- bolic reaction which is dependent upon the formation of a sub- stance in darkness the katabolism of which takes place upon mechanical stimulation and becomes evident to observation as the energy of light. BIBLIOGRAPHY. Agassiz, A., ’74.—Embryology of the Ctenophore. Mem. Amer. Acad. Arts and Sci., vol. x, pt. 2, no. 3, pp. 357-398, 5 pl. Auiman, G. J., ’62.—Note on the Phosphorescence of Beroé. Proceed. Roy. Soc. Edinburgh, vol. iv, no. 57, pp. 518-519. Cuun, C., ’80.—Die Ctenophoren des Golfes von Neapel und der angrenzenden Meeres-abschnitte. Fauna und Flora des Golfes von Neapel. Monographie 1, xvii + 313 pp., 18 Taf. PancerI, P., ’72.—La luce e gli organi luminosi dei Beroidei. Atti R. Accad. Sci. fis. e mat. Napoli, vol. v, no. 20, 15 pp., I tav. AStuUDY OF THE INHERITANCE OF DICHROMATISWM INC UINA PAPPONICA. BY ISABEL McCRACKEN. Stanford University, California. Wirth 1 Pirate anp 3 Ficures IN THE Text. Tr. INTRODUCTION. This paper contains a statement of breeding experiments with a certain species of leaf- beetle, Lina lapponica, which have been carried on this year (1904) in the Entomological Laboratory of Stanford University. Lina lapponica is a small beetle of the family Chrysomelide. Both larvz and adults feed from early spring until late in the fall on willow or poplar leaves. ‘The females are, for the most part, considerably larger than the males, although intergrading sizes occur. The thoracic length of the smallest males is about 1.5 mm., abdominal length 6 mm., thoracic width 2.5 mm., abdominal width about 4 mm. ‘Thoracic length of the largest females 1s about 2 mm., thoracic width 3 mm., abdominal length 7 mm., abdominal width 5 mm. ‘The wing covers in both males and females may be entirely black, (PI. 1, Fig. 6), or brown with fourteen black spots (Pl. 1, Fig. 4). The eggs are elongate, yellowish, and laid side by side upon the leaves of the plant furnishing food for both larvz and adults. ‘The life of each individual, in the early generations of the season, occupies from three to six days in the egg stage, from fifteen to twenty days (with two moults) in the larval stage, and from four to eight days in the pupal stage. ‘The adult stage varies from twenty to thirty days. ‘The adult stage of later genera- tions is of longer duration. Each female produces from four to six broods a season, each brood containing from thirty to forty individuals. The number of generations in a year under normal 118 Isabel McCracken. outdoor conditions is unknown, but under laboratory conditions at least five generations may be secured. The object of the present experiment was to observe through several generations the behavior of the particular differentiating character, color, with the view of testing for this insect Mendel’s principles of dominance and segregation. The particular circumstances that make Lina lapponica favor- able material for this study are these: 1. Both sexes are dichromatic. 2. The sexes are easily distinguished on account of the differ- ence in size. 3. Individuals may be mated for life, or males of one brood may be allowed to mate freely with females of another, thus secur- ing diversity of partners (the plan without doubt pursued in nature), while securing the same lineal record for the offspring. 4. Life habits are adapted to laboratory conditions. 5. At least five (probably more) generations may be reared in a single season. This work was not begun until the first generation of Lina for the present year had come to maturity. However, the four succeeding generations studied offer some interesting and instruc- tive data that will be supplemented another year when an earlier brood will be secured. Ii. CHARACTER OF THE MATERIAL USED IN THE EXPERIMENTS: For the initial study, about 1000 individuals in the last larval stage were collected from willows between April 20 and May 4, 1904. From these a total of 600 adults were secured, the rest having fallen prey to a parasitic fly. ‘This lot contained a repre- sentative number of males and females, and the dichromatic extremes of color. Individuals in one of the two color and pattern series have, as previously stated, wing covers with ground work of brown, dotted with fourteen black spots (Pl. 1, Fig. 4). There is considerable variation in shape, size and coslebrenee of spots, but no apparent variation in ground color. Individuals representing this color type are referred to in this paper as S. In the other series the individuals are wholly melanic or black (PI. 1, Fig. 6). ‘These are here referred to as B. In each series the Peaenca is similar, Dichromatism in Lina Lapponica. 11g having a median dorsal area of black covering one-half of the dorsal surface, and marginal areas that are white when the insect first emerges, and turn a copper-red about six days later. A single black spot is present within each marginal area. For the purposes of this year’s study no account was taken of the variation within each series, namely, the fluctuating variations, and their behavior in heredity, as this demands a special series of experiments. I began the present series of experiments with the notion that the melanic variety was the possible result of a coalescence of all the spots in the spotted variety. A study of the color develop- ment at ecdysis, however, revealed the fact that the dark pigment giving the melanic series its color is superimposed on the spotted condition. ‘he wing covers of an adult that has just slipped out of its pupal case are at first white (PI. 1, Fig. 1). Within a few minutes the spotted areas, first the anterior, then the middle, and lastly the posterior are dimly but distinctly indicated as light drab color against the white background (Pl. 1. Fig. 2). The spots deepen gradually to a dark drab, and the ground color becomes a light drab (PI. 1, Fig. 3). In the melanic series these two steps, namely, pigmentation of the spotted areas and pigmentation of the rest of the wing cover, occasionally take place almost simultane- ously, the spotted areas then becoming so obscured as not to be apparent. ‘heir presence in every doubtful case, however, was determined by holding the wing cover between the light and the observer. From the drab stage development proceeds in one of two directions. In one series of individuals, including both males and females the spots deepen to black, and following quickly upon this change, the drab ground color gives way to brown pigment against which the fourteen black spots are clearly marked (PI. 1, Fig. 4). In the other series, the spots as before deepen to black, and the drab gives way to black pigment (PI. 1, Fig. 5). This soon overshadows and totally obscures the spots from surface view (Pl. 1, Fig. 6). Their presence may still be demonstrated, however, by holding the wing to the light. It appears, therefore, that in Lina lapponica we have to deal in its dichromatism with a case of ‘‘substantive discontinuous? variation.” Each individual is either melanic, B, or not melanic, S, all individuals alike being spotted. 1Bateson: “ Materials for the Study of Variation.” 120 Isabel McCracken. ‘The question arises, ‘ How do these extremes of color and pattern behave in heredity ?”’ Having no known pure bred stock to begin with, my first attempt was to breed out by selection the alternative color from each of the two extreme lines. Succeeding in this, I hoped to have on hand material for testing the validity of Men- del’s laws of “dominance and segregation”’ for this species. In pursuance of this the following breeding experiments were devised and carried out, and the results recorded in detail. In the suc- ceeding tables summaries only are given. III. METHOD OF EXPERIMENTATION AND RESULTS. Experiment 1.. To determine relation of first generation from laboratory reared adults to the color types sand Bo (Pablew ) Experiment 2. To determine relation of second generation bred from similars to color types S and B. (Tables II and III.) Experiment 3. ‘To determine relation of individuals bred for two generations from similars to the color types S and B. (Tables IV and V.) Experiment 4. To determine relation of offspring of extremes, having on each side pure heredity for at least a generation to the color typesS and B. (Tables VI, VII and VIII.) Experiment 5. To determine in what generation from mixed parentage the alternative characters breed pure. (Tables [X and X.) Experiment t. ‘The generation with which this work was begun was collected in last larval instar from willow trees in a certain locality in the neighborhood of Stanford. ‘These were caged in the laboratory, and thereafter fed upon poplar and willow leaves until pupation. As soon after emerging as the wing color was established, namely, in about an hour, the adults were placed in one of four breeding cages as follows: Cage 1. Black @’sand black 9’s only. Cage 2. Spotted <’s and spotted 9’s only. Cage 3. Black o’s and spotted 9’s only. Cage 4. Spotted @’s and black 9’s only. Sex was determined by size. Individuals not exhibiting extremes of size, namely, individuals not easily distinguished as to Dichromatism in Lina Lap ponica. Ion sex were discarded. By this means individuals were limited to mates of a definite color without forced mating. A pair once mated, however, were mated for life, as they were then removed to a 1 x 4 inch shell vial and numbered. Here they lived and reproduced for the rest of their lives. Each mass of eggs oviposited was removed to a 12 ounce breed- ing jar for further development, and given its parental number. In this way 288 individuals were mated, representing 144 crosses. Of these, ten pairs produced no eggs, although copulation took place several times, nineteen pairs produced eggs that failed to develop, nine pairs produced eggs that went through pre-embry- onic development, but failed to hatch, and 106 pairs produced eggs that hatched in from three to six days, the offspring reaching maturity in about twenty-five days from the date of hatching. The 106 pairs represented the following matings: 57 pairs of SS XS 9 19 pairs of Ba X BG 14 pais of BS xX 5S 16 pairs of SS X BQ Table I, compiled from the records of individual broods, gives a summary of the data obtained. We find, therefore, that in a lot of 57 * S’s mated with a lot of 57 2 S’s without respect to ancestry, that over one-half reproduce their kind, while the others produce mixed broods (32:25). The mixed broods are made up of individuals representing the extremes of color only—no blends—in the proportion 890 S : 280 Bor 3.25 :1 3B Also in a lot of 19 @ B’s and 19 9 B’s, the parents chosen at ran- dom without respect to ancestry, two sorts of broods are produced, namely, broods true to parent color and mixed broods, in the pro- portion 14 pure: 5 mixed. The mixed broods are made up of individuals representing the extremes of color only, in the pro- portion 18: 1.7 B. Therefore, from parents chosen at random, but similarly mated, two sorts of broods are obtained, broods true to parental type, or pure broods, and mixed broods, the preponderance of individuals being in each case on the side of the color type of the immediate parents. Records of individual broods show that this condition Isabel McCracken. 122 TABLE I. "IOJOD [eI -uaieg 0} any, sjenpra SHANE OY ac Toheednhe ENOL 2455 1029 Producing B Broods ~ only. “IO[OD) [eUorTe | 0} INIT, Spoorg suronp -O1g Sileg toquinyy [ej0T, *Aquiny “PI 0} ~poreay syen “PpIAIpuy doquinyy [%IOT, *pooig RB Ul JoquINNY adseiaAy 32 Producing S Broods only. 1049 gio 6872 *paurerqo spooig jo Toquin yy [239°.L “sie Suey jo raquiny [eI0 7, Color Character of Matings b BS XBE 30 31 35 29 217 16 14 c SAOXBe d BS XS 106 Total .. Seats *Spoolg paxtyy ip ™~ ul g : § jo uonrodorg a ot So | Ze A | Swot | = = 8 fae en feral g 23 ee Ome pes y s = Boe ‘spoorg aing suronp BESS -O1g sileg Jo wag pg seal: wr “spooig paxil oa ur g jo oquiny [eIO, | go *SpOOlg pextf & 8 ul g jo Jaquinyy [PIO], Se *‘spoorg paxly Jur inate -INpoIg sieg jo raquinyy N ee = eee Saree a n 538 ns og Iter 10) ers a nm S39 == | | | | Total Total Number Mixed Broods. | | | Number SinS S Broods. Total Number Broods. = _ + + sm (ne) m™ oO _ _ om CO aA a Ww Oo my Ww oO t+ WY No Yo) om) oO + m4 oc Le) = _ Ot OF mwm ax rome) nm Coy as} Dichromatism in Lina Lapponica. 123 obtains for the total number of offspring of each pair, as well as for the sum total in both series. Dissimilar parents produced on the whole mixed broods with preponderance of individuals in the S color type. Records of individual broods show that that preponderance was maintained in every S # X B 9 cross but four, namely, in 12 out of 16 Tasie II—B xX B. (See also Table VII.) = , | Broods Utilized Ea one | 80 SELON BREE (Note-book as | E Numbers). ig 2 : | 2 £2 Par 2 = J Q 3 B. S Gs 10a x 127.9) || 25 | 460 | 379 | 839 | all none Fe 2S XTO.S | 18 314 243 | 557 | ~ alll none En 200 292 31 570 AOTS | A 1O37) «eal none d 208X 392 | 24 376 | 348 | 724 | all none e 2005 xX 40:9 | Bre 59 Al | too. «6|~s all =s|_—snone -f 4080X 392 | & 88 83. |) 70 |) @all -2iemone g 1100'X 559 | 7a Aas 6 III 231 all = none b 55h X1109 | 21 299 I5E) |e. 58o all | none AOD x. 59°O) | I 14 20 34 all | none fa 50c. 4629. 22 373 330 | 94 i712 all none Total 157 26735) 23120 )|) 4085.‘ ||) Vaal none crosses, and in every S 2 X B & cross but two, namely, in 12 out of I4 crosses. Experiment 2. My next effort was to see if I could by selection bring the two differentiating characters to breed true in certain lineages. I, therefore, selected ten broods from among the pure broods (broods true to parents) produced by B x B and ten broods from among the pure broods produced by S x S._ All the males of one brood were placed in a breeding jar with all the females of 124 Isabel McCracken. another brood of the same type. Each mass of eggs oviposited was removed to a separate jar for development. Tables II and III show the results in full. “The numbers in the first column are simply my distinguishing numbers of broods utilized for the crossings. ‘The second column represents, not the total number of egg masses produced, but the number of broods which the facilities for handling permitted me to rear. TasLe III—S X S. (See also Table VI.) See corel ae Sires | | & a | 3 2 = Offspring Reared ye 2 | Ree ite Broods Utilized. = Bg 3 2 lean = Pee Broods. : Si Sa sg) 6) Sy f| 33 Be fae ee met 9 ame B91. BoP BOG XajO 2) | 414) Only OS.) © 72 vTO5")) | Oat AGs has ag bi TOG 5309 |) 6 2 4 123 93 | 216.| -7o | 27 | 119 CL PREOQS I2G 2 1 4. I 2 gl BE) |, t42 ZI 21 go da 12900 X 1109 | 5 3 2 112 68 180 gI 25 64 e 1440'X1349 8 8 Oo USOe mE 5 |) 2050s 205 fo) Ff 1940)X 1442 | 6 6 O 104 7S | - 182-2102 fe) Bee stex 799" 19 9,0] 10 A£0G 274. | 083>.\4 228 78 | 284 De gs <32'9 "tg aro 9 ALT} 293 | 16845) “2g0l) "Oqn me 240 META SX TICS i 3 2 I 63 BE le OAR) . IGT 5 28 (7 LEO e pC 1t2 9S erg! eur 3 2457 172 |. 4E7 ;| Body |Raas 71 ol Rover! Minera Ase 188 | 52 | 36 | 1801 | 1227 | 3028 1662 | 345 1021 In Table II we find a total of 4985 individuals, representing a total of 157 broods, all coming true to parents in the second generation, the grandparents having been selected at random, except for color type (7. e., regardless of ancestry). In Table III we find two matings, namely, e and 7, producing broods entirely like parents, but every other mating produced either mixed broods, or some pure and some mixed broods. If we leave out of consideration the evidently pure lineage-matings produced Dichromatism in Lina Lapponica. 125 by e and f, the proportion of the pure to the mixed broods, stands as 38 pure to 36 mixed in the aggregate, or, 51 +per cent : 49 — per cent, a relation not materially differing from that obtained in Table I from random matings within the color type S. In the mixed broods the aggregate proportion of S : B is 1021 : 345, or 2.98 8:1 B. ‘Table I shows the relation of S : B resulting from random S x S crossings to be 3.25 :1 B. TasLe IV—BxB. = os fe Broods Utilized (Note- = Es G | | Beale Meee wd a = a | ie | by g is s io | Z Z Zi Se as eae pe ie 1 PSs @ “226 xXSs1 9 4 4 fo) 4a |, 40 | 81 | all Oo b 819'X219 5 5 Oo 635) 5G0) | Srg2ni/ call O é 289139 5 5 O 652,54 | . HEROS) all Geto AauLIa hx 28:0 7 7 O G4. |./85. | 179) } all fo) Cee X35.2 5 5 fo) aoe |) 6G: |) tad) caller bao 5c X17 2.) | 6 6 Oo |, 98) 96; |, Tose) alll Oo Bo e326: 039" | TO» | TO O. |.75 >| ag0)) gma all O fee OSX 321% Ng 25 pra O @2). |b. yi nora sell il 550 fo arse 47 2 | San 5 O 95 | 74) 169 | all O i 470X312 ° 4 | ‘a Oo 607 55 setae lial fe) | | Metalic: 56 | oe | 864. | 753.| 1607 |\ all fo) 97 7% | 168-}, -all fo) f= 120o1K139.2 5 all O 109 | 90 199 | all fo) b 1312 X1390' 3 all fo) 50 36 86 | all fo) 1 1610'X1569 2 all fe) 22 28 50 |_ all O qf §1612-X1560' I all fo) 21 14 26h e-all O Pe a7 ISS? 4 all fo) 74 Gael eran sald fo) I 37 OS XI8 5S 2 all fe) 40 36 76) | all O m.) 1029 X 1306" 2 all “xe 27 35 62 | all fo) Wotallsss 2.2. 52 all O Obs |e O55) 1570. laale | 0 made between similars from mixed broods, columns g and Io, to obtain data for comparison with Tables II and ILI. It will be remembered that Table II represents results of B x B that had bred true to B x B parents, and Table III represents results of S x S that had bred true toS x S parents. Tables IX and X represent results of matings of B x B and S x S, respectively, between individuals from mixed broods, 1. ¢., from broods that had not bred entirely true to parentage. gy Isabel McCracken. We find upon inspection that, as in Tables II and III, B x B produces offspring like parents, while S x S under similar condi- tions produces in part pure broods, and in part mixed broods. In the initial experiment, adults were isolated according to color type, as already stated, as soon as color type was established or in the morning succeeding the night during which adults had issued. Since mating does not take place for several days after the adults issue, I considered this a sufficient safeguard against Taste X—SXS. (From SS, Same Parentage as in Table IX.) g S 3 2 2 a 12 Bila se eel i : | = g ae] eI Mixed Broods Utilized 50 S & 5 Bande (Note-book See Sls S 2 wn Numbers). re) 2 a) 8 os 3 g Eee ues ae Ss nemes as 2A, fe) Sa, oS = iz S| Ee| § £3) 3 Zz Aer) i= 3 Q a a B.S AN ORS S100 OF 5 2 125-1 ‘T10"| -233>|| “168.420. ego DINAQ3(o X100.S 1 t CH Ale es 27 19 46 fo) 7 | 30 £ eITZowI28 Of, 2 Te Am a 39 28 67 35 @ | 123 d “1137 Oo X128.5" | 76 2 4 134 96 | 230 58) | 54 | 219 e. 1299. X13900"| - 4 O I 24 16 40 @ |. 'ar2).1 398 fF LOLO OS T1506", |, Sat O I 20 2g | Az O 72. | eAO Tie (18 | 8°) 10 | 372] 296] 668] 0 108 | 299 mating before isolation. It has been Rieetadty: to me je aH possibility exists that some of the adults escaped notice until mating had taken place. If this were the case, it would account for the discrepancy in results between Table I, 6, and Table IX. While the data in Table X are recognizably insufficient, they point in the same direction as results drawn from previous tables of S x S matings; that is, that S x S, when presumably not far removed from B progenitors produces two kinds of broods— broods wholly like the parents and mixed broods. ‘Table [IX shows conclusively that B x B produces offspring true to parents in the first generation from similar parents, leaving the discrepancy between this table and Table | to be accounted for as suggested. Dichromatism in Lina Lapponica. 133 A number of matings were made between similar individuals, the offspring of dissimilar parents. “The broods chosen for these matings were taken from those represented in Table I, c and d. TasLe XI—S*xS. s | oe na | © : : St lise! 3 Total in . Color of Parents Bo | 8 4 g | ay Mixed Broods Utilized. and re} | As = | 5 Reacts Grandparents. S | 6 x me) | | & : £82] Hee esses | Ee) Omens ce eee | Pin Ae S| .Oti| eee| ese hea: CS) oe ES a5). P | | | a 2'X569 | SXB-—G.P.{| ?° 3 7 | 217 | 141 | 358 | 100 | 190] 68 SyGs-ibs, | | - 6 56S X22 \\ oy R—c.P.(| 8 6 | 246 | 159 | 405 | 142 224 39 {Sx Ss—P | c 600 X 107 2 | BX S—G.P 3 _ 3 Crd) GME Say I kG) me) Ga Wn ee ae le n6,\e nee | 10 ) 2 2 2 I | I 7 ERGs = eae 4 7 5 asst WS Se | e 840'X 1089 | Be Sleeps (le= I I 29) \|5) 237) Skate) |e 2o mle {Sx S—P | | | f 1080 X84Q | BX S—G.P. 3 2 I 59 | 39 OPS || GN eo G a — == = _——— es | Motallencemecr: Wetegcenterscnrs chet ctene cyan 36 16 20 | 700 | 489 | 1189 | 422 612 | 155 | | i In each case the dissimilar parents had produced mixed broods. The following diagram shows the method of mating for data of color inheritance of first generation from mixed parentage: Parents Sa x B 9 Sox Be iS ic ar ONG! Swe He Ofispring: 3 x ; Bg , ZA e 134 Isabel McCracken. Table XI shows results of S x S matings from both S «x B ¢ and B x S 2 parentage, while Table XII shows results of B x B matings from the same parents. The data in these tables add to the evidence of previous tables that B behaves like a Mendelian recessive, reproducing its kind, while S behaves like a Mendelian dominant, reproducing both its own kind, S, and the recessive, B. TasLe XII—B x B. a ee 2 Color of Parents alee Sa @ Broods Utilized. | and as | ae | | | Grandparents. x | c x S ag alee g ¢ | ge | Z| i Zz Zz S| Se eal Beas = & BX B—P [ } | | | | a 240'X569 l\sxB—G.P.{ eel 3 fo) 56 | 42 ee all | o pelt J 6a) fe) NPS GU etl \| | 8 | b 560X249 SX B_-G.P 2 2 ° a5 2 HA ezr ey asec one es Bee se | 220 | alt | c OX 107 |\BX S—G.P 7 7 ro) 132 | | 220 | a fo) |{BX B—P d 1070'X60Q | BX S—G.P.| 3 3 ° 61 | 23 | 84] all | o | (BX B—P. e 845X108 9 | BX S_G.P 2 2) ° 43 220 65 | all ae | | | |{BXB—P. | E Sai | f 1080'X*84Q l\Bxs—GpP | 2 al Zen | Ona 4 es Onl asGaa alli Hino ces | | | =— | | — i lz = ae sBokale ree shcd hae os Sheree | 19 +2439! vel yice | 361 | 233 594 | all | ° At date of writing, the available material for the fifth generation matings is very much reduced, being confined to fifteen B broods pure bred for four generations and five S broods pure bred for the same length of time. “These have shown no tendency to mate, and it is hoped that they will hibernate and form a nucleus for next year’s work; failing this, the experiment will be continued with the first outdoor individuals to appear in the spring. Dichromatism in Lina Lapponica. 135 SUMMARY. 1. No amount of crossing between the two characters in ques- tion accomplishes any disintegration or breaking up of either one. These are absolutely fixed with reference to each other in this species. (Tables I and VIII.) 2. Inthe offspring of a cross between the two characters, either both characters, or only one, the spotted, appears. (Tables I and VIII.) (Data on the latter point are insufficient.) 3. Cross-bred B’s, namely, B’s appearing in a cross between the two opposing characters, transmit B only to the offspring when similars are bred together. The B character is, therefore, stable, or self-perpetuating in the first generation. (Tables II, IV, VII, TX, XT.) 4. Cross-bred S’s transmit both opposing characters to the offspring, the offspring likewise transmitting both characters, though bred from similar parents. (Tables III, VI, X.) . In the third generation from similar parents, S’s appear to g P Pp breed true. (Table V.) While this summary shows no exact parallelism to Mendelian results, it is in accord with Mendelian principles in the following features: 1. Character S of S x B parentage behaves like a dominant when mated with S._ It appears always in greater numbers than character B (with the exception noted in Table I, b) and its broods fall into two categories, 7. ¢., pure broods (each individual being similar to the parents) and mixed broods (broods made up in major part of S, in minor part of B). Its behavior when mated with B must be further noted, I believe, before we can say assuredly that it is a “fully normal Mendelian dominant” in all respects. 2. Character B behaves like a Mendelian recessive in that from its first appearance (with the exception noted in Table I, 6) it reproduces B only. Que wsetouthie “segregation ” of characters in the germ cell, or “ purity of the germ cell,” it 1s evident that only one of the two opposing characters in question is called into activity in the somatic cells capable of expressing it, namely, the cells of the wing covers, and that as far as the experiments extend, individuals in each series appear at once, or eventually, to breed true. 136 Isabel McCracken. In conclusion it appears from this single year’s observations that dichromatism and variability are two distinct characteristics as represented in this species. ‘he heredity of the dichromatism is such that if any barrier should interpose to intercrossing between the two types, each would eventually become permanently estab- lished. Fig. Fig. Fig. types. Fig. Fig. Fig. In this seems to lie its only’ evolutionary significance. EXPLANATION OF PLATE. . Lina lapponica, immediately after casting pupal skin, showing absence of pigment in elytras. . Lina lapponica, 10 or.1§ minutes after casting pupal skin, spotted areas outlined in elytras. . Lina lapponica, 15 or 20 minutes after casting pupal skin early drab stage of both S and B . Lina lapponica, about 45 minutes after casting pupal skin, S type (mature). . Lina lapponica, 20 or 25 minutes after casting pupal skin, later drab stage of B type. . Lina lapponica, about 45 minutes after casting pupal skin, B type (mature). DICHROMATISM IN LINA LAPPONICA. Isapet McCracken. Mary Wellman del. Tue JourNAL or ExprriMENTAL Zo6 LoGy, vol. il. EVOLUTION WITHOUT MUTATION? BY CaB DAVENPORG: Professor de Vries having found in nature races that seem to have arisen suddenly, fully formed, and having repeatedly observed mutating individuals that breed true, declares, in his “Mutationstheorie,” for the universality of mutation as the method of phylogenetic differentiation. He says (1901, p. 139): a gradual origin of elementary species is not yet known toe very many cases are known in which species have suddenly made their appearance. “Nach der Mutationstheorie sind die Arten nicht durch allmahlige, wahrend Jahrhunderte oder Jahrtausende fortgesetzte Selection entstanden sondern stufenweise, durch plotzliche, wenn auch ganz kleine Umwandlungen.” The mutation theory as a sufficient theory of evolution has many supporters. Bateson has long urged a theory of this sort as a result of his studies, particularly on the data collected in his “Materials for the Study of Variation,” 1894. In his recent book “Evolution and Adaptation” Morgan adopts de Vries’ views. Now it seems to be a common characteristic of men of science when they have discovered a real and large truth to insist on its universality. But, particularly in biology, this tendency 1s fraught with danger on account of the complexity of the phenom- ena involved. [am quite convinced (and have, indeed, for more than a decade in my university lectures contended) that mutation or sporting plays an important part in evolution. I yield to no one in admiration for the work of the genial author of “Die Mutationstheorie,” a work that has placed experimental evolution on a solid basis:as a science and which has thus rendered a service to biology with which Darwin’s only compares. Yet, I think, the acceptance of mutation as a method of evolution should not pre- vent us a conceding the force of any evidence that selection of "Read before National Academy of Sciences, Chicago, November, 1903. 138 C. B. Davenport. trivial or individual variations has had something to do with the origin of species. For what are species? ‘They are assemblages which differ in one or more of their characters to so pronounced a degree as to meet the more or less hazy ideals of the systematist. Now it is quite cer- tain that such differences may arise in several ways. I propose to present certain evidence indicating that the differences between certain recognized species are of the order of accumulated individ- ual variations and not of the order of mutations. Such evidence will then go to prove that evolution may, in some cases, take place without mutation. Evidence as to the method of origin of species should be looked for in a group where closely related species are found to-day. Any interspace of distribution or time should be searched to see whether there are any graduated, transitional forms, or whether, on the contrary, a sudden change of character occurs. ‘There are, in general, two kinds of series available for examination: one is the geographical, the other the paleontological series. At the present day two “species”’ are often found occupying two more or less distant regions of the continent. If we examine the inter- vening territory shall we find all gradations between the species or shall we find a sudden transition from one to the other? We should expect the sudden transition only if mutation is the sole method of origin of species; on the other hand, if fine intergrada- tions appear these would speak for evolution by trivial variation. So, too, if a series of fossils connecting one species A with a second B are examined a sudden transition or a gradual one will be found as mutation has or has not acted in the case in question. The facts of geographical variation are well illustrated in the broad territory of North America. Our eastern song sparrow, to cite a single example, was formerly thought to be replaced bya distinct species on the Pacific Coast in the vicinity of San Fran- cisco, by a small light-colored desert species in Arizona and by a large dark species in Alaska. Later collections from inter- mediate localities revealed intermediate forms so that the different geographical forms are now regarded as varieties of one wide- spread species showing fine gradations in coloration and structure from place to place. In speaking of the results of the study of geographical x ariation of land birds in America, Newton (’93-'96, p- 343) says: “ The great fact was established that, given a species Evolution without Mutation. 139 which had a wide range on a continent, the variation exhibited by individuals from different localities is generally so considerable that it is hardly possible to predict its amount while almost every intermediate form may be found if the series of specimens be large enough.” To get additional data relating to geographical variation | have made quantitative studies of races of Pecten inhabiting different geographical regions. My first example will illustrate the fact of geographical variation where the extremes are not usually called species.. Pecten opercularis occurs on the coast of Europe from the Lofoten Islands to the Canary Islands and throughout the Mediterranean Sea. Ihave studied (1903) specimens from three localities on the coast of Great Britain: at Eddystone Light, the Irish Sea, and the Firth of Forth, at north latitudes 50° 15’, 54° 18” and 56° 05’, respectively. I find that the individuals from the ends of the series are the most unlike and that those from the intermediate latitudes are intermediate in most of their dimen- sions, as the following table shows: Eddystone. Irish Sea. Firth of Forth. Maximum dorso-ventral diameter 70 mm. 77 mm. 80 mm. Ratio ant.-post. to dorso-ventral diameter when latter = 67 mm. . 1.067 1.061 1.039 Ratio hinge length to antero-pos- CEHIOM CiaMEter..+5/.dsas ces o's 0.507 0.483. 0.473 Half globosity at length of 53 mm. 0.151 0.151 0.132 Relative length of ears to hinge... = shortest longest Average number of rays..........| 17.478 18.101 17.673 Standard deviation of rays....... ¥,000=2).020) | 1407422020 | 1 Diz 22.019 Coefficient of variability......... Le fe: 5-93 6.32 This series shows that there is a geographical variation and that the transition from one extreme to the other may be gradual. The second example has to do with the Pectens of the East coast of the United States. On the shores of Long Island is a species of scallop known as Pecten irradians. On our Gulf of Mexico coast occurs a second “species” —P. gibbus. For the 140 C. B. Davenport. cA purposes of this paper these two form units may be considered distinct species although some persons, considering facts like those here presented, would regard the two as varieties, but this difference in view does not affect our argument. When shells from Long Island and the Gulf Coast at Tampa, Fla., are com- pared they are found to differ in color, the lower valve of the Gulf shells lacking the blue of the more northern shells and being white or white and red. ‘They differ quantitatively as follows: Long Island. | Tampa. Average number of rays, R. valve...) 16.48+ .08 to 17.35+.02 | 20.512+ .030 Average globosity of shell when | antero-posterior diameter = 57-62 TIANA os ai'eis'n legen kw 'pteiae 5 CT .283+ .o10 |) 2is)O16 Average excess of antero-posterior | diameter over dorso-ventral when latter == 56-00 MMmes acer +6.1 mm. | +1.5 mm. Taken together these three pairs of characters seem satis- factorily to differentiate the two species. But when we study shells from Cape Hatteras (Morehead, N. C.) we find here a form unit in many respects constituting a link connecting the two species. [he color is quite intermediate. ‘The number of rays is 17.3, which is intermediate between some Long Island localities and Tampa, but more like Long Island. The globosity of the shell is much like that of ‘Tampa, being 0.319 for shells of a length of 5mm. ‘The range of globosity is such as largely to bridge the gap between the means of the Tampa and Long Island lots. ‘The average excess of antero-posterior diameter is 2.5 mm. for 5g mm. shells; thus intermediate between the 1.5 mm. of Tampa and the 6 mm. of Long Island. Finally, important evidence is afforded by a series of fossils from the Pliocene or late Miocene of the Nansemond River (James River system) at Jack’s Bank near Suffolk, Va. ‘These fossils are Pectens' closely related to P. irradians and known 1Now deposited at the University of Chicago. Evolution without Mutation. I4I as Pecten eboreus of Conrad. I gathered shells from three layers, at I foot, 4 to 6 feet and 15 feet above tide water. As there were relatively few from the middle layer these shells are not considered here. ‘These layers represent periods of time, the upper layer of molluscs having lived latest. First, let us examine the number of rays in the right and left valves from the bottom and top layers and, for comparison, in recent shells (1900) from Morehead, N. C., in default of living Pectens from a nearer locality. Both averages (A) and indices of variability (0) are given, although the former alone are of special importance in this discussion. ‘The number of shells measured in each case 1s given in the column headed n. NuMBER OF Rays. Right Valve. Left Valve. n A o n A o Lowest tier..... 164 22.478 + .059 1.118 + .042 138 21.674+ .070 1.223 + .049 Wipperstiek-rrr 163 21.693 + .058 1.104 .041 1940) 220-019-065 1.121 .046 Morehead...... 449 17.307 + .O17 0.821+ .021 558 17.228 + .028 0.982 + .020 ’ Transverse diameter Groxrosity or VALYE—. ce... ————— -WHEN Dorso-vENTRAL DIAMETER IS 57-61 MM. Dorso-ventral diameter Right Valve. Left Valve. n A o n A o Lowest tier.... 13 -1481 + .0013 -0072 + .0009 8 -1975 + .0039 -0164+ .0028 Upper tier..... 17 -1579- .0017 -O107 + .0012 8 .2063 + .0019 .0078 + .0017 Morehead..... 75 +3303 + .0012 -0158 + .0008 129 .2800-+ .oo10 -O161 + .0007 Ratio or ANTERO-POSTERIOR TO Dorso-vENTRAL DIAMETER WHEN LATTER Is 57-61 MM. Right Valve. Left Valve. n A o n A 0 Lowest tier.. 10 1.0960 + .0049 0232 + .0035 7 1.0750-+ .0033 -O131 + .0023 Upper tier... 25 I .0800+ .0043 -0321 + .0030 25 1.0560+ .0038 -0283 + .0027 Morehead... 52 1.0411 + .0024 .0258 + .0017 127 1.0459+ .0015 .0261 + .ool! The number of varieties (7) is fairly satisfactory for deter- mining the number of rays because this quality is independent of the size (age) of the shell and consequently shells of all sizes were taken. The proportions of shell diameters, on the contrary, change greatly with age; moreover, it seemed desirable to take shells of the same size from all series whether the modal size was small or great. Consequently 7 is small in the ratio determina- tions and the probable errors correspondingly high. Despite the large size of the probable errors there is a significant difference in 142 C. B. Davenport. the shells of the three epochs; the shells from the bluff being, how- ever, more like each other than like those of Morehead. The series show that the number of rays in both right and left valves has diminished since the Pliocene and that the reduction had made progress during the interval from the lowest deposits in Jack’s Bank to the uppermost deposits. “They show also that the change in question has been of the quantitative order rather than of the qualitative or mutational. Again, the shells have been becoming more globose. For, the ratio of transverse diameter of either the right or the left valve to its dorso-ventral diameter has increased. Although the recent P. irradians is twice as globose as the fossil P. eboreus the later fossil deposits show a change from the earlier in the same direction and make it probable that a quantitative change that was in progress in geological times has continued to the present time. Finally, both valves have been getting more nearly circular— the antero-posterior diameter becoming more nearly equal to the dorso-ventral one. Here, again, there is a quantitative change in a character; not the introduction of a new one. Apart from a certain bleached appearance of the shell and its less weight (both due, in part at least, to postmortem changes) the fossil shells differ from the recent ones, so far as I can see, in no other respects than the three enumerated above. It seems justifiable, therefore, to conclude that the evolution from the one species to the other has been without mutation and solely by graduated variation. SUMMARY. The process of evolution has taken place by various methods and not always in the same way. It is no more justifiable to maintain that all evolution is by mutation than that evolution has always proceeded by slow stages. The best evidence for slow evolution is found in wide-ranging species which while differing greatly at the limits of their range exhibit all gradations in peenediare localities (Melospiza, Pecten); also in fossil series (Pecten eboreus and P. irradians) where the change from one horizon to the next is of the quantitative order. ‘Thus evolution may take place without mutation. Station for Experimental Evolution, Cold Spring Harbor, N. Y., January 24, 1905. Evolution without Mutation. 143 LITERATURE CITED. Bateson, W., ’94.—Materials for the Study of Variation. London: Macmillan, 598 pp- Davenport, C. B., ’03.—Quantitative Studies in the Evolution of Pecten. III. Comparison of Pecten Opercularis from Three Localities of the British Isles. Proc. Amer. Acad. Arts and Sciences, XXXIX, 123-159. Nov. DE Vries, H., ’01.—Die Mutationstheorie. Versuche und Beobachtungen tber die Entstehung von Arten im Pflanzenreich. Leipzig: Veit & Co. 1901. 648 pp., 8 Taf. Newton, A., ’93-96.—A Dictionary of Birds. London: A. & C. Black. 1088 pp. MOSAIC DEVELOPMENT IN ASCIDIAN EGGS. EDWIN G. CONKLIN. With 82 Ficures. ieeNormal’Developmentjcsan-ide. ase) ha PART ae eR ee ar ae ete, SPRY ee tk 146 ive O bjectsvandel Methodstoty Pxpermmentys..y este. 1s ice eee ls = tee = lee alti stale re tonal 151 TEs ARES Gli Ted orsiatanOal Gea. opae BOSC Srp tor Sonar SOoEn SH ene ATO nC Boar Dp oouoors aadcGot 155 1. Right or Left Half lByedloin Css cmnac eee oe ACO ane Or SsbA io IeAmmooe nce ao Conon < 157 (OBEN sn Sie paloprere oC ome UR robe Sir at boo One Gober OLED ae ocd inbas Aae 157 PraGAstnil atone ce eels a steve ee oes soos Bilin alata ego srayare ate Grech bl staiatelsieletage Ble 163 Cus MODIMAtlOnt OLVALVANe ae shat. oi yetricllcuciaee eset anda s See Re sores, ae ete olapsleterers res (q) BNeuraltPlate“andtSense Organs) 2). 6/12. ee eee ects > see | seeiiee ters 168 @)eNotochord ease cits Galera =o os ncle meee cai steepest 5 tote sieeee mane 169 (3) Muscles and Mesenchyme ... SE ae ee EEN 169 A, Aides OW eine LN IAC, a ones coohb con a SuacogMneae ae ecmown gouLt Gono paebo reac 170 sisal Noliciatsie la alba Dsl a Oe Nee ravine GO e Eee Sonn te cae tae qn atnao Ove se ootteonimadbe 175 aw Posterior Elalbabinbry Gs: scvieis)eci piece whee © Wales ise -eg cist Fs a sqisvare sla rede sbelensiaabeneommaperen gana 179 iio MOMEGNe Lea INOS inane aoc das abt cub Unryetcrein ni eietocnineheoce podece sgucabs 183 GapEiohtbsom Sixteemeli el malbryOse ce ote si) ves cehene ae shape cael odele ose ateeetetey terete aeaiate ey arse 189 7a Anteniorsandebosternion Halt Gastrula’ 52 o25.. 05 2-2 sere nee ea Par eee ieee 193 iVeeOthenExpenmentall Work onytite AScidtam Bp. acc icsie)atetel olelelare aelaleneiel state atsray tee elepee ata 197 Ve Cl EV oe pate secrets ee Oem 5 5 Orne ca RIGA SAO a CAS toa nina Meni onan hae 198 DH Gastuila Gecetap et shyscdese ow veel, sears Fe Swed Sahee che, HUA n eae ease et ecettaleko ie epson seeracaee ae 199 ap ALEING Soo nck GnoSUe rb spepode apes Aen oMeouoodsc oad Sonor Us cor msggobon ce cdae 200 a» Neural Plate and Sense @rgans) socct +. ani nies aisle neon ialsl< ern slap haste 203 BapNotochord ea. :a-tiasarsk wits wat ave sis eal ob Frost ae eater eck eaerse uae es elev lene take 204 ea Musclessand) Miesenchyme: - Satine o: sore acini eicierey menses) fe eeetererele ps ie eter 205 WeeRepulattonmeAsctdtaneh cesar dmb m bry OMe. ele oe) ahatactae tele ete aCateies «leecher ke reece 206 VI. General Conclusions ........ Be OD eR ee Rh ae Peace nator Goma aacieae 209 Hen Orp anh OLN ErS UD StAN GS) ssreretc\o/eiersy= ete let Wel emi aie lees Chae rad seek ea reer eke 209 Padlbocalizationsor@aoplasmic Substancesa ni. cei. s atria tas ete elmer eet eee 210 3. Cleavage and Mica zations # < ico era eckh aes RA gs AE ee el eee 213 4. Determinate and Indeterminate Cleavage and Development ...................... 214 SUMMA) oc Sk conende cde u CORP ONEd Gono DOOD Ad COLON SMA DOGS ObnwE Scds. pattie ae letoneioh a aera halo atone Pegs 216 TU renerey rete Cotirega lo a aie ose tho elas ace ERR OG Cashel bes re Latin & Sibir oo Cae rcaenO Gran eorciraiio acre coon 221 In almost every instance in which fragments of eggs or isolated blastomeres have been found to be capable of giving rise to entire larvee the substance of the unsegmented egg is apparently undiffer- entiated and the cleavage cells are so nearly equal and homo- geneous that it has not been possible to trace the lineage of individ- 146 Edwin .G. Conklin. ual blastomeres throughout the development. ‘The most notable exception to this rule is found in the case of ascidians. ‘That the cleavage of the egg in these animals is constant in form and differ- ential-in character and that specific blastomeres are destined in the course of normal development to give rise to specific parts of the larva has been demonstrated by Van Beneden and Julin, Chabry, Castle, and many others. Chabry (87) also showed, in one of the earliest experimental investigations dealing with he potency of cleavage cells, that individual blastomeres of Ascidia aspersa always develop into those parts of the larva which they would produce under normal conditions. On the other hand, Driesch (’95) discovered, some eight years later, that in Phallusia mammilata individual blastomeres up to the 4-cell stage at least are capable of giving rise to entire larvz and this conclusion was afterward confirmed by Crampton (’97) in the case of Molgula manhattensis. Since the results of Chabry were thus flatly con- tradicted by these later investigators and as they have been de- fended by no one who has actually experimented on these eggs‘ these results have been generally discredited and the ascidians are now commonly regarded as belonging to that group of animals in which the early cleavage cells are equipotential. ‘The ascidians, therefore, should afford an excellent opportunity of determining the exact method by which an egg fragment or isolated blasto- mere gives rise to an entire larva, since in this case it 1s possible to Fallow the lineage of individual cells until they enter into larval organs; furthermore, they should afford means of testing the justice of the distinction which has been proposed (Conklin, ’97) between determinate and-indeterminate types of cleavage, and finally they should throw light upon the significance of the high degree of differentiation which is known to exist in the early development of these animals. I. NORMAL DEVELOPMENT. I have recently (’05') shown that these differentiations of the ascidian egg are much greater than has heretofore been supposed; in the unsegmented egg of Cynthia (Styela) partita at least five distinct kinds of ooplasm- can be recognized. ‘These are, (1) the ‘Several persons, viz: O. Hertwig ("92), Roux (’92), Weismann (92), Barfurth (93) have discussed Chabry’s work from a critical point of view. Mosaic Development in Ascidian Eggs. 147 deep yellow protoplasm which later enters into the muscle cells of the tail of the larva; (2) the light yellow material which becomes mesenchyme; (3) the light gray material which forms the chorda and neural plate; (4) the slate gray substance which becomes endoderm, and (5) the clear transparent protoplasm which gives rise to the general ectoderm. All of these substances are recog- nizable in the egg before the first cleavage and immediately after that cleavage they all occupy their definitive positions in the egg, the yellow protoplasm forming a yellow crescent around the pos- terior side of the egg just dorsal to the equator, the light gray substance forming a gray crescent around the anterior border of the egg, the slate gray substance lying at the middle of the dorsal hemisphere and between the two crescents, while the transparent protoplasm is chiefly localized in the ventral hemisphere of the egg. In these positions and from these substances the organs and germinal layers specified arise. At the first cleavage of the egg all of these substances and areas are equally divided, since this cleavage lies in the plane of bilateral symmetry of the egg and future embryo. The second cleavage plane is perpendicular to the first and separates the gray crescent in front from the yellow crescent behind; the cells of the anterior quadrants are therefore very unlike the posterior ones and the two can always be distinguished ata glance. (Fig. 1.) ‘The third cleavage is equatorial and separates four clear ventral cells from four dorsal ones which contain the yellow and gray crescents and the deep gray material. (Fig. 2.) ‘The ectoplasm is now com- pletely segregated in the four ventral cells but the other ooplasmic substances are not as yet located in separate cells, though from the time of the first cleavage onward their locations and boundaries are perfectly sharp and distinct. At the fourth cleavage each of the eight cells divides, thus giving rise to sixteen cells (Fig. 3) and at the fifth cleavage these are increased to thirty-two. During the fifth cleavage the substance of the gray crescent is segregated into four cells (A*’, A®*, Fig. 4) at the anterior border of the egg, while the yellow crescent comes 1The system of cell nomenclature employed in this paper is similar to that used by Castle (96) and is fully explained in my work on the cell-lineage ('05'); in brief 4 and a designate cells of the anterior half of the egg, B and b those of the posterior half, the capitals being used for cells of the vegetal (dorsal) hemisphere, the lower case for those of the animal (ventral) hemisphere. Corresponding cells of the right and left sides receive the same designation, except that those of the right side are underscored. 148 Edwin G. Conklin. NorMat DEVELOPMENT oF CYNTHIA PARTITA, 4-CELL TO 64-CELL STAGES; X 333. The yellow crescent which surrounds the posterior half of the egg dorsal to the equator is stippled. The gray crescent around the anterior border of the egg is left unshaded. The boundary between the clear protoplasm and the yolk is indicated by a crenated line. The polar bodies (shaded by vertical lines) lie at the animal or ectodermal pole. Fig. 1. Four-cell stage from the animal pole, the yellow crescent showing through the egg. Fig.2. Telophase of the third cleavage (8-cell stage), from the left side. Fig. 3. Twenty-cell stage from the animal (ventral) pole. Fig. 4. Twenty cells, transitional to the 24-cell stage, from the vegetal (dorsal) pole. The gray crescent is now segregated in the two pairs of cells A®*, A®4; the yellow crescent will be localized in separate cells at the close of the division which has already begun in the cells B®. Figs. 5 and 6. Ventral and dorsal views of the same egg in the 64-cell stage. The yellow and the gray crescents each consist of a double arc of cells; the anterior arc of the gray crescent (A7.4, A7.8) is composed of neural plate cells, the posterior arc (A73, A%-7), of chorda cells; only two pairs of cells in the yellow crescent (B’-4, B7-§) are muscle cells, the others are mesenchyme. The pair of cells A7-6 also gives rise to mesenchyme. All the other cells of the dorsal hemisphere (Fig. 6) are endodermal. All the cells shown in Fig 5, except those of the yellow and gray crescents, are ectodermal. Mosaic Development in Ascidian Eggs. 149 150 Edwin G. Conklin. to occupy six cells (B°*, B**, B**) around the posterior border (the spindles which lead to the formation of these six cells are indicated in Fig. 4). These thirty-two cells are increased to sixty-four at the next cleavage (Figs. 5 and 6); during this cleavage four chorda cells (A7*, A”*) are separated from the four neural plate cells (A™, A™*®, Fig. 6), while the six cells of the yellow crescent have given rise to twelve, four of which are muscle cells (B74, B78) and eight mesenchyme Ge BeBt Bt). At the same wmeson additional pair of mesenchyme cells a(t" )is separated from a pair of endoderm cells in the anterior quadrants. This is the only mesenchyme cell derived from the anterior quadrants. At this stage all the substances of the germ layers and of the principal organs of the larva are gathered into separate cells, but although this segregation into separate cells comes relatively late in the cleavage these substances have been definitely localized in certain regions of the egg from the time of the first cleavage. Subsequent cleavages lead to changes in the shape of the embryo but produce no changes in this localization. In the gastrulation the endoderm cells are depressed and are overgrown in front by the chorda cells and these in turn are covered by the neural plate cells; similarly the mesenchyme cells overgrow the endoderm at the posterior border of the blastopore, while the mesenchyme cells are overgrown by the muscle cells, and finally the latter by the ectoderm. (Figs. 7-10.) Inthe closure of the blastopore the anterior (dorsal) lip grows posteriorly until it covers most of the dorsal face, while the muscle cells form the lateral boundaries of the blastopore. (Figs. 9, 10.) In this over- growth of the dorsal lip the chorda cells which originally lay at the anterior border of the egg are carried back into the posterior half of the embryo, where by interdigitation they form the chorda. The neural plate cells are also carried back with the chorda nearly to the posterior end of the embryo. ‘The ventral (posterior) lip of the blastopore then grows forward over the remnant of the blastopore and the neural plate is rolled up into a tube which closes from behind forward. The muscle cells become arranged in three rows on each side of the chorda; in front of the muscle cells is a mass of small mesenchyme cells, while a double row of endoderm cells ventral to the chorda constitutes the cord of ventral or caudal endoderm. (Figs. 11 and 12.) Finally the tail of the larva elongates greatly and becomes coiled around the body of the Mosaic Development in Ascidian Eggs. 151 larva within the egg membranes, and about twelve hours after the fertilization of the ege the larva may hatch and become free swimming. However, in a considerable proportion of cases the larva never hatches but undergoes its metamorphosis within the egg membranes. Il. OBJECTS AND METHODS OF EXPERIMENT. This brief review of the normal development! shows that there is a remarkable degree of differentiation and localization of the substances of the egg and embryo and it seems to render necessary some further explanation of the results of the experiments of Driesch and Crampton; certain it is that the egg is highly differ- entiated and if portions of this differentiated ooplasm may give rise to portions of the larva which they would never produce under normal conditions it is important to know the steps by which this is accomplished. With this object in view I spent the summer of 1go4 at the Ma- rine Biological Laboratory at Woods Hole, Mass., experimenting on the eggs of Cynthia (Styela) partita and of Molgula man- hattensis; | was unable to obtain Ciona intestinalis, the normal development of which I had studied during the previous summer, and my experimental work is therefore limited to the two species first named. Most of my work was done on the egg of Cynthia, which is a better object for experimental work than that of Mol- gula, owing to its greater size and the more brilliant coloring of its different oéplasmic substances. Enough work was done on Mol- gula, however, to show that the development of isolated blasto- meres is the same in this genus as in Cynthia. All the experiments performed had for their purpose the testing of the potencies of the various substances and blastomeres of the egg. Injuries to the unsegmented egg of whatever nature, whether produced by sticking, cutting or shaking the eggs, invariably inhibited all further development. I have therefore been unable to test the developmental potencies of the different kinds of odplasm of the unsegmented egg. But inasmuch as these substances are the same in appearance and localization before and 1For a more detailed account of the normal development of these ascidians the reader is referred to my previous papers on the “Organization and Cell-Lineage of the Ascidian Egg” (o5"), and on “Organ-Forming Substances in the Eggs of Ascidians” (’os”). 152 Edwin G. Conklin. NorMaL DreveLopMENT oF CYNTHIA PARTITA, GAsTRULA TO TADPOLE; X 333. ‘The neural plate or tube is finely stippled, the chorda coarsely stippled; muscle cells are shaded by vertical lines, mesenchyme by transverse lines. ; Figs. 7 and 8. Ventral and dorsal views of a gastrula (180-cell stage), showing T-shaped blastopore, neural and chorda plates, mesenchyme and muscle cells. Most of the cleavage cells are in the ninth generation. Figs. 9 and 10. Two views of the same gastrula from the dorsal pole; Fig. 9, showing the super- ficial cells, Fig. 10, those at a deeper level. The overgrowth of the dorsal lip of the blastopore and the approximation of the muscle cells of each side toward the median plane have reduced the blastopore to a longitudinal groove in the posterior half of the embryo. The ectoderm cells are in the tenth genera- tion and there are in the entire embryo about 360 cells. Fig. 11. Dorsal view of an embryo in which the neural plate (n. p.) is closing to form the neural tube (n.t.) Beneath the nerve tube is the notochord and on each side of the latter is shown a row of muscle cells(ms.) At the posterior end of the muscle rows is the caudal mesenchyme, at their anterior end the trunk mesenchyme (m’ch.) Fig. 12. Young tadpole viewed from the left side, showing three rows of large muscle cells (ms.) along the side of the notochord (ch.); dorsal to the latter is the nerve tube (n. t.); anterior to the muscle rows is the trunk mesenchyme (m’ch.); ventral to them is the ventral or caudal endodem (v. end.) Mosaic Development * ~~ =o Ges Z BS." Gy anat Nee eee 154 Edwin G. Conklin. after cleavage begins it can scarcely be doubted that their poten- cies are also the same. Hundreds of experiments involving many thousands of eggs were made upon the various cleavage stages. ‘The methods of experimenting which I employed were essentially like those used by Driesch and Crampton, viz: the eggs in the 2-cell, 4-cell, 8-cell or later stages were strongly spurted with a pipette, or were shaken in a vial, and thereby some of the blasto- meres were frequently injured while others were uninjured and continued to develop. The injured blastomeres were rarely killed, as was shown by the fact that they remained transparent and entire for a day or more, whereas dead cells soon become opaque and disintegrate. ‘These injured cells never again divide and sections show that their nuclei are frequently broken and their chromosomes scattered. Cells are more likely to be injured during nuclear division than during rest. The fact that these injured cells never again divide though they remain whole within the chorion and preserve their characteristic color and structure makes it possible to determine at all stages just what cell or cells have been injured. Whether or not the presence of these injured cells within the chorion may influence the development of the uninjured cells will be considered later. Attempts to completely separate individual blastomeres by the use of Herbst’s calcium- free sea water were not successful, probably owing to the presence of the chorion and to the close union between the blastomeres! In addition to this method of experimentation which yielded hundreds and thousands of eggs in which one or more of the cape meres had been injured I also cut eggs and embryos 1 in two with knives made from small needles. In no single instance was | able to get fragments of unsegmented eggs to develop; in the gastrula stages | was more cnecesstall being able to cut gastrula in two in the manner described by Driesch (03) and observe the subsequent development. I have not attempted to repeat the various ingenious methods of injuring blastomeres which were devised aaa employed by Chabry, since they are necessarily slow and difficult of application and yield but a small number of injured eggs, whereas by simply spurting or shaking the eggs one may injure blastomeres in an enormous number on eggs which can then be sorted out and classi- hed according to the character of the injury; furthermore the ease and certainty with which the identity of injured blastomeres of Mosaic Development in Ascidian Eggs. 155 Cynthia may always be determined renders unnecessary such experiments as Chabry’s on the individual cleavage cells. If one desires to trace with accuracy the lineage of individual blastomeres, whether in normal or experimentally altered develop- ment, it is essential that a large quantity of material should be available. In even the most favorable material the lineage of the later stages can be successfully studied only by the aid of fixed and stained material and without a large number of eggs it is difficult if not impossible to secure all the stages of dev elopment. Furthermore it 1s desirable that a considerable number of eggs of every stage be available for study, since the liability to error decreases with the number of cases studied. Accordingly, in addition to the study of living eggs during successive stages after their injury, many eggs were Sikes fixed at brief intervals and were afterward stained and mounted entire or sectioned. For this purpose I have found Kleinenberg’s picro-sulphuric acid followed by my picro-hematoxylin to give the best results. Entire eggs so prepared show cell outlines, nuclei and karyokinetic figures much more plainly than in the living condition; on the other hand the yellow crescent is less distinct since the yellow pigment 1s extracted by alcohol; nevertheless this crescent may always be recognized by its peculiar staining qualities and it therefore affords a never failing aid in Stentationi lit RESULTS OF EXPERIMENTS: In undertaking this work it seemed to me scarcely possible that all of these strikingly different kinds of odplasm, each with its own peculiar developmental history and destiny, were neverthe- less morphogenetically alike, as might be concluded from the results of Driesch and Crampton. On the other hand a possible escape from this conclusion was suggested by the fact that although the cleavage cells are strikingly different from one another, the isolation of the odplasmic substances in them is not quite com- plete; almost all of the yellow protoplasm is contained in the yellow crescent; but a small amount of it is found around the nuclei of all the ae most of the gray substance is contained within the dorsal hemisphere, but a small amount of it occurs in the ventral cells also; most of the clear protoplasm is found in the ventral hemisphere but a small quantity is also found in the dorsal cells. 156 ,; Edwin G. Conklin. It therefore seemed possible that the production of a complete larva from any one or two of the first four cells might be due to the replacing of a missing substance by the greater development of the trace of that substance contained in the cells in question. “hus the anterior quadrants which lack the yellow crescent might, perhaps, regenerate it from the small amount of yellow perinuclear protoplasm which they contain, and correspondingly the posterior quadrants might regenerate the lacking gray crescent from the small amount of gray substance which they contain. In the light of the work of Driesch and Crampton either there must be ies regeneration, or the substances which appear so different must after all be each and all totipotent. However the solution of this problem has turned out to be much simpler than I had supposed possible, viz: 1solated blastomeres do not give rise to entire larve, as claimed by Driesch and Crampton, but on the contrary each blastomere produces only those parts of a larva which would arise from it under normal conditions. The development ts, 1n short, a “mosaic work.” Since the first cleavage is bilaterally symmetrical each of the first two blastomeres con- tains one-half of each and all of the substances of the egg and correspondingly the half larva which develops from one of these blastomeres contains portions of every larval organ. Owing to the fact that the cells which arrse from an isolated blastomere close over the injured surface these partial embryos are rounded in form and many of the one-half larve resemble superficially whole larvz of half Size, but in no case are they complete. When the anterior or posterior quadrants of the 4-cell stage are killed nothing even remotely resembling a normal larva is ever pro- duced. My results are therefore directly opposed to those of Driesch and they agree in all essential respects with those of Chabry. The partial embryos and larve obtained in these experiments may be classified as right or left, anterior or posterior, dorsal or ventral, or composite forms. Furthermore they may be known as half, quarter, eighth, sixteenth, etc., embryos, according as they are produced from blastomeres of the 2, 4, 8, 16, etc., cell stages; however, the character of the embryo depends entirely upon the region from which the isolated blastomeres come and not upon the number of such blastomeres. « Mosaic Development in Ascidian Eggs. 157 1. Right or Lejt Half Embryos (Figs. 13-33, 36-46). a. Cleavage. When the right or left half of an egg is injured in the 2, 4 or 8-cell stage, the other half continues to segment in a normal manner, provided it was not also injured. I have traced the cell- lineage of these right or left half embryos up to the eighth genera- tion of cleavage cells (the 112-cell stage of normal eggs), while I have determined the lineage of many individual cells as late as the ninth or tenth generation (218-360 cell-stage). ‘The cell-lineage of these half embryos 1s essentially like the right or left half of a normal egg, except that the direction of arcing and consequently the position and size of some of the blastomeres may be slightly altered. This alteration in the direction of cleavage is most evident in cases where the egg was injured in the 2-cell stage, and it is prob- ably due to the fact that the uninjured blastomere in such cases becomes nearly spherical in shape, and does not remain hemi- spherical as inthe normal egg. Owing to this fact the median pole of certain cleavage spindles, 7. ¢., the one next to the original median plane, is shifted toward the middle of that plane. The resulting mass of cells is, therefore, more nearly spherical than in the half of a normal embryo. (Figs. 13-20.) If the injury occurs in the 4-cell stage or later, the change in the direction of the early cleavages is not so evident as alien it takes place in the 2-cell stage. In case.one of the blastomeres was injured at the close of the first cleavage, the direction of the karyokinetic spindles of the second and fchicd cleavages are entirely normal, since in both these cases they lie parallel with the first cleavage plane, Fig. 13; but in the fourth cleavage in which one pole of the spindles lies nearer that plane than the other, the median pole is shifted toward the middle of that plane and consequently the cells formed along the median plane come into closer contact with one another and the cell aggregate is more nearly spherical than in the mght or left half of a normal 16-cell stage. (Figs. 14, 15, 21, 22.) These results entirely agree with those of Chabry and Crampton. The fifth cleavage of the right or left half embryo is also like the normal except in the direction of a few of the divisions; e. g., Fig. 16 is nearly normal but in Fig. 17 the division of the cell 158 Edwin G. Conklin. DEVELOPMENT oF RiGHT BLASTOMERE OF 2-CELL STAGE. Figs. 13-20. Successive stages in the development of the same right half embryo, the left blasto- mere having been injured in the 2-cell stage; drawn at intervals of about five minutes. Here and elsewhere the yellow protoplasm is indicated by coarse stipples. Fig. 13. Right half of 8-cell stage, posterior view. A small amount of yellow protoplasm surrounds the nucleus of the ectoderm cell b‘.2.. The position of the cells shows that the ventral ends of the third cleavage spindles diverged from the first cleavage plane in the posterior quadrant and converged toward that plane in the anterior quadrant, just as in the normal egg. (See Conklin, ’o5'.) Fig. 14. Right half of 16-cell stage, anterior view. The yellow crescent is seen through the cell B*-1. In the normal egg of this stage the cells A®-! and a*-3 lie more nearly in front of the cells A5.2 and a®-4. Fig. 15. Same stage as preceding posterior view. In normal eggs the cells B®-2 and b*-4 Jie nearly behind the cells B®“) and b®-* and not on their median side. Fig. 16. Right half of 30-cell stage, dorsal view. A®? and A®4 are cells of the gray crescent; B*.? and B¥.?, cells of the yellow crescent. Fig. 17. Right half of 34-cell stage, posterior view. In normal eggs the cell B®-4 lies on the lateral border of B38, Fig. 18. Same stage as preceding, dorsal view. The cell B®-! normally lies between B®.3 and A®.!. 160 Edwin G. Conklin. DeEvELopMENT oF RiGuT BLASTOMERE OF THE 2-CELL STAGE; ALSO OF R1GHT AND Lerr BLASTOMERES oF THE 4-CELL SraGe. Figs. 19, 20. Same embryo as that shown in Figs. 13-18. Fig. 19. Right half of 46-cell stage, posterior view; the yellow crescent cells are not quite normal in position. Fig. 20. Right half of 48-cell stage, dorsal view. The caudal endoderm cells (B’-! and B’-?) have been shoved away from the median plane by the cell B7.5. Figs. 21,22. Fixed and stained preparations of half embryos in the 16-cell stage. Fig.21. Right half embryo, posterior view. Fig.22. Left half embryo, ventral-posterior view. Figs. 23,24. Successive stages of one and the same half embryo, the left half having been injured in the 4-cell stage, dorsal view. Fig. 23. Right half of 16-cell stage. Fig. 24. Right half of 32-cell stage. The cleavage is like the right half of a normal egg in every respect. Mosaic Development in Ascidian Eggs. 161 162 Edwin G. Conklin. B®? into B** and B** is almost at right angles to its normal direction. In other cases, as is shown in Fig. 24, this cleavage is normal in direction, and I am, therefore, of the opinion that the condition shown in Fig. 17 and the later stage of this same egg shown in Fig. 19 may be due to some slight injury to the developing half of this egg. In Fig. 18, which is a dorsal view of the same egg in the same stage as Fig. 17, the cells A®? and A** have moved in toward the median plane as compared with Fig. 16, though in this - respect, also, the corresponding stage shown in Fig. 24 is quite normal. ‘This shifting of the anterior dorsal cells toward the median plane is shown again at the next cleavage (the sixth), of this egg. (Fig. 20.) The seventh cleavage, which is shown in Figs. 25 and 26, is also normal except for the direction of a few of the divisions. The cells which constitute the yellow and gray crescents are in all respects like the right half of a normal egg. However the position Ofte cells Aland Alsip. 25. and che direction of division in several of the ectoderm cells shown in Fig. 26 are not quite normal. In conclusion therefore it may be said that the cleav age of one of the blastomeres of the 2-cell stage or of the right or left blasto- meres of the 4-cell stage, is like that of the corresponding half of a normal egg, except in minor details. Even these minor differences are not always present and when they are they do not alter the localization of the odplasmic substances. In every case the dis- tribution of the yellow, the gray and the clear substances to the different blastomeres is the same as in the right or left half of a normal egg; the cells of the yellow crescent, for example, form only the right or left half of a normal! crescent, and the same is true of the gray crescent and of the other substances of the egg. Even the small amount of yellow protoplasm which is found around the nuclei of the posterior ectoderm cells b*?, Fig. 13, is perfectly normal in its occurrence and subsequent distribution. I have elsewhere (’05") shown that the localization of different ooplasmic substances in the ascidian egg precedes cleavage and that cleavage and localization are here “relativ ely independent of each other; these experiments show that in both cleavage and localization the development of the right or left half of an ascidian egg is a ““mosaic work,” for the slight ; amount of regulation, which 1s Reitested i in the changes in fhe direction of certain cleavages, and the consequent closing of the embryo in no way alters ane Mosaic Development in Ascidian Eggs. 163 histological character of the cleavage cells nor their developmental tendencies. 6. Gastrulation. In the development of the right or left half of an egg the process of gastrulation sometimes occurs in an unusual manner. The most frequent modification of the normal process is that shown in Figs. 27, 29, 30, where the endoderm cells are not infolded but come to protrude above the level of the other cells, thus forming exogastrula. In later stages these endoderm cells must become infolded for it is a rare thing to see exogastrulz or any indication of an original evagination of endoderm cells in any of the cultures of older embryos. By what process these exogastrule right them- selves I have not been able to observe, but I think it probable that this like normal gastrulation is accomplished by overgrowth of the ectoderm cells and change of shape of the endoderm cells. Sometimes when the endoderm cells are evaginated other por- tions of the blastula wall invaginate. In this way false gastrulz may arise in which the infoldied cells are not endudecnel but ectodermal, as is clearly shown by their histological structure. (Fig. 63.) While some embryos in the gastrula stage show such abnormali- ties as those which have just been described in other cases the gastrula is strictly a half one, as is shown in Fig. 31, and it seems to me probable that exogastrulz or false gastrule only arise when the surviving half of che egg has been slightly injured. ‘These half gastrulz contain just one-half of all He cells of the normal gastrula and the position of the various cells and organ bases 1s essentially like that which occurs in the right or left half of a normal gastrula; the cells of the yellow crescent lie along one side only of the blastopore groove; the neural plate and chords cells each form half of the arc which is normally present in the anterior lip of the blastopore, while the closing of the open side of the astrula, which is turned toward the injured cell, is chiefly accom- plished by the overgrowth of the ectoderm cells of the ventral Sides pig. 31.) Except, therefore, for this tendency of the cells along the injured side to come together, these half gastrulz are strictly partial and the gastrulation no less than the cleavage may be regarded as an illustration of mosaic development. 164 Edwin G. Conklin. ’ Ricut or Lerr Harr Empryos; 64-Certs to Gastruta. Figs. 25,26. Fixed and stained half embryos; spurted in the 2-cell stage and fixed 2 hours later. Fig. 25. Right half of 64-76-cell stage, dorsal view. The neural plate cells (A’-7, A8.8, A815, A8.16) have just divided, the chorda cells (A7-3, A7-7) are dividing. The position of the cells A7-1, A7-2 is slightly abnormal. (v. Fig.6.) Fig.26. Left half of 64-76-cell stage, ventral-posterior view. Figs. 27,28. Right half of embryo in about 180-cell stage; spurted in the 4-cell stage and fixed 2 hours later. Fig. 27. Dorsal view; the large endoderm cells lie above the level of the other cells and form an exogastrula; some of the yellow cells (stippled) still lie at the surface while others are covered by endoderm cells. Fig. 28. Ventral view of similar embryo. Figs. 29, 30. Living right half embryos, dorsal view, showing the endoderm cells forming exogas- trulz and the yellow crescent cells at the surface. 165 1c Development in*Ascidian Eggs. Mosa 166 | Edwin G. Conklin. Ricur or Lerr Harr or Turee-Quarter Empryos; GastrutA to Tapporr. Drawn FROM Fixep AND STaInED Materia. Fig. 31. Right half gastrula of about 220-cell stage; spurted in the 4-cell stage and fixed 3 hours later. The neural plate, chorda and mesoderm cells are present only on the right side and in their normal positions and numbers. Fig. 32. Left half of young tadpole, dorsal view; spurted in the 4-cell stage, fixed 5 hours later. The notochord is normal except for size and number of cells; the muscle and mesenchyme cells are present only on one side; the neural plate is abnormal in form but not in position. Fig. 33. Right half of young tadpole, dorsal view; spurted in the 4-cell stage, fixed 44 hours later (slightly younger stage than Fig. 32). The notochord consists of a small number of cells which are interdigitating; muscle cells and mesenchyme lie on the right side of the notochord, but not on the left, though the muscle cells have begun to grow around to the left side; the neural plate is normal in posi- tion but not in form. Fig. 34. Right-posterior three-quarter embryo, from the right side. The left anterior cells (A*-1, a4-2) were killed in the 8-cell stage and the embryo fixed 5 hours later. The posterior half of the embryo is normal, but the left half of the anterior part is lacking and the neural plate is abnormal and has not formed a tube though sense spots are present. Fig. 35. Left-anterior three-quarter embryo, dorsal view; the right posterior quadrant (B*) was killed in the 4-cell stage and the embryo fixed 6 hours later. The anterior half of the embryo is entirely normal. The muscle cells are lacking on the right side though they have begun to grow around the hinder end of the notochord. The posterior portion of the trunk mesenchyme is found only on the left side, but its anterior portion, which is derived from the cells 47.6 and A?.6 (Fig. 6) of the anterior quad- rants is present on both sides. In the region of the injured cell the notochord and neural tube are curved away from that cell. Fig. 36. Left half embryo, from left side; spurted in the 4-cell stage, fixed 6 hours later. The dorsal lip of the blastopore is being overgrown by the ventral (posterior) lip. Muscle cells and mesenchyme are found only on the left side. The neural plate is abnormally folded, but still open; sense spots are present. . Mosaic Development in Ascidian Eggs. 167 ‘is . 168 Edwin G. Conklin. c. Formation of Larva. A considerably later stage in the development of the half embryo is shown in Figs. 32, 33 and 36 (Figs. 34 and 35 are three-quarter embryos and will be described later); of these stages Fig. 33 1s the youngest and Fig. 36 the oldest. In all of these figures the blasto- pore has already closed and the chorda cells have given rise to a fusiform notochord, which lis in the posterior half of the embryo. The blastopore closes chiefly by the posterior growth of the dorsal (anterior) lip, as in the normal gastrula. With the formation of the notochord the posterior half of the embryo becomes elongated and narrower than the anterior half and the developing tail bends around toward the injured side. (Figs. 32, 33.) ‘The anterior half remains large, the posterior half becomes long and narrow; the latter portion contains the notochord and muscle cells, the former the gastral endoderm, mesenchyme and most of the neural plate. gine general superficial appearance of an embryo of this stage 1s very similar to a normal one, but a more detailed study shows many differences. (1) Neural Plate. ‘The neural plate occupies in the main its normal position, that is, it lies along the first cleavage plane on the dorsal side, next to the injured cell. In this position the: plate becomes folded and ultimately comes to contain a vesicle (the sense vesicle) though the steps by which this vesicle is formed are always irregular and abnormal. (Figs. 36-40.) The anterior por- tion of ches plate is usually doubled over posteriorly while the posterior portion is folded forward (Figs. 36, 39, 40) and in this way a vesicle is finally formed. The tail of the embryo grows around toward the injured side so that the concave side of the embryo is median or dorsal, the convex side being lateral or ventral. In the younger, normal larvae the concave side is ventral, the convex dorsal. In these half larvae the nerve plate lies along the concave side, a con- dition which is the reverse of what is found in the normal larva. (cf. Figs. 12 and 36.) In the older half larve there is almost always found one or more pigmented sense spots in the neural plate or sense vesicle. (Figs. 36-40, 45, 46.) These pigment spots appear within cells of the neural plate and, as [am well convinced, always within definite cells, though owing to the abnormal foldings of the neural plate they do not always occupy exactly the same Mosaic Development in Ascidian Eggs. 169 positions. Furthermore these sense spots may be more numerous than in the normal larva, as shown in Figs. 45 and 46, probably owing to the fact that the cells which form the pigment and which normally lie on the margins of the neural plate do not come to- gether to form two spots as in normal larve, but remain separated so that several such spots are formed. (2) Notochord. The chorda cells grow back into the posterior half of the embryo and the cells here interdigitate in the normal manner, finally forming a linear series of cells. (Figs. 32-46.) The notochord, which is at first relatively short and thick, Fig. 33 becomes later very much longer and more slender, Fig. 40, Sida in all respects it has the appearance of a normal Horace: save that it evidently contains a smaller number of cells. “The position of the notochord of the half larva is always slightly abnormal; it never lies along the original median plane (first cleavage) as in normal larvae, but its anterior end is diverted away from that plane and toward the lateral border of the larva. (Figs. 32, 33> 37,41.) This position is that which the chorda cells, which arise in the anterior lip of the blastopore and which grow posteriorly around the mar- gin of the blastopore, would naturally assume. (cj. Figs. 31 and 33.) What it is which causes the chorda cells to interdigitate in their characteristic manner is a question difficult to answer; it certainly is not dependent upon the crowding together of chorda cells from the right and left sides since it occurs normally when the cells of one side only are present; on the other hand it must depend upon a certain amount of lateral compression of the chorda cells since it occurs very rarely if at all in the anterior half larva in which the ectoderm and mesoderm of the tail are lacking. (3) Muscles and Mesenchyme. — In these right or left half embryos and larvz the muscle and mesenchyme cells are present on one side of the notochord; here they occupy their normal posi- tions, the muscle cells giving rise to three rows of cells along the lateral border of the notochord and the mesenchyme forming a group of small cells anterior to the muscle rows. (Figs. 32, 33, 36.) In later stages the muscle cells slowly extend over to the side of the tail on which they were originally lacking; this takes place espe- cially at the hinder end of the tail, the overgrowth taking place around the end of the notochord and over its ventral side. In this way the right or left half embryo or larva tends to become com- plete, but I have never seen a case in which three rows of muscle 170 Edwin G. Conklin. cells were found on both sides of the notochord. Indeed, I am not at all sure that this extension of the muscle cells around the end of the notochord is accompanied by any increase whatever in the number of muscle cells or in the number of rows of cells. The latest stage in which I can positively identify the three rows of muscle cells is shown in Fig. 36. In this larva the muscle rows lie nearer the ventral side than in aonmal larvae (see Fig. 12), and they are evidently extending over the ventral surface toward the opposite side. In later stages the muscle cells become much elongated, but I have not been able to determine the number of rows present. | have found it still more difficult to decide whether the trunk mesenchyme ever extends over to the side on which it was originally lacking, but I believe that this takes place only to a limited extent, if at all, and that Chabry was right when he afhrmed that only one atrial invagination 1s formed in these right or left half embryos. 2. Three-Quarter Embryos (Figs. 34-35). In connection with the right or left half embryos I shall here consider three-quarter embryos, which, of course, include the whole of the right or left half. “Iwo such embryos are shown in Figs. 34 and 35. In the former the left anterior quadrant was killed in the 8-cell stage; in the latter the right posterior quadrant in the 4-cell stage. “Che embryo in which the cells of the anterior quadrants were uninjured (Fig. 35) is perfectly normal in its anterior half; its posterior half, however, lacks those parts which would have developed from the cell which was injured. ‘This embryo is younger than the one shown in Fig. 34 and no sense spots are present, but the sense vesicle is closing in a normal manner. ‘This figure well shows that a part of aa5 trunk mesen- chyme is derived from the anterior quadrants, and indeed from the pair of cells A7*, Fig. 6, while a portion of it comes from the posterior gains. as may be seen by comparing the right and left sides of Fig. 35. “The muscle cells are entirely lacking on the right side, the substance which would have formed them being located in the injured cell B%; they are shown growing around che end of the notochord as in the half embryo “Shon in Fig. 33: The notochord and nerve tube are apparently full sized, te. is explained by the fact that they come from the anterior quadrants, but owing to the lack of the right side of the tail they are somewhat dioomeds in form. Mosaic Development in Ascidian Eggs. 17! Rieut or Lerr Harr Larvar. Fixep AND STAINED MATERIAL. Figs. 37-40. Four half larve from eggs which were spurted in the 2-cell or 4-cell stage and fixed 22 hours later. These larve are still within the egg membranes though at a corresponding age normal larve are undergoing metamorphosis. Fig. 37. Right half larva, ventral view. The tail which is elongated is turned down toward the dorsal side; the sense vesicle also lies on the dorsal side and is here seen through the embryo. The muscle cells are chiefly on one side of the notochord but have grown over to the other side at the posterior end. Fig. 38. Left half larva, dorsal view. The neural plate with sense spots is partly covered by the end of the tail. The mesenchyme is found only on the left side. Fig. 39. Left half larva from the left side. The neural plate is folded so as to form a nearly closed sense vesicle, in which are two sense spots. Fig. 40. Left half larva viewed from the left side. The neural plate is partially closed, but is abnormal inform. In all of these larve the neural plate lies on the concave side. Tye Edwin G Conklin. Ricut or Lerr Harr Larvar Drawn From Livine SpecIMENS FROM 12 Hours (Fics. 41, 42) To 2¢ Hours (Fias. 45, 46) Arrer THE Injury oF ONE oF THE First Two BLasToMeEREs. Fig. 41. Posterior-dorsal view. Fig. 42. Same embryo, posterior ventral view. In both these figures the muscle cells are found chiefly on one side of the notochord, but they have grown over to the opposite side at the end of the tail. Fig. 43. Right half larva from right side. Fig. 44. Left half larva from left dorsal side. The yellow crescent on the injured blastomere apparently occupies dif- erent positions with respect to the larva in these two figures, but it is by no means certain that the convex side of the larva is morphologically the same in the two figures. Figs. 45, 46. Two views of one and the same left half larva. Fig. 45, from the dorsal side; Fig. 46, from the left side, showing two sense spots on the dorsal and two on the ventral sides. The neural plate is continuous between these spots on the side next to the injured blastomere. Mosaic Development in Ascidian Eggs. 174 Edwin G. Conklin. In Fig. 34 the left anterior quadrant was killed and the posterior portion of this embryo is normal save only for the fact that the notochord and nerve tube are smaller than usual, which is ex- plained by the fact that the substance of these organs 1s derived from the anterior quadrants; three rows of muscle cells are found on both sides of the tail. ‘The anterior half of this embryo, on the other hand, is quite defective; the neural plate is irregularly folded and has not formed a sense vesicle, although sense spots are present. I have seen and studied many three-quarter embryos sim- ilar to those shown in Figs. 34 and 35 and they all show, as do the right and left half embryos, that where part of the substance which would normally form an organ is destroyed the organ which develops is defective, whereas if all or any organ- forming! substance is lacking the organ to which it would ‘normally give rise is also lacking. So far as I have observed these partial larve never escape from the egg membrane, and in this my observations accord with those of Chabry and Driesch, and although I have kept them alive until a period after the normal larve have undergone metamorphosis [ have never observed this transformation in them. In conclusion then I find that the cleavage and gastrulation of these half or three-quarter embryos is partial and the resulting larva incomplete although the notochord is well formed and there is a tendency on the part of some of the cells to grow over and close up the open side of the larva. However, this regulation never leads to the formation of a complete larva; the neural plate may close, but it forms an abnormal sense Genel: at the end of the tail the muscle cells extend over toward the injured side, but they do not form three rows of cells on each side of the notochord as in the normal larva; the mesenchyme likewise does not develop along the injured side and it 1s probable that only one atrial invagination 1s formed. Furthermore not a single cleavage cell nor any one of the odplas- mic substances ever gives rise to parts or organs which it would not normally produce; the notochord, for example, invariably comes from the chorda cells, the sense vesicle from the neural plate cells and both these structures from the material of the gray crescent; the muscles always come from the muscle cells and these from the sub- stance of the yellow crescent; the ectoderm, fromthe ectoderm cells and ultimately from the clear protoplasm; the endoderm, from the Mosaic Development in Ascidian Eggs. 175 endoderm cells and these from the deep gray material of the egg. In spite therefore of the regulation which is apparent in the closing of the open side of the embryo, and in the formation of a whole notochord and of an imperfect sense vesicle, the various odplas- mic substances of the unsegmented egg and of the different blastomeres are not totipotent but each shows in these experi- ments, as well as in normal development, that it is differentiated to give rise to one, and only one, particular kind of tissue. 3. Anterior Half Embryos (Figs. 47-52). The anterior and posterior half embryos show even more clearly than do the lateral ones the mosaic character of the develop- ment of these eggs. When the posterior half of an egg is killed in the 4-cell or 8-cell stage the anterior half continues to develop as if the posterior half were still living. ‘The cleavage 1s in all respects like that of the anterior half of a normal egg; the gastrula- tion is essentially the same, but the later development is modified in many important particulars. Figs. 47 and 48 are ventral and dorsal views, respectively, of one and the same living embryo of the 76-cell stage, in which the posterior dorsal cells, B*?, containing the yellow crescent, were killed in the 8-cell stage. None of the cells of the ventral hemi- sphere were injured ad consequently the cleavage of these cells is quite normal; thirty-two ectoderm cells are present, all of which have entirely normal positions, shapes and sizes. (cf. Figs. 5 and 47.) ‘The anterior half of the dorsal hemisphere 1s also entirely normal (cf. Figs. 6 and 48); eight chorda cells are shown forming an arc which bounds anteriorly the six endoderm cells and which is flanked on each side by the anterior mesenchyme cell, A’. The number, size and position of each and all of these cells is the exact counterpart of what is found in the normal embryo, and, although the outlines of the neural plate cells were so indistinct in the living specimen from which this figure was made that I could not draw them, there is every reason to suppose that these cells like all the others in this embryo conform to the normal type. In the posterior half of the dorsal hemisphere all the parts which would have developed from the cells B*1 and B** are entirely lacking; there are neither mesenchyme, caudal endoderm, 176 Edwin G. Conklin. Anterior Harr anp THreE-QuarTeR Empryos; 76 Certs to METAMorRPHOSIS. Figs. 47, 48. Anterior-ventral three-quarter embryo of the 76-cell stage (v. Figs. 5 and 6); the dorsal posterior cells B*-1, containing all of the yellow crescent, were killed in the 8-cell stage. The ventral ectoderm cells (Fig. 47) are quite normal both in position and number (cf. Figs. 5 and 47); the anterior dorsal cells are also normal, but the posterior dorsal cells (muscle, mesenchyme and caudal endoderm) are entirely lacking. (cf. Figs. 6 and 48.) Figs. 49-51. Three views of one and the same anterior half embryo of about the 250-cell stage; spurted in the 4-cell stage and fixed 2 hours later. Fig. 49. Dorsal view, superficial focus, showing the neural plate. Fig. 50. Dorsal view, deeper focus, showing two rows of chorda cells besides several ectoderm and endoderm cells. Fig. 51. Dorsal view, still deeper focus, showing the cells of the ventral ectoderm. Fig. 52. Anterior half embryo, dorsal view. Spurted in the 4-cell stage, fixed 22 hours later. The yellow crescent is plainly visible in the injured cells. Sense spots are present but the neural plate never forms a tube. The chorda cells lie in a heap at the left side. There is no trace of muscle sub- tance or of a tail in this anterior half embryo. This embryo is from the same experiment as Figs. 37-40; normal larve of this stage are undergoing metamorphosis. Mosaic Development in Ascidian Eggs. Tay NG 178 Edwin G. Conklin. nor muscle cells. Unfortunately this particular embryo was not followed through the various stages of development until it gave rise to a larva and none of the older stages which I have studied have shown precisely this type of injury, 7. e., the destruction of the yellow crescent without injury to the ectoderm cells of the posterior half. . In many other cases which I have seen all of the posterior half of the egg was injured in the 4-cell stage. I have followed the development of the surviving anterior halves of such eggs as late as the stage of the metamorphosis of the normal larvz; the develop- ment of such blastomeres is always partial. Figs. 49, 50 and 51 represent three views of one and the same anterior half embryo of about the 250-cell stage; in all the fgures the embryo is viewed from the dorsal side, but in Fig. 49 the focus is high and only the ectoderm and neural plate cells of the dorsal surface are shown; Fig. 50 is a median optical section showing chorda and endoderm cells surrounded on the anterior side by ectoderm; Fig. 51 represents the ectoderm of the ventral surface which 1s visible at a deep focus. ‘This half embryo is exactly like the anterior half of a normal one in the formation of the neural plate, the chorda plate, the general ectoderm and gastral endoderm, in the overgrowth of the dorsal lip of the blastopore, even in the position, shape and size of the individual cells. (c7. Figs. 9 and 10.) Finally in Fig. 52 there is represented an anterior half embryo 22 hours after the posterior cells were killed, and at a stage when normal larve of corresponding age have already under- gone metamorphosis. ‘The ectoderm has not yet inclosed the embryo on the side next the injured cells, and this rarely happens in anterior or posterior half embryos. ‘The neural plate has not rolled up nor invaginated to form a tube, though it is slightly depressed along its median line; two sense spots are present though there is no sense vesicle. The large rounded chorda cells are irregularly scattered along the posterior border of the embryo, where they project beyond the ectoderm; they never form a noto- chord. There is no trace of yellow crescent substance nor of muscle cells in these anterior larvz and no indication whatever of a tail. They are, therefore, altogether unlike the normal larve and they afford complete.and convincing evidence that the anterior blastomeres of the ascidian egg are not totipotent but rather that the development is a mosaic work. Mosaic Development in Ascidian Eggs. 179 4. Posterior Half. Embryos (Figs. 53-58). All that has been said of the mosaic-like development of the anterior half of the egg is equally true of the posterior half. The cleavage progresses in normal fashion up to the time of the closure of the blastopore. Figs. 53 and 54 represent posterior half em- bryos of the 32-cell and 76-cell stages, respectively. “The former is entirely normal and the latter 1s normal in all respects save that a single pair of cells, B**, is larger than in the normal embryo. The clear, the yellow and the gray substances of the egg are distributed exactly as in the posterior half of a normal embryo. ‘The clear ectoderm cells lie on the ventral side and only two of them appear in the dorsal view shown in Fig. 54 (the two clear cells at the pos- terior pole). In Fig. 53 the gray endoplasm 1s contained in two cells (B**) and in Fig. 54, in four (B™, B7*); these cells give rise to the strand of caudal endoderm. ‘The yellow crescent consists at the 32-cell stage of a single arc of yellow cells (Fig. 53) which then, by division, become a double arc of fourteen cells (Fig. 54); the inner arc consists of eight mesenchyme cells and the outer of six muscle cells. In all these respects these posterior half embryos are entirely like the posterior half of a normal embryo. But while the pregastrular stages of these posterior half embryos are like the normal, the gastrulz and later stages show many interesting modifications. Figs. 55, 56, 57 are three views of one and the same posterior half embryo, the normal embryos of the same stage being young tadpoles like Fig. 11. In all of these figures the embryo is viewed from the dorsal side; Fig. 55 shows the ectoderm cells which cover the dorsal surface; Fig. 56, the muscle cells which le below the ectoderm on the dorsal side; Fig. 57 is an optical section at a still deeper level showing the caudal endoderm and mesenchyme. Fig. 58 is another posterior half embryo of similar age seen from the ventral side, showing the yellow mesoderm cells on each side of the caudal endoderm. The gastrulation occurs between the stages shown in Figs. 54 and 55. The caudal endoderm and the surrounding arc of mesen- chyme, shown in Fig. 54, invaginates; the muscle cells come to lie above (dorsal to) the mesenchyme cells and finally the latter are overgrown by the ectoderm in the manner shown in Fig. 8. In normal embryos the posterior part of the blastopore is closed chiefly by the growth of the anterior lip; in the latter stages of 180 Edwin G. Conklin. Posterior Harr Emsryos; 32°Certs to Tappore Stace. Fixep ANp STAINED PREPARATIONS. Fig. 53. Posterior half of 32-cell stage, dorsal view. The cleavage is altogether normal. Spurted in the 4-cell stage, fixed 1 hour later. Fig. 54. Posterior half of 76-cell stage (cf. Fig. 6); spurted in the 4-cell stage, fixed 2 hours later. Two rows of yellow crescent cells are present, the inner being mesenchyme, the outer muscle cells; the anterior pair of mesenchyme cells (B®) are larger than normal. There are two pairs of caudal endoderm cells (B7-1 and B7.2). A pair of ventral ectoderm cells is visible in the midline behind. Figs. 55-57. Three views of one and the same embryo; spurted in the 4-cell stage, fixed 4 hours later, normal embryos being in the stage represented by Fig. 11. Fig. 55. Dorsal view of the super- ficial ectoderm. The notch in front represents the notch in the ventral lip of the blastopore. Fig. 56. Same view, deeper focus, showing the muscle cells beneath the ectoderm; these cells are continuous from side to side, there being no chorda inthe midline. Fig. 57. Same view, still deeper focus, showing the double row of ventral endoderm cells in the midline, and on each side of this a mass of mesenchyme cells. Fig. 58. Ventral view of posterior half embryo of the same stage as the preceding, showing the muscle and mesenchyme cells beneath the ectoderm and on each side of the strand of ventral endoderm. Mosaic Development in Ascidian Eggs. ISI 182 Edwin G. Conklin. gastrulation a blastopore groove is left in the posterior half of the embryo, on each side of which lie the muscle cells. (Fig. 9.) By the continued growth of the anterior lip this groove is shoved to the posterior end of the embryo and the rows of muscle cells are tilted up from an antero-posterior to a vertical position. Later, when the notochord is formed, the muscle cells come to lie alongside of it, thus forming the three rows of muscle cells on each side. Finally the ectoderm of the posterior lip of the blastopore, which has, up to this stage, formed a notch at the end of the blastopore groove, grows forward and reduces this groove to a minute pore. Owing to the absence of the anterior lip of the blastopore, and of the notochord and the neural plate, the later stages in the develop- ment of these posterior half embryos is much altered. In the first place the blastopore groove and the muscle cells are not pushed to the posterior end of the embryo. ‘Then the muscle cells on each side of the blastopore groove are not kept apart by the notochord but come into contact forming a continuous layer of muscle cells across the dorsal side. (Fig. 56.) “The blastopore groove, therefore, disappears by the fusion of the lateral lips of the groove and the ectoderm cells grow over the whole dorsal surface; the only trace of the blastopore groove which 1s left is a slight notch in the ante- rior border of the embryo. (Figs. 55, 56.) “The ectoderm never entirely incloses the posterior half embryo on the side next the injured cells, but the endoderm here comes to the surface as shown in Figs. 57 and 58. No trace of notochord, neural plate nor sense spots ever appears in these posterior half embryos, and what is more remarkable a tail is never formed but the embryo always remains rounded in form, as shown in Figs. 55-58. It is quite evident that the elonga- tion of the tail of the normal larva, together with the elongation of the individual muscle cells and perhaps also the arrangement of these cells in three rows on each side, is dependent upon the pres- ence and elongation of the notochord. Perhaps one reason why a normal notochord is never formed in the anterior half embryo is due to the fact that the ectoderm does not completely inclose the embryo, so that the chorda cells in their growth crowd out of the open side and hence become free and scattered. In conclusion, the study of anterior or posterior half embryos establishes in a most convincing manner the fact that the develop- ment of individual blastomeres of the ascidian egg 1s a mosaic work. Mosaic Development in Ascidian Eggs. 183 These blastomeres give rise only to those tissues and parts of an embryo which would come from them normally. Nothing even remotely resembling a complete normal larva is ever produced from the anterior or posterior quadrants of the egg. 5. Quarter Embryos (Figs. 59-70). The development of individual blastomeres of the 4-cell stage furnishes additional confirmation of the mosaic theory as applied to ascidian eggs; in every instance individual blastomeres give rise only to those parts or organs which they would produce in normal embryos. Quarter embryos generally show more abnor- malities and variations than half embryos,—probably owing to the more severe injury which they have suffered, which often affects the surviving quarter of the egg. The cleavage of these quarter eggs 1s normal in every detail, save that the position of the cells is sometimes slightly altered; the rhythm of cleavage and the size and quality of oy cells is the same as in the corresponding quarter of a normal egg. In Fig. 59, which corresponds to the 16-cell stage of the berate egg, each of the surviving quadrants has divided twice; in Fig. 60 the left posterior quadrant of a 44-cell stage is shown and in both of these figures the size, quality and position Oe the cells as well as the rhythm of division and the distribution of the different odplasmic sub- stances is entirely normal. Fig. 61, which is the right anterior quadrant of the 76-cell stage, is normal in every respect, save for the position of the endoderm cells which are here displaced toward the first cleavage plane. The mesoderm cells in the nght poste- rior quadrant, shown in Fig. 62, are not normal in position; the two caudal endoderm cells (lying next the first cleavage plane) are, however, normal and the ectoderm cells are normal save that they show a tendency to grow inward at the first and second cleavage furrows and thus surround the embryo. In particular, attention should be directed to the yellow crescent and caudal endoderm cells in Fig. 60, and to the neural plate and chorda arcs in Fig. 61, which are similar in every respect to the quarter of a normal embryo at these stages. ; I have already commented upon the fact that the quarter embryo shown in Fig. 63 is a “false gastrula” since the invaginated cells are ectodermal, probably neural plate cells, while the larger endo- 184 Edwin G. Conklin. Quarter Emeryos; 16 Certs to YounG Tappore Stace. Fixep AND STAINED PREPARATIONS. Fig. 59. Left anterior and right posterior (diagonal) quarter embryos of the 16-cell stage, ventral view. Fig. 60. Left posterior quarter embryo of the 44-cell stage, posterior view. Fig. 61. Right anterior quarter embryo of the 76-cell stage, dorsal view, showing the neural plate and chorda cells of the right side. : Fig. 62. Right posterior quarter embryo, of about the 180-cell stage, dorsal view (cf. Figs. 7, 8); spurted in the 4-cell stage, fixed 24 hours later, showing 6 muscle and 2 caudal endoderm cells. Fig. 63. Left anterior quarter embryo, dorsal view; spurted in the 4-cell stage, fixed 5 hours later. An invagination of the ectoderm cells has the appearance of a gastrula, but is probably the invagination of the neural plate. Fig. 64. Left anterior and right posterior (diagonal) quarter embryos, dorsal view; spurted in the 4-cell stage, fixed 5 hours later. Muscle cells are found only in the posterior quarter. 185 Mosaic Development in Ascidian Eggs. 186 Edwin G. Conklin. Quarter Empryos; Younec Tappote to Meramoreuosis StaGes. Fixep AND STAINED PREPARATIONS. Fig. 65. Left anterior and right posterior (diagonal) quarter embryos, dorsal view; spurted in the 4-cell stage, fixed 5 hours later. The anterior quarter shows thickened ectoderm cells, probably neural plate. around the endoderm cells; in the posterior quarter are 8 muscle and 3 caudal endoderm cells. Fig. 66. Left anterior and right posterior (diagonal) quarter embryos from the right anterior side, the dorsal pole being above; spurted in the 4-cell stage, fixed 22 hours later. In the posterior quarter the muscle and mesenchyme cells form a solid mass; in the anterior quarter the chorda cells project freely over the dorsal surface and the neural plate is partially infolded and contains three sense spots. Fig. 67. Right anterior and left posterior (diagonal) quarter embryos, dorsal view; spurted in 4-cell stage, fixed 22 hours later. Fig. 68. Left anterior and right posterior (diagonal) quarter embryos, dorsal view; spurted in 4-cell stage, fixed 22 hours later. In this and the preceding figure the chorda cells (Ch.), neural plate (n. p.) and sense spots are found only in the anterior quarters; the muscle, mesenchyme and caudal endoderm cells, only in the posterior quarters. Figs. 69, 70. Right anterior quarter embryos, dorsal side above; spurted in 4-cell stage, fixed 12 hours later. These embryos show free chorda cells, neural plate and sense spots, but not a trace of muscle cells. Mosaic Development in Ascidian Eggs. 187 188 Edwin G. Conklin. derm cells remain on the rounded surface of the embryo. 1! have not observed in detail the process of gastrulation in any of these quarter embryos, but it is evident that there is no considerable gastrula cavity and that the endoderm cells are chiefly overgrown by the ectoderm, as shownin Fig.65. Ultimately the endoderm and mesoderm are largely overgrown, though in this case, as in the half embryos, the ectoderm does not entirely inclose the embryo on the side next to the injured cells and through the opening thus left some of the endoderm cells may protrude. Although the localization of ooplasmic substances and of organ bases is usually the same as in the quarter of an entire embryo, in some cases there are dislocations of these substances and bases which are probably due to injury of the surviving quar- ter. Thus in the left anterior quarter, shown in Fig. 64, large endoderm cells lie at the surface next to the first cleavage plane; in the same quadrant of another egg shown in Fig. 65 the neural plate cells lie at the periphery of the quadrant and chiefly on the left side, instead of along the median plane as in normal embryos. I have seen many other instances of such dislocations but they are all of such nature that they can be interpreted as due to slight injury to the surviving blastomeres. In not a single instance are parts derived from a blastomere which would nor- mally have come from another cell. The anterior quarter embryos are always recognizable by the presence of the neural plate and, in later stages, of the sense spots. The neural plate usually remains at the surface and 1s not infolded, but in some cases it 1s invaginated through at least a portion of its area, though a sense vesicle is not formed. (Figs. 63, 66.) In all later stages one or more sense spots appear in the plate. (Figs. 66-70.) The neural plate always lies along the dorsal side of the embryo, though it may be shifted more or less from the median plane. (Figs. 65-70.) The chorda cells are found exclusively in the anterior quadrants and in later stages they protrude to the exterior along the injured side where they are found as scattered cells in the perivitelline space. (Figs. 66-70.) In no case, save one, have I seen any indication that these cells form a rod- -shaped notochord, and this case (Fig. 72) was that of a living embryo in which it is possible that the notochord-like structure was really composed of gastral endoderm and hence not a true notochord at all. It is evident that the chorda cells are unable to give rise to a Mosaic Development in Ascidian Eggs. 189 notochord when once they have escaped and have become free, a certain amount of compression being necessary to bring about the characteristic interdigitation which leads to the formation of a rod- shaped notochord.* The posterior quadrants can be distinguished in all eggs at all stages by the presence of the yellow crescent substance or cells. In early stages, as I have shown, these crescent cells are normal in position and character; in later stages the yellow cells fill the whole interior of the embryo. When once these cells have been -inclosed by the ectoderm I have been unable to recognize any constancy in their position and arrangement. As in the posterior half embryos, a tail is never formed in these posterior quarter embryos and the muscle cells are never elongated, both these features evidently depending upon the presence of a notochord. The caudal endoderm cells are found in most, if not all, of these posterior quarter embryos as a single row of yolk laden cells which lie along the first cleavage plane (Figs. 65-68), the position which they normally occupy. T hese quarter embryos show in the most unmistakable manner that the development is strictly partial, and that an individual blastomere never gives rise to parts which it would not produce in the entire embryo. Among the hundreds of quarter embryos which I have studied both in the living condition and as stained and mounted preparations I have never seen a single one which even remotely resembled a normal larva. 6. Eighth or Sixteenth Embryos (Figs. 71-76). When eggs are spurted or shaken in the 8-cell and 16-cell stages a great variety of abnormal forms are produced, a few of a hice are shown in Figs. 71 and 73-76. Without exception, however, the same principles apply here as in the case of half and quarter embryos, viz: a given blastomere or group of blastomeres produces only those parts of an embryo or larva which would develop from it under normal conditions. Fig. 71 represents an embryo derived from the dorsal anterior eighth of an egg (the cell 4") 14 hours after the injury. Normally this eighth gives rise to neural ples chorda, gastral endoderm, and a small amount of 1Chabry, however, figures (his Fig. 18) a partial embryo with a rod-shaped notochord lying outside the embryo in the perivitelline space. : 190 Edwin G. Conklin Partiat Empryos FROM IsoLATED BLAsToMERES OF 8-CELL oR 16-CEeLL StTaGes. DRAWN FROM Livine SPECIMENS. Fig. 71. Right anterior dorsal eighth embryo, 14 hours after injury, showing endoderm, chorda, and neural plate cells with sense spots. Fig. 72. Right anterior quarter embryo, 14 hours after injury, showing chorda, neural plate, and sense spots. Fig. 73. Posterior ventral quarter embryo derived from the cells b‘.?, 6.2 and containing no endo- derm and only a small amount of yellow protoplasm which was derived from the perinuclear plasm of the cells b+”, Fig. 13. Fig. 74-76. Three views of a partial embryo derived from 7 cells of the 20-cell stage, viz: 2 (B*.?), 2 (b®-4), 1 (a5-4), 2 (a5*3). (cf. Figs. 3 and 4.) The embryo consists of an outer layer of clear ectoderm and of a mass of yellow mesenchyme cells derived from the cells B®, but it is wholly without endoderm. Fig. 74, Ventral view; Fig. 75, Posterior; Fig. 76, Postero-dorsal. 1gI Mosaic Development in Ascidian Eggs. od eae me Ke Lge: fe ( oe Cae rr 192 Edwin G. Conklin. mesenchyme derived from the cell A”®. Inthe embryo shown in Fig. 71 the neural plate cells are clearly shown around the periphery af the figure and two of the cells contain sense spots. ‘The chorda, endoderm, and mesenchyme cells are shown internal to the neural plate, but | am unable to distinguish in this embryo between these . three kinds of cells; they are all more or less yolk-laden as in the normal egg. Owing probably to the fact that no ventral ecto- derm cells are present the neural plate is not pushed up onto the dorsal face and there are no evidences of gastrulation, although normal embryos of a corresponding age have already reached the full larval development. ‘That this failure to gastrulate is not due to the slower development of the egg fragments as compared with the entire egg is shown by the degree of histological differen- tiation of the aeural plate and sense spots, the latter appearing normally only i in the fully formed larve. Fig. 72 is a quarter embryo of the same age as the preceding, derived from the cells 4*1, a‘? of the right anterior quadrant. ‘The ventral ectoderm cells have here pushed the neural plate cells up onto the dorsal face of the embryo, while the chorda cells (?) lie along the median and transverse furrows. Four sense spots are present in the neural plate. Fig. 73 1s also a quarter embryo of the same age as the pre- ceding, derived from the two posterior ventral cells b*?, 5*?. This embryo consists entirely of ectoderm which 1s arranged in a single layer of cells around a central cavity, the blastocoel. ‘There has been no gastrulation and the embryo contains neither endo- derm nor mesoderm. 4, b>4, a3, a3 and a4. (See Fig. 3.) This embryo consists entirely of an outer layer of clear eetadenm cells, inclosing at. its posterior end a mass of small mesenchyme cells; it contains no endoderm. It is an interesting fact that the mesenchyme cells are here inclosed by the Soden showing that some process in the nature of gastrulation must have taken place. A great many other partial embryos, produced from one or more leeromicses of the 8, 16 or 32-cell stages, have been studied Mosaic Development in Ascidian Eggs. 193 but they all illustrate the principle that a blastomere never gives rise to any other structures than those which it would produce in a normal embryo. 7. Anterior and Posterior Half Gastrule (Figs. 77-82). In a recent publication Driesch (’03) has maintained that an alteration in the capacity for regulation occurs in the ascidian development between the early eae the late gastrula stages. When the open cup- -shaped g eastrule of Phallusia were cut in two transversely into anterior ind posterior halves, each of these halves developed into “einer vollstandigen kleinen Appendi- cularie, welcher Organe niederer Bedeutung (Otolith, Augenfleck) eventuell fehlten.”” However, when the elongated gastrule were cut in two transversely a head developed fronts one piece and a tail from the other, “so deutlich und sharf begrenzt und ausgebildet, als habe man eine fertige Appendicularie er durchschnitten. Considering the Pale which [ have obtained on the develop- ment of the two anterior or two posterior cells of the 4-cell stage of Cynthia the conclusions of Driesch seemed most remarkable and I therefore undertook to repeat his experiments upon Cynthia. Gastrulz of the stage shown in Fig. 8 were cut in two with a sharp knife made from a needle, under a Zeiss binocular dissecting microscope. With the power used the individual cells of the yellow crescent could be plainly seen and it was always easy to determine the exact boundary between the anterior and posterior halves. In every instance the section was made as close as possi- ble to this boundary (second cleavage plane) and so as to leave all of the yellow cells in one of the pieces. Owing to the presence of the chorion the experiment was not an easy one to perform, since the chorion would frequently slip under the knife, or the egg move within the chorion. Nevertheless in one day I succeeded: in cutting in two about thirty of these early gastrule; ten of these lived for twenty hours or longer after the operation, the others were too badly crushed to survive. Four of these which survived the operation are shown in Figs. 77-82, the drawings having been made from nineteen to twenty hours after the operation. Every one of these ten surviving embryos was a partial one and, although I was unable to determine their structure with the same amount of detail as in the case of stained and mounted preparations, it was DE. goa” Ede GuGenin ANTERIOR AND PosterioR Harr GastTRULAE. Figs. 77-82. Partial embryos derived from gastrule of the stage shown in Fig. 8, which were cut in two transversely so as to leave the whole of the yellow crescent in one half. The chorion is shown as a line around the embryos. Figs. 77, 78. Dorsal and ventral views respectively of one and the same embryo, drawn 19 hours after the operation. A mass of cellular debris lies between the two half embryos; the endoderm cells are chiefly contained in the anterior half, the mesenchyme and muscle cells are entirely confined to the posterior half. Neither half at all resembles a normal embryo or larva. Figs. 79, 80. Ventral and postero-dorsal views of another embryo, 194 hours after the operation. The crescent of yellow cells is entirely confined to the posterior half and neither half resembles a normal larva. Fig. 81. Anterior and posterior half embryos 20 hours after the operation. Fig. 82. Pos- terior half embryo from the postero-dorsal side, 20 hours after the operation. The anterior half is degenerating and is shown only in dotted outline; the posterior half contains all of the yellow cells and practically no endoderm. At the stages represented by all these figures the normal embryos have already undergone their metamorphoses. Mosaic Development in Ascidian Eggs. 195 196 Edwin G. Conklin. quite evident that not one of them resembled in any respect what- ever a normal larva. In some cases both halves survived, as shown in Figs. 77-81, in other cases one half only survived. In all cases the surviving halves became rounded in form after the operation, the more seriously injured cells being crowded out of the embryo and forming a cellular mass of débris within the cho- rion. In every instance the surviving halves remained within the chorion, which was sometimes infolded as shown in Figs. 77-80. Each half was surrounded by a layer of clear ectoderm cells; the yellow cells were always found exclusively in the posterior half, the gray endoderm cells largely in the anterior half. Nothing resembling a notochord or neural tube ever developed in either half and no structure resembling a tail was ever formed. In fact these half embryos produced by cutting the early gastrule in two were altogether like the anterior and posterior half embryos which I have already described. (cj. Figs. 77-82 and Figs. 47-58.) ‘These results were so deGnibe- and conclusive that I did not continue the experiments and | regret now that I did not also cut gastrule in two along the median plane, though there is no reason to doubt that the results would be the same as in cases where one of the first two blastomeres is killed. Comparing these results with those of Driesch, only one of two explanations is possible. Either Phallusia must differ most fundamentally from Cynthia, or Driesch must have mistaken the median for the transverse plane in these cup-shaped gastrule. That the former possibility is not probable is evidenced by the fact that the cell-lineage of all ascidians so far studied is essen- tially the same; feethermone my results as to the development of anterior and posterior halves of the egg of Cynthia are confirmed by my experiments on Molgula, as well as by Chabry’s experi- ments on Ascidia aspersa. ‘There 1s every reason to believe that what 1s true of these three genera 1s also true of Phallusia. On the other hand there are certain evidences that Driesch may have mistaken the transverse plane for the median; on p. 56 he says, “ Aber auch an der Bechergastrula kann man die kunftige Mediane und also auch die Hauptrichtungen senkrecht zu ihr unterschieden: es verlaufen namlich die Zelltheilungsgrenzen des Ektoderms dieser Objecte so, dass sie gerade in der Medianen eine tiber die ganze Oberflache fortgesetzte, nur sehr wenig gebrochene Ein- hewabeaie bilden (S. z. B. Castle, Fig. 62, 71) euleae ohne Weiteres Mosaic Development in Ascidian Eggs. 197 schon bei schwacher Vergrosserung kenntlich ist; schneidet man also in der Mitte und senkrecht zu dieser Linie, so zerlegt man auch die Bechergastrula in ‘vorn’ und ‘hinten.’”’ It is true that the median plane is marked out by a nearly straight line, though Castle’s figures to which Driesch refers show this line between endoderm a not between ectoderm cells, but any one who has studied these embryos knows how difficult it is to determine the median plane in this way, especially in living material. Even in stained and mounted preparations it would not be a sure guide, much less could it be relied upon in the study of living gastrula. Whether the median plane appears as a straight line or not depends entirely upon whether that plane lies directly in the line of vision, and conversely some of the transverse planes of cleavage may appear as straight lines if they lie in the line of sight. “hus Fig. 7 shows several transverse rows of ecto- derm cells which in the hinder part of the embryo are curved back in the middle and forward at the sides, but if the embryo were rotated forward so that the polar body were brought to the highest point these transverse rows would appear nearly straight. I am convinced therefore that the half gastrula from which Driesch obtained apparently normal Lave were right or left halves and not anterior and posterior ones as he supposed. Whether these larvz were really normal, 7. e., whether they had the organs of both the right and left sides, cannot be determined from Driesch’s figures or descriptions, since he seems to have considered that the only evidence required to show that a larva is complete is that it should have a head and a tail. The fact that Driesch always obtained partial larve from the anterior and posterior halves of an elongated gastrula, where the chief axis is unmistakable, requires no comment. IV. OTHER EXPERIMENTAL WORK ON THE ASCIDIAN EGG. Chabry’s (87) contribution on the normal and teratological embryology of ascidians contains not only the most careful and complete experimental work which has ever been done on the ascidian egg but it is at the same time such an excellent analytical treatment of the: normal development that it deserves to rank as an embryological classic. The experimental part of his paper was based upon an unusual knowledge of the normal and patho- 198 Edwin G. Conklin, logical development of this species and it was carried out with a delicacy and precision of method which has never been surpassed. Add to this the fact that the work was undertaken with clear insight into the principal problems involved and at a time when almost no other work of this sort had ever been done’ and its right to rank as one of the great works in experimental embryology seems assured. Considering these facts it is surprising that this work should have received so little attention and that it should have been so widely misunderstood or discredited. Chabry’s extensive experiments deal with right and left half embryos, anterior and posterior two-quarter embryos, and various forms of three-quarter, one-quarter and two-quarter diagonal embryos, and in all of these I find that my results are in the main in accord with his. ‘The points in which my work is more detailed than his concern the presence and distribution of the various ooplasmic substances and the more accurate study of some of the later stages, made possible by the use of fixed and stained material. That the substance of the mesodermal crescent was seen by Chabry as early as the 32-cell stage is evident from his description of the mesoderm cells, which in wens aspersa are greenish (‘‘verdatre’’) in color and which he recognized when only hres were present on each side. Neither Driesch nor Crampton speak of having observed any of these odplasmic substances and neither of them studied the later stages by means of fixed and stained material. 1. Cleavage. Chabry showed that in rhythm of cleavage and in the size and character of the daughter cells the isolated blastomeres of Ascidia behave as if they were still part of the normal ege, while he described in great detail the changes which take place in the facets between cells. Crampton’s conclietne are very similar; he found that “an isolated blastomere of the Molgula egg segments as if still forming a corresponding part of an entire embryo. The cleavage phenomena are strictly partial, as regards the origin of cells, the inclination of cleavage planes, and especially in respect to the rhythm of segmentation.” Driesch, on the other hand, found in Phallusia that there was no fixed relation between the 1See Roux, Ges. Abhand II, p. 958. Mosaic Development in Ascidian Eggs. 199 cleavage planes of the surviving half and the dead blastomere; that after the third cleavage the cells occupy very different posi- tions from the normal (Tetraeder, Halbtetraeder); that divisions may be equal or unequal at the fourth cleavage, and finally that the cleavage could not be regarded as partial (“halb’’) nor entire (es ganz ”) but “regellos-solid.” The evidence which Driesch brings in support of this conclusion is of little value since it is plain that he was unable to orient these cleavage forms and did not know from what part of the original egg they came nor from what pole they were viewed. My observations on the cleavage of isolated uninjured blastomeres of the egg of Cynthia ees: and extend the conclusions of Chabry and Crampton that the cleavage of such blastomeres is unaltered save for slight changes in the direction of some of the divisions; they are opposed to the conclusions of Driesch that the cleavage of such blastomeres is inconstant and irregular. 2. Gastrula. Chabry figures four gastrule from isolated blastomeres, viz: his Figs. 108, 114, 129 and 130. O. Hertwig, who copies Fig. 129 in his book, “Die Zelle”’ (’98), says that it is a normal ty ypical gastrula. Similarly Korschelt and Heider, who also copy this figure in their text-book (’02), affirm that it is a normal small gastrula. However, these authors bring no particle of evidence to the support of this bare assertion; Chabry himself nowhere says that any of the gastrule figured by him are normal and the figures themselves do not show that such is the case. On the other hand I can positively affirm that a normal entire gastrula is never formed from an isolated blastomere of the egg Sor Cynthia. In the absence of any evidence in favor of Hertwig’s and Korschelt and Heider’s interpretation and in the face of this positive evidence against it I think it may safely be assumed that Chabry’s figures are not those of normal typical gastrule. Crampton expressly says that he did not carefully observe the process of gastrulation in the embryos derived from isolated blastomeres of the Molgula egg, but Driesch says that the process of gastrulation may be easily observed in Phallusia, that a typical ascidian gastrula is formed and that the closure of the blastopore takes place in the normal manner. “Alles sind verkleinerte Aehnlichkeitsbilder der Processe an normalen Eiern, welche stets vergleichen wurden.” 200 Edwin G. Conklin. However, it 1s quite evident from the observations of Van Beneden and Julin, Chabry, Castle and many others that something more than a mere invagination is necessary to constitute a normal gastrula. ‘The ascidian gastrula is bilaterally symmetrical and its anterior and posterior portions are very unlike; furthermore all the principal organs of the larva are here represented by cells of peculiar structure and localization. In order to determine whether a gastrula is normal or not all of these features have to be considered, and this Driesch has not done. 3. Larva. It is somewhat surprising that doubt should have been expressed as to whether Chabry obtained half embryos or whole embryos of half size from one-half of the ascidian egg. He again and again declares that lesion of a single cell up to the 16-cell and probably up to the 32-cell stage always causes a “hemiterie,” or monster. (Chabry, pp. 246, 249, 250, 257, 258, 261, etc.) He even enters into a calculation of the number of kinds of monsters which may be produced by injuries to the cleavage cells. He says that if at the 8-cell stage each cell is capable of Poor different kinds of modifica- tion (certainly less than the reality), the number of modalities of this stage is 4° (= 65536) of which only one is normal. In this way teks arises that ““admurable and infinite variety of monsters” to which he repeatedly refers. He says expressly, p. 289, “ De la on tire aisement la conclusion (que je ne crois valable que pour l Ascidie et les animaux, dont les blastomeres sont différenciés de bonne heure), que chaque blastomeére contient en puissance cer- taines parties dont sa mort entraine la perte irrémédiable et que les différentes parties de animal sont préformées dans les différ- entes parties de l’oeuf.’’ Again on p. 299 he says, “On ne saurait donc conclure avec sécurité de |’oeuf d’Ascidie a celui des autres animaux, mais, en ce qui concerne celui-ci, il est exact de dire qu'il se comporte comme s’il contenait en puissance un seul adulte déterminé et que chaque partie de l’oeuf contint une partie de cet adulte.”” ‘This same conclusion is repeated again and again so that as Barfurth (’93) and Driesch (’95) have said there can be no question as to what Chabry believed that his observations and experiments proved. Mosaic Development in Ascidian Eggs. 201 The statements of Driesch and Crampton are even more posi- tive and explicit that whole larve are formed from any one or more of the first four blastomeres. Driesch (p. 405 in sum- marizing his results uses, in part, the very words of his conclusions regarding the value of the cleavage cells in the echinoderm egg: “Aus isolirt tuberlebenden Blastomeren des Ascidieneies ent- wickelt sich nicht ein halber (resp. viertel, drei viertel) rechter oder linker (resp. vorderer oder hinterer) Embryo, sondern stets ein ganzer von halber Grosse, dem allerdings (meist) gewisse Organe von minderen Bedeutung (Otolith, ein Haftorgan) fehlen.”’ Crampton neither figures nor describes the larve obtained from isolated blastomeres of Molgu'a, but he says, p. 55, “Enough of the later development has been ascertained o prove that a larva arises wh ch resembles the normal larva, except as regards its smaller size and certain minor defects. My results, therefo: e, are entirely confirmato y of those of Driesch upon Phallusia.”’ Chabry first discovered that larvze from one of the first two blastomeres were superficially like normal larve in that they had head and tail, notochord, neural plate and sense spots, but he showed that they also lacked the organs distinctive of the missing side, viz: one papilla, one or more sense spots and one atrial invagination. It ‘s surprising therefore that neither Driesch nor Crampton undertook to prove that the larve obtained by them from one of the first two blastomeres were really complete. One looks in vain in their papers for any evidence that the organs characteristic of that side which would have developed from the dead half (muscles, mesenchyme, papilla, atrial invagination) are present in the surviving half. Chabry further showed that the type of embryo derived from the anterior or posterior two-quarters of the egg was very unlike that derived from the right or left two-quarters, while the one- quarter embryos were Shik more unlike the normal; n each of these cases he found that the development was strictly partial, only those parts arising from a blastomere which would develop from it in the normal “embryo. In the face of these conclusions of Chabry’s neither Driesch nor Crampton advance any evidence in favor of their claim that the anterior and posterior quadrants of the egg as well as the right or left may give rise to a larva. Cha- bry’s figures and descriptions show plainly what my work proves that nothing even remotely resembling a normal larva is ever pro- 202 Edwin G. Conklin: duced from any portion of an egg which does not include the whole of the right or left half. In my opinion Driesch and Crampton have not studied nor taken any account of anterior or posterior half embryos, but only of right or left ones. The question whether these embryos were actually complete will be considered when we come to deal with the various larval organs. Both Driesch and Crampton make the claim that single blasto- meres of the 4- cell stage of the ascidian egg may give rise to entire larve. This isa emacial test of their views, for while it is possible and | believe practically certain that all their “complete larvae of half size”? were derived from the right or left halves of the egg and so included portions of all the various ooplasmic substances, this explanation could not apply to their quarter embryos. Driesch figures a larva with all the principal organs (his Fig. 16), which he says is derived from one of the first four blastomeres. How- ever, In size it is as large as any of the half larve which he figures, and I have no doubt that it is such. Crampton figures correctly the early cleavages of one of the anterior quadrants and he gives two figures of quarter larve, probably of an advanced stage; these figures, however, show no structure whatever save that there is an outer layer around the embryo. ‘There is absolutely no evidence that these embryos are complete. Crampton calls attention to the fact that the long axes of these quarter embryos “are approximately parallel to the principal dorso-ventral axis of the original egg,” a fact which I alsocanconfirm. (See my Figs. 66,69, 70.) He does not, however, determine the fact, which He apparently assumes, that the long axes of these quarter embryos correspond to the long axis of a normal embryo. ‘This is actually not true, as | have shown; the long axes of the quarter embryos are not antero-posterior in aincerion but dorso-ventral and there has not therefore been any shifting of the axes nor of the odplasmic substances of these quarter embryos. Whether a larva derived from the right or left half of the egg is complete or not can be determined only by a study of the various systems of larval organs. It is evident that parts of all organs which are normally formed along the median plane (first cleavage plane) would appear in an embryo derived from one of the first two cleavage cells, even if the development were strictly partial; the really decisive test as to whether such an embryo is complete Mosaic Development in Ascidian Eggs. 203 or not must be found in the study of those organs which do not lie along the median plane. a. Neural Plate and Sense Organs. Chabry says that he never saw a partial embryo in which the neural plate had invaginated; on the contrary the nervous system always remains spread out in the form of a layer or plate; this plate occupies the face of the embryo which is morphologically median in position (its normal location), while the sense spots consist of pigmented cells which are superficial in position and which lie near the base of the tail. ‘This agrees very closely with my observations, though I have frequently seen the neural plate invaginate by an irregular process. ‘The eye is said by Chabry to Be formed on the right side normally, but the fact that it may appear in the left half embryo leads him to conclude that its rudi- ment exists in the left half of the egg also. He thinks that the otolith comes only from the right posterior cell. I have not determined the exact cell origin of the sense organs in the normal larva, but in the partial larve they are homed only from the anterior quadrants and from either the right or left sides. I have not been able to distinguish between the eye and the otolith in the partial embryos of Cynthia. Driesch says nothing of the neural plate nor of the manner in which the nervous system is formed in his small larve, though he mentions the fact that “the sense vesicle with the eye and otolith are not formed in the typically clear manner characteristic of the normal development.” He found the eye spot almost always present, the otolith very seldom and he concludes that it makes no difference in the presence or absence of the sense organs whether the embryo has developed from certain cells of the 4-cell stage rather than from others. Since Driesch expressly states that he never raised a quarter embryo beyond the stage of his Fig. 16, at which stage the sense organs have not appeared, and since Seether his fisures nor descriptions give any evidence that he has distin- guished anterior or posterior quadrants from right or left ones, it would be interesting to know how he could determine that sense organs might be formed from any quadrant of the egg—a result entirely contrary to my observations. 204 Edwin G. Conklin. b. Notochord. Chabry supposed that the notochord arose from both the anterior and posterior quadrants of the egg. Castle (’96) held that a single pair of cells of the posterior quadrants, B®, “the posterior eid fundament,”’ were the only cells of the posterior quadrants which entered into the formation of the notochord. I am of the opinion that this cell is a mesenchyme and not a chorda cell (see Conklin, ’ 05'), but even if it should be found to be a chorda cell it is only one cell of nine on each side of the mid line which give rise to that structure, while eight ‘pairs of chorda cells come from the anterior quadrants. Cerin it is that no trace of a notochord ever arises from the posterior cells when they are isolated, whereas chorda cells always arise from isolated anterior cells, though a notochord is rarely formed in such cases. Chabry describes (p. 294, Fig. 118) an anterior two-quarters embryo in which a naked chorda was seen in the perivitelline space outside the body of the embryo; such a case somewhat resembles the one shown in my Fig. 72. However, in every other instance which I have observed the chorda cells of an anterior embryo do not give rise to a notochord, but after escaping from the body of the embryo lie free in the perivitelline space as scattered cells. (Figs. 52, 66-70.) But while a notochord is rarely or perhaps never formed in an anterior embryo and never in a posterior one, it 1s invariably found ina right or left one, and the figures of Chabry and Driesch as well as my own show that the process of formation 1s essentially the same as in a normal embryo. Chabry indeed believed that the notochord was primitively double and that half of it arose from each lateral half of the egg. He speaks of the fact that in Ascidia and Botryllus it is composed of a double row of cells and Crampton also refers to the fact that in the normal ascidian tad- pole there are two rows of chorda cells, whereas Driesch has well said that in its fully formed condition the ascidian notochord is normally composed of a single row of cells. _[ find, as did Driesch, that the notochord of a tateral embryo 1s formed by interdigitation, just as in the normal embryo, but I also find, as opposed to Driesch that the notochord is never formed from any cells save the chorda cells which come from the posterior part of the gray crescent. Furthermore, my observations show, as did Chabry’s, that the Mosaic Development in Ascidian Eggs. 205 formation of a tail is dependent upon the development of a noto- chord. c. Muscles and Mesenchyme. Chabry paid no particular attention to the number and location of the muscle cells in his partial larve, though he frequently speaks of their presence as being proved by the ‘twitchings of the tail; these movements are less energetic than in normal larve and, as a consequence, partial larvze do not escape from the egg mem- branes. Driesch also found that partial larve rarely hatch, probably because of their weak muscular movements, but he, too, paid no attention to the number and position of the muscle cells. Owing to the brilliant color of these cells in Cynthia they are recognizable at all stages; in the partial larve they are found only along one side of the notochord, where they form the characteristic free: rows of cells, whereas the muscle cells of the opposite side are entirely lacking. In the oldest larvze.a few of the muscle cells extend around the end of the notochord to the side on which they were lacking. I havenot beenable to determine whether the num- ber of muscle cells is actually increased during this process or merely rearranged, but I believe that the whole process consists in the moving Ri certain cells over to the side on which they were lacking, Saco any increase in their number. ‘This is part of that process of regulation which begins with the rounding up of the surviving blastomere after the other one has been eile In fact, this very extension of the muscle cells around the end of the notochord begins 1 in this rounding up of the surviving blastomere and in that slight change in the dieabion of division which causes the median celle of the “yellow crescent to lie nearer the middle of the first cleavage plane than in the normal egg. (Fig. 15.) Chabry found (p. 308, Fig. 132) only one atrial invagination and one organ of fixation (papilla) in right or left half embryos. Driesch did not determine the number of atrial invaginations but he does call attention to the fact that but one papilla is present in embryos from isolated blastomeres. I have not observed the formation of the atrial invaginations or of the papilla in Cynthia; even in the normal larve they are inconspicuous at the time of the metamorphosis and | have not studied them before that period. However, the areas of trunk mesenchyme in which the atrial invaginations appear, are conspicuous areas of clear, slightly 206 Edwin G. Conklin. yellow, protoplasm in front of the muscle rows on each side of the tail; these areas may be recognized in the early cleavage stages and in no case are both these mesenchyme areas present in right or left half embryos. It is almost certain, therefore, that only one atrial invagination is formed in such embryos. We find, therefore, that those parts of the larva which normally lie on the right side are missing in a left half embryo and those which normally le on the left side are not found in a right half embryo, whereas unpaired organs which lie along the median plane are represented in both lateral half embryos. ‘This 1s exactly what might be expected from a study of the organization of the egg since the substances, which give rise to median organs, are found along the median plane in both right and left blasto- meres, whereas the materials which give rise to organs of the right side are found only in the right blastomere, those which give rise to organs of the left side, in corresponding positions in “he left blastomere. Neither Driesch nor Crampton attempt to show that a larva from the right half of an egg has the organs of the left side and this is the whole question at issue; if it does have these organs it 1s a complete embryo; if it lacks them it is a partial embryo, even if it does have a head and a tail. Chabry found that a larva from one of the first two blastomeres had a head and tail and median organs, but that it did not have the organs of the missing side and this conclusion I can entirely confirm. All of the muscle substance (myoplasm) and most of the mesen- chyme (chymoplasm) is localized in the posterior half of the egg, and corresponding to this distribution we find that an anterior half embryo entirely lacks muscles, though it may have a small amount of mesenchyme (that derived from the cell A**), whereas a posterior half embryo contains a large number (probably the full normal number) of muscle cells and most of the mesenchyme. V. REGULATION IN THE ASCIDIAN EGG AND EMBRYO. It is well known from the work of Loeb (’92) and L. Schultze (99) that the brain of Ciona will be regenerated when extirpated in the adult animal, and that the siphons will be restored when they are cut off. Driesch (’02) has also shown that Clavellina has extraordinary powers of regenerating almost all lost parts. Mosaic Development in Ascidian Eggs. 207 This power of regeneration in the adult is in striking contrast with its lack in the egg and embryo and requires some explanation. It should not be overlooked that such injuries to the egg and embryo as have been described in the preceding pages are prob- ably more extensive and far-reaching than any which are capable of being repaired in the adult. As Chabry says the destruction of one of the first two blastomeres is the same in its effect as the destruction of the right or left half of the body of an adult. The destruction of the anterior half of the egg is similar to the total loss of the nervous system and notochord of the larva; while the death of the posterior half corresponds to the destruction of the whole of the muscular system and most of the mesenchyme of the larva, since in each case the specific substance which alone gives rise to these organs is destroyed. ‘Therefore these injuries are probably much more extensive than any which have been practiced on the adult animal. Furthermore, I am of the opinion that the extremely rapid development of the ascidian egg and embryo may itself act as a check on regulation. In Cynthia and Ciona the fully formed larval stage is reached in about twelve hours after the fertilization of the egg, and these larve usually undergo metamorphosis into the adult form within the next twelve hours. In Molgula the development is even more rapid. It seems to me probable that the restoration of the parts of the missing right or left half of a larva might be fully accomplished if the larval life were longer. In a right or left half larva one day old the ectoderm cells have closed over the injured side, the notochord is complete, the neural plate has invaginated, although abnormally, and the muscle cells have begun to grow over from the uninjured to the injured side. There is here evidence of considerable regulative ability and it seems to me possible that, with more time before the metamor- phosis, complete rows of muscle cells might be found on both sides of the tail and that the mesenchmye cells might grow over to the side on which they are lacking and an atrial invagination appear in them. Inasmuch as the only form of regulation shown by the ascidian egg or embryo 1s this overgrowth of cells from the uninjured to the injured side, it is probable that no amount of time would ever sufce to produce an entire larva from the anterior or posterior half of an egg or from a quarter or any smaller portion. As a 208 Edwin G. Conklin. matter of fact there is not the slightest indication in an anterior half embryo of any attempt to restore the missing myoplasm or muscle cells, nor does a posterior half embryo show any tendency to form chorda-neuroplasm or neural plate or chorda cells. So far as observation and experiment show, each odplasmic substance is capable of giving rise only to one particular kind of organ or tissue. ‘The question may be raised whether the presence of the injured blastomere within the chorion may not influence the development of the surviving cells and possibly prevent regeneration. In this and in all previous experimental work on che ascidian egg these injured cells have been left within the chorion in contact with the surviving cells and in this respect all work on these eggs has been done eee similar conditions. Owing to the presence of the chorion it is practically impossible to remove the injured cells, and [ am therefore unable to furnish an experimental test of the influence or lack of influence of these cells upon the surviving ones. However, there is sufficient evidence, I think, to show that it is not the presence of these cells which prevents regeneration. Contact with the injured cell might be expected to hinder or pre- vent the closing of the surviving half along the injured side, but it is just this form of regulation, and this only, which is manifested by these eggs. “The presence of the injured cells can have nothing to do aay the failure of the anterior half embryo to form a tail, or the posterior half embryo, a head; on the other hand, I have shown conclusively that the development of a tail is dependent upon the presence of a notochord, and the formation of a head upon the presence of the gastral endoderm and neural plate. “The only possible influence of the injured cell upon the surviving one would be to limit the form- regulation; but as I have said fee it does not do. It is Heese that the presence of the injured cell should prevent the myoplasm from giving rise to other organs than muscles, or the chorda- -neuroplasm to other organs than chorda and neural plate. These injured cells are rarely killed, but they remain transparent and entire, although quiescent; they do not decay and form a nidus for bacteria and rT am convinced that their presence does not materially influence the development of the surviving half nor limit its powers of regulation. Mosaic Development in Ascidian Eggs. 209 VIE (GENERAL CONCLUSIONS: The conclusions which follow from these experiments are so obvious that they need but little emphasis here. Not only is the fact established that individual blastomeres give rise only to those parts of an embryo which they would produce under normal conditions, but the cause of this is clearly indicated. ‘The devel- opment of the ascidian egg is a mosaic work because individual blastomeres are composed of different kinds of ooplasmic material; this mosaic work is not merely a cleavage mosaic but also a mosaic of germinal substances, several of which are recognizable before cleavage begins. 1. Organ-Forming Substances. I have elsewhere shown that at least five distinct kinds of odplasm are recognizable in the egg of Cynthia before the first cleavage and that all of these substances are localized in their final positions as early as the close of that cleavage. In these experiments I have not been able to isolate the different odplasmic substances in the unsegmented egg, but after the second or third cleavages several of gece substances may be isolated and in such cases each substance gives rise only to a definite kind of tissue or organ, and apparently it has no power to produce any other kind. The myoplasm produces muscle cells only; the chorda-neuro- plasm, only chorda and neural plate cells; the chymoplasm, only mesenchyme; the endoplasm and ectoplasm only endoderm and ectoderm, respectively. Whenever an isolated blastomere lacks any of these substances, the embryo which develops from that blastomere lacks the corresponding organs. Accordingly the potencies of individual blastomeres are dependent upon the odplas- mic substances which they contain; the prospective value of any blastomere is not primarily a faction of its position, but rather of its material substance. The reason that the anterior quadrants of the egg never produce muscle cells is evidently due to the fact that they totally lack the yellow myoplasm; the fact that the posterior quadrants never produce a neural plate or chorda, is evidently due to the complete | absence of the chorda-neuroplasm in these quadrants; the cells of the ventral (animal) pole produce only ectoderm, without a trace of endoderm or mesoderm,—evidently because these cells are composed almost entirely of clear ectoplasm. 210 Edwin G. Conklin. Experiment confirms, therefore, what observation of the normal development plainly indicates that these strikingly different odplas- mic substances are not totipotent, but that as early as the close of the jirst cleavage and probably much earlier, they are differentiated for particular ends, and that 1f they develop at all they give rise to organs of a particular kind. T hese matertals are, therefore, “ organ- jorming substances” and the areas of the egg in which they are localized are “ organ-forming regions.” I need not here point out the similarity between this conclusion and the well-known theories of Sachs and His, nor the differences between my results and the commonly accepted view that the egg is composed of ‘simple undifferentiated protoplasm” or that ‘“‘cleavage is a mere sundering of homogeneous materials capable of any fate,” or that “the prospective value of a blastomere is a function of its position.”” Whatever may be true of other animals these things are certainly not true of ascidians. While there are few, if any, other cases known in‘which the differentiations of the odplasm are so striking or so numerous as in the egg of Cynthia there can be no doubt that organ-forming sub- stances are present in the eggs of many animals. In particular the works of Fischel (’97, ’98, ’03) on the Ctenophore, of Boveri (01) on Strongylocentrotus; of Wilson (’04) on Dentalium and Patella and of Conklin (’03) on Physa, Planorbis and Limnza have shown that distinct kinds of protoplasm are present in these eggs which are destined in the course of development to give rise to particular germ layers or organs. In the light of these discoy- eries it can scarcely be doubted that the general cause of mosaic development is to be found in the presence in the egg or blasto- meres of distinct kinds of protoplasm, or of organ-forming substances. 2. Localization of Oéplasmic Substances. The three principal substances in the egg of Cynthia, viz: the clear, the yellow and the gray, are already present and localized in the odcyte before it escapes from the ovary. The yellow (mesoplasm) forms a peripheral layer around the entire egg; the clear (ectoplasm) is the ‘clear achromatic substance within the germinal vesicle; the gray (endoplasm) constitutes most of the Mosatc Development in Ascidian Eggs. 2 Ta remainder of the egg.'| For the sake of brevity this earliest form of localization may be described as concentric or spherical, although the germinal vesicle does not lie exactly in the center of the egg but is slightly eccentric toward one pole. During maturation and fertilization this concentric localization gives place to a polar or radial form. Immediately after the entrance of the spermatozoon into the egg the peripheral layer of yellow mesoplasm flows rapidly to the lower pole where it collects in the form of a cap; the clear ectoplasm which escapes from the germinal vesicle at first lies at the animal pole where it surrounds the maturation spindles but after the entrance of the spermatozoon it also flows to the lower pole where it collects into a layer or stratum just above the mesoplasm; the gray endoplasm after these movements occupies almost all of the upper half of the egg. The egg at this stage appears to be radially symmetrical, the three principal substances being arranged in strata at right angles to the egg axis. Soon after the entrance of the spermatozoon this radial form of localization gives place to a bilateral one; the sperm nucleus and aster move up to the equator of the egg along one meridian which further development shows to be the median plane on the posterior side; the clear and yellow substances also move to the posterior pole along with the sperm nucleus and the yellow substance here forms a crescent around the posterior side of the egg, just below the equator. At this stage the egg is bilaterally symmetrical, there being but one plane which will divide equally all of the odplasmic substances. Finally during the first cleavage this early bilateral localization is changed into the definitive localization which is characteristic of all stages up to the late gastrula. “The yellow crescent remains in the position which it occupied before the first cleavage and here gives rise to muscle and mesenchyme cells; the clear protoplasm comes to occupy most of the ventral hemisphere and gives rise to ectoderm; the gray substance occupies the dorsal hemisphere in ‘Although I have not been able to isolate these various odplasmic substances before cleavage begins and, therefore, can bring no experimental evidence to prove that they are organ-forming substances at this early stage, it nevertheless seems probable that materials which are identical in color and texture with the organ-forming substances of later stages, to which they directly give rise, are also similar in potency. There is no apparent reason for believing that these strikingly different kinds of odplasm of the ovarian egg are any less distinct or more nearly totipotent than during the cleavage stages. 212 Edwin G. Conklin. front of the yellow crescent and its anterior portion becomes the gray crescent of chorda-neuroplasm, while its posterior portion 1s the deep gray endoplasm which gives rise to the gastral endoderm. ‘The form of localization of these substances, therefore, undergoes marked changes during the fertilization and first cleavage; it 1s concentric in the odcyte, polar or radial immediately after the entrance of the sperm, bilateral just before the first cleavage, and definitive at the close of the first cleavage. I have elsewhere (’05') shown reason for believing that even in the stage of radial localization in the egg of Cynthia there 1s prob- ably some structural peculiarity of the egg which determines that _the path of the sperm shall le in one meridian rather than in another and therefore that the median plane of the embryo and its posterior pole are not determined by the chance movements of the sperm within the egg. Similarly the basis for polar or radial localization is present in the ovarian egg in the slight eccentricity _ of the germinal vesicle toward the animal pole, though the odplas- mic substances are largely localized in concentric form at this stage. Iam unable to determine whether any structural basis for bilateral localization exists in the ovarian eggs of ascidians, but inasmuch as the localization invariably becomes bilateral at a later stage 1t seems necessary to suppose that there is some such intrinsic determinative factor. In almost every group of animals the chief axis of the egg is already marked out in the odcyte, the pole toward which the ger- minal vesicle is eccentric becoming later the animal pole of ‘ies ege and the ectodermal pole of the embryo. Despite this eccen- tricity of the germinal vesicle the localization of odplasmic sub- stances in the odcyte of ctenophores, nemertines, echinoderms and ascidians is chiefly concentric, the polar localization of these substances first appearing during the maturation and fertilization. On the other hand Wilson (04) has found a markedly polar localization of the ooplasm 1 in the odcyte of Dentalium; while it 1s probable that in the odcytes of insects and eephelapods the local- ization is bilateral in form. Boveri (01) found that distortion of the egg of Strongylocen- trotus after the formation of the equatorial zone produced no change in the polar stratification of the egg nor in the potencies of its different substances. Wilson (03), Yatsu (04) and Zeleny (04) have discovered that fragments from any part of the egg of Mosaic Development in Ascidian Eggs. 21g Cerebratulus before maturation may give rise to entire larvex; whereas this is not usually the case after maturation and fertiliza- tion, the potencies of the substances at the animal and vegetal poles being different. It is evident that during the concentric stage of localization section of an egg in any plane would leave samples of all the ooplasmic substances in each piece; in the stage of polar-radial localization any section of the egg parallel with the egg axis would leave samples of all the odplasmic substances in each piece; in the bilateral stage, only the right or left halves would contain parts of all the eabee nies! Probably one important rea- son why parts of eggs may give rise to whole embryos in some cases and not in others may Bevronndl in the fact that at the time of the experiment the form of localization may have been concen- tric in some cases, radial in others and bilateral in still others. (See Boveri, 01; Wilson, ’04*.) 3. Cleavage and Localization. / In previous publications ('05', 05° 2) | have pointed out the fact that localization precedes cleavage in the ascidian egg and that the localization pattern does not closely coincide with the cleavage pattern. On the other hand there is normally a constant relation between particular cleavage planes and the various ooplasmic substances. The first cleavage always lies in the median plane and bisects all the odplasmic substances; the second is transverse to the median plane and separates the yellow crescent from the gray one; the third cleavage 1s at right angles to the two preceding ones and separates the clear ectoplasm of the ventral hemisphere from the different substances of the dorsal hemisphere. Probably in no other animal is the cleavage so constant and so perfectly bilateral as in the ascidians and yet even here the position and direction of the cleavage planes is less constant than the form of localization. Among annelids and mollusks, as is well known, the cleavage 1s typically spiral and in many cases it is radially symmetrical. This radial symmetry of cleavage does not indicate, however, that the localization of odplasmic substances is also radially symmet- rical, for in some cases this localization is known to be bilateral and this is probably true in all cases. (See Conklin, ’05', pp. 90-92.) The relation of the cleavage planes to this bilateral organization 214 Edwin G. Conklin. is very different in cases of spiral and of bilateral cleavage, and consequently the results of killing any one or more of the first four blastomeres may vary in different cases; in general there is less likelihood of obtaining an entire embryo from an isolated blasto- mere of spiral cleavage than from one of the first two blastomeres in bilateral cleavage. In other cases the cleavage planes bear no constant relation to the planes of localization. ‘Thus in the frog’s egg the first cleavage may lie in the median plane or at varying angles to this plane and Brachet ('04) has recently shown that the character of an embryo derived from one of the first two blastomeres depends entirely upon the relation between the first cleavage plane and the median plane of organization. It is probable that the bilaterality of organization is no more perfect in ascidians than in annelids, mollusks or amphibians, but the bilaterality of cleavage is much more perfect. Accordingly, each of the first two blastomeres of the ascidian egg always con- tains half of every odplasmic substance, in the frog’s egg it may or may not contain half of these substances, in the annelid or mollusk it never does. I agree therefore with Brachet (04) and Wilson (’041, ’04?) that the varying results of experiments on the potencies of blasto- meres are due in part to the varying relations of cleavage to local- ization, and in part also to the different types of localization (concentric, radial, bilateral) in different eggs. 4. Determinate and Indeterminate Cleavage and Development. In a great many animals belonging to phyla as widely separate as Ctenophora, Polyclada, Nemertinea, Nematoda, Rotifera, Annelida, Mollusca, Arthropoda ‘and Tunicata the cleavage of the egg is constant in form and differential in character and under normal conditions, definite cleavage cells always give rise to defin- ite structures of the embryo or larva. For this type of cleavage I proposed several years ago (’97, 98) the designation “ deter- minate.”” In a few animals the cleavage 1 is known to be extremely irregular, as in Pennaria (Haregitt, ‘O4)s Renilla (Wilson, *84), and probably also in planarians (Hallez, ’87; Stevens, ’o4), while in other cases it is unknown whether the cleavage is normally con- stant and differential or not (Echinoderms); in still other cases Mosaic Development in Ascidian Eggs. 215 the planes of cleavage bear no constant relation to the planes of localization, as in the eggs of some of the vertebrates (frog, fish). For all such cleavages I proposed the name “indeterminate.”’ but at the same time I was careful to state that this was “‘to be under- stood as applying only to the cleavage, for in its main features and results the development of all animals is determinate; that is, predictable. Even in cnidaria, echinoderms and _ vertebrates there appears successively a blastula, gastrula, larva, and adult of determinate form and character” (’98, p. 21). But while the cleavage is indeterminate in some cases there is reason to suppose that there is a definite organization of the egg in all animals—in short that the organization of the individual is determinate at all stages from the egg to the adult. Even in such an egg as that of Pennaria it is certain that there must be deter- minative factors somewhere, if not in the cy toplasm then in the nucleus, which determine that the egg shall dev elop into a Pennaria rather than into some other animal; and it is further evident that these determinative factors must be present in the cytoplasm at a relatively early stage, if not at the very beginning of development. In the echinoderm egg, which was at one time supposed to be homogeneous or isotropic, Boveri (’01) has shown that a polar- radial localization of at least three distinct morphogenetic sub- stances takes place immediately after maturation, and in this case, as in the ascidians it is probable that there is an earlier concentric localization of these substances in the odcyte. Since these three substances are localized in zones or strata, one above the other, around the chief axis of the egg, they are all present in each of the first four blastomeres of the egg, each of which may give rise to an entire embryo; but when they are isolated each is found to be strictly limited in its potentialities. While therefore there are several groups of animals in which the cleavage is indeterminate there are few or none in which the odplasm is isotropic; on the contrary in almost every phylum the eggs and blastomeres show differentiations and localizations of the odplasm which are of morphogenetic value. “Everywhere,” as Fischel (’03) has well said, “the fundamental principle of normal development is a mosaic work.” But while Fischel supposes that “only the materials for the primitive organs of the embryo are preformed in the egg cell and that the material substratum for the differentiation of the special organs is probably first formed during 216 Edwin G. Conklin. the later stages,” I find that in the ascidian egg all the principal organs of the larva are represented by distinct organ-forming substances which are localized in their definitive positions and proportions as early as the close of the first cleavage. There is a world-wide difference between such results as these and those which were reached by Driesch and some of the earlier workers in this field. Wilson (’04) has recently expressed the opinion that “had the experimental analysis of cleavage been first undertaken in the case of such a determinate type as that of the gasteropod or annelid and had Roux not handicapped his theory with a purely speculative hypothesis of differentiation, which proved to be untenable, the whole discussion would have taken a different course; and I believe it would from the first have been recognized that the mosaic principle holds true in greater or less degree for every type of development, not excepting the most ‘indeterminate’ forms of cleavage.” Considering the fact that such highly determinate forms as the ascidian and the cteno- phore were studied in some of the earliest experiments on the potency of cleavage cells, | am of the opinion that the course which this discussion took was not primarily due to the fact that work began on relatively indeterminate forms. On the other hand I am convinced that the whole trend of opinion on the organization of the egg and on the potency of cleavage cells would have been different 1f those who did this work had been more familiar with the normal development of the forms studied, and in their zeal for the experimental method had not discarded the old and approved method of observation. It has taken such careful observers of normal processes as Boveri and Wilson to apply most successfully, the experimental method to the problem of the organization of the ego, and the results of such work constitute a well- ee, ed tribute o>’ to the permanent value of the work of Roux. SUMMARY. I. Normal Development. 1. Inthe ovarian egg of Cynthia (Sty ela) partita there are three strikingly different lated: of odplasm, viz: a superficial yellow layer, a centcal gray area, and a large transparent g verminal vesicle. At this stage the localization an these sliseeantes is approximately concentric. Mosaic Development in Ascidian Eggs. 217 2. During maturation and fertilization the yellow substance flows rapidly to the vegetal pole where it forms a superficial layer or cap; the clear substance escapes from the germinal vesicle and flows toward the vegetal pole where it forms a stratum above the yellow cap; the gray substance occupies the animal half of the egg. The localization at this stage is polar-radial. 3. [he sperm nucleus which lies in the center of the yellow cap moves to the posterior pole of the egg and the yellow and clear substances move with it. The yellow material here forms a crescent which lies with its center at the posterior pole and its arms extending forward on each side about halfway around the egg; the clear substance forms a band just above and internal to the crescent; the gray substance occupies the remainder of the egg. At this stage the localization is bilateral. 4. The first cleavage furrow appears in the plane of bilateral symmetry and divides each of the odplasmic substances equally. At the close of this cleavage the clear substance occupies the ani- mal (ventral) half of the egg; the gray substance lies at the middle of the vegetal (dorsal) pole while around the posterior border of the dorsal hemisphere is the yellow crescent and around its anterior border is a light gray crescent. This is the definitive localization of these substances, and in these positions the clear material gives rise to ectoderm, the gray to endoderm, the yellow crescent to - muscles and mesenchyme, and the gray crescent to chorda and neural plate. 5. The second cleavage is transverse to the antero-posterior axis and separates the gray crescent from the yellow; the third ‘cleavage separates the clear protoplasm of the ventral hemisphere from the various substances of the dorsal hemisphere. i i Ex periments. 6. Individual blastomeres were injured by spurting or shaking the eggs in the 2, 4, 8, or 16-cell stages. “The surviving blastomeres were then studied both in the living condition and after being stained and mounted. 7. Cleavage. Isolated blastomeres always segment as if they still formed part of the whole, except that the direction of some of the cleavages is slightly altered so that the resulting cell mass is more nearly spherical than in the normal egg. ‘These alterations 218 Edwin G. Conklin. in the direction of cleavage and the consequent closing of the injured side are more apparent in isolated blastomeres of the 2-cell stage than in those of later stages. 8. Gastrulation. In right or left or anterior halves, gastrula- tion usually proceeds as if the fragment still formed part of the whole; even though the gastrula may be rounded in form the location of the different substances shows that it is strictly partial. Not infrequently isolated blastomeres give rise to exogastrule, which ultimately right themselves. In posterior halves and in quarter embryos, gastrulation does not at all resemble the normal process, either in methods or results. g. Right or Left Half Embryos. A lateral half embryo is usually laced along the injured side; it has a head and a tail; a typical notochord, which is formed only from the chorda cells of the surviving es and which is therefore composed of half the normal number of cells; an atypical neural plate and sense vesicle, formed only from the typical neural plate cells of the surviving side; a typical mesenchyme area in which the atrial invagination of one side 1s formed and three typical rows of muscle cells on one side of the notochord, but none along the injured side. In the latest stages to which these lateral embryos were reared (corre- sponding to the period of metamorphosis in normal larve) the muscle cells have begun to grow around the hinder end of the noto- chord to the side on which they were lacking; but in no case are the three rows of the normal embryo present on this side. Prob- ably only one atrial invagination and one papilla are ever formed in these lateral embryos. ‘These are therefore half embryos in which some cells have grown over from the uninjured to the injured side, but in which absolutely no change has taken place in the potency of the individual cells or of the different odplasmic sub- stances. 10. Anterior Half Embryos. Embryos derived from the two anterior quadrants of the egg have no trace of muscle cells nor of muscle substance; although the normal number of chorda cells are present they rarely if ever form a notochord but usually escape from the body of the embryo and lie scattered in the perivitelline space; the neural plate cells are present in normal number and position but the plate rarely, if ever, invaginates to form a sense vesicle; in late stages sense spots are formed from certain cells of the neural plate; cells of the gastral endoderm and general ecto- Mosaic Development in Ascidian Eggs. 219 derm are frequently present in their normal positions and num- bers. A tail is never formed in these anterior embryos and they bear no resemblance whatever to a normal larva. 11. Posterior Half Embryos. Embryos derived from the two posterior quadrants have no trace of notochord or of chorda cells, neural plate, sense vesicle, sense spots, or gastral endoderm; they contain a mass of muscle and mesenchyme “oak anda gable row of caudal endoderm cells, as in the normal embryo. ‘There is no indication of a tail or head, the embryo remaining rounded in form as long as it lives. 12. Three-Quarter Embryos. Embryos derived from three of the first four blastomeres are more nearly perfect than are half embryos, but they always show defects corresponding to the missing blastomere. If an anterior blastomere is killed, the neural plate and sense vesicle of the resulting larva are atypical and the notochord lacks the normal number aS cells; if a posterior cell is killed, the muscle and mesenchyme cells are lacking along one side of the tail. 13. One-Quarter Embryos; Two-Quarter Diagonal Embryos. Embryos derived from any one quadrant or from two diagonal quadrants of the egg are always very defective. “They never have a notochord, though if they come from anterior quadrants they may give rise to scattered chorda cells in the perivitelline space; there is never a sense vesicle, though if they are from an anterior quadrant a neural plate and sense spots are present. ‘The poste- rior quadrants always contain muscle, mesenchyme and caudal endoderm cells, but never a trace of notochord, neural plate nor sense spots. [he embryos are always rounded, there being no distinction of head and tail, and in no respect do they resemble normal larve. I4. Eighth and Sixteenth Embryos. When blastomeres are injured in the 8-cell or 16-cell stages a great variety of abnormal forms are produced. Ventral blastomeres give rise only to rounded masses of ectoderm cells in which there is no trace of endoderm or mesoderm; posterior dorsal cells give rise only to muscle, mesenchyme, and caudal endoderm; anterior dorsal cells to neural plate, chorda, and gastral endoderm. 15. Anterior and Posterior Half Gastrule. When cup-shaped gastrule of the stage shown in Fig. 8 are cut in two transversely so as to leave all of the yellow cells in one half and all of the chorda 220 Edwin G. Conklin. and neural plate cells in the other a notochord, sense vesicle, or tail is never formed and nothing resembling a normal larva develops from either half. The anterior half never contains muscle cells; the posterior half contains many muscle and mesen- chyme cells, but evidently no chorda or neural plate cells. III. Conclusions. 16. My results confirm and extend those of.Chabry, but they are fundamentally unlike those of Driesch; I agree with the work of Crampton as to the cleavage of isolated blastomeres but cannot agree with him that whole embryos or larvz are ever formed from isolated blastomeres of the ascidian egg. 17. Regulation in the ascidian egg and embryo is limited to the closing of the embryo and the consequent extension of certain cells from the uninjured to the injured side; and also to the forma- tion of a typical notochord and an atypical sense vesicle in right or left half embryos. One odplasmic substance never gives rise to another nor does a given type of cell ever produce cells of another type or organs of a diferent kind than those which would arise from it in a normal embryo. ‘The fact that the power of regulation is apparently greater in the adult ascidian than in the egg or embryo may be deceptive; the injury to the egg which wipes out completely certain ooplasmic substances may be really greater than any which may be repaired in the adult. Furthermore it is possible that the very rapid development of ascidians may act as a check on regulation. 18. [hese results prove that at least five of the substances which are present in the egg at the close of the first cleavage, viz: ectoplasm, endoplasm, my yoplasm, chymoplasm, and chords neuroplasm, are organ-forming substances. ‘They develop, if they develop at all, into the organs which they would normally produce; and conversely, embryos which lack these substances, lack also the organs which would form from them. Although I have been unable to test the potencies of these substances before - cleavage begins, there seems to be no reason for supposing that they are ever totipotent. ‘Three of these substances are clearly distinguishable 1 in the ovarian egg and I do not doubt that even at this stage they are differentiated for particular ends. Mosaic Development in Ascidian Eggs. 221 19. A possible explanation of the fact that all the fragments of an immature egg may give rise to entire larvae, whereas the pro- portion which gives rise to larva steadily decreases after matura- tion and fertilization, may be found in the fact that before matura- tion the localization.of odplasmic substances is usually concentric, after maturation and fertilization, polar-radial; while just before or shortly after the first cleavage the localization may become bilateral. Also the fact that isolated blastomeres may give rise to whole embryos in some animals and to partial ones in others may be due to the varying relations of cleavage planes to localiza- tion planes. If at the close of the second cleavage the localization is still radially symmetrical, each of the first four blastomeres would probably be capable of giving rise to an entire larva; if the first cleavage invariably lies in the plane of bilateral symmetry, as In ascidians, each of the first two blastomeres might be capable of giving rise to an entire larva (though my work shows that this would not necessarily happen); if the cleavage planes do not coincide with the planes of localization, as in mollusks and anne- lids, isolated blastomeres would not give rise to entire larve. (See Wilson, ’04}, ’04?.) 20. The development of ascidians is a mosaic work because there are definitely localized organ-forming substances in the egg; in fact the mosaic is one of organ-forming substances rather than of cleavage cells. ‘The study of ctenophores, nemertines, anne- lids, mollusks, ascidians and amphibians (the frog) shows that the same is probably true of all these forms and it suggests that the mosaic principle may apply to all animals. (c7. Fischel, Wilson.) Pip VATURE Sehr’ BarFurty, D., ’93.—Halbbildung oder Ganzbildung von halber Grosse? Anat. Anz., vill. BENEDEN, VAN ET JuLtn, ’84.—La segmentation chez les ascidiens dans ses ee arpors avec l’organization dela larve. Arch. de Biol., v Boveri, [H., ’01.—Ueber die Polaritat des Seeigeleies. Verh. Phys. Med. Ges., Wurzburg, xxxiv. ‘o1.—Die Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus lividus. Zool. Jahrb., xiv. Bracuet, A., ’04.—Recherches expérimentales sur |’oeuf de Rana fusca. Arch. de Biol., xxi. 222 Edwin G. Conklin. * Caste, W. E., ’96.—The Early Embryology of Ciona intestinalis, Flemming L. Bull. Mus. Comp. Zool., xxvii. Cuasry, L., ’87.—Contribution a l’embryologie normale et teratologique des Ascidies simples. Journ. Anat. et Physiol., xxiii. Conk in, E. G., ’97.—The Embryology of Crepidula. Jour. Morph., xiii. *98.—Cleavage and Differentiation. Woods Hole Biol. Lect. 03.—The Earliest Differentiations of the Egg. Science, xvii. ’05'.—The Organization and Cell-Lineage of the Ascidian Egg. Jour. Acad. Nat. Sci., Philad., xiii. ’05’—Organ-Forming Substances in the Eggs of Ascidians. Biol. Bull., viii. Crampton, H. E., ’97.—The Ascidian Half-Embryo. Ann. New York Aca. Sci. x Driescu, H., ’95.—Von der Entwicklung einzelner Ascidienblastomeren. Arch. Entw. Mech., 1. ’o2.—Studien tber das Regulationsvermogen der Organismen. 6 Die Restitutionen der Clavellina lepadiformis. Arch. Entw. Mech., XIV. ‘03.—Ueber Anderung der Regulationsfahigkeiten im Verlauf der Ent- wicklung bei Ascidien. Arch. Entw. Mech., xvii. FiscHEL, A., ’97, ’98.—Experimentelle Untersuchungen am Ctenophorenei. I and II Arch. Entw. Mech., vi, vii. ’03.—Entwicklung und Organ-Differenzirung. Arch. Entw. Mech., xv. Hattez, P., ’°87.—Embryogénie des Dendrocoeles d’eau douce. Paris. Haraitt, C. W., ’04.—The Early Development of Pennaria tiarella. McCr. Arch. Entw. Mech., xviii. Hertwic, O., ’92.—Urmund und Spina Bifida. Arch. Mik. Anat., xxxix. 798.— Die Zelle; 11. Jena. KORSCHELT UND HeipeErR, ’02.—Lehrbuch der vergleichenden Entwicklungs- geschichte. Allgemeine Theil. Jena. Loes, J., ’92.—Untersuchungen zur Physiologischen Morphologie der Thiere, vi and vii. Roux, W., ’95.—Gesammelte Abhandlungen tber Entwicklungsmechanik der Organismen. Bad. II, Leipzig. ’92.—Ueber das Entwicklungsmechanische Vermogen jeder der beiden ersten Furchungszellen des Eies. Verh. d. Anat. Ges. zu Wien. ’03.—Ueber die Ursachen der Bestimmung der Hauptrichtungen des Embryo im Froschei. Anat. Anz., xxiii. Mosaic Development in Ascidian Eggs. 223 ScHULTZE, L.S.,’99.—Die Regeneration des Ganglions von Ciona intestinalis L. und uber das Verhaltnis der Regeneration und Knospung zur Keimblatterlehre. Jena. Zeit., xxx. Stevens, N. M., ’o4.—On the Germ Cells and the Embryology of Planaria Sim- plicissima. Proc. Acad. Nat. Sci., Philad. WEIsMANN, A., ’92.—Das Keimplasma. Jena. Wi1son, E. B., 84.—The Development of Renilla. Philos. Trans., clxxiv. ’03.—Experiments on Cleavage and Localization in the Nemertine Egg. Arch. Entw. Mech., xvi. ’04'—Experimental Studies on Germinal Localization, I and II. Jour. Exp. Zodl., 1. ’04?,—Mosaic Development in the Annelid Egg. Science, xx. Yatsu, N., ’04.—Experiments on the Development of Egg Fragments in Cere- bratulus. Biol. Bull., vi. ZELENY, C., '04.—Experiments on the Localization of Developmental Factors in the Nemertine Egg. Jour. Exp. Zodl., 1. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM . OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Direcror—No. 164. DIMORPHISM AND REGENERATION IN METRIDIUM. BY CW i. ECAVERING: WITH 2 FIGuREs. The experiments on which this paper is based were begun at Harvard University in the winter of 1901-02 and continued at the United States Fish Commission Laboratory at Woods Hole in the summers of 1902 and 1903. To Prof. E. L. Mark, Director of the Harvard Zoological Laboratory, and Drs. H. M. Smith and Francis B.. Sumner, directors in successive years of the Fish Commission Laboratory, I wish to express thanks for the accommodations kindly provided for the work. At the suggestion of Dr. W. E. Castle, to whom I am deeply indebted for guidance and help throughout this investigation, I undertook to discover if the dimorphism which occurs in Metri- dium marginatum Miulne-Edwards, 1s perpetuated in accordance with some hereditary law, perhaps related to the law of Mendel. So far at least as concerns asexual reproduction, it soon became evident that this is not the case. Further studies have shown that the dimorphism is due to a peculiar method of development in asexual reproduction. The dimorphism of actinians was noticed and described more or less completely by Thorell (’59), Dixon (’88), McMurrich (89), and Carlgren (’93), but the first full discussion of it was made by Parker (’97), and the only extensive experimental study thus far made with a view to discovering the exact nature of the anomaly is that of Carlgren (’04). The dimorphic condition referred to is this: The polyps of a given species may have either one or two (rarely three) siphono- glyphs. With each siphonoglyph, as shown for Metridium by Parker (97), there is invariably associated a pair of directive mesenteries. 226 GSW Fab The siphonoglyph is a groove in the cesophagus, covered with cilia, which tend to set up a current of water into the polyp. The relative abundance of monoglyphic, diglyphic and_ triglyphic Metridia, as indicated by observations of Torrey (98), Parker (99), and myself, is shown in Table I. Monoglyphic polyps, it will be observed, predominate 1 in all localities examined, though the proportion varies within wide limits. ‘The large and small TABLE I. ; Total No. Number Number Per cent Eocality. Counted. Diglvyphic. Monoglyphic. Monoglyphic. BYP BYP BIYP Newport, RekA@Parker))2 00.5.5: 131 53 Va 59 Oakland,/Cal (Vorrey).c.. 0.22. 200 43 157 78.5 Dalene Miasssae ee ac Sete we. ok 465 57 399 85.8 Exch Viasieer pea tis,« Shrae nies ne & 670 53 611 92.6 Woods Holes Mass. i. i200: 123 15 108 87.8 polyps from the same locality occur with about the same pro- portion of monoglyphic to diglyphic forms. ‘There is no dis- coverable correlation with any variation in color or in structure, save the constantly associated pair of directive mesenteries and perhaps certain irregularities of the non-directive mesenteries. These structures have been investigated by Parker (’97, ’99). He finds the diglyphic character to he associated more often with regularity in the number and arrangement of the non-directive mesenteries. But the variations in the mesenteries are not con- formable to any known laws and cannot be regarded as of specific importance. My first experiments were directed toward discovering whether monogly phic and diglyphic polyps produce, in asexual teproduc- tion, each itsown sortonly. The result obtained gave an emphatic negative to this hypothesis. Each sort was Faind to produce, by asexual methods, both monoglyphic and diglyphic polyps, monoglyphic individuals predominating among the young in both cases. ‘Two methods of inducing multiplication were employed. The anemones were, in somé cases, divided into halves by a vertical cut, so as to produce artificially specimens equivalent to those Dimor phism and Regeneration in Metridium. 227 supposedly produced by fission in nature. ‘The greatest success, however, was attained when fragments were cut from the basal portion of a polyp, as in the natural process of basal fragmen- tation. In either case the number of siphonoglyphs in the parent polyp was determined, so that the fragments from monoglyphic and diglyphic parents might be kept separate. Diglyphic and monoglyphic parent polyps could be distinguished either by inspection externally, when they were fully expanded, or by cutting the contracted polyp across after removing frag- ments from its base. ‘The fully regenerated young were stupefied by means of magnesium sulphate and fixed in chromic acid (1 per cent) then imbedded in parafhn and sectioned. Staining the sections with haematoxylin and eosin made it possible, with a low power of the microscope, to decide the various questions on which the interpretation of a polyp’s structure depends. Fragments cut from monoglyphic polyps produced twenty- eight monoglyphic and five diglyphic individuals. (See ‘Table II.) Three of the diglyphic polyps were simple, 1. ¢., having a single cesophagus to which the two pairs of directive mesenteries were attached; two were double, 1. e., having a divided cesophagus, each branch of which was connected with a different pair of direc- tive mesenteries, as if a portion of cesophagus had been produced in connection with each pair of directives, but the two portions had failed to unite. There were also twelve polyps regenerated from fragments cut from monoglyphic parents, which up to the time lhe they were examined had developed no mesenteries which could be recognized as directives. It is probable, however, for reasons which will presently appear, that most, if not all, of these would have become unmistakably monoglyphic polyps had they been given a longer time for regeneration. Fragments cut from diglyphic polyps produced in all thirty- six monoglyphic and ten diglyphic polyps, all simple, as well as two polyps whose character was indeterminable. These experiments show conclusively that each sort of polyp can produce the other by asexual methods; but, it will be observed, the diglyphic parents produced a somewhat greater proportion of diglyphic young, and the question at once arose whether this might not be due to an hereditary influence. A more careful study, however, of the regenerated polyps showed that such was 228 C. W. Habn. not the case. It was found that in the monoglyphic polyps the directive mesenteries arose regularly from the mew or regenerated portion of the body -wall, and chat in the diglyphic polyps, likewise, one of the two pairs of directives arose in this same position, but the other arose from the old or parental tissue. ‘This observa- tion at once suggested a different explanation of the dimorphism, -urz: that it was due not to the monoglyphic or diglyphic nature of the parent polyp, but to the character of the fragment from which regeneration took place. If this fragment contained a pair of directive mesenteries the polyp produced would be diglyphic, since it would retain the pair of directives derived from the parent and gain a second pair through regeneration. If, on the other hand, the parent fragment contained no directive mesenteries, then the regenerated polyp would be monoglyphic, containing only the pair of directives produced in the new tissue. To test this hypothesis a more detailed examination of the polyps was made, the monoglyphic polyps of different parentage being compared with each other, and, likewise, the two lots of diglyy phic polyps with each other. In determining which is the regenerated portion of a polyp no single Chace can be relied upon. Usually considerable familiarity is necessary to enable one to distinguish regenerated from parent tissue. The latter, as seen in cross-section, is usually characterized by deep folds of the ectoderm into which small V-shaped points of the mesogloea extend. (Figs. 1 and 2.) The external surface in this side is more regular and evenly curved; the mesenteries are quite regular, especially is this true of the secondaries and tertiaries in relation to the primaries. The primaries arising from the old portion of the body-wall are longer than those arising ‘from the regenerated part. The mesogloea on the regenerated side is not of uniform thickness or contour and does not conform as regularly with the folds of the ectoderm, when such exist. Regenerated directives and old directives usually differ in length and thickness when viewed in cross- sections, the former usually being short and thick. (Figs. 1 and 2.) These conditions, however, vary greatly, but when the evidence from one criterion is uncertain that furnished by other criteria is usually conclusive. The results of the detailed examination made are incorporated in Table II. From this it will be seen that the monoglyphic Dimor phism and Regeneration in Metridium. 229 polyps derived from monoglyphic parents had the directives developed in new tissue in all except one of twenty-six cases, in which the limits of old and new tissue could be recognized. In that one case the directives clearly were attached to the old or [¥ Fic. 1. Cross section of a diglyphic polyp produced by a fragment cut from the foot-disc of a diglyphic parent in such a way as to include a pair of directive mesenteries. The parental directives lie in the upper half of the figure, the regenerated directives in the lower half. The ectoderm is stippled in the regenerated portion of the body-wall. (From a camera lucida drawing, histological details being omitted.) Fic. 2. Cross section of a monoglyphic polyp produced by a fragment cut from the foot-disc of a diglyphic polyp so as not to include directive mesenteries. ‘The regenerated portion of the body-wall is indicated, as in Fig. 1, by stippling of the ectoderm. ‘The single short pair of directive mesenteries is attached to this portion of the body-wall. (From a camera lucida drawing, histological details being omitted.) parental part of the body-wall. It would seem, accordingly, that in this exceptional case no directives had been produced in the new tissue. Had this taken place the polyp would have been diglyphic, with the directives arranged exactly as in the twelve 230 C. W. Habn. diglyphic polyps mentioned in the fifth column of able II. This peculiar monoglyphic polyp may be considered the result TaBLeE II £ i eS Ce) Cee ci alee - - (exer ois BS te teams ae SS Pehle saat deer ok ae Sie Sm visa e 49.4 |/0 8 Parents. Young. CS oD 2 oe: 4 eso 8 a a Totals. elisa 4S a A= | faa] = ° = A = a) = a) a 5) AE ” 2 5 << 0 Pe | Cy l=! I Soiites 1 = oe 32 =O |S 0ee = 04 | E4p s = | = fences ss) an | M j M 25 I 5 — 2 28 ieee rstes | ! DN ae? | ga poe 2adoable ian 5 | | / | a ave? | etek if M 24 | I | | lio 36 iL D | —- | — 9 ig —_- IO Character of polyps produced by basal fragments cut from monoglyphic and from diglyphic parents respectively, the relation of the cut to the parental directives being unknown in a majority of cases. M = monoglyphic; D = diglyphic. of the union of the cut edges of the parental fragment without regeneration of a directive system. Such a result occurs not infrequently i in the case of fragments containing only non-direc- tive mesenteries, as well as of those, like this one, which contain a pair of directives. In the former case wholly aglyphic polyps are produced. ‘Three such were observed in these experiments. Carlgren (04) has shown, in the case of Sagartia, that such a result 1s obtained most often when the parental fragments are of relatively large size. It is probable that the same is true in Metridium, though precise observations on this point are wanting. The monoglyphic polyps of diglyphic parentage, like those of monoglyphic parentage, had the directive mesenteries in all cases except one (out of a total of twenty- -five) in the regenerated area. In that one case the single pair of directives was clearly received directly from the parent polyp and no new directives had been regenerated. Likewise in the case of the diglyphic polyps no difference was recognizable between those of monoglyphic parentage and those of diglyphic parentage. In all cases except possibly one there was a pair of directives in the old tissue, and one in the new. ‘There were three diglyphie polyps of this sort Dimor phism and Regeneration in Metridium. 231 derived from monoglyphic parents, and nine derived from diglyphic parents. [he possible exception mentioned was a diglyphic polyp of diglyphic parentage which had two pairs of directive mesenteries, both apparently 1 in the new tissue. Yet the limits of the old tissue could not in this case be located with certainty, and it is possible that one of the two pairs had really been deriv ed directly from the parent fragment. Otherwise we must suppose that regeneration had taken place in such a way as to produce simultaneously out of new tissue two pairs of directive mesen- teries. [hat sucha thing probably occurs sometimes is indicated by the observation once in a great while of a trig/lyphic individual, a condition which would be reached if a fragment already con- taining a pair of directive mesenteries acquired two more by regeneration. [he triglyphic condition may, however, arise in a mercer way, viz: by laceration of a digly phic polyp, which then produces in the area of regeneration a new or third siphono- glyph system. It still remains to account for the fact shown in Table II that more diglyphic polyps are produced by digylphic than by mono- glyphic parents. A moment’s reflection will show that this is not difficult. If pieces are cut at random from the bases of polyps without reference to the position of the directive mesenteries, it is evident that directives are likely to be included in the fragment removed, twice as often when the parent polyp is diglyphic as w ahem it 1S monoglyphic, since the diglyphic polyp contains two directive sys- tems on opposite sides of the body, whereas the monoglyphic polyp contains only one. Accordingly we should expect the proportion of diglyphic polyps regenerated to be about twice as great in one case as in the other. ‘The observed proportions are not greatly at variance with this expectation. In order to test more fully and directly the hypothesis already presented,—that the condition of a regenerated polyp, whether monoglyphic or diglyphic, depends on whether the parental frag- ment did or did not contain portions of the directive mesenteries,— advantage was taken of the fact that in the experiments summarized in ‘Table II certain fragments had been cut from the bases of parent polyps in such a way as to include a pair of directive mesenteries, and others had been cut in such a way as not to include directives in the fragment removed, the two lots having been reared separately. In the former lot unfortunately the 232 C. W. Habn. mortality was high, because of unfavorable conditions in the aquarium in which they were placed, and only three polyps survived. Further, two of these were insuthciently regenerated to show the character of the new mesenteries, but the third was clearly a diglyphic polyp with one pair of directives in the old tissue and one in the new, as expected. The fragments cut so as to exclude directives did somewhat better. [en polyps were reared from them. Eight of the ten were clearly monoglyphic, with the directives always in the new tissue; a ninth polyp was insufhciently regenerated, but it had a pair of mesenteries on the regenerated side, which gave some indications of being directives. If so, this polyp is similar in character to the eight previously mentioned; if not; it 1s aglyphic. The tenth polyp was digly phic, but quite asymmetrical in char- acter, one of the two pairs of directive mesenteries arising close to the boundary between the old and the new tissue. It 1s impos- sible to say whether a pair of directives was accidentally included in the fragment from which this polyp developed or whether there arose simultaneously two areas of regeneration, each of which produced a pair of directive mesenteries. This direct experiment, incomplete though it is, supports the hypothesis based on the experiments previously described. It indicates that the dimorphism found in Metridia asexually pro- duced is not dependent upon the monoglyphic or diglyphic char- acter of the parent poly p, but upon whether the parent fragment does or does not contain a portion of a siphonoglyph system. It harmonizes, likewise, with the observations of Carleren ('04) on regeneration in Sagartia and other actinians and supports the idea advanced earher by Carlgren (’93) and supported by Parker (97), that the dimorphism on actinians is an incident of asexual reproduction. As a control of the experiments examination was made of nine spontaneously regenerated polyps collected at Lynn, Mass. This yielded results closely similar to those obtained from the artificially regenerated i One of the polyps was inde- terminable; one was diglyphic, with one pair of directives in the old and one in the new tissue; and seven were monoglyphic.. Of the seven monoglyphic Solyps, five had the directives attached to what was unmistakably the regenerated portion of the body- wall, while in the remaining two ald and. new tissue could not be Dimorphism and Regeneration in Metridium. 233 distinguished on account of the advanced state of regeneration of the polyps. The frequent occurrence of asexual reproduction in Metridium explains the prevailing asymmetry of individuals in this species, regenerated diglyphic polyps in particular being rarely sym- metrical. It is usual to find the mesenteries arranged with more primaries and secondaries on one side of the plane passing through the siphonoglyphs than on the other. ‘This condition is to be explained by the fact that fragments, either spontaneously pro- duced or formed artificially by random cuts from the base of the foot-disc, arise without any definite reference to the parental mesenteries which traverse that region. Hence, if the directive mesenteries chance to be nearer one end of a fragment than the other or if the new directives are formed nearer one edge of the regenerated area than the other, an ‘asymmetrical polyp results. The idea which has been advanced in the foregoing pages 1s capable of giving an explanation also of the great variation in the numerical proportions of monoglyphic sid diglyphic poly ps in different localities. (See Vable I.) If we suppose that in cer- tain localities, like Newport, R. I., or at particular seasons of the year sexual reproduction is favored, regular hexamerous diglyphic polyps should at such places or seasons be relatively more abun- dant. Torrey (’02) correctly explains as due to asexual repro- duction patches of similarly colored sea-anemones, but the occur- rence of diglyphic individuals among polyps asexually produced does not, as he supposes, show that the diglyphic character has been inherited as such, but rather that in these particular cases the parental fragments happened to include a directive system. The diglyphic hexamerous polyp of Aiptasia, described by Andres and cited by Torrey (’02) as “evidence to be explained,” may be explained on the same basis. The dimorphism which, according to Torrey (’02), occurs in polyps produced by budding from Ene column of sea-anemones is doubtless capable of explanation i ingbet similar wa ive production in the experiments here described of polyps with a divided cesophagus and perhaps, in other cases, of two directive systems formed simultaneously in the regenerated tissue are worthy of notice as giving a clew to the origin of double mon- sters and of triglyphic polyps. Both of these abnormal conditions well known in collections of polyps, doubtless arise in spontaneous 234 C. W. Habn. asexual reproduction, since the processes leading up to them have been observed in regeneration artificially induced. In view of these facts it is improbable, as has been supposed by several investigators, that double Metridia are a stage in a process of repro- duction by longitudinal fission. The older view that they are genuine monstrosities seems better supported, but not the view chat they are due to coalescence, as was once thought to be the case. From long strips cut from the margin of the foot-disc and including a half or more of its circumference, polyps with two or three distinct oral discs have several times been obtained in these experiments. ‘This result throws still further light on the origin of double monsters. SUMMARY. The dimorphism which occurs in Metridium is due not to alternative inheritance of the diglyphic and monoglyphic condi- tions, but to the frequent occurrence of asexual reproduction. This takes place spontaneously by basal fragmentation and may readily be induced by cutting off pieces of the foot-disc. Whether a particular fragment produces a monoglyphic or a diglyphic polyp depends, not on the monoglyphic or diglyphic condition of the parent polyp, but upon whether the fragment does or does not contain some portion of a directive system, for a directive system is regularly produced in the regenerated portion of the young polyp. Accordingly, if the portion derived from the parent already contained a directive system, the young polyp will have two such systems and will be diglyphic. But if the parental fragment contained no directives, the young polyp will have only one directive system, that produced in regeneration, and will be monoglyphic. Not only the dimorphism of Metridium, but also its prevailing asymmetry and extreme variability in number and arrangement of mesenteries can be explained by its method of development in asexual reproduction. ‘Trigly phic polyps and those with two or more oral discs or with double or branched cesophagus or devoid of siphonoglyphs are abnormalities due probably to regeneration from fragments of unusual size or shape, as compared with the fragments normally produced in spontaneous basal fragmentation. Dimor phism and Regeneration in Metridium. 225 BIBLIOGRAPHY. CaRLGREN, O., ’93.—Studien uber nordische Actinien. Kongl. svenska Vet.- Akad.) Handi Ne ., Bas 25, No. To, 148 pp. 10 Eat, ’o4.—Studien tber Regenerations- und Regulations-Erscheinungen. Kongl. svenska Vet.-Akad., Handl., N. F., Bd. 38, No. 8, 84 pp. 229 ip., 10°F at: Dixon, G. Y., ’88.—Remarks on Sagartia venusta and Sagartia nivea. Sci. Proc. Roy. Dublin Soc., N. S., vol. 6, pp. 111-127. McMoraicu, J. P., °89.—The Actinaria of the Bahama Islands, W. I. Jour. of Morph., vol. 3, no. 1, pp. 1-80, pl. 1-4. Parker, G. H., ’97.—The Mesenteries and Siphonoglyphs in Metridium margina- tum Milne-Edwards. Bull. Mus. Comp. Zool. Harvard Coll., vol. 30, no. 5, pp. 259-273, I pl. *99g.—Longitudinal Fission in Metridium marginatum Milne-Edwards. Bull. Mus. Comp. Zool., Harvard Coll., vol. 35, no. 3, pp. 41-56, 3 pl. 2 TuHore.t, T., ’59.—Om den inre byggnaden af Actinia plumosa Mull. Ofversigt Kongl. Vet.-Akad., Forhandl., Arg. 15, 1858, pp. 7-25, Tab. 1. Torrey, H. B., ’98.—Observations on Monogenesis in Metridium. Proc. Cal. Acad. Sci., ser. 3, Zool., vol. 1, pp. 345-360, pl. 21. *o2.—Papers from the Harriman Alaska Expedition. XXX. Anemones, with a Discussion of Variation in Metridium. Proc. Wash. Acad. Sci., vol. 4, pp. 373-410, pl. 34, 35. From the Rudolph Spreckles Physiological Laboratory of the University of California. THE EFFECT OF VARIOUS SALTS UPON THE SUR- VIVAL OR THE. INVERTEBRATE HEAR BY CHARLES G. ROGERS. WitH 1 Prater. The cause which underlies the rhythmic contraction of the heart has been the subject of much controversy. In recent years the importance of the inorganic compounds of the blood has been acknowledged by most physiologists, but the role of each of these different salts in originating and maintaining rhythmic contrac- tions has caused aagh digevesian: Up to the present time almost all of the work done upon the physiology of the heart has concerned vertebrates alone. It was, therefore, a pleasure to follow the kind suggestion of Dr. Loeb and use the heart of an invertebrate as the subject upon which to conduct a series of experiments which may furnish an answer to the following questions: What 1s the influence of the various salts, found in the blood, upon heart action? And, is this influence the same in the crab as in the heart of the verte- brates? I wish to express my thanks to Dr. Loeb for suggesting the problem and for many kind criticisms during the course of the work. : METHODS. In the study of the problem the hearts of the marine czab Brachynotus nudus were employed. ‘This crab is found very abundantly along the western shore of San Francisco Bay, be- neath rocks, between tide marks. ‘The crabs may be kept in the laboratory for a considerable period without serious deterioration and hence prove to be an admirable form upon which to work. In carrying out these experiments three general methods of procedure have been employed, of which two, however, were 238 Charles G. Rogers soon discarded. ‘The first consisted in carefully isolating the hearts in watch glasses, each containing about ten cc. of the solution to be tested, and making observations upon the number and quality of the beats developed. “This method was employed only during the early stages of the work as it did not permit of any accurate estimate of the amount of work done by the heart. The second method was that of carefully suspending the hearts by means of delicate glass hooks in connection with light recording levers and allowing them to trace upon smoked papers the records of their contractions. In these experiments the hearts were moistened with the solutions by means of camel’s hair brushes. Oxygen, of course, 1s easily taken up from the air, but the effects of the various constituents of the solutions are difficult to deter- mine with this method as the amount of solution in contact with the heart is small and variable. One can not be sure at any given time that the solution has replaced the body liquid normally present. In view of this fact a third method was employed. This was similar to the second except that the hearts were im- mersed in tubes, each containing about 30 cc. of the solution. While the general results of the first method agree with those of the third they are disregarded in the final summing up of the work as lacking in sufficient accuracy. The second method failed to give uniform results. “The author feels that the results obtained by the third method are far more reliable than those obtained by either of the preceding ones, hence they form the basis of the following report. EXPERIMENTS A. What ts the Optimum Concentration of Salt Solution which will Favor the Rhythmic Activity of the Heart? Botazzi! has measured cryoscopically the osmotic pressure of the blood of many of the marine invertebrates and has found that it is practically the same as that of the sea water. ‘The average depression of the freezing point in the body liquids of inverte- brates is given by Hamburger? as —2°.2Q, and the Capea of the freezing point of the sea water 1s given by Hober as —2°.3. 'Botazzi. Archives Italiennes de Biologie, xxviii, 1897, p. 67. "Hamburger. Osmotischer Druck und Jonenlehre in den Medicinischen Wissenschaften. Effect of Salts Upon the Invertebrate Heart. 239 The NaCl solution having the same osmotic pressure contains 3-783 per cent of NaCl, or is a solution of about 2 m. concentra- oe. In bays and in the mouths of rivers the somuce pressure of the sea water becomes greatly modified. Dr. Loeb! has shown that animals taken from the waters of San Francisco Bay thrive in solutions with an osmotic pressure approximately equal to that of a? m. NaCl solution. The animals upon which the present work was carried out were collected at a point about three miles north of the Golden Gate where the water sweeps by at both flood and ebb of tide in strong currents. At flood tide the water has nearly the same concentration as the water of the open ocean, but at ebb tide it is much freshened by the water of the Sacra- mento River. __ A series of experiments was made to determine the concentra- tion of a solution of NaCl which would favor the rhythmic con- traction of the heart. For this purpose a 8 m. NaCl solution was employed and this was diluted with distilled water in varying amounts. It might be expected that the beats, if any at all appeared, would continue longest in that concentration of NaCl which most nearly approaches the normal concentration of the blood of the animal. As a result of a long series of trials it was found, that a % m. and a 5% m. ohio of NaCl acted most favorably. Both of these concentrations were employed in the further work. B. Is NaCl Essential for Maintaining Rhythmic Contractions? In considering the question whether any substance is essential for the origination of rhythmic contractions the following con- dition must be kept in mind: In order to demonstrate that a substance 1s necessary for the origination of rhythmic beats in a muscle we must employ a muscle which does not beat rhythmi- cally when it is removed from the body. Dr. Loeb’ has shown that this is true in the case of the center of the bell of the medusa Gonionemus, and was able to demonstrate that NaCl is essential for the origination of rhythmic contractions in this muscle. ‘The ‘Loeb, J. Pfluger’s Archiv., vol. xcvii, 1903, p. 394. Also University of California Publications, Physiology, vol. i, No. 7, pp. 55-69- Loeb, J. American Journal of Physiology, vol. iii, 1900, p. 383. 240 Charles G. Rogers. ventricle of the heart of the turtle also does not exhibit rhythmic contractions when it is removed from the body of the animal and in this case also Lingle’ was able to show that the development of rhythmic contractions depended upon the presence of NaCl. Lingle also emphasized the necessity of a large supply of oxygen. Overton’ working independently, and apparently not having seen the reports of the work already mentioned found that in the absence of NaCl, e. g., in a pure sugar solution muscle does not respond to electrical stimuli. In the present work we are dealing with a heart of a single chamber and one that continues to beat when it is removed from the body of the animal. We can, hence, only raise the question whether the heart of the crab will continue to beat for a long time in the absence of NaCl while with this salt present it will continue to beat for a much longer period. We may also raise the question whether when the contractions of the heart have ceased in some solution lacking 1 in NaCl the addition of NaCl will cause rhythmic contractions again to take place. In order to show whether the hearts of the crabs depend upon the presence of NaCl to maintain rhythmic contractions they were immersed in solutions lacking in this salt, but in which the osmotic pressure was kept approximately at the normal height by means of cane sugar. In some experiments no oxygen was added to the solution in others hydrogen- peroxide was added, following the experiments of Lingle, and in others a current of gaseous oxygen was allowed to bubble through the solution. As the result of these experiments it was found that the fresh hearts of the crab do not cease beating at once when placed in a pure sugar solution and that the length of time during which such contractions may continue is somewhat extended by the presence of oxygen. In no case, however, did the heart in such a solution continue to beat for more than one hour and in the very great majority of cases not more than twenty minutes. ‘There seemed to be no great difference in the action of the hearts when the concentration of the solutions varied between 2m. and 3 m. In a sugar solution the beats are at first not weakened but very soon they lose in strength and soon cease altogether. When no oxygen is ‘Lingle, D. American Journal of Physiology, vol. viii, p. 75 ff. “Overton. Pfluger’s Archiv., Bd. 92. Effect of Salts Upon the Invertebrate Heart. 241 added to the solution the beats are of regularly decreasing ampli- tude until finally no contraction is visible. When the oxygen supply is ample it frequently happens that the last beats have perhaps one-third of the amplitude of the normal contraction, but they occur at regularly increasing intervals until they cease altogether. In some cases irregularities of beat occur. “These may be due to injuries received by the heart when it was removed from the body of the animal or from a deficient supply of oxygen. A very marked effect of the cane sugar is the great increase a muscular tone which occurs in all heatis immersed i in such solutions. If, as has been held by some, NaCl is the substance which is necessary for the dev elopment of rhythmic contractions we should find upon adding NaCl to the sugar solution that the length of time during Which 4 heare will continue to beat will he lengthened as we increase the amount of the salt, up to the limit of ‘hie con- centration in which this salt exists in the sea water. In order to test this varying amounts of ? m. NaCl were added to a 3m. cane sugar solution and it was found that as the proportion of NaCl in the solution increased the hearts beat for a longer time. The following examples will illustrate: No. 222—25 cc. $m. NaCl plus 75 cc. $ m. cane sugar beat for 26 minutes. No. 213—50 cc. $ m. NaCl plus 50 cc. 2 m. cane sugar beat for 35 minutes. 3 3 No. 228—75 cc. $m. NaCl plus 25 cc. # m. cane sugar beat for 70 minutes. (The above experiments were made without adding any extra oxygen to the solutions.) In a pure $ m. NaCl solution the hearts beat on the whole longer than in the mixtures of NaCl and cane sugar. It now becomes of interest to know whether other substances than the NaCl have the power to aid in the maintenance of rhythmic contractions when added to a solution of cane sugar. On account of the importance of calcium, potassium and magne- sium for marine animals we naturally turned first to these in order to answer the question. Small and varying amounts of the chlorides of these metals were added to solutions of cane sugar and records made of the heart contractions under the influence of these solutions. 242 Charles G. Rogers. ON Bay Effect of the Addition of Calcium Chloride to a Sugar Solution. Small “amounts 1(.5 (ce: to 13-00 cc.) of a s,m... 2m on aoe calcium chloride solution added to 100 cc. of 2 m. cane sugar solution in which a heart may be aiaierced modify very materially the action of the heart. “The beats become more uni- form in quality and occur at more regular intervals than when the heart is immersed in a pure sugar solution. At the same time it seems probable that the length of time during which a heart will continue to beat in such a solution is somewhat lengthened. Tt is dificult to make a definite statement with regard to this last point, however, on account of large individual variations in the actions of different hearts. A very characteristic effect of the addition of calcium is seen in the gradual retardation of the beats, the contractions coming at regularly increasing intervals until they stop altogether. In some cases instead of this retardation we may find a progressive decrease in the amplitude of the beats while the rate remains fairly constant. If larger amounts of calcium chloride be added to the solution there is a very evident poisonous effect exerted upon the heart by the salt and in a pure calcium chloride solution the effect becomes very marked, the hearts continuing to beat for only a very few minutes even though a large supply of oxygen be available. D. The Effect of the Addition of Potassium Chloride to a Sugar Solution. The addition of small amounts of a ,°; m. solution of potas- sium chloride to a solution of cane sugar in which hearts are immersed brings about a marked increase in the amplitude of the contractions. At first the beats occur much more rapidly than in-the pure sugar solution and these contractions are very powerful. After the first series of very strong contractions, which lasts for only a few minutes (eight or nine at the most) comes a series of contractions of nearly normal amplitude but somewhat more rapid than usual. Following these but coming more slowly is another series of exceedingly strong contractions which 1s finally followed by a rapid decline in the amplitude of the beats. Effect of Salts Upon the Invertebrate Heart. 243 Coincident with this decline is the characteristic increase of muscular tone generally associated with the action of the sugar solutions. A particular feature of the action of solutions con- taining potassium is that a single muscle twitch occupies much less time than when the muscle is immersed in a pure sugar solution, or even in other solutions in which the amount of potas- sium is much less. E. The Effect of the Addition of Magnesium Chloride to a Sugar Solution. When small amounts of a ~; m. solution of magnesium chloride are added to a solution of cane sugar in which the hearts are immersed it is found that the quality of the contraction becomes modified although the length of time during which the heart will continue to Bek; is not alncsedk: The first contractions are usually stronger than normal. After a short series of these powerful beats the beats lost strength and became somewhat irregular, and finally were weak and rapid, with occasional strong contractions scattered among the much weaker ones. F. The Effects of the Addition of Calcium and Magnesium to a Sugar Solution. Small amounts of 3 m. CaCl, and a 3 m. MgCl, solution added to a solution of cane sugar have a marked influence upon the action of a heart immersed in such a solution. ‘The beats become more regular as to time and intensity and the average length of time during which the heart will continue to beat is greater than in the solution with either one salt alone. G. The Effect of Sodium Chloride and Calcium Chloride. _ While in a pure NaCl solution the heart tracings are very similar to the fatigue curves of voluntary muscle, as has been noted by other observers upon other hearts, the addition of a slight amount of a ? or ;4 m. solution of calcium chloride exerts a profound influence upon the heart action. ‘The cardiac 2.44. Charles G. Rogers. contractions become more regular in time and amplitude and last for a longer time than when the heart is immersed in pure NaCl alone. This may be due to one of two causes: either the calcium is necessary in itself for the long continuance of the con- tractions or it may be necessary to counteract the poisonous effects of the NaCl. A record of a ‘single experiment will indicate the trend of results. The heart was teannetsedia in a solution contain- ing 100 cc. 7; m. NaCl plus 3 cc. 7 m. CaCl,, ‘These propor- tions are the same as regards these two salts as were used later in the optimum solution. In this solution the beats continued fora period of more than two hours, probably for more than three but owing to a mechanical defect the heart tracing is imperfect. ‘The record indicates however the main fact which is to be demon- strated—that the addition of calcium chloride to a solution con- taining sodium chloride renders that solution less harmful. In no case did a heart continue to beat for so long a time in a pure sodium chloride solution as in the experiment just mentioned. Whether any other salt may be found to fully take the place of the calcium chloride in the solutions | am at present unable to say. Up to this time none has been found. H. Will NaCl Restore to Activity Hearts Which Have Ceased Beating in Other Solutions? A heart immersed in a solution containing 100 cc. 3 m. cane sugar and .5 cc. 3 m. CaCl, beat regularly for a oe oF hfteen minutes, the beats becoming gradually retarded during that time. During the next fifteen minutes only one contraction was recorded. At the end of half an hour the heart was immersed in a 5% m. NaCl solution and rhythmic contractions began at once and con- tinued for about an hour and a half, becoming more rapid and of less amplitude toward the end of the series. It ought to be stated that in this case the response was unusually prompt and long continued. In another case an immersion for 50 minutes in a solution con- taining 100 cc. ? m. cane sugar, .§ cc. 7, m. CaCl, .75 cc. 2m. MgSQ,, and a slight amount of hydrogen peroxide sufhced to bring the heart to a standstill. The heart was then immersed in a solution of 3%, m. NaCl and after a latent period of twelve a —— Effect of Salts Upon the Invertebrate Heart. 245 minutes contractions were resumed. At first these contractions were very weak and of little amplitude, but they gradually became stronger, and later diminished in the manner common to hearts immersed in a pure solution of sodium chloride. The substitution of what is termed later in the paper the “opti- mum solution” failed to restore rhythmic contractions in hearts which had ceased beating in a sugar solution. A large number of experiments were made to discover if hearts which had ceased beating in a pure NaCl solution could be made to beat again by some other solution. In no case were beats resumed after they had stopped in a pure NaCl solution. ‘This might seem to indicate that in this case irreversible compounds are formed in the tissues of the heart under the influence of the sodium chloride which will not allow rhythmic activity to proceed. J. The Effect of Hydrogen Peroxide and of Oxygen in Solutions. During the course of the experiments it became evident that in some cases at least the failure of the heart to respond to the solutions was due to an insufficient supply of oxygen. Even when well aerated the solutions contain less oxygen ae does the blood by which the hearts are normally surrounded. Lingle! found that the addition of small amounts of hydrogen peroxide to his solutions aided very materially in the lone continuance of the heart beats. My own experiences confirm Aas results in this re- gard. In fact it seems safe to say that without a good supply of oxygen the heart beats are impossible. In many experiments made with sodium chloride as the princi- pal agent there was noticed a very marked loss of tone as the heart continued to beat. At first this was-attributed entirely to the action of the NaCl but later it seemed more probable that the loss of tone was, partly at least, due to the lack of oxygen. Hearts beating 1 in a solution lacking in oxygen show marked fatigue in a short time and finally cease entirely to beat. Such hearts may be revived and again caused to contract rhythmically by simply adding to the solution in which the heart is immersed a little hydrogen peroxide. ‘The.following experiment will illustrate the point in question: When a heart was immersed in what I have 'Lingle. Loc. cit. 246 Charles G. Rogers. shown elsewhere to be the “optimum solution” plus hydrogen peroxide or plus g gaseous oxygen it would continue to beat for a period ranging from 20 to 30 hours. ‘To show the effect of the oxygen a heart was immersed in such a solution lacking 1n oxygen. At first the beats were quite strong but became weaker rapidly and within forty minutes had ceased entirely. When the heart had been in the solution for fifty minutes it was immersed in another solution of the same composition but containing hydrogen peroxide. After a latent period of about an hour and a half con- tractions were again resumed, becoming gradually stronger till they had tench’ a maximum which was steadily Serres When this heart had been beating steadily for one and three- fourths hours it was again Prana in the solution lacking in oxygen. ‘The contractions were almost immediately slowed ‘and were later reduced in amplitude. After being in this solution for fifteen minutes and the beats had become very feeble the heart was again placed in the solution containing the hydrogen peroxide. After a few minutes of weak contractions it again feeoncd and continued to give maximum contractions for some hours. The exact length of time during which the beats continued was not taken. If instead of adding hydrogen peroxide to the solution we allow a current of gaseous oxygen to bubble through it, taking care that the bubbles do not cause sufhicient agitation of the solinion to mechanically stimulate the heart to contraction, we find that the heart will make use of the oxygen held in the solution and con- tinue to beat for a long time. (See Fig. 4.) K. The Effect of Van’t Hoff’s Solution. Van’t Hoff has given us the formula showing the relative pro- portions of the various salts in the sea water. Calcium 1s, ac- cording to his statement, the only considerable variant. The other ale exist in the following proportions: NaCl 100, KCl 2.2, MgCl, 7.8, MgSO, 3.8. If, as has been supposed, these salts exist in the blood and body liquids of the crab in the same proportions in which they are found in the sea water and the heart of the crab derives its stimulus from such a solution, then an artificial solution containing these salts Effect of Salts Upon the Invertebrate Heart. 247 in the proportions mentioned should be as favorable for the con- tinuance of rhythmic contractions as the blood itself provided we add to it the amount of calcium which 1s usually found in the sea water in which the animal occurs. Analyses of the sea water showed that the amount of calcium chloride present in the sea water of San Francisco Bay is about one for every one hundred of sodium chloride. The addition then of a corresponding amount of calcium chloride to the Van’t Hoff solution should render that solution favorable for the life of the hearts. A series of experi- ments with solutions containing sodium, potassium and mag- nesium in the proportions stated and with calcium as a variant were made. The amount of calcium chloride used ranged from .5 cc. to 3.25 cc. for every 100 cc. of NaCl of the same molecular concentration (,%; m.) As the result of these experiments it seems safe to say that the heart of the crab will continue to beat for a long time only 1 in a solution which contains a greater propor- tion of calcium chloride than the sea. water. ne the following experiments the sodium chloride, magnesium chloride, magnesium sulphate, and potassium chloride were employed in the propor- tions stated above, the concentration of the solutions being ;; m. Various amounts of ;% m. calcium chloride were aided as indicated: Exp. 349—1!.0 cc. CaCl, to 100 cc. NaCl, heart beat for 50 minutes. oe “cc “ec oe “ce “ee Exp. 384—1.5 cc. go minutes. “ce “ee oe “ “ “ ee 6 hrs. or more. Exp. 387—2.0 ce. oe “cc “ oe oe “ce “ce Exp. 513—3.0 ce. 18 hours. “ce “ce oe ce ee ee oe 30 “ec Exp. 515—3.25 cc. *(See Figs. 1, 2 and 3.) The above figures do not give an adequate idea of the improve- ment in the action of the hearts caused by the increase in the amount of calcium in the solution. ‘There is a very marked im- provement in the quality of the beats, and the regularity of the contractions in addition to the increase in the length of time during which the contractions may continue as the greater amounts of calcium are added. ‘That a part of the effect of the calc1um con- sists in neutralizing the poisonous effect of the KCl in the solution was shown when the amount of KCl employed was less than that called for by the formula. In such cases the amount of calcium 248 Charles G. Rogers. chloride needed to make a well balanced solution was less than when the full amount (2.2 cc.) was used. When we employ a solution containing sodium, potassium and magnesium in the proportions in which they exist in the sea water ate add a sufhcient amount of calcium to neutralize the poisonous effects of these salts we find that the beats. exhibit a remarkable uniformity of contraction which is long continued. When more than 3:0 cc.of calerum chloride is sidlded to 100 of sodium chloride we Gal that the amplitude of the contractions 1s lessened, but the beats are slower, more nearly the normal rate, and continue through a longer period than in any of the solutions containing less of the caleiee Le Phe Ejfect of Sea Water as a Nutrient Solution. In Van’t Hoff’s solution of ;‘; m. concentration we have been using the various salts in the proportions in which they exist in the sea water, as it is found in the bay. If the concentration of the various salts in the blood of the animal is the same as in the sea water by which they are normally surrounded we should find that when a heart is immersed in sea water it would beat as well as in our artificial solution. “The water used in this series of experiments was taken from the open ocean and hence of higher concentration than the water of the bay. In order to reduce the osmotic pressure of this water to about that of the water of the bay it was diluted with distilled water. It was found that the most satisfactory results were obtained when 85 cc. of sea water and 15 cc. of distilled water were used. But even this dilution did not give a solution which was so favorable for the action of the hearts as was the artificial solution. In the diluted sea water all the hearts behaved like hearts immersed in solutions containing too much NaCl or too little CaCl,, As we have already shown the sea water contains about one part of calcium chloride for every one hundred of sodium chloride. But the artificial solution which had been found most favorable for the long continued heart action contained at least three parts of calcium chloride to every one hundred of sodium chloride. If now we add to the diluted sea water small amounts of calcium chloride so as to raise the proportion of this salt to about that which we have in our Effect of Salts Upon the Invertebrate Heart. 249 artificial solution we should have a solution which would be equally as good as the artificial solution in its action upon the heart of the crab. ‘This was indeed found to be the case. The heart beats became more regular and the length of time during which the heart would continue to beat was much increased. ‘The solution now proved to be in every particular the equivalent of the artificial solution. ‘This suggests the possibility that the con- centration of the CaCl, in the blood of the crab, and also the concentration of this same salt necessary for long continued heart action 1s higher than that of the sea water. M. The Role of Sodium Bicarbonate and Sodium Hydrate in Artificial Solutions. It was found during the course of the experiments that the addi- tion of small amounts of sodium bicarbonate to the solutions em- ployed had a very beneficial effect upon the action of the hearts. For a time it was thought that this substance was in itself a neces- sary component of the liquids intended to favor rhythmic activity. Dr. Loeb! has shown that the sea water is practically neutral in reaction. How the bicarbonate could affect the action of the heart was a puzzle until it was remembered that very small amounts of free acids in artificial solutions may exert very in- jurious effects. ‘The role of the bicarbonate in neutralizing any free acid that may be present in the solutions throws a new light upon the subject and makes its presence desirable. It has the power to neutralize acids and yet is not itself alkaline in reaction. It is therefore possible to have solutions containing an excess of this substance without affecting the neutrality of the solution. By thus neutralizing any free acid which may be present we make the conditions most favorable for heart activity, and add for the proof of the fact that the body liquids are neutral in reaction. The addition of small amounts of ~, NaOH will have exactly the same effect. Care has, of course, to be taken not to add too much of this substance as the solution must not be too alkaline in reaction. ‘Loeb, J. Archiv. fiir die gessammte Physiologie, Bd. 99, 1903, p. 637. 250 Charles G. Rogers. N. The Substitution of Another Metal jor Sodium. Dr. Loeb! has shown that in the case of the skeletal muscles which are made to give rhythmic contractions by means of elec- trolytes, it 1s possible to substitute in the place of the sodium another metal, especially lithium. Up to the present time no one has succeeded in making such a substitution in the case of cardiac muscle. A large apnier of experiments were made using lithium chloride in the place of sodium chloride in the Van’t Hoff solution. In no case was it found that rhythmic contractions would con- tinue in such a solution for a longer time than they would in a pure sugar solution. SUMMARY AND CONCLUSIONS. 1. The blood of the crab studied, Brachynotus nudus, probably has the same concentration as the average of the sea water of the bay. 2. On account of the fact that the heart does not cease beating when it is removed from the body of the animal it is impossible to determine that any substance is essential for the origination of rhythmic contractions. It has been demonstrated, however, that such contractions will not long continue when NaCl is absent. Calcium, potassium and magnesium each have an important influence upon the heart contraction. 3. The balance between the salts entering into the composition of the artificial solution, and presumably of the blood also, is a very delicate one and can be determined with great accuracy. 4. Sodium chloride has the power to restore rhythmic contrac- tions in hearts which have ceased beating in some other solutions. 5. Hearts which have ceased beating 1 in a pure NaCl solution do not again beat when placed in a solution lacking in NaCl. 6. The presence of a supply of oxgyen in the solutions is neces- sary for rhythmic contractions. Oxygen may be supplied by adding small amounts of hydrogen peroxide to the solution or by allowing oxygen gas to bubble through it. 7. The solution most favorable for rhythmic contractions of the crab’s heart was found to have the following composition: ‘Loeb, J. Festschrift fiir Professor Fick, 1899. Effect of Salt U pon the Invertebrate Heart. 251 100 parts NaCl, 7.8 parts MgCl, 3.8 parts MgsO,, 2.2, parts KCl, 3-25 parts CaCl, all of 7; m. concentration and oxygen. In case the oxygen is added in the form of the peroxide, ES digm bicarbonate or sodium hydrate must be added to neutralize the acid introduced with the peroxide. 8. Sea water to which has been added calcium chloride acts in the same way as does the artificial solution. g. The normal circulating fluid of the crab must contain a larger proportion of calcium than does the sea water. 10. Lithium chloride can not be substituted for sodium chloride in the artificial solutions. 252 Charles G. Rogers. EXPLANATION OF PLATE. All records read from right to left. The time marker indicates intervals of thirty seconds. Fig. 1. .Record of a heart beating in a solution containing 100 cc. $ m. NaCl, 2.2 cc. $ m. KCl, 7-3 cc. 2 m. MgClo, 3.8 cc. $ m. MgSOu, 1 cc. $ m. CaCl, .75 cc. § m. NaHCOgz and oxygen. It willbe noticed that the first beats were strong, but became rapidly weaker, then slower, finally ceasing in less than an hour from the beginning of the experiment. The curve is characteristic of all hearts beating in solutions in which the amount of Na is too great, or the Ca too small. Fig. 2. Record of a heart beating in a solution similar to that mentioned above, except that 2 cc. % CaClo were employed. This heart beat for a much longer period than that in the previous experiment. Section } of the record was taken two hours from the start a; section c four hours and section d six hours. Fig. 3. Record of a heart beating in a solution similar to those used in the above experiments except that it contains a larger amount of CaClo, viz: 3 cc. $ m. solution. The beats maintain their first strength and rate of contraction for a long period. Section 6 is taken four hours from the beginning of the record, section c seven hours, section d eleven hours, section e eighteen hours and section f twenty hours. At the point x in section d a couple of drops of hydrogen peroxide were added to the solution in which the heart was immersed. Its effect is shown in the ensuing stronger and more rapid contractions. Fig. 4. Portion of the record made by a heart beating in a solution similar to those above, but con- taining 3.25 cc. $m. CaClz. The beats shown at a are characteristic for hearts beating in such a solution and under ordinary conditions will continue for periods of thirty hours or more. At the point b the solution containing oxygen was replaced by one which had been heated to expel the gases in solution and then cooled to the same temperature as the solution first employed. The effect of the lack of oxygen is Shown in the regularly decreasing force of contraction. At c the heart was again placed in the solution containing oxygen, and after a short time regained its former strength and continued beating for a number of hours. ‘This experiment was repeated frequently with similar results. a EFFECT OF SALTS UPON THE INVERTEBRATE HEART. Cuarces G. Rocers. iat ae c Ye Tuer JourNaL or ExperIMENTAL ZoOLoGy, vol. ii Fig. 4 SUDIES ON REGULATION. VII. FURTHER EXPERIMENTS ON FORM-REGULATION IN LEPTOPLANA, BY Cave GEE D:: Witu 34 Ficures. The present paper is devoted to a consideration of certain phases of the process of form-regulation in Leptoplana, which, although of great interest when viewed in the light of the conclu- sions reached in previous papers on Leptoplana (Child, ’o4a, ’o4b, ’04c), do not in themselves afford sufficient data for these conclusions. Considered at this time they serve to confirm and extend the conclusions already drawn from other data. A. TYPICAL CHANGES IN PROPORTION DURING REGULATION. During the process of form-regulation of pieces in many of the lower animals certain changes oF form occur which involve not only the new parts but Aa the old fully differentiated regions. Under normal conditions these changes consist in approximation to the typical proportions of the species. A description of these changes in Planaria, where they are considerable, has been given by Morgan (’00) who has applied to them the term “morphal- laxis.” I have shown recently (Child, ’o2, ’03) that similar changes in Stenostoma are at least in part the result of traction upon the, parts in certain directions in consequence of the charac- teristic motor activity and I have obtained strong evidence as yet unpublished, that the same factors are concerned in Planaria. But the term “morphallaxis”’ has been applied to phenomena which, in my opinion, are wholly diverse and therefore, although I have employed it in certain previous papers (Child, ’o2, ’o3a, "03b), it seems preferable to use some less vague term. Driesch (or) considers “morphallaxis” identical with his “Restitution durch Umdifferenzierung,” but morphallaxis may occur without 254 C. M. Child. b any “‘redifferentiation’ 99): Some term is necessary to denote those changes of form in the Turbellaria and other groups which are primarily mechanical and connected with motor activity. There is no fundamental difference between such changes in the new parts and in the old. Both, as well as many other regulative phenomena, may be included under the head of mechanical regulations (Child, ’o2). The fact that the typical proportions usually result is merely incidental. I have shown (’02) that in the absence of the loco-! motor tensions the result may be exactly the reverse. Until opportunity offers for a more extended discussion of this matter IE prefer to designate these changes merely as changes in propor- tion. : During form-regulation in Leptoplana changes in proportion similar to those occurring in Planaria and Stenostoma occur. In pieces containing the cephalic ganglia changes are considerable, though not as great nor as rapid as in Planaria and Stenostoma, a diffcrerice which is evidently due to the fact that the tissues of Leptoplana are less soft and plastic than those of the other forms mentioned. ‘The changes consist in relative elongation and re- duction of the transverse diameter, especially cowed the posterior end. In order to make clear my point of view in these experiments it is necessary to refer briefly to earlier experiments on Stenos- toma and Leptoplana (Child, ’02, 03a). In the case of Stenos- toma I found that the changes in form and proportion of the pieces during regulation, 7. ¢., the elongation and the change from cylindrical to conoidal form were due primarily to ches tension upon the tissues consequent upon the use during locomotion of the posterior end as an organ of attachment. It was possible to inhibit or retard the change in proportions by preventing the pieces from attaching themselves to the substratum (Child, ’03a). In Stenostoma the changes in proportion are much more rapid than in other forms examined, being completed in many cases in twenty- four to thirty hours. In my first paper on Leptoplana (Child, ’o4a) a brief descrip- tion of the method of locomotion was giv en, certain points of which must be recalled to mind. In creeping, Leptoplana uses the margins of the head region for drawing the body forward, as in the case of Gonionemus (Morgan, Studies on Regulation. 255 while the margins of the body from about the middle or a little anterior to it, to the posterior end are employed as organs of attachment, the most posterior part, the “tail’”’ being most fre- quently used in this manner. ‘The body of the animal is fre- quently subjected to tension during creeping and I believe that, as in Stenostoma, this tension is of considerable importance in determining the general form. Its effect upon the newly formed regenerating parts has already been discussed (Child, ’osb, "O4c). We have now to consider the changes in proportion of the old parts during regulation and to discuss the role of mechanical factors in these changes. Since the changes are slow it is imprac- ticable to control them by preventing the animals from attaching themselves to the substratum, as was done in the case of Stenos- toma, but modification of the changes is possible by certain meth- ods which will be described below. In Stenostoma (Child, ’o2) the posterior region of the body 1s subjected most frequently and in greatest degree to tension, but in Leptoplana the lateral margins of the body as well as the posterior end are used for attachment, so that very frequently only the anterior portions of the body are subjected to tension, the whole posterior portion being attached. However, the posterior end 1s usually the first part to attach itself and the last to be released, hence in the long run it is undoubtedly more stretched than other parts. It is probable that the outline of the body of Leptoplana is due in large part to the mechanical factors connected with attachment and locomotion. A form which like Stenostoma uses only the posterior end and the median ventral region for attach- ment must be slender and only slightly tapering in the definitive condition, while on the other hand a species which uses the mar- gins of the body for attachment will be broader and the decrease in breadth toward the posterior end will depend upon the relative frequency with which the lateral margins and the posterior end alone are used for attachment. In Leptoplana the posterior end is still the principal organ of attachment and the body possesses a tapering form as it must according to mechanical principles if it is plastic. But in such forms as Stylochus where the whole margin is used as an organ of attachment and the posterior end is used with no greater frequency than other parts the form of the body becomes ovoid or almost circular. (See Child,’o4a.) ‘The 256 C. M. Child. more frequently the lateral margins are extended laterally and attached, the broader does the body of the worm become. When a part of the body of Leptoplana is isolated from the other parts it is necessary to consider the particular conditions in each case before we can understand the changes that occur. ‘The mechanical conditions connected with locomotion will differ according to the region of the body from which the piece is taken, since different regions show different behavior as regards attach- ment and, what is more important, the kind and degree of change in the direction and amount of tension to which the tissues are subjected, will differ greatly according to the regions of the body involved, the amount and kind of movement and various other conditions. Usually observation of the pieces during locomotion is the only satisfactory method, for this is the only way of deter- mining how a particular piece uses a certain part or when it begins to use the regenerated tissues in locomotion. All figures are drawn from careful measurements made when the specimens were fully extended, the following measurements being made in each case, where the parts mentioned were present: length of animal or piece, distance from anterior end of head to middle of groups of eyes, distance from anterior end of head to anterior end of pharynx, length of pharynx, length of new tissue, width of head at widest part, width of head at level of eyes, width of body at posterior end of phary nx, width of body I-2 mm. anterior to cut surface, 7. e., just anterior to the region which has contracted in consequence of the cut, width of new tissue at cut surface, one or two other measurements of width of new tissue at different levels according to form of this part. While the speci- men was under observation figures were drawn from the measure- ments in order that local curvatures and other special features not indicated by the measurements might be recorded; the extent of the intestinal branches in the new tissue was also indicated in the figures. Tle Ex periments. From some twenty-five series of experiments concerning the changes in proportion four have been selected which show the results obtained after section at different levels of the body. In order to permit direct comparison the more important measure- ments of these series are grouped in tabular form. -All measure- Studies on Regulation. 257 ments given are reduced to millimeters. ‘The table gives simply the measurements of the pieces concerned; not of the whole animal from which they were taken. ‘The first measurements of TABLE OF MEASUREMENTS. S cs \ = 4 dics! mee |e g 5 = oF Vile Se os : see il gee | 5S hvac itl} AES OS te | Salers (CONCEUMSIONS. My experiments consist in cutting singly unfertilized eggs by horizontal section at two different periods and in treating the enucleated fragments thus obtained with a solution of CaCl.,. By these experiments I think I have established the facts, (a) that at the period of the metaphase of the first maturation mitosis cytasters can arise at any point of the egg,’ but (>) that prior to the fading of the germinal vesicle cytasters never arise. (cf. foot- note on p. 304.) In all the cytasters developed in enucleated fragments there is a central group of dark staining bodies, which I do not hesitate to identify as multiplied centrioles. It is to be regretted that I did not find in any enucleated fragment either a single centriole or one in division. ‘This, however, does not etic the general conclusion for the reason that centers of exactly the same nature as those in enucleated fragments are found in the nuclear division figure in whole CaCl, egg. ‘My experiments show that cytasters appear in the vegetative half. It was, however, impossible to test experimentally whether the cytasters develop in the vicinity of the first maturation mitotic figure. Nevertheless it will not be unreasonable to infer that they may so arise there from the fact that the whole CaCls egg has many cytasters near the animal pole as well. Centrosome in Enucleated Egg-Fragments. 309 My cutting experiments were performed at two periods, one immediately after release and the other an hour and a half later. From these we can by no means determine exactly when the cytoplasm acquires the power of producing the centrioles and ray system. What brings about the change in the characters of the cytoplasm during this interval? In all probability the inter- mingling of the nuclear fluid and cytoplasm during the time of fading of the germinal vesicle gives to the cytoplasm the aster producing power. A striking difference between the matured and immature cytoplasms has been described by many observers. Delage emphasizes the fact that during this period, when the cytoplasmic maturation takes place, the eggs become fecundable both in Strongylocentrotus (99) and in Asterias (o1.) Wilson verifies this phenomenon in the eggs of Cerebratulus (’03, p. 417). Spermatozoa can enter immature eggs freely, but they remain undeveloped. (O. and R. Hertwig,’89, p. 199; Wilson,’96, p. 149.) In immature cytoplasm not only is the development of the sperm nucleus and ray system inhibited, but also the centrioles do not arise in eggs, even if they are treated by salt solutions. Morgan (’99) noticed that cytasters did not develop either in the ege of Sphzrechinus or of Sipunculus before maturation begins, and I was told by Professor Wilson that he observed the same fact in the MgCl, egg of Toxopneustes. Leaving open for the present the question how the nuclear fluid acts upon ithe cytoplasm we can at least say that the matured cytoplasm is ready to produce or, in other words, has the power to form centrioles as well as rays as a result of certain stimuli, this being in our case a CaCl, solution. As to the origin of the centrioles in the cytasters there are two possibilities besides the one just mentioned. First, as suggested by Meves the centrioles might multiply in the cytoplasm during the growth period of the egg and become the centers of the cytas- ters under the action of a salt solution. Such an assumption is not in contradiction with what has just been said, that asters do not develop in unmatured cytoplasm even when the spermatozoon brings a centriole into the egg, since the centrioles might be present, but incapable of producing asters until the germinal vesicle fades. “There is another possibility similar to the above, namely, that centrioles may be present as such in the nucleus and, at the dissolution of the germinal vesicle, escape into the cytoplasm 310 Naohidé Yatsu. where they acquire rays and thus give rise to the cytasters. Apart from the fact that neither of these assumptions is supported by any direct observations they contradict the definition of the cen- trosome as given by Boveri (’01, pp. 132, 162, etc.) that the organ is single (or double by anticipation). A multiplication of cen- trioles capable of producing centrosomes 1s nowhere known to take place unless it be in abnormal or degenerating cell, such as the giant cells or the oligopyrenous spermatozoa. It may be said that centrosomes (centrioles) arise by the enlargement of ultra- microscopical granules or plastids that coexist with the visible astral centriole. ‘This is quite possible, but if visible centrioles may thus arise in addition to the visible ones already existing and independently of them centrosome formation de novo in the ordinary sense of this ex pression is demonstrated none the less. The results of my cutting experiments, therefore, I believe, lead us to the unavoidable conclusion that the centrioles are formed de novo, as Wilson maintained. In conclusion one word about the nature of the sperm centriole. One might be readily led to infer from what I have said that the sperm centriole in the normally fertilized egg may arise in the same manner as those found in the cytasters. In Cerebratulus, at least, this is not the case, for | have been able to show that the centriole, as such, is actually brought into the egg in the middle . piece of the spermatozoon. Detailed evidence in support of this statement will be published hereafter. VII. SUMMARY. 1. When subjected to the action of a solution of CaCl, enu- cleated fragments of unfertilized egg of Cerebratulus lacteus, obtained by cutting the eggs singly at the metaphase of the first maturation mitosis, develop true asters containing central bodies. The corresponding nucleated fragments show the typical matura- tion spindle. 2. Cytasters do not, however, appear in enucleated fragments from unfertilized eggs before the fading of the germinal vesicle. 3. The central bodies of the cytasters developed in enucleated fragments are centrioles identical in structure with those in the nuclear asters of whole eggs similarly treated. Centrosome in Enucleated Egg-Fragments. 260 4. Centrioles, therefore, can be produced de novo in the matured cytoplasm (7. ¢., after the dissolution of the germinal vesicle). Zodlogical Laboratory, Columbia University. January 23, 1905. Vili Site RATU RE Boveri, [H., ’01.—Ueber die Natur der Centrosomen: Zellenstudien, Heft 4. *02.—Das Problem der Befruchtung. DeExacE, YVES, ’99.—Etude sur la mérogonie: Arch. Zool. exp. (Ser. 3), 7. ’o1.—Etudes expérimentales sur la maturation cytoplasmique et sur la parthénogénese artificielle chez les échinodermes: Arch. Zool. exp. (Ser: 2),0: FiscuEer, M. H., anp OstwaLp, W., ’05.—Zur physikalisch-chemischen Theorie der Befruchtung: Arch. f. d. ges. Physiologie. 106. Hertwic, O. anv R., ’87.—Ueber den Befruchtungs- und Teilungsvorgang des tierischen Eies unter dem Einfluss ausserer Agentien: Jen. Zeit. 20. Hertwice, R., ’02.—Die Protozoen und die Zelltheorie: Arch. Protistenkunde 1. Meves, F., ’02.—Ueber die Frage ob, die Centrosomen Bovert’s als allgemeine und dauernde Zellorgane aufzufassen sind: (a) Verhandl. der anat Gesell. in Halle. b) Mitteilungen Aerzt. Verein Schlesw.-Holst. Jgate; Ne G: ’03.— Ueber oligopyrene und apyrene Spermien und uber ihre Entstehung, nach Beobachtungen an Paludina und Pygaera: Arch. mikr. Anat. 41. ; Morean, T. H., ’96a.—On the production of artificial archoplasmic centers: Rept. of the Am. Morph. Soc., Science N.S. 3, No. 54. *96b.—The production of artificial astrospheres: Arch. Entwm. 3. ’98.—The effect of salt-solutions on the unfertilized eggs of Arbacia: Science N.S. 7, No. 164. ’99.—The action of salt-solutions on the unfertilized and fertilized eggs of Arbacia and of other animals: Arch. Entwm. 8. ’o0.—F urther studies on the action of salt-solutions and other agents on the eggs of Arbacia: Arch. Entwm. Io. PETRUNKEVITscH, A., ’04.—Kiinstliche Parthenogenese: Zool. Jahrb. Supple- ment 7. Vejpovsky, F. anp MrAzek, A., ’03.—Umbildung des Cytoplasma wahrend der Befruchtung und Zellteilung, nach den Untersuchungen am Rhynchelmis-Eie: Arch. mikr. Anat. 62. 312 Naohidé Yatsu. WassILiEFF, A., ’02.—Ueber kinstliche Parthenogenesis des Seeigel-Eies: Biol. Centbl. 22 No. 24. Witson, E. B., ’96.—The Cell: 1st Ed. New York. ’o1.—A cytological study of artificial parthenogenesis in sea-urchin eggs. Experimental Studies in Cytology I: Arch. Entwm. 12. ’03.—Experiments on cleavage and localization in the nemertine egg: Arch. Entwm. 16. ‘o4.—Cytasters and centrosomes in artificial parthenogenesis: Zool. Anz. 28. Yarsu, N., ’04.—Aster formation in enucleated egg-fragments of Cerebratulus: Science N.S. 20, No. 521. From the Biological Laboratory of Bryn Mawr College. Aes TUDY OF Crh GERM CELLS OF APHIS; ROSA AND APHIS G2NOTHER A BY N. M. STEVENS. Associate in Experimental Morphology, Bryn Mawr College, and Research Assistant, Carnegie Institution of Washington. : Witu 4 Pirates. ieee MiatertalwandiMethodser ete oe ces aii fs bys laos Gece Mya Malara a eRe Seminar oeleens 314 lig Earthenopenctica Developments. jr\an ciel ccc eteveier= 2 a. @ els eiebotsie) a cistccke ete tie sitet ay Sete oles 314 Ape Demmale BlsTOw ay Ns meals tate eARACoc erate eae ee Ata Rn er ateeers aictedta rte ane ee ere 314 mE ALEY ITO sc Ae pcheaeagreeeyceetinte ean Conese A ORE eaah tate a aie Nacsa eve Rr iter aie ate. 316 Heeb AW inten ope sta nencrels cueyataer soir eines Braictys aaa atts tae eee e aie Mie nTee) eease 318 teambanly Development, and. Growthen qm ciee eee ares cieae ols iclsrerseate sre ays arose Toei 318 Dis meV ACUIT a GON tks fia roxers foyer Noise tareyvoqare' l= sh spailae sp sgh BNO UC ae oh nasil a eioutuetave ie niall aera ete 320 iTWViewe S PERTN ALO DENESIS: cc scr-teues eva 's ate ete Pet edc yok To 9afatoa vo reer ey FIST Tora © Rates erat eee 321 Vise enerall Miscussioniss< Hace as cts Whe ies ms ae A oe re See ee oe cha a 228 1. Mendel’s Law, and the Individuality of the Chromosomes............-...--.-+-- 323 Zee Maturation sobs bartieno PeneeiGish Poss pete reeeNstetistetats rie tenet nana 324 25 e JD) etermin ation” Off SEX snacoi toretofe vane pues taney tate ers Sesser rete Sposa ataters eee eins ae 326 Wile Summary. /of Results) savers 21-1 torte erase ervoeie tern cies Se rete oe Se oars eer ee eats ee 329 This work was undertaken in connection with a series of experi- ments on sex-determination in Aphids. On approaching the subject, it was found that veLy little is known concerning the ovo- genesis and spermatogenesis of these insects. Blochmann (’87) showed that the parthenogenetic egg gives off one polar body, and the winter egg two; but I find no account of the number of chro- mosomes or the meno of reduction in either egg, nor is there any published work on the spermatogenesis of Aphids. ‘The present paper will be devoted mainly to the germ-cells, a few points in the early stages of parthenogenetic development being considered incidentally. ' This paper was awarded the prize of one thousand dollars offered in 1904 by the “Association for Maintaining the American Woman’s Table at the Zodlogical Station at Naples, and for Pro- moting Scientific Research among Women.” 314 N. M. Stevens. [MATERIAL AND ME TREO DS: Sections of Aphids from various host plants—the rose, English ivy, honeysuckle, alder, arrow-head, C‘nothera, and hop, were examined; but Aphis rose proved the most satisfactory for study of the ovogenesis of both parthenogenetic and winter eggs, and Aphis cenotherz for the spermatogenesis. Both the Aphids and the winter eggs were killed and fixed with Gilson’s aceto-chloroform-sublimate mixture, which gives far better results than any other method tried. Very small Aphids were embedded whole; larger ones after removing the head and thorax. Sections were cut from 5 » to 7 » thick, and stained with Delafield’s hematoxylin and orange G, or with Heidenhain’s iron- haematoxylin. ‘The former method of staining gives remarkably good differentiation for ordinary purposes. The latter is more satisfactory for the study of ovogenesis and spermatogenesis. Many slides stained at first with Delafield and orange, were re- stained with iron- hematoxylin for more careful study. li PARTHENOGENETIC S DEVE TORMENT: T. Female Line. Most of the stages in ovogenesis of parthenogenetic eggs and also early segmentation stages were obtained from sections of the unborn young of the viviparous female. For more advanced segmentation stages and early embryos very young Aphids were sectioned. All the species examined have twelve ovaries in two groups as described by Balbiani (69-72). Oogonial mitoses were observed, but offered nothing of especial interest as the division figures are identical with other embryonic mitoses. The resting odcyte, before the growth stage of the ovum begins, ‘has a very large nucleus in proportion to the size of the cell (Figs. I and 2). A ‘large nucleolus occupies the center of the nucleus, and in addition to this the nucleus contains a linin reticulum and irregular masses of material, presumably chromatin, which does not take the haematoxylin stain at this stage. One at a time the od¢ytes at the posterior end of the ovary enlarge and push out into the oviduct, remaining, however, con- The Germ Cells of Aphis. 215 nected by a stalk with the central core of the ovary (Fig. 1). As many as three eggs may be thus connegted by stalks with the cen- ter of an ovary,—one egg just passing into the oviduct, another in maturation stage, and a third in 8 to 16-cell stage. As the odcy te increases in size, the cytoplasm changes its staining reaction, coloring somewhat deeply with Delafield; and the nucleus shows various changes. ‘The nucleolus disappears and the chromosomes become stainable at an early stage (Figs. 1, 3, 4, 5). As the egg rapidly enlarges the nucleus approaches the periphery at one side of the oval egg (Fig. 6). Just before maturation, clear vesicles appear in the cytoplasm and soon fuse forming large irregular spaces filled with a clear non-staining substance, presumably yolk material, separated from the general cytoplasm (Blochmann). Fig. 7 shows the equatorial plate of a polar spindle in metaphase. There are 10 chromosomes of 5 different sizes. ‘This is the so- matic number, and there is therefore no synapsis or conjugation of chromosomes and no reduction in the maturation of the female parthenogenetic egg. [he same number (10) and the same varia- tion in size (5 pairs) is shown in the segmentation spindles and equatorial plate of Fig. 12. The maturation spindle in anaphase is well shown in Fig. 8; also the lacunar spaces in the interior of the egg. The polar body 1 is at first completely extruded from the egg and separated from 1 it as seen in Figs. 8 and g. It is distinctly a ‘polar body not a “polar nucleus. mm Soon, however, it comes to lie within the boundary of the egg among the segmentation nuclei (Figs. 10 and 11). In such a stage as in Fig. Ti, the polar body is easily recognizable by its alot cytoplasm and deeply- staining mass of chromatin. It lies in a sort of vacuole in the egg-cytoplasm. I have been able to follow the polar body as far as the stage shown in Fig. 13. The cytoplasm can no longer be distinguished from that of the e ge; the chromatin mass 1s irregular in outline, usually stains less deeply and appears to be degenerating. I therefore feel sure that the polar body takes no part in the development of the embryo. Its inclusion within the egg 1s probably due merely to mechanical conditions, 7. ¢., to the pressure of the walls of the oviduct upon it, as it lies on the side of the oval egg. There is only one point of especial interest in the later develop- ment, and that is the relation of the young embryo to the vitellaria. 316 | N. M. Stevens. ‘This is shown in Figs. I4and 15. Fig. 14 shows an embryo which has just begun to paler in yolk (the ‘ eee Dotter” of Will, 30) At the base of the embryo as figured are two. conspicuous cells (b) which apparently guard a valvular opening in the wall of the oviduct, and recall the four guard-cells at the inner end of the embryonic pharynx of Planaria simplicissima. At the lower focus of the section the valve is slightly open and a small amount of yolk material has entered (Fig. 14, a). In Fig. 15 the valve is widely open and yolk cells are being taken into the embryo. Whether the embryo actively sucks in the yolk, or the yolk cells themselves are the active agents, it is impossible to tell, but the former seems more likely. Will (89) describes the secondary yolk as forming 1 in connection with the follicle epithelium and then being taken into the gastrula. In speaking of the work of Wit- laczil (°84), he says, ““Von seiner ganzen Darstellung ist nur das eine richtig, das der secundare Dotter yon Follikelepithel seinen Ursprung nimmt,” and later he says, “ Diese innerhalb der Epi- thelverdickung producirte Dottermasse ist es nun welche in das Fi eintritt und demselben den sogenannten secundaren Dotter liefert. Dass es sich dabei nicht um eine Einwanderung von Zellen e The close resemblance in structure between the secondary yolk and certain large cells in the body cavity of the mother was easily observed in the sections, but the relation between the embryo and the vitellaria described above was first seen in connection with the egg of Aleurodes, a related form, where the relation is much more conspicuous than in the Aphid. It was later traced with certainty in the winter egg and in the parthenogenetic embryo of the Aphid. No variation could be detected in the development of ova which produce winged parthenogenetic individuals. The winged young can often be distinguished before birth, and some of the same brood may be winged, others @pterous. “The winged par- thenogenetic individuals are ne he their appearance seems to be conditioned by the amount or the quality of the food supply. 2. Male Line. The mothers of the males are apterous and cannot be distin- guished externally from the apterous females which produce female offspring. ‘The ovaries show no difference in structure, unless it may be in size and number of the odcytes, and only one The Germ Cells of Aphis. 317 polar body is given off. In studying the development of male! eggs, one is at a disadvantage so far as the amount of available material 1s concerned, because only eggs connected with the ova- ries of females which contain embryos large enough to be recog- nized as male can be utilized, while in the female line the best eggs for study are found in abundance in the larger embryos. No polar spindles were found in the few male eggs obtained. Figs. 17 and 18 show the polar body as it appears in eggs containing 4 and 8 nuclei. ‘There is no indication of fusion of ean polar haces In Fig. 18 the chromosomes are not so fully fused as in Fig. 17, but this is the condition at a corresponding stage in the female eggs. Examination of the segmentation spindles in irale embryos and of spermatogonial mitoses makes it certain that the full somatic number of chromosomes is present until we come to the spermato- cytes when reduction occurs. ‘This is what we should expect had it not been claimed by Castle (’03) that the female character which is usually dominant in parthenogenetic insects is removed from the egg with the second polar body, thus allowing the reces- sive male element to assert itself. ‘Tienes is no evidence in my material of any difference between the maturation of the female parthenogenetic egg and that of the male egg, and until I can procure more material and examine the point further, I shall assume that the method of maturation of all parthenogenetic Aphid eggs is the same, 1. ¢., only one polar body is produced and there is no reduction of chromosomes. There is no mixture of male and female young in the offspring of one individual: certain apterous females produce only females, either winged or apterous, and others produce only winged males. The ovarian odcytes, eggs and polar bodies in the male line (Figs. 16, 17, 18) are noticeably larger than those figured in the female line (Figs. 1-12), but this may be due to the fact that the drawings were made from different species; the former from Aphis Geiothere: the latter from Aphis rose. ‘The difference 1s one of size not of structure, and much the best and most abundant male material was obtained from Aphis cenotherz, where partheno- genetic reproduction was wholly replaced by the sexual method early i in October, and young males of all ages were abundant. 1Male is used here merely in the sense that these eggs produce males. : @ 318 N. M. Stevens. Ill. THE WINTER EGGS. I. Early Development and Growth. ‘The material for study of the winter eggs was obtained by bring- ing into the laboratory rose twigs with broods of sexual females on the leaves. Adult males were usually found on the same leaves. [hese young females develop more rapidly in a warm room under glass, and soon begin to lay the fertilized eggs. “The winged mothers of the sexual females were sectioned for the study of the ovaries of the sexual female embryos, and young sexual females for early stages in the development of the winter egg. Fig. 19 shows an ovary from such an embryo. It is considerably larger than the ovary of the ordinary parthenogenetic female embryo (Fig. 1), and in every case these ovaries show a large num- ber of degenerating odcytes at the posterior end (Fig. 19, a). These cells are more or less shriveled and stain deeply and irregu- larly. Whether this is simply degeneration of a large number— at least half—of the odcytes of an ordinary parthenogenetic ovary in order that the remainder may have room for growth, or whether the parthenogenetic ovary may contain originally both eggs capable of parthenogenetic development and others capable of development only after fertilization, and the former degenerate in order that the latter may develop, I am unable to say. The number of odcytes in the twelve parthenogenetic ovaries is cer- tainly more than double the number of young ever produced by an individual, so that the latter supposition might be possible. Two cases observed in dissection, but never duplicated in fixed material, might support either view, and certainly tend to show that the ovary which produces parthenogenetic embryos and the ovary which produces winter eggs are originally identical. In two individuals ege-strings and winter ovaries with developing eggs were found associated in both groups of ovaries. “Che num- ber in each varied—in one individual one group contained 5 eg strings and one winter ovary, the other group 3 egg-strings be 3 winter ov aries; in the second individual each group contained 5 winter ovaries and one egg-string; on one side there was one parthenogenetic ovary with eggs and embryos (Fig. 20, c and e), on the other side was an egg-string consisting of two partheno- genetic embryos, a winter ovary and a young winter egg (Fig. 21, a, bande). ‘These individuals with mixed ovaries were found in The Germ Cells of Aphis. 210 the greenhouse January 23, 1904, on a small rose bush which had lost its leaves and was nearly dead as a result of serving as a food plant for several generations of Aphids. They were the third generation from a winged female. ‘Iwo or three others escaped dissection. Unfortunately only freehand sketches were made, and no other such cases were observed. One of these sketches is reproduced in Fig. 20 and a part of another in Fig. 21. Similar observations were recorded by Bonnet (’45) and by Leydig (’50), and Kyber (’15) observed sexual forms on the willow in June and on ripening grain in midsummer, but did not connect both par- thenogenetic young and winter eggs W ith the same individual. The oocytes in the winter ovaries increase immensely in size before any eggs are given off, and the large nearly spherical ova- ries are easily distinguished in dissections from the minute oval parthenogenetic ovaries (Fig. 20, a and c). The first egg is given off after the birth of the sexual female. It is not my purpose to describe the growth period of the winter egg in detail. ‘The nucleus, at first central, gradually moves to the periphery at one side, usually nearer the anterior end of the egg as it lies in the oviduct. When the egg has reached about one- half its ultimate size, yolk material from the vitellaria 1s taken in at the posterior end of the egg. There seems to be no such definite opening as in the case of the parthenogen- etic embryo and of the egg of Aleurodes. Fig. 22 is an oblique section through an egg which plainly howe the rela- tion between the yolk within and that without the egg. Entrance seems to be effected between any of the cells of the follicle epithe- lium, which is merely a part of the oviduct. Here again one wonders which is the active agent, the egg or the yolk celles but it is impossible to tell. In the case of the embryo one is inclined to believe that it may actively suck in the yolk as the planarian embryo does, but there is nothing to indicate that the egg could have any such power. In the case of Hydatina senta, Lenssen (98) describes the yolk cells as penetrating the egg by their own activity. The entrance of yolk seems to be associated with a definite size of the ovum and would therefore occur at a definite point in the oviduct. ‘The relation between the oviduct and the volk gland may therefore be a more definite one than the sections show. [Entrance of yolk must be effected very quickly as cases where the process can be demonstrated are very rare; and one 320 N. M. Stevens. finds no such intermediate stages as would necessarily appear if this secondary yolk were formed within the egg as described by Balbiani (’69-’72). The outlines of the yolk cells are lost and only fragments of nuclei are found (Fig. 22), while in Aleurodes several whole yolk cells with nuclei intact enter the oviduct below the egg and are later included in the posterior end of the egg. The chromatin in the egg during its growth period offers no favorable conditions for study. In earlier stages it does not take chromatin stains, and in later stages the chromosomes are spheri- cal and mingled with nucleoli of similar form and staining qualities. 2. Maturation. ‘The earliest stage found in the laid egg was that shown in Fig. 23—the equatorial plate of the first polar spindle, showing 5 chromosomes, the reduced number, of the same relative form and size as the chromosomes of the 5 pairs in Figs. 7 and 12. The manner in which the egg chromosomes are paired is not evident, but the two divisions appear to be longitudinal and iden- tical with the maturation divisions of the spermatocyte where it is quite certain that they are paired longitudinally, and probable that the first division separates the paired chromosomes. Fig. 24 shows the first maturation spindle in metaphase. One chromosome appears in both figures (stippled in 6). In Fig. 25 the first polar body and the second polar spindle in metaphase are figured. A part of a chromosome appears in a and parts of two in the spindle of b. In Fig. 26, a later stage is seen; the chromo- somes of the first polar body are massed together, those of the second (not yet separated from the egg) show the comparative size relation of Fig. 23, their position indicating a longitudinal division; the chromatin ne the egg nucleus is becoming diffuse and less stainable. The spermatozoon enters at any point, more often near the pos- terior end of the egg, and leaves a train of cytoplasm behind it, as it traverses the yolk preceded by an aster. Fig. 27 shows the male and female pronuclei, the former distinguished by the cyto- plasmic path (indicated by arrows). I have never found the first segmentation spindle in my material nor indeed the divisions immediately following, though the resting nuclei of these stages have frequently been observed. In the later segmentation stages The Germ Cells of Aphis. 321 it is impossible to distinguish the individual chromosomes in the spindle; they are crowded together and often are so united 1 metaphase as to resemble a spireme in one plane. IV. SPERMATOGENESIS. Nearly all of the material for the study of the spermatogenesis was obtained from the CEnothera. On the rose the young males are never met with in large. numbers while on the Génothera an abundance of all sizes may often be found on the still-blossoming tips of the flower spikes, while the sexual females are scattered over the leaves and stalks. Here also were found a few mothers of the males. ‘The few young males from the rose showed the same number and relative size of chromosomes. The testes, as seen in dissections, have 6 lobes corresponding to the 6 ovaries in each group. ‘The lobes are much larger than the ovaries, and as the spermatogonia are somewhat smaller than the oogonia, the number of mitoses leading to the spermatocyte must be several more than in the case of the oogonia. Only spermat- ogonial divisions are found in the embryos, and the last such division often, if not always, occurs after birth. Fig. 28 shows a resting spermatogonium; Figs. 29 and 30 spermatogonia just before division. In Fig. 30, g chromosomes of characteristic form and size can be recognized, one of the smallest not being visible. “The equatorial plate, as also in most somatic mitoses, 1s too crowded for distinguishing either number or form of chromosomes. The resting spermatocyte of the first order (Fig. 32) does not differ materially in appearance from the resting spermatogonium (Fig. 28) and indeed can often be recognized only by its relation to later stages. ‘There is no evidence of so long a growth stage as in most forms. Closely associated with the first maturation mitosis and probably immediately preceding it are found the stages shown in Figs. 33 and 34. In Fig. 33, a, 8 of the 10 chro- mosomes are to be seen scattered through the nucleus; in 4, c and d, chromosomes of the same form and size are seen paired longi- tudinally. Fig. 34 suggests the synapsis stage described for many insects. [his stage is of much more frequent occurrence than that shown in Fig. 33, and appears to follow that, and immediately precede the first spermatocyte division which is shown in Figs. 35-39. Fig. 35, a, b and c show the 5 chromosomes in the equa- 322 N. M. Stevens. torial plate of the first maturation spindle. All are connected by linin threads, and every possible arrangement of the 5 chromo- somes is found in different cells. In c the longest chromosome is seen to be double and as a side view of the spindle in metaphase always shows the chromosomes double, it 1s probable that they come into the mitotic figure in that condition from the preceding conjugation stage. A second longitudinal split to form a tetrad cannot be detected at this stage. In Fig. 36, a side view and an oblique view of the equatorial plate show the double chromosomes. ‘Two stages of the anaphase appear in Figs. 37 and 38. There is always one “lagging”? chromosome in this division, and it cer- tainly is not usually the longest, as I at first thought likely. In fact 1t appears in most cases to be either the second or third in size. After the two spermatocytes of the second order are fully formed, as shown in Fig. 39, the two daughter elements of this “lagging” chromosome are still connected by a thread extending through the cytoplasm of each cell. “This phenomenon seems to be a peculiar characteristic of one of the chromosomes in this particular division. I have never seen an exception, nor have I ever seen anything similar in the second spermatocyte division or in fact in any other mitosis. hs If, as I have supposed, this first maturation division simply separates paired chromosomes, it is possible that the pair that shows this peculiarity has a different linin connection from the others. In Fig. 35, it will be seen that the 5 chromosomes are united by linin threads into a chain with free ends. A side view (Fig. 36) shows two pairs connected by two parallel linin threads. Nowif one of the end pairs always has its two elements connected by a single thread, we might expect such figures as 37, 38 and 39. Fig. 40 is the equatorial plate of the second maturation mitosis, showing again the 5 chromosomes of characteristic form and size. Fig. 41 ‘shows one of a few fortunate sections showing the daughter chromosomes in anaphase, and removing all doubt as to the kind of division, longitudinal or transverse. Judging from analogy in other insects, I fully expected to find one longitudinal and one transverse division, but was soon con- vinced that both are longitudinal, and from such figures as 23 and 26 that the same is true in the maturation division of the winter egg. This point will be more fully discussed later. The Germ Cells of Aphis. R28 The chromosomes retain their individuality in the spermatids for some time, but finally become massed together to form the sperm-head (Fig. 42). The development of the spermatozoon from the spermatid appears to be very simple, the head being formed mainly from the chromatin and the long, rather thick tail from the cytoplasm. None of the accessory structures described for other insects are present. No centrosome has been detected in any mitosis, and asters have been seen only in connection with the sperm-nucleus in the egg, the pronuclei, and the segmentation spindles of the winter egg. ‘There is no trace of anything that could be called an “accessory chromosome” (McClung, ’02). The nucleolus appears to be of the same character throughout, appearing in resting cells and disappearing in mitosis. ‘That it is not a chromatin nucleolus, or karyosome, is shown by the fact that the chromosomes in many cases are visible before it disappears (Fig. 29) and with the Delafeld-orange combination the nucleolus invariably takes the orange stain while the nuclear reticulum stains with the haematoxylin. V. GENERAL DISCUSSION. I. Mendel’s Law, and the Individuality of the Chromosomes. It appears that in the Aphids studied there is a series of 5 chro- mosomes of different shape and size in the germ cells of the sexual generation: the maternal or egg-series 1s exactly equivalent to the paternal or sperm-series. The chromosomes show the same rela- tive form and size throughout the maturation divisions. ‘The two series of chromosomes meet in fertilization, and throughout the parthenogenetic generations, both female and male, we find the double serfes, 10 chromosomes of five different sizes. In the spermatocytes, and presumably in the odcytes, the chromosomes of the double series are paired, and in one of the maturation divi- sions, apparently the first, the paired chromosomes are separated. Supposing that the different chromosomes have different physio- logical values, or represent different hereditary characters, as maintained by Boveri (02), we find in the behavior of the chro- mosomes in the germ cells of the Aphid exactly the conditions re- quired by Mendel’s Law of Heredity. The characters represented by the 5 constantly different chromosomes would be segregated at 324 N. M. Stevens. each recurrence of sexual reproduction, giving germ cells pure with respect to each of the 5 different characters or sets of corre- lated characters represented by the five chromosomes. During the whole series of parthenogenetic generations the same paternal and maternal series of chromosomes 1s maintained by longitudinal division, there is no amphimixis and no apparent chance a varia- tion unless it be a change in dominance of certain characters, due to external conditions; fn example, the winged-character and the sex-character. The constant recurrence of this single or double series of chro- mosomes of the same relative form and size, is one point more in support of the hypothesis of the individuality of the chromosomes, strongly advocated by Rabl (’85) and Boveri (’87, ’88, ’91, ’02). Recent papers by Sutton (02) on Brachystola and Baumgartner (04) on Gry ‘lus show similar form and size relations of chromo- somes; but in the Aphid one has the advantage of working with a smaller number, where each individual chromosome can be dis- tinguished from all others of the series appearing in a winter egg or a spermatocyte. 2. Maturation of Parthenogenetic Eggs. In comparing the results of various authors on this subject, one meets with great variations in different parthenogenetic forms. In the drone bee (Blochmann, ’88—’89) two polar bodies are found, and Petrunkewitsch (’o1) states that reduction occurs in the second maturation division, the normal number of chromosomes probably being restored by a subsequent longitudinal splitting of the chro- mosomes without mitosis. In Liparis dispar, Bombyx mori and Ocneria dispar (Platner, ’88—’89, Henking, ’92), two polar bodies are given off by the occasional parthenogenetic eggs and both sexes are produced. Weismann (’91), in attempting to ‘bring these cases into line with his views of maturation and fertilization, says, “The nucleoplasm of certain eggs possesses a greater power of growth than that of a majority ha the eggs of the same species, ai in the case of the bee every ovum possesses a power of growth sufhcient to double its nuclear substance.”’ Petrunkewitsch’s explanation of the presence of the normal number of chromosomes in somatic cells of the male bee sounds more like an echo of Weismann’s argument than like the result of actual observation. The Germ Cells of Aphis. 325 In Rhodites rose (Henking) there are two maturation divisions but no reduction of chromosomes in eggs that produce females. Males are rare and the maturation of the male egg 1s not known. In Artemia salina (Brauer, ’94) either one polar body only is formed, or a second division occurs and the resulting nucleus con- jugates with the egg nucleus. Here again the male and male generations seem not to have been distinguished. Weismann and Ischikawa (’88) found only one polar body in the parthenogenetic eggs of several rotifers and crustaceans, no statement being made in regard to the male generations. Mrazek (97), and Erlanger and Lauterborn (’97) found that in Asplanchna, a rotifer, the parthenogenetic female eggs gave off one polar body, while the parthenogentic male eggs formed two, and there was no indication of a union of the second polar body with the egg nucleus. In Hydatina senta (Lenssen, ’98) the first maturation division in parthenogenetic female eggs goes only as far as the metaphase; there is no reduction, and the chromosomes (10 or 12) fuse to form the egg nucleus. In the male egg reduction occurs, 5 or 6 chro- mvusomes appearing in the polar plates of the spindle. In Hyda- tina it is supposed that the first maturation division 1s suppressed in both the parthenogenetic and the sexual eggs. In the Aphid only one polar body 1s given off in the parthenogenetic ego—male or female—and there is no evidence of reduction in either male or female parthenogenetic egg. Thus we find a series, beginning with forms where partheno- genesis is either occasional or continues for only one generation, eohen maturation appears to follow the usual course for fertilized eggs; and ending with Hydatina senta where the whole process 1s practically suppressed in the parthenogenetic female eggs, and the Aphid where one maturation division without reduction remains in both male and female parthenogenetic eggs. We are thus led to question the importance of the second polar body in deter- mining the male sex, also to question the view that parthenogenesis is due to the suppression of one or both maturation divisions, and to suggest that the various degrees of suppression of maturation phenomena in parthenogenetic eggs may be a more or less simple and perfect adaptation to a necessity of continued parthenogenetic reproduction, 7. ¢., the retention of the full double series of mater- nal and paternal chromosomes throughout the parthenogenetic 326 N. M. Stevens. portion of the cycle. The whole subject needs further investiga- tion from the standpoint of the determination of sex. 3. Determination of Sex. (a3 As the male sex cells of the Aphid contain no “accessory chro- mosome,’ McClung’s theory of sex-determination need not be discussed in this connection. The question naturally arises whether the “accessory chromosome” is to be found in any of the parthenogenetic insects or crustaceans. Castle (03) in his recent paper on “The Heredity of Sex” attempts to place the sex-character in the same category with other hereditary chracters and to apply to it the principles of Mendel’s Law of Heredity—dominance and segregation. He says on page 198, “A study of sex-heredity in parthenogenetic animals shows (1) that in such animals the female character uniformly dominates over the male whenever the two are present together,” and on page 199, “ With a single exception, we know that in unin- terrupted parthenogenetic reproduction, as. ‘it qoccurs im the Daphnide and Rotifere at certain seasons, the parthenogenetic ege forms only one polar cell, and the animal dew lopmne from such an egg 1s invariably female, or more correctly # (2), the male character being recessive,’ and further on, “At the return to sexual reproduction, the parthenogenetic mother produces eggs which form a second polar cell, and from such eggs only males develop. It is clear, then, that in the second maturation division the female character has been eliminated from the egg, for were it still there, it must from its nature dominate.” As an exception Castle cites Rhodites rosz, in which according to Henking the parthenogenetic eggs produce two polar Wodice: but no reduction occurs, and therefore no segregation of sex characters. Castle assumes that the occasional egg which pro- duces a male, does, in some way, eliminate the female character. Hydatina senta is also cited: according to Lenssen (’98) no polar body is formed and there is no veut in the female egg, while in the male egg one maturation division occurs with reduction. Castle supposes in the latter case that the first maturation division is regularly suppressed as Sobotta (’99) has tal to be the case in the mouse. In the Aphid only one polar body is formed in the female and The Germ Cells of Aphis. 327) also in the male parthenogenetic egg, and there is no reduction of chromosomes in either case. If the sex character resides in one of the chromosomes, it is certainly not eliminated from the male egg. How shall we harmonize the conditions in the Aphid with Castle’s theory? We cannot argue as he does for Bombyx mori, Ocneria dispar and all normally dicecious animals that there is no uniform dominance of one sex over the other. How then shall we account for the appearance of males, if all parthenogenetic Aphids are sex-hybrids with the female character usually dominant? The only possible argument seems to be that certain favorable conditions (warmth and abundant food) determine that the female sex-character shall continue dominant, the male sex-character recessive; while certain other conditions (not yet definitely deter- mined) cause the male sex-character to become dominant and the female character recessive. A similar change in dominance may be imagined to account for the presence or absence of wings in both the parthenogenetic and the sexual generations. According to this theory we must suppose all of the oogonia to be alike in their hereditary characters—all sex-hybrids as well as hybrids in respect to other maternal and paternal characters. In addition, we must suppose that in parthenogenetic eggs, which undergo no reduction, dominance of certain characters can be reversed by external circumstances. ‘This may of course occur at a very early stage in the history of the germ-cells, making the eggs at the time of maturation virtually male or female. ‘The case cited of two Aphids, each containing both partheno- genetic and winter ovaries (Fig. 20), and also showing that a parthenogenetic ovary may, after giving off parthenogenetic eggs, change to a winter ovary, the sexual form, is strong evidence that the ovarian oocytes of the Aphid may be affected by external con- ditions. Whether the degeneration of certain odcytes in all ovaries which are to produce winter eggs, can be considered evi- dence that there are two kinds of eggs, those that produce partheno- genetic young and those that require fertilization in order that they may develop, is not clear. Beard (’02) says, in discussing parthenogenesis, “When in a parthenogenetic form, a long series of one sex appears, the eggs of the other sex must have been either delayed or suppressed.”’ According to Beard’s theory we must suppose that in the Aphid there are both male and female eggs, or at least such germ-cells, 328 N. M. Stevens. produced, and that all the male eggs are suppressed in the female generations while all the female eggs are suppressed in the male generations. ‘There is no histological evidence of any such degen- eration of the germ-cells. In trying to fit the Mendelian theory of dominance, as elaborated by Castle, to the sex-conditions in Aphids, we meet with a peculiar contradiction in the fact that the same external conditions lead to the production of males and of sexual females. On the Gtnothera this condition is very conspicuous, for in the autumn partheno- genetic reproduction is completely changed over into the sexual method of reproduction. Certain apterous individuals are pro- ducing male offspring, and at the same time or slightly earlier other winged individuals are producing the winter egg-layers. Three generations at least are involved in the winter egg produc- tion—an apterous generation followed by a winged generation and that by the apterous sexual females. Inthe case of the males only two generations are necessarily involved, an apterous gen- eration and the generation of winged males. ‘The food conditions which probably lead to the change in method of reproduction, may therefore differ in degree, the earlier conditions starting the sexual female line, and later conditions the male line. In favor of this argument is the fact that on the rose a scattered genera- tion of sexual females is often met with before there are any males and before the regular female sexual generation appears. It is perfectly evident that histological study of the germ-cells combined with observation of the living insects has not settled the question of sex-d Mo STEVENS: WitH 21 Ficures. While enjoying the hospitality of the Hopkins Seaside Labora- tory at Pacific Grove, Cal., the past summer, I made a few experi- ments to test the powers of regeneration of the red accelous flat- worm, Polychcerus caudatus, which abounds there in shallow tide-pools on the underside of stones and shells and on Ulva. The object of the experiments was a comparison of the regenera- tion of this form which has no definitely differentiated organs— eyes, central nervous system, pharynx, etc.—with the more highly organized fresh-water Planarians, as well as with the results of Schultz ('02) and Child ('04) on Leptoplana and other marine forms which show very incomplete anterior regeneration. Method. In most of the experiments, the worms were cut into three nearly equal parts as in Fig. A, a—b, c—d. ‘These parts will be spoken of as head-pieces, middle-pieces and tail-pieces. ‘The material was kept in covered glass dishes, somewhat shaded, and the sea-water was changed morning and evening. Regeneration in general was much slower than in fresh-water Planarians. The animals are very sluggish normally, and the pieces moved but little even when disturbed by changing the water, the head-pieces, however, being much more active than the middle-pieces and tail-pieces. ‘The tail-pieces continued to deposit eggs for several days as freely as did the entire worms, and the eggs developed normally. N. M. Stevens and A. M. Boring. B Cc D B ie: a aD a- b {), y H eS -b c-- da ee Vos -d 0 P [ f ane A.—Whole worm showing planes of section. B—D.—Head-pieces after 2 weeks’ regeneration. E.—Head-piece after 4 weeks’ regeneration. F.—Middle-piece after 2 weeks, showing ventral union of anterior edges (e—f), V of new tissue (g—e—h), and posterior regeneration. G.—Middle-piece after 2 weeks, showing anterior regeneration where the edges have not united as in F. H.—Middle-piece after 19 days, showing heteromorphic tail. L.—Middle-piece after 4 weeks, showing more advanced anterior regeneration of the type shown in F. M.—Middle-piece after 4 weeks, showing anterior regeneration of the type shown in G. N.—Posterior regeneration of middle-pieces, showing super- numerary appendages. O—P.—Regeneration in tail-pieces, 2 weeks. R—S.—Lateral regeneration, 4 weeks, T—X.—Young worms, still in jelly, with appendages just developing. Regeneration in Polycherus Caudatus. 53/7 Head-pteces. These pieces very soon began to produce new tissue at the cut surface as in other Planarians. Among the 40-50 pieces in a series, at the end of two weeks, the stages me posterior regeneration shown in Figs. B, C and D were found with all Tateericdinte stages. A rounded mass of new tissue of considerable size forms posterior to the cut surface, a—b, before the characteristic notch and appendage appear. Continued regeneration adds to the length of the new part while the old part decreases in width and the whole piece gradually assumes the typical form. ‘The notch, at first broad and shallow, becomes deeper and narrower, and the appendage longer. The new part assumes the characteristic pigmentation of elie adult tail-region, and a digestive region forms anterior to the line of section, a—b. Regeneration of fhiese pieces was not followed longer than four weeks, when most of the pieces had assumed the form shown in Fig. E, where, if one compares with Figs. A and B, morphallaxis is very apparent. Middle-pieces. In these pieces posterior regeneration proceeded ‘somewhat differently. New tissue appeared along the whole of the cut surface, but was so distributed as to form a median notch from a very early stage. One or more appendages appeared earlier than in the regeneration of head- “pieces. Figs. F and G show the usual amount of posterior regeneration ter two weeks, and Figs. H, L, M and N after four weeks. In all of these pieces the notch is still much broader and more widely open than in the typical form A. ‘The multiple appendages shown in the figures were at first thought to be a peculiarity connected with regeneration; but examination of many normal worms showed that, though one appendage is the typical structure, still all the variations observed in regeneration are to be found in normal adult worms. ‘These variations are, however, far more frequent 1 in regeneration, and more frequent in middle-pieces than in head-pieces, where, as a rule, only one appendage develops. ‘These observations sug- gested a comparison with the formation of the tail-region in the embryo. Figs. T and X show two young worms, ten to twelve days after the eggs were laid, and still in the jelly which enveloped the eggs. ‘he appendage has appeared but not the characteristic 338 N. M. Stevens and A. M. Boring. notch. Posterior regeneration in head-pieces (Figs. B and C) follows more nearly the embryonic method of tail development than does that of middle-pieces, where regeneration from the beginning seems to be based on the adult form of the tail-region which has been removed. Anterior regeneration varied greatly in different lots of material and in different pieces of the same series. ‘There are, however, two distinct types. In most cases the cut anterior end, a—J, folded together ventrally and the portions on either side of the median line united as shown in Fig. F, e—j. In the first set of pieces no anterior regeneration occurred while the material was under observation. In another set, in which all the pieces regen- erated better, a few at the end of two weeks showed a V of new tissue between the united cut edges, Fig. F, g—e—h. At the end of four weeks such pieces had developed as in Fig. L, g—e—A, and later some of them produced typical worms. As the union of the cut edges, as in Fig. F, e—, appeared to hinder regeneration in many cases, an attempt was made to prevent the union of the edges or to remove the hindrance later on. Pieces were cut as in Fig. A, x—y, or with a sharper angle, but the cut edges still curled under and united as before. Cutting the line of union was equally unsuccessful. “There were a few pieces which contracted at the anterior end without folding under and uniting; - these regenerated as shown in Figs. G and M, and in due time produced worms of typical form. Anterior regeneration was, however, in all cases less rapid than posterior. One piece produced a hetero- morphic tail, Fig. H. ‘This individual did not crawl normally, but half crawled, half swam with great difficulty on its back or side. ‘This was the only case of heteromorphosis observed. T ail-preces. Anterior regeneration of tail-pieces was of the two general types described for middle-pieces and illustrated in Figs. O and P. In general it was less rapid and less complete than in middle- pieces. Lateral Regeneration. A few worms were cut longitudinally in various ways. Regen- eration occurred along the whole cut surface as in other forms, the new material being distributed in proportion to the amount Regeneration in Polycherus Caudatus. 339 temoved. Figs. R and S show the result in two cases of diagonal section, as in Fig. A, o—p, and i after four weeks. In Fig. R the posterior end on the regenerating side is in approximately the same condition as in cases of entire posterior regeneration. In Fig. S, an abnormal notch and appendage has developed near Ss, as though the notch and appendage were a necessary accompani- ment of posterior regeneration without regard to the presence of the same structure in the old part. “This phenomenon also recalls the supplementary | heads and tails described by Morgan (’or) and others, as appearing on long obliquely or longitud. nally cut surfaces. General Discussion. The results of the experiments show that in Polychcerus cauda- tus anterior regeneration at different levels may proceed much as in many fresh-water forms (Figs. G, M and P), or it may be pre- vented or delayed, not by muscular contraction and union of the muscle bands, as described by Schultz oe but by a folding under and union of the cut edges. (Fig. F, e—f.) That such union of the cut edges 1s not an insuperable h’ndrance to regenera- tion in this form is proved by such cases as are shown in Figs. FE, L and O, where regeneration begins with the formation of a V of new tissue and ends with the production of a typical head- region. In Polychcerus there is no axial gut (Bardeen, ’or), nor is there a central nervous system to influence regeneration (Lillie, ’oo; Child, ’04). The fact that head-pieces, which are more active, regenerate more rapidly than middle-pieces or tail-pieces, might be held to support Child’s theory that “there is a close parallelism between the rapidity, amount and completeness of regeneration and the characteristic activity of the part concerned;” but the difference in rate of regeneration and morphallaxis 1 is not propor- tionate to the difference in activity, for head- “pieces are easily stimulated into activity by changing the water or jarring the dish, while middle-pieces and tail-pieces hardly move at all during the first two weeks unless violently disturbed. The difference in activity is great, while the difference in rate of regeneration is comparatively small. So far as regeneration in Polychcerus has been tested by these experiments, it seems to be largely a question of “organization”’ 340 N. M. Stevens and A. M. Boring. and “totipotence” of material (Morgan, ’o4) modified in many cases by the folding under and uniting of the anterior cut surfaces. It is the intention of the authors to supplement this work with further experiments during the coming summer. PARC His ROLOGY. BY A. M. BORING. Wirth 2 PLares AND I FiGuRE IN THE TEXT. After working on the external features of the regeneration of Polychcerus caudatus in California during the past summer, Miss Stevens brought back to Bryn Mawr some preserved material— the whole flatworms and pieces that had regenerated for varying lengths of time. ‘The simplicity of structure and the lack of any great differentiation of tissue, made it a matter of interest to work out the histological side of the regeneration of this form, in order to see whether it differs in any essential points from the method of regeneration in more highly differentiated forms, such as Planaria simplicissima, described by Stevens ('o1), and Planaria maculata, worked out by Curtis (’02) and Thacher (’02). Technique. The material had been fixed in a mixture of corrosive sublimate and acetic acid, the regenerating pieces at the end of one, two, five, seven, ten, fourteen, and twenty-eight days. After being hardened in the alcohols, and embedded in parathne, the whole worms were sectioned in transverse and sagittal planes, and the regenerated pieces in transverse, sagittal, and frontal planes. ‘The sections were stained in Delafield’ s hematoxylin, followed by orange G. This combination gives a good differentiation of the various tissues. [he reproductive pall stain purple, the mucus blue, the nuclei of the parenchyma cells brown, the parenchyma itself pale yellow, the muscle cells deeper yellow, and the cilia usually form a slightly stained border at the margin of the sections, in parts of which the separate cilia can be distinguished. Regeneration in Polycherus Caudatus. 341 Normal Structure. Before describing the process of regeneration, it seems necessary to describe the normal structure, as this differs essentially from that of other Planarians, and has not been described in detail. Fig. K is a sagittal section of a whole worm showing the general outline of the form and the location of the different openings; » 1s the digestive opening, r the female repro- ductive opening, and p the penis. It also shows the position of the cells that secrete the jelly in which the eggs are laid /. Fig. I is a transverse section taken near the anterior end of the worm (Fig. K, a—), showing the testis cells ¢, maturing spermat- ozoa s, mucus m, the parenchyma nuclei 7, and the ciliac. Fig. 2 1s a transverse section through the middle-region (Fig. K, c—d), showing in addition, € egg cells o, the irregular digestive region d, containing some food f, and the digestive opening x. Fig. 3 is a transverse section near the posterior end (Fig. K, e—f), showing besides the foregoing features, the female reproductive opening r, and the jelly gland 7. In these three sections, certain distinctions between the dorsal and ventral sides can be seen. Most of the mucus lies on the dorsal side. There are more nuclei on the ventral side than on the dorsal, and there is a marked aggregation of nuclei at the lateral edges of the ventral side. By comparing Fig. 4, a piece of the dorsal margin of a transverse section (similar to Fig. 2), with Fig. 5, a piece of the ventral margin, an ditional difference appears, that of ae arrangement of the muscle fibers. On the dorsal side, they are more regularly arranged, forming an outer circular and an inner longi- tudinal layer, while on the ventral side, Chete 342 N. M. Stevens and A. M. Boring. are no distinct layers. [he apparent difference in the length of the cilia in these two figures (4 and 5) may be due to their being matted together in Fenton There is no definite ectoderm or endoderm. ‘The cells com- posing the mass of the body are the parenchyma cells, irregularly spindle-shaped, with large nuclei. (Fig. 4, 7.) In many places, the outlines of these cells are so indefinite that it appears as though they merged into one another, forming a syncytium studded here and there with nuclei. Among these parenchyma cells are mucous cells, which have similar nuclei, but contain masses of a blue-staining secretion. (Fig. 4, m.) On the outer edge, where one would expect to find a definite ectoderm, these parenchyma cells are ciliated (Fig. 4, ©); and stain a little more deeply, perhaps due to a cuticular secretion; but in no other way is the outer layer of cells different from the cells making up the mass of the body. This outer layer is not even arranged regularly, for the nuclei are at varying distances from the base of the cilia, and at irregular distances apart. ‘The cells of the digestive region (Fig. 2, d)—it is not definite enough to be called a digestive tract—do not differ in any respect from the other parenchyma cells. In_ places pieces of crustaceans, which have been taken in as food, are found in between the cells near the digestive region (Fig. 2, q), showing that this cavity is continuous with the spaces between the loose parenchyma cells. At the opening of the digestive region (Fig. 2, x), a few of the cells are sometimes ciliated (Fig. 2, c) like the ectodermal parenchyma cells. Muscle fibers are scattered throughout the parenchyma, but are accumulated especially among the ectodermal parenchyma cells (Fig. 4, g), around the female reproductive opening, and in the penis, of which they are the chief constituent. “They vary much in size, in fact, so much that in sections stained with iron hzmatoxylin and orange G, some take the black and some the yellow color. The reproductive cells are more distinctly differentiated than the other cells in these flatworms. ‘They are not grouped into ovaries or testes, but they lie in definite positions among the paren- chyma cells, and are discharged through definite openings, guarded by muscle cells having a sphincter-like arrangement. The testis cells (Fig. 2, 7) extend ‘along the lateral edge from near the anterior end to the penis which is an external muscular organ. (Fig. Regeneration in Polycherus Caudatus. 343 KP): The ege cells (Fig. 2, 0) lie on each side of the median ventral line, extending from the region of the digestive opening back to the female reproductive pore. Just in front of this pore lie the cells which secrete the jelly in which the eggs are laid. (Fig. 3, 7.) This form has no central nervous system, no eyes or other sense organs, and no excretory system. Regeneration. The regeneration of this form is as simple as its structure. The worms were cut into three pieces as stated in Part I, a head-piece, a middle-piece, and a tail-piece. In the regeneration of Planaria simplicissima and of Planaria maculata, the old ectoderm stretches over the cut surface in a thin layer, but the regenerative process in Polychcerus caudatus is more like the regeneration after natural fission in Planaria maculata, as described by Curtis (’02), where the exposed surface simply heals over and embryonic cells migrate to that region and form the new tissue. In Polychcerus, the cells at the cut end secrete a cuticular substance and develop cilia. Sections of most of the pieces fixed two days after being cut, show short cilia at the cut end (Fig. 6, c,) and the cells stain a little more deeply at the base of the cilia. In the five day sections, the cilia have reached their normal length. (Fig. 7, c.) By this time there is also a decided accumulation of nuclei at the regenerating end. Fig. 7 shows this, and a comparison of Fig. 7 with Fig. 6 clearly shows the progress of regeneration. [his accumulation is not due to cell division, either in nate regenerating end, or the old part. Cell division has been carefully looked for throughout the work, and the one or two cases which might possibly be interpreted as prophases of mitosis lose all significance from their rarity and the entire absence of actual mitoses; neither has any evidence of amitosis been discovered. Many of the nuclei in Fig. 7 have their long axes pointed toward the end, and the cells, as far as their outline can be made out, point in the same direction, indicating a streaming of parenchyma cells toward the regenerating region. In the whole worm, the parenchyma nuclei are accumulated on the ventral side and especially toward the lateral edge. In Fig. 9, a sagittal section some distance lateral to the median line, the accumulation of nuclei on the ventral side is continuous with the 344 N. M. Stevens and A. M. Boring. accumulation at the regenerating end 1, suggesting this accumu- lation as the chief source of the iG. in the new part. Some of the cells come from the dorsal side, but the evidence from the exami- nation of many sections is convincing that the majority come from the ventral side. The muscle cells must develop from the parenchyma cells 17 situ, as they appear below the ectodermal parenchyma only in pieces which have been regenerating several days. (Fig.8,¢g.) In Fig. 6, a section of a piece before the accumulation of nuclei had begun, some fibers appear scattered irregularly through the parenchyma near the end, but these are probably old fibers, as this section shows no definite layer of muscle fibers below the ecto- dermal parenchyma at the regenerating end. The seven-day and ten-day sections show an increase in the length of the new part, but no other new points. In two weeks, most of the new tissue has taken on the loose parenchymatous character of the old part, as shown by the spaces in the tissue and the more scattered position of the nuclei in Fig. 10,4only the extreme end of the regenerated tissue still having the nuclei in close proximity and the cells densely packed together. (The dotted lines in Figs. 9-12 show approximately the boundary between old and new parts.) In the regeneration of one of the oldest head-pieces, a new digestive opening has formed. (Fig. 11, x.) It is in all respects like the opening in a full sized worm, being situated about halfway between the anterior and posterior ends, and opening directly from the digestive region to the exterior. Middle-pieces of this age have the old digestive opening, but some distance posterior to this, at the base of the new tissue, there 1s an accumulation of parenchyma and muscle cells, as in Fig. 12, 7, which can be recognized as the anlage of the penis, for the sperm has moved down near to this anlage. Anterior to the penis is a slight indenta- tion r which may indicate the anlage of the female genital pore. Sections show that anterior regeneration is always slower than posterior; there is less new tissue at the anterior end than at the posterior, and it keeps its compact character and accumulation of nuclei longer than the posterior, as shown by comparing Fig. 7, an anterior ead with Fig. 8, a posterior end of the same age. In some pieces in which the anterior end folded under to form a pocket, as described in Part I, no regeneration can be seen in the Regeneration in Polycherus Caudatus. 345 oldest stages, but in a few of these, the growth of new material between the united edges can be seen in section. (Fig. 13, v.) By studying the whole series of sections, this region can be identi- fied as the place where the cut edges united. The accumula- tion of nuclei shows new tissue to be regenerating on both sides of the line of union. A few cases of lateral regeneration were studied, but the sec- tions showed no divergence from anterior and posterior regenera- tion. The regeneration of Polychcerus caudatus is an excellent example of the remolding of the old tissue in a piece of an organ- ism, into the tissues and form of the whole organism, without the assistance of cell division by mitosis or amitosis. ‘This is what Morgan calls morphallaxis. Other flatworms in which regenera- tion has been worked out histologically, Planaria simplicissima and Planaria maculata, show a proliferation of new cells at the cut end, as well as the changes of form due to morphallaxis, but in Polychoerus the new part is formed wholly of cells which migrate from the old part. Regeneration in this form is, therefore, an example of morphallaxis, pure and simple. Bryn Mawr College, Pa. April 19, 1905. LITERATURE. BARDEEN, C. R., ’o1—On the Physiology of Planaria maculata with Especial Reference to the Phenomena of Regeneration. Am. Journ. of Physiology, vol. v, 1go1. CuiLp, C. M., ’o4a.—Studies on Regulation. IV. Some Experimental Modifica- tions of Form Regulation in Leptoplana. The Journal of Exp. Zoology, vol. 1, No. 1, 1904. ’04b.—Studies in Regulation. V. The Relation between the Central Nervous System and Regeneration in Leptoplana: Posterior Regeneration. Jbid., No. 3, 1904. ’o4c.—Studies in Regulation. VI. The Relation between the Central Nervous System and Regulation in Leptoplana: Anterior and Lateral Regeneration. Jbid., No. 4, 1904. Curtis, W. C., ’02.—Life History, Normal Fission, and Reproductive Organs of Planaria maculata. Proc. Boston Soc. of Nat. Hist., vol. xxx, No. 7, 1902. 346 N. M. Stevens and A. M. Boring. Lituig, F. R., ’o1.—Notes on Regeneration and Regulation in Planarians. Am. Journal of Physiology, vol. vi, 1gor. Morean, T. H., ’01.—Regeneration. Columbia Univ. Biol. Series, No. 7. New York., 1901. ’o4.—An Analysis of the Phenomena of Organic ‘Polarity.’ Science, N.S., vol. xx, No. 518. Stevens, N. M., ’o1.—Notes on Regeneration Planaria lugubris (simplicissima). Archiv. fiir Entw., Bd. xiii, H. 3, rgor. Tuacuer, H. F., ’02.—Regeneration of a Pharynx in Planaria maculata. Am. Naturalist, vol. xxxvi, No. 428, 1902. EXPLANATION OF PLATES. Figs. 1, 2, 3, 9, 10, 11, 12, 13 were drawn with Leitz oc. 2, obj. 3, camera lucida. Figs. 4, 5, 6,7, 8 were drawn with Leitz oc. 2, obj. 1-12, camera lucida. Figs. 1 to 8 are reduced one-half. The following lettering is used in all the figures: c, cilia; c, cilia half developed; d, digestive region; f, food in digestive region; g, muscle fibers; 7, jelly gland; m, mucus; 1, nuclei of parenchyma cells; 0, Ova; p, penis; q, food among parenchyma cells; r, female reproductive opening; s, sperm; #, testis cells; v, new material; «, opening to digestive region; y, appendage. Pirate I. Fig. 1. Transverse section of whole worm near anterior end. (Fig. K, a—b.) Fig. 2. Transverse section of whole worm near middle, through the digestive opening. (Fig. K, c—d.) Fig. 3. Transverse section of whole worm toward posterior end, through female genital pore. (Fig. K, e—f.) Fig. 4. Portion of dorsal margin of transverse section. Fig. 5. Portion of ventral margin of transverse section. Fig. 6. Sagittal section of anterior end after 2 days’ regeneration, showing developing cilia. Fig. 7. Sagittal section of anterior end after 5 days’ regeneration, showing accumulation of nuclei. Fig. 8. Sagittal section of posterior end after 5 days’ regeneration, showing appearance of muscle fibers. Prare II. Fig. 9. Lateral sagittal section of middle-piece after 5 days’ regeneration, showing accumulation of nuclei, 7. Fig. 10. Median sagittal section of middle-piece after 2 weeks’ regeneration, showing tissue with the loose character of the old. (Exceptionally rapid regeneration.) Fig.11. Sagittal section of head-piece after 4 weeks’ regeneration, showing the new digestive opening. Fig. 12. Sagittal section of middle-piece after 4 weeks’ regeneration, showing anlage of penis and of female genital pore. Fig. 13. Transverse section of middle-piece (4 weeks) with a ‘‘pocket,” showing triangle of new material, v. REGENERATION IN POLYCHGRUS CAUDATUS. N. M. Stevens anp A. M. Borne. PLATE I. A. M. Boring del. Tue Journat or ExperIMENTAL ZodLoey, vol. ii. os 9 uu « : i Kal } 4 REGENERATION IN POLYCHERUS CAUDATUS. N.M.Srevens anv A.M. Borinc. PLATE II. A.M. Boring del. , THE JourNAL or ExPerIMENTAL ZoOLocy, vol. ii. HE REE AI@N OF THE DEGREE OF INJURY TO FHE RATE OF REGENERATION? BY CHARLES ZELENY. I. INTRODUCTION. It is a common belief that an increase in the degree of injury to an animal lowers its vitality and thereby diminishes its capacity for repairing sustained injuries. It is certainly true that if an animal is mutilated to a degree so great that it can barely survive the operation a rapid rate of regeneration of the parts is not to be expected, though there 1s little direct evidence in favor of this statement. ‘he general view that injury to an increased number of organs implies a decrease in the rate of regeneration of each, however apparent it may seem at first sight, needs further exam- ination. The data to be given below prove very conclusively that the view is an erroneous one, for it is shown that the animal with the greater number of removed parts regenerates each part more rapidly than does the one with the lesser number of removed parts. In the summer of 1902 the author performed some experiments on the fiddler crab, Gelasimus, which showed that when both chela are removed each of the regenerating buds grows more rapidly than does the single one in the cases where only one chela is removed. ‘The rate of moulting of the animals is likewise greater in the individuals of the former group than in those of the latter. [he difference was naturally more plainly made out. in the female individuals which have chele of equal size than in the male individuals which have chelz of unequal size. The results are, however, not as conclusive as they might have been, had the number of individuals been greater and had a greater length of time been available for the experiment. 1Contribution from the Zoélogical Laboratory of Indiana University, No. 68. 348 Charles Zeleny. In the winter and spring of 1902-03 with the above results in mind two groups of experiments were undertaken to further test this point. A comparison of the rate of regeneration of the arms in five series of the brittle-star, Ophioglypha, with one, two, three, four and five removed arms respectively, showed that excepting the case where all five arms are removed and in which the animals were dead or dying before the completion of the experiment, a series with a greater number of removed arms regenerates each arm faster than does a series with a smaller number of removed arms. ‘hus with an increase in the degree of injury there is more than a corresponding 1 increase in the total amount of regeneration In a given time. In the Crustacean, Alpheus, a result similar to that for Gela- simus was found but with the addition of a quantitative deter- mination of the actual rate which was not possible for Gelasimus because of the slow rate of moulting in the latter. ‘The Alpheus data are, however, complicated by the fact that the two chelz are of Maegae size and undergo a reversal upon removal of the larger one.t| The number of individuals available for the final comparison was likewise small because a large proportion of the specimens cast their chela accidentally during the course of the experiment.’ It seemed desirable, therefore, to test the results in a more con- clusive way upon a form which does not have the complications found in Alpheus and Gelasimus. “The common crayfish, Cam- barus propinquus, has chela which fulfill the requirements of such a form. ‘They are equal in size and similar in character, are cast off at a definite breaking joint upon injury to their nerves and the animal does not readily throw off its appendages as a result of the necessary handling incidental to the course of the experi- ment. In one series the right chela alone was removed. In the other series the two chelz and the last two pairs of walking legs were removed. ‘The resultant data show very conclusively that in the series with the greater degree of injury each chela regenerates more rapidly than the single removed chela of the series with the 1Przibram, ’o1, Arch. Entw. Mech., xi; Wilson, ’03, Biol. Bull., iv; Brues, ’o4, Biol. Bull., vi; Zeleny, ’05, Journ. Exp. Zodl., 11. 2The description of the preceding experiments is given in Journ. Exp. Zodl., vol. ii, No. 1, Apr., 1905, pp- I-102. Rate of Regeneration. 349 lesser degree of 1 injury. Likewtse the members of the series with the greater injury moult more rapidly than those of the series with the lesser 1 injury. 2. METHOD. The specimens used in the experiment were collected in a small brook about a mile and a half from the Indiana University campus at Bloomington. ‘They were all taken from a part of the brook not exceeding two hundred feet in length and it is probable that the general conditions of the environment to which they had been subjected were similar for all up to the time of capture on October 11, 1904. About 150 specimens were obtained at this time and Fig. I. Diagram showing a bottle as arranged for the reception of one of the crayfish used in the experi- ment. See text description on page 350. from this lot 77 of the individuals ranging in thoracic length from ten to twenty millimeters were selected and divided into two groups which were made as nearly as possible equivalent in point of size of individuals. In series A which comprised 36 individuals the right chela was removed at its breaking joint. In series B which comprised 41 individuals the two chele and the last two pairs of walking legs were removed in a similar manner. Except for this difference in the degree of injury the two series were consistently treated alike throughout the whole course of the experiment. ‘The series with the greater injury (Series B) was purposely given the greater number of individuals in anticipation of a greater death rate in this series. Both males and females were included in each series. 350 Charles Zeleny. The crayfish were kept in individual wide-mouthed bottles, which were inclined at a slight angle to the horizontal and were covered with pieces of cheese cloth held in place by rubber bands (see Fig. 1). “The crayfish were fed every fifth day on frog meat or beef, the water being changed immediately after the meal. Under these conditions no difficulty was experienced in keeping the animals alive. ‘The few deaths recorded during the course of the experiment were for the most part due to neglect in changing the water immediately after the meal. Such a suspension of ordinary care is especially liable to be fatal when occurring soon after a moult. ‘The operation on the majority of the crayfish was performed October 12, 1904, and the experiment was closed April 20 after an interval of 181 days. A small minority comprising 16 individuals was operated on two days later and kept until April 22, the interval being likewise 181 days. 2.) BATA: The records of the experiment include the sex of the animals, the date of moulting, and the length in millimeters of the thorax and of the chelz after each moult. ‘The size of the regenerating walking legs of each individual in Series B is approximately expressed in my notes in fraction of the legs which are bein replaced. ‘The latter data are, however, not given in the tables reproduced in the present paper. In these tables (pp- 352 to 358) the moulting time is given in days after the operation. ‘The thoracic length is the distance in millimeters between the posterior edge of the thorax and the base of the thoracic spine. ‘The chela length is the greatest length in millimeters of the next to the last segment of the chela, the propodite. The data are given in Tables I, I], II]and1V. ‘The males and females are separated because the rate of moulting and of regener- ation was found to be different in the two sexes. “Lhe individuals in each table are arranged in order of thoracic length after the first moult. In the columns giving the original lengths, 7. ¢., the lengths before the operation, blank spaces indicate that the meas- urements were not taken. In the other columns a blank space indicates that the animal had not moulted when the experiment was closed, 181 days after the operation. In the last column the Rate of Regeneration. 351 number after “died” is the interval in days between the operation and the time of death. A comparison of the rate of moulting in the two series is given in Table V. This table is derived from Tables I to IV and gives the number of male and female individuals which had moulted in each series 95 days, 130 days, and 181 days after the operation. The first column under each moult gives the number of individuals which have moulted, the second the number which have not moulted, the third the number which have died, and the fourth the per cent of the living which have moulted. The data for the rate of regeneration as derived from ‘lables I to IV are given in Tables VI and VII. ‘Table VI gives the male individuals of the two series and Table VII the female individuals. In these two tables the individuals are arranged in order of moult- ing, those moulting first being put at the head of the list. ‘The specific amount of regeneration (Sp. Amt.) is the amount per unit of thoracic length at the end of the first moult. ‘The specific rate of regeneration (Sp. Rate) is the amount of regeneration per unit of thoracic length per day. EXPLANATION OF TABLES. Series A = Series with right chela alone removed. Series B = Series with the two chelz and the last two pairs of walking legs removed. In Tables I, II, III and IV the individuals are arranged in order of the thoracic length as determined after the first moult. In Tables VI and VII they are arranged in order of the date of the first moult. In Series B the specific amount of regeneration and the specific rate are the averages of these quantities for the two chele of the individual. % a52 Charles Zeleny. TasLe I.—Series A. Males (181 days after operation). Original. First Moult. Second Moult. Cat. Re = a Saniora g s | s sles Remarks. Meise sal @ | eos | culmea esetad Papa cs ve FT| eLOO ii a7 OM; |e — | — | — |} Died 157. 806 86 | 11.5 | 4.6 O27: || 104 |11-2 | Stor 15 O16 7408) 1% of 3; —)]—] = ity nee Mea tsied he ae) OW oF: P< SSA Lee N57 7.6)|| "044. |-12:6 || 5.8 | 7-3'|| Died 1409. 74a || 02/00) 2 7.0)|| 108 "| 12.24) 5.9) ecu tamed. 5a O.Q) ani 0) Fase Ms Boulatae7. || 0.3 8.6 ||. * — | — | — |} Died 73. (210m arene & 72) Tz s6:0 OF 14g a SLAs 87 200i| 10-6 FROM CAT | TE-7 Se 804 | .. = 92: 1 -14:001F0-G2 |)" 0:01 eae » 2 ee ee FOZ 4-1) “9.1 || 107 | 15.2))-6:85)| O:9i|\ei7oO | 15-1 )e70 | ToLo ion ae PVE LORS | The Oe7 8.9 PRE NOAH 9-3) 137. 15-07) 7-347) 10-8 FASO ENS Onl LUl-Oy| 127 || LO:6)|°7 20). 4) Rona 8% >. oh os FS t eget i220. FOON TOs (7.6) smaco%|) * — | — | — || Died 112. FOE A 1G. 08) TtO4|!116 | 16:6)|-7.0,) 10.8 (52) \ UO 22 stele EI" | 07.7) 8:0 | | 2T350 FOS | 15.7) T3166 | 1924 | 5.25) 1224 Rate of Regeneration. 353 Tae I].—Sertes A. Females (181 days after operation). Original. First Moult. Second Moult. ae cel r r ee Cat.| § 2 ES 5 a3 a g 5 23 od Remarks. Kase sol A la \2o).Soll a) =e |e@ol as Ola LO. 2 Orda t as" || Io | 4-01) One SOF UE ohsa | 140 | 112) 2:8" 5:6 ApS AIPES © | O25 |) TO: |-U4so | 0.0.) Ore *3d Moult. 786 P| rae OS | Tae kate) Gia) | roe | iene 2 785 8.0 || 104 | 14.6] 6.3 | 8.3 || 180 | 14.0] 6.4 | 7.9 a GAS one Nees I? LOO) -15.0ncO4 | G.1 || TOBaleEked |. 72.0: leGar Foxe 8:0) 2: Toll 108 | 15.241 16-0.5| 16:6 | 738) 14.0) 7:0 I, 108 ||-15.4 1) 6:9) |) “9:0 7O9-| 14.0) 9.6) 165 | 15.7)'1)6:0 | g:4 POON E52) 0:0 Ne142 | 15.0808 | 10.4 796 | 15.1 | 10.2 || 133 | 16.2| 6.7 | 10.6 TE ee) tote al ns || Died 154. FSU eUSe7e\ $0.5 |] 05a. 27:05 0.2 Tier FOS. || 10:0) 102 Il, =... a oe Bri S054 |o60.2." Tet 167 | 27.0 | °6.1 | 10.5 FSSE LT MEDION mae | ae | oe. GAO | L7-Or| 10.2 18D \er8.0 |-720: | Tao TG peels @ 73) | ee | yee F5On) LOO lUrsr ltads |e8.8: | Ozer | make 354 Charles Zeleny. TaBLe HI.—Sertes B. Males (181 days after operation). a | First Moult. Second Moult. Third Moult. on | See mem ee | eM als ull Merete Se ese a el No 2 | &| 2 |SSlee| | 2 |SSlee| | 2 |Seleg| saa BH |Al BB |/#0/HU/] Ale lx0laol aA |] B leo] ao] EB —|—-— — 7 erie | Se ee ee ee 78 | ne ’ 53 | 11-4 | 4-4 | 4.4 * == | = |) = jl Died Ga. 789 | av || stittey |) Es | als LOS SUT ON Neda Gia s a2 *Missing. 739 | zeeh || ae) Geet Gyo) 109 | 12.8 | 6.4 | 6.3 | 792 \ 2ut W neler Geo) Lee Es = | a | ee == | = | S| = Jt AD ge 803 ; | 42 | 13-5] 5.6 | 5.0 | 106 | 13.9 | 6.6 | 6.4 | 176 | 14.5] 6.9 | 6.6 801 | 65 | 14.3 | 6.65 | 6.75 BV. oe _ 740 760} 04.7) |6:8) ||) 6:7 142 | 15.6| 8.6 | 8.6 764 WSalerag lezen i\rer 142 | 15.0] 8.3 | 8.4 780 | 44 | 14.8) 7.2 | 6.8 ni WGA || GE |) Gyn 790 ASalietigas al O27, sO 748 83 | 15.8 | 6.4 | 6.3 | ete | ee e. 758 | FUE | ye he 97 | 16.8] 8.4 | 8.3 * — — | — | *Died 174. 732 | 69 | 16.5 | 6.4 | 6.3 125 | 16.9| 8.0 | 8.0 | TES | 95 | 16.9 | 7.6 | 7.6 note) |) 3k) || GG? || Ge 765 | TAY Nl Agfa tose) || ie? 9) == = | = | | = |) = | DesloR Rate of Regeneration. TasBLeE IV.—Series B. Females (181 days after operation). oe First Moult. Second Moult. inal. a me Akeeee less allt dW ESS ls nel 2 | &| 2 |Szlszl| £12 |Szle2 H A AH |MO/HO A lH |HO}/HO 734 27 | 11.8] 4.9 | 4.9 || 72 | 12.7] 5.9 | 5-9 741 29 cea erate Py meester || ts 794 37 5-3 | 5-0 || 103 | 13.0] 5.6 | 5.7 TER: MN oe Atel erz Only shal ged: * = | = 802) | 11-8") * = |=] = a |e | 788 I a6 a AS sTAn2 Sage nh s6 747 31 6.2 | 6.1 Sau ae2s || “Fae eTEO 795 35 Bo! |] Sev 81 | 14.1 | 6.0 | 6.0 787 Geo Whoo | EHO | Rea I wee | tito |) ee) Bere) || oe B77 PESeOn |e Ost les-Om|lieLzall ena Saler7-Om moro TT \\ Heyok || sae | vAsoy | Ce G2) I ae | Tie | ey || (oe 793 33 | 14.0] 5.3 | 5.4 || 116 | 14.4] 6.8 | 6.9 FF OLA 7 LOS em5.00|0 520) | Sey 148) |r 6.2) 7G |n76 TEN co BP | on |) Ops |) Ge | Wer || aoe) || 75 |] eG Tee \| We |) wees WIGS) oe ae 168 | 16.6] 5.6 | 6.4 TB |) alse |) EY TERS Gs) |i] ek POS Geo || Gee WEG) | oo | wap | aye gt) se III) aisle |] HOY GS |) 9B OR |) ae 52) |) 16;9))|16:7, ||| (6:9) Il TOW 172011 7a" 74 TSS eLOst || t20e 191723) 6:81 |720) ||| 169) 1/00 7-9) || 8.011 Sa 757 BAUIEE 7-30 52ON || 7-000! LOOM ETS 25 mS.S ses ST een LOAN U7 a5 07 -ON| ts -2ai|( 120) | euse Tee Ses oO 756 | 16.6] 143 | 17.8} 7.0 | 6.9 Be ae Hie LOA LS al \eL7/-801) 7-201 17-0) 138) leuSe7a | mScon|eOez 782 | 18.0| 148 | 18.8] 7.0 | 7.4 a an ae 750 | 17.6] 147 | 19.0} 6.8 | 6.3 || 179 | 20.2 | 8.9 | 8.7 766 | 19.7] 161 | 19.8] 5.6 | 6.1 Third Moult. S : aes P| 2 es a me Wea * aie |e 128 | 14.7 | 6.4 * Eas ae Gi |) Ee | * = ee * = ace 180 | 19.0] 9.3 355 Remarks. *Died 73. *Lost in moulting. *Died 95. *Died 17. *Died 166. *Died 166. **Lost. *Died 174. *Died 146. 356 Charles Zeleny. iter V.—Rate of Moulting. 95 Days after Operation. | y | First Moult. Second Moult. | | : aa lear; % | 3 EB) + | 88 2 | Boe Bat a Gee lg t= ce a dy eae = Os & oye) BS 5S S 5.0 o i OS = Ze al Oe = Ze al ae | | SeriesA G..... 7 ete) O | 41.0 fo) TO) \ea VW O Deltes Diiciueecs). 5p] 0 |) Yas t0e0 I 13 I ee: DELIES A Ory. o5!: I | 18 O | isis) O 19 O Oo SeriesB @ ..... 12 | 12 | 1) |) 5250 4 20 |, te 130 Days after Operation. First Moult.** Second Moult. i ‘ a0 | * = : x OSIM ges meer 5 g Sl ee amt Bi Lace nS Os e os ‘s Ors ° © ,O o Oo ) te) 5 bo 2 Ze fa = = rarer Il {| ae . | RemessAN Gila... — — = — I 14 Zz | 6.7 Denies nc! : son — 7 6 2 | 53n8 STS de. OU 5 14 Oo 26.3 brs fe) Bae penesis. Of: .2| 19 6 I 76.0 12 12 2 | 50.0 St Days after Operation. Second Moult. Third Moult. eer ck : wal % = | aoe 2 G) kS cg 3 eee eae! Os 3 {Sl we Os SC) © Oo o bo } a0 By iO = Ze a = = Ze fa v= DEnIES ANG. «ha | 6 8 3 42.9 14 3 fe) DERES BG... 10 3 2 76.9 10 4 g.1 DemestAy OV s.1.2 3 3 15 I TOeg I iy I 5.6 Sectesy is) Os... 21 3 2 87.5 3 17 6 15.0 + Without having moulted. * Of living individuals. ** A comparison of the males is not valid here because all those of Series B had already moulted 95 days after the operation. +f} One individual of Series BQ moulted a third time 128 days after the operation. Rate of Regeneration. 357 TaBLe VI.—Rate of Regeneration (1). Males—First Moult. Series A. | Series B. Cat. ¢ & E 5 | Cat. x fs E 3 eee aly.e le eee te |e *746 3 e es Pe 1 a3 on eee rs oM Ojai alee" kO7 SOOSZEH 730 |. 385 hia 2: || eg .0132 VASE sO 1327.1. 2400 0879 || 789 | 34 | 11.0] .418 0123 F378 | egta TORO, | > 4 gu 0061 | $03") a7 ng.5, | gos |) G004 FaOn\e 72. “ I4e2 | 422 0059 || 17758 | 44 | -- ae: ‘s 806 | 86 | 1r.5 .400 10047 || 780.) 444-| 14.8 fe 2473 .O107 8040/92) || TAZ" | 9.463 -0050 || 792 | 44 | 13.4 . 388 .0088 Poul tOs, 1525 | | 1432 -0041 || 790 BS? “WUE AS ee te .0090 7540100: | 16.0 472 foo45 ||, “Som Obs. | T4271). 409 .0072 GOR NAOT A 5.29 455 200425 ||! “72REEGG. | 1OrG) No. 285 | B.0056 7Ad | 108 11173:3 | .4440) eogr || “FOS as | 17250) 471 |) se065 52a a ara 3452 SO0dO) |) = FAO gleZO: | 14. Tei ARO .0060 FOU eh1O) || TORSO | 2422 .0036 FAS eO3) D5 a8 | 402 .0048 Te 037. | T5e0n| 2459 || 30034, |) Pye 84) Vea 7) ee. 483) 70057 Pasialszien|) LOsOn||) 2427 -0032 || 773 | 95 16.0 | ©3450" |" 20047 2*708) 4 t00 -| 10.4. ae Soh | ee as 776 | 181+ BAe sliuntohe ma 444 .0049 | a i wD: 435 .0080 = 003) |=2.0003 +.006 |+ .0005 | For explanation of Tables VI and VII, see pp. 350, 351. *, | No visible regeneration has taken place. ** The stump of the operated leg was diseased for a long time after the operation and the data are therefore not included with the others. + The plus sign after 181 indicates that the animal had not moulted when the experiment was closed. TT The animal moulted a second time before measurements for the first moult were taken. 358 Charles Zeleny. TasLeE VII.—Rate of Regeneration (2). Females=First Moult. Series A. Series B. | : @ | re) o < E 5 . alia Usk = ca ee ah San ik Sine eee pith tL A H D a (a) = | n n PSO on al Tae ce 1788 | 1 Ry S| ay Fos tose eae! 432 SOO42,.||| =e. 7A D775 4) MTS | 415 .O154 FAS (mLOO | aS A Ol «| 3.427 0040 | Fat | S207 WG 395 .0136 738 | LOS! || su5 ie: .448 | 0042 | TAD. 52 12,03). 2022 O140 775) 119 | 14.0 429 -0036 || 6747 | 31 | 13-5 | -456 O147 FIO NP 232% |) 00,2 414 | .0031 FOG) 32" ere e ON, a dOs .0127 mot | 135 | 11.0]. .418 | .0031 | 787 | Son) || haa 412 .0129 807 | 140 |glt.2 7336 | e024 1! 993 fs a3 3ha RAO 382 .O116 799 | 142 | 15.9 | .427 | .0030 | 757 | 34 | 17.3} * ‘i 759 | 144 | 18.8) .378 | .0026 705°) (250 \ag5 || aAoR or16 FSi at aass | 17 see 2305) peroooaelll a maned! | 37 | 12.4) -415 O12 784 203) || 152 2K BOS |i 6024. | NeSeco.| -a7. lanspoml age OII7 700 1 865. ‘| -15s71/en 382 | Wooze? lea seco | Gugi)) sedas 0077 805) 107. | 17.0350 OO2T lf 7ST \WOA I) W775 * + 770 | 181 |\18.0 |, .389 | oo2r. | 779 108, | 15.01], 3737) eas 783 | 154+ | eet | eal eeegza aig |r 8a 300 emeooas 798 | 181+ 749 | 117 | 46.4) 375 .0032 753 | Cou T{ 2 \vay ets. 443 .0038 707 | 181+ | || 755 | 121 | 17-3 | -399 | .0033 | 730 |nleae (escord 425 | .0032 ire Se vee a i sto aja lees 750 |\tae | 27.8) 4.2007 |" soo 75° |, TAZ \alOrOn | 1.845 0023 792) || 148. | 1848, | 3382 .0026 766 | 161 | 19.8 es si | | 802 | 17+ Ay. Thy 5.5 a . 400 .0030 | Av. 20 | | 403 .008 3 easess| 5: .. |+.006 |+.0001 cases |+ .004 |+ 0007 T No visible regeneration has taken.place. ** These cases are not included in the general result because one chela in each is much smaller than the other probably as a result of secondary injury. ** Chela deformed and small. No measurement taken. < (Plus sign), see under Table VI. Rate of Regeneration. 359 “4. RESUEES. Rate of Moulting. A comparison of Series A with Series B shows that the individ- uals of the latter, the ones with the greater degree of injury, moult sooner than those of the former, the ones with the lesser degree of injury. The data for the rate of moulting are collected in Table V in which ts given the number of individuals of each series which E J WW’ 7 iy SET V2 3 W 2 1 ile W 3 W' 4 iar Fic. 2. Fi, 3. Diagrams to illustrate the comparative degree of injury in Series A (Fig. 2) and Series B (Fig. 3). CC! = chele. Wr to W4, W'1 to W'4 = walking legs. Plain lines = uninjured legs. Barred lines = removed legs. had moulted once, twice or three times 95, 130, and 181 days after the operation. ‘The males and females are considered separately in each case because the rate was found to differ in the two. The more rapid rate of moulting of the series with the greater degree of injury 1s evident throughout. Ninety-five days after the operation only seven out of the seventeen male members of the series with 360 Charles Zeleny. the lesser 1 injury (Series A), or 41 per cent, had moulted while at the same time all those of Series B had moulted. Likewise 95 days after the operation only one out of the nineteen female mem- bers of Series A or 5.3 per cent had moulted while thirteen out of the 25 living members of Series B, or 52.0 per cent, had done so. At the same time the only individuals that had moulted a second time belonged to the series with the greater injury. Of these one is a male and the other four are females. One hundred and thirty days after the operation Series B shows a similar advantage over Series A. Only five out of the nineteen females, or 26.3 per cent, of Series A had moulted once or more as against nineteen out of twenty-five, or 76.0 per cent, of the living females of Series B. At the same time only 6.7 per cent of the males and 5.3 per cent of the females of Series A had moulted twice while 53.8 per cent of the males and 50.0 per cent of the females of Series B had done so. ‘The one individual that had moulted a third time belongs to the series with the greater injury in accordance with the general rule for the other moults. The final data as collected 181 days after the operation, when the experiment was closed, show the same advantage of Series B over Series A. ‘Thus only 42.9 per cent of the living males and 16.7 per cent of the living females have moulted twice while 76.9 per cent of the living males and 87.5 per cent of the living females of Series B have done so. For the third moult at the same time there are none of the males and only one female, or 5.6 per cent of the living females, in Series A as against one male, or 9.1 per cent of the living males, and three females, or 15.0 per cent of those living, in series B. The general result is very clear. ‘The individuals of Series B moult more rapidly than those of Series A. Emphasis must be again laid on the fact that Series B differs from Series A only in the greater degree of injury in the former. All other conditions are as nearly alike as possible in the two cases. Specific Amount of Regeneration. The amount of regeneration of the right chela at the end of the first moult divided by the thoracic length gives a quotient which may be called the specific amount of regeneration or the amount per unit of thoracic length. It is a fairly constant quantity for the Rate of Regeneration. 361 individuals of one sex in a series and is equal in the two series. (See Tables VI and VII, pp. 357 and 358.) The amount of regeneration of the right chela at the end of the first moult is therefore the same no matter what the degree of injury may be. The average specific amount of regeneration for the males of Series A at the end of the first moult is .444 (+.003). For the males of Series B at the same time it 1s .435 (+.006). ‘The differ- ence between the two is just equal to the sum of the probable errors and therefore cannot be considered as significant. Like- wise the females of Series A have an average specific amount of regeneration equal to .400 ( +.006) and those of Series B an aver- age of .403 (+.004). The difference is less than the sums of the probable errors and 1s therefore not significant. These results show very definitely that the specific amount of regeneration of a removed chela at the end of the first moult after the operation is a constant which 1s not affected by the time of the moult, the size of the animal, or the degree of other injuries to the individual. Four of the individuals, which moulted very soon after the operation, three within the first day and one in three days, are not included in this statement. None of the individuals moulted in the interval between three and twenty-seven days after the operation so that it is not possible to say to what degree the state- ment holds true for this period. For all periods above 27 days up to 181 days when the experiment was closed the specific amount of regeneration is a fairly constant quantity for the first moult after the operation. Specific Rate of Regeneration. The specific amount of regeneration of the right chela divided by the number of days between the date of operation and the first moult gives the specific rate of regeneration. The specific rate of regeneration is the amount of regeneration per unit of thoracic length per day. The average specific rate of regeneration of the two chelz in the series with the greater injury (Series B) is greater than that of the one removed chela in Series A. This is brought out very definitely in Tables VI and VII (pp. 357 and 358). For the males the values of the specific rate are .0049 ( +.0003) for Series A and .0080 ( +.0005) for Series B. For the females the corresponding 362 Charles Zeleny. values are .0030 ( +.0001) for Series A and .0083 ( +.0007) for Series B. In each instance there 1s a very striking advantage of the series with the greater injury over the one with the lesser ‘injury. ‘This amounts to 63 per cent for the males and 177 per cent for the females. “The individuals with the two chelz and the last two pairs of walking legs removed as compared with the individuals in which the right chela alone is removed regenerate the right chela more rapidly than do the latter. ‘This takes place notwithstanding the fact that at the same time they have also to regenerate the left chela at the same rate as the right one and also the last two pairs of walking legs. ‘The individual therefore which has the greater amount of material to regenerate regenerates each part faster than does the individual with the smaller amount of removed material. Relation between Rate of Moulting and Rate of Regeneration. The fact that the specific amount of regeneration is a constant for all individuals of a sex makes the relation between the rate of regeneration and the rate of moulting a very close one. One of three possibilities in the relation of the two must be the true one. The more rapid rate of regeneration of the limbs may be the cause of the acceleration of the moulting or the opposite may be the case or finally the two phenomena may be co-ordinate and only indirectly related. If the first is the case the erowing limb- buds in pressing against their chitinous envelopes more vigorously 1 in Series B must be supposed to act as the stimuli for the increase in the rate of moulting. If the second possibility is true the first result of the operation is an acceleration of the rate of moulting which secondarily affects the rate of regeneration. Finally it is possible that the stimulus of the removal of the limbs acts directly and independently upon both regeneration and moulting processes. 5. DISCUSSION. e In opposition to the very common belief that an increase in the degree of injury to an individual implies a lowering of its ability to repair the sustained injuries the experiments on the several forms mentioned above' have shown that with an increase in the 1Gelasimus, Alpheus, Ophioglypha, Cambarus (see pp. 347-362). Rate of Regeneration. 363 number of removed legs or arms there is an increase and not a decrease in the rate of regeneration of each. ‘This striking fact must be reckoned with in any theory bearing on the nature of regeneration. It would be premature to attempt to build up a constructive theory on the basis of the few facts so far discovered. Enough, however, 1s clear to make profitable the mention of the bearing of the facts on some of the more common theories of regeneration. 1. Observers who have had to do with the responses of animals to adverse conditions have pointed out again and again the pecu- liar fact that the response to such adverse conditions is in a direc- tion advantageous to the animal. ‘The results of the present ex- periments may therefore be taken as another instance of such a response. ‘lhe crayfish with the greater number of removed legs and the brittle-star with the greater number of removed arms respond to the greater injury by an increase in the rate of regenera- tion of each member. Obviously the animal with the greater number of removed appendages has more need of the absent organs than does the other. It therefore may seem very plain to some that the rate of regeneration is greater in the one case because there 1s more need Br a rapid replacement i in that case. Itis but a step further to the familiar statement that the cause of any need in an organism may be taken as a sufhcient cause for the fulfill- ment of that need. ‘There are unfortunately a few who have been and will continue to be satished with superficial explanations of this character. For these the problem of regeneration 1s con- sidered solved when the naive statement is mands that if a part of an animal is removed it is obviously more advantageous to the animal to regenerate a new part than not to regenerate one. 2. The suggestion may be made that the animal with the greater number of appendages gone, exercises the regenerating ones more vigorously than does the animal with the smaller num- ber gone. As a result of this greater activity the regenerating appendages grow more rapidly in the former case than in the latter. In support of this idea it may be said, for instance, that the animal which still has one uninjured chela concentrates its chela-functions upon that organ, thereby lessening the activity of the regenerating bud. On the other hand the animal with both chela gone having no uninjured chela upon which to concentrate its ghela: functions exercises to their full extent the developing 364 Charles Zeleny. functions of the new buds. ‘Thus each of the new buds grows faster than the single bud of the other animal because each gets more exercise of its parts than does the latter. Unfortunately observations made upon the individuals of the two series did not show any difference between them as regards the activity of either the old or the new parts. It is, however, very hard to judge differences in activity in animals like the crayfish in the present experiment, for the specimens are observed only when disturbed by the presence of the observer. ‘Uhe individuals with one remain- ing chela under these circumstances naturally often put themselves in a defensive attitude threatening the observer with their unin- jured chela. The members of the other series having no chela cannot do this. A strong individuality was found in the members of both series. Daily bee on of the individuals in the experi- ment for 181 days with but a few gaps enabled me to make out striking differences in the activities of members of a single series. These individual differences in function were not correlated with any differences in the resulting regeneration as far as I was able to decide. Though there is no evidenke one way or the other from the present instance, the comparative activities of the organs in future experiments of a similar character should be carefully observed. On the other hand there seems to be great danger in carrying the idea too far for it is inconceivable to me how the attempt of an animal to exercise a function for which it has no morphological background can lead to the formation of a structure furnishing the necessary background. Does not such a state- ment of the case come dangerously near to the other statement that “‘the cause for the existence of a need is a sufhcient cause for the fulfillment of that need?” A mystical attempt to function resulting from the need to function has been supplied, that is all. 3. The difference in the mechanical redistribution of food materials which results from the difference in the extent of the injury may be supposed to cause directly the greater rate in the one series. A discussion of the assumptions which must be made in order to explain the facts on this basis will be interesting. Before going on it will be well to recognize the fact that the difference in activity of the parts in the two series as formulated above under the second suggestion (pp. 363, 364) is supposed to lead to a difference in the distribution of food materials which in turn brings about the difference in rate of growth. ‘The same must be oid of all Fic. 4. = Fic. 6. Fic. 5. Diagrams to illustrate the distribution of food materials of a constant amount (K), to the limbs of an unoperated crayfish (Fig. 4), a crayfish of Series A (Fig. 5), and a crayfish of Series B (Fig. 6). 366 Charles Zeleny. other attempts at explanation. The factor that they bring up may be supposed in every case to cause a difference in food- material distribution which in turn causes the difference in rate of growth. Under the present head it is therefore pertinent to dis- cuss only the supposition that the direct mechanical disturbance of the channels of food-distribution shunts the materials off in such proportions into the new channels as to make probable an explanation purely on these grounds. At the very beginning the pure assumption must be eile that the total amount of food materials elaborated and involved in chela-building in our case is a constant (K) in all individuals regardless of the character of the injury. The source and distribution of the food stuffs may be indicated by the diagrams shown in Figs. 4, 5 and 6. ‘The facts to be explained are that chela C in Series A oie 5) regenerates less rapidly than either chela C or Ct in Series B (Fig. 6). The assumption according to the hypothesis now being tested is that the rate of growth and regeneration is determined ‘by the amount of food material, an increase in the amount of food material going to a part determining the increase in rate of growth or regenera- tion of that part. ‘The assumption is also made that the same kind of material is used in the growth of an uninjured chela as in the regeneration of a removed one and that the total amount of this food-material being distributed is a constant (K). The distribution of the food-material may be represented for the three cases given in Figs. 4, 5 and 6 by the following formule: K= C+C'+D+D!+ E+E! (Unoperated’ series, Fig. 4.) =rep. bud C+C'+D+D!tE+E. (Series A, Fig. 5.) = reg. bud C+ reg. bud C'+ D+D!'+reg. bud E+reg. bud E'. (Series B, Fig.6). Now the rate of regeneration of chela C in Series B is greater than that of chela C in Series A. ‘Therefore according to the pres- ent hypothesis the amount of food material going to the former chela bud is greater than that going to the latter or reg. bud C (in senies|b) > tee. bud ©(inioeries A). ‘Therefore reg; bud'C' + D 4D! ree. bud E+ ressbudt > @' + D+ Dkk. (Series B). » (Series A). But D + D' is the same in the two series. ‘Therefore reg. bud C!+ reg. bud E+ reg. bud E1< C'+ E+ FE} Rate of Regeneration. 367 or the regenerating buds of the chela and walking legs receive less food material than do the same organs when uninjured. As the total amount of food material to be distributed is by hypothesis constant it follows that when a greater number of appendages is removed the surplus of material is greater. The surplus of material is therefore greater in Series B than in Series A and crowds upon both the regenerating parts more vigorously than in the latter. “The regenerating buds in Series B, the series with the greater injury therefore grow more rapidly than those of Series A. Evidently when the degree of injury becomes great enough to dis- turb the mechanism of production of food material so that the amount of the latter is diminished and there is no longer a con- stant quantity K, the present statement cannot hold. 4. The results of the experiments on the relation between the degree of injury and the rate of regeneration of the crayfish and the brittle-star bring out very strongly an. essential difference between crystals and organisms. In the former no matter what the number of removed parts the growth of each in a nutrient solu- tion is entirely independent of the number or character of other removed parts in the same crystal. In the organism on the other hand there is no such independence. No part of the organism can be removed without affecting all other parts. ‘This difference may, however, be due to the difference in the nature of the food- supply and not to an essential difference in the structures them- selves. In crystals the food material is external and practically inexhaustible. Each part is thus independent of restrictions due to amount of food material. 5. The stimulation of the nerve of a leg or arm as a result of the injury to that member may be supposed t to induce the processes leading to its regeneration. If it is assumed that an increase in shinmulation causes more than a corresponding increase in intensity of such processes there will result a greater rate of regeneration in an animal with a greater injury an in one with a lessees injury. Physiologists have found that in general the curves for increasing stimulus and increasing response are not parallel. In some cases and this is especially true near the lower limits of the curves, the response curve runs up faster than does the stimulation curve. The organism possesses some inertia in every case and it is neces- sary to overcome this before any response at all is obtained. Hav- ing passed the lower limit, however, there is a very rapid upward 368 Charles Zeleny. rise of the response curve in many instances. May not the acceleration of the regeneration rate with an increase in injury to the animal be explained on a similar basis? 6. Ina series of experiments on the opercula of the Serpulid worms it was shown that when the large functional operculum is cut off near the middle of its stalk the small rudimentary oper- culum of the opposite side develops into a functional operculum while the remaining stalk of the old functional drops off at its base and in place of it a new rudimentary operculum is developed. ‘The final result of the operation is therefore a reversal in position of the opercula. When the rudimentary operculum is cut off a. new rudimentary is regenerated in its place. When the whole head region of the animal is cut off the two opercula which are regenerated are equal in size and resemble the old functional one.* It seems therefore that when one of the regenerating buds gets a start over the other it holds the latter back at the rudimentary stage. On the other hand when both buds have an equal start two opercula of equal size are developed. A retardation stimulus must be assumed to be given out by the functional operculum which holds the rudimentary operculum in check. When the organ is removed the retardation stimulus is likewise removed and the rudimentary operculum is enabled to develop into a functional one. The same method of reasoning may be applied to the case of the regenerating chelz of the crayfish. Each uninjured chela may be assutned to exert a retarding influence upon the growth or regeneration of all the others. When only one chela is removed the number of uninjured limbs remaining is greater than when the other chela and the last two pairs of walking legs are also removed. ‘The retardation influence in the former case is there- fore greater than it is in the latter and correspondingly the rate of regeneration in the former case is smaller than it is in the latter. The term “retardation influence or stimulus” is undoubtedly a very vague one. It may perhaps be best considered as a nervous 'The results obtained for the chele of Alpheus are essentially similar except in the case with both chele removed where the regenerating chele are not alike. The difference here is probably due to the fact that the removal of both chele at their breaking joints leaves a basal stump on each side and is not a total removal as in the corresponding case of the Serpulid opercula. However, the resulting chele even here show an approach toward similarity. (See Przibram, ’or, Arch. Entw. Mech., xil; Wilson, ’03, Biol. Bull., iv; Zeleny, ’05, Journ. Exp. Zodl., ii.) Rate of Regeneration. 369 influence exerted either upon the other organ or organs directly or else upon the mechanism for carrying food Aredials to those organs. The retardation influence is brought out in a somewhat different light when considered as a manifestation of the inertia of the organism. This idea also tends to unite the two suggestions of positive stimulation as a result of injury to the snp aa the negative or retardation stimulus exerted by the uninjured organs. The former acts after the operation in overcoming the inertia of the organism. ‘The latter on the other hand is merely a mani- festation of the same inertia acting before removal. The crayfish data when taken alone furnish no evidence in favor of either one of these two views as opposed to the other. ‘The experiments on the Serpulid opercula and Alpheus chelz, however, cannot be as well explained by the positive stimulation theory as by the retardation view. The foregoing speculations are evidently of but small direct value. ‘Their purpose is accomplished if they have emphasized the importance of the discovered relation between the degree of injury and the rate of regeneration in any general theory of regen- eration. 6. SUMMARY. A comparison was made of the rate of regeneration and the rate of moulting in two series of crayfish with different degrees of injury. In one series the right chela alone was removed. In the other series the two chelz and the last two pairs of walking legs were removed. It was found that the rate of regeneration of each chela in the series with the greater injury is greater than that of the single removed chela in the series with the lesser injury. Likewise the rate of moulting of the animals is greater in the former series than in the latter. Indiana University, May 31, 1905. STUDIES ON CHROMOSOMES. I. THE BEHAVIOR OF THE IDIOCHROMOSOMES IN HEMIPTERA.* BY EDMUND B. WILSON. With 7 Ficures. In studying the spermatocyte-divisions in Lygzus turcicus and Coenus delius, and afterward in several other genera of Hemip- tera, my attention was directed to the fact that the number of chromosomes appeared to vary, polar views of the equatorial plate showing sometimes seven chromosomes, sometimes eight (cf. Figs. 1b, 11, 2a, 21, 3d, 37, etc.). Montgomery, in his extensive comparative paper of 1901, describes and figures a similar varia- tion in a number of cases, including Coenus delius and Euschistus tristigmus COI, pp- TOK, 166), and in the latter case considered it as a result of variations in the synapsis of the two “ chromatin nucleoli” which he supposed might either conjugate to form a bivalent body kefore the first division (in which case this division 'This paper is based on a study of some very fine series of sections of the testes of certain Hemiptera, prepared six or eight years ago by Dr. F. C. Paulmier, in connection with his valuable paper on the spermatogenesis of Anasa tristis (’99). Part of the original Anasa sections, with a number of series of the testes of some other insects, were given to the cytological cabinet of the Columbia laboratory at that time; some of the best of the remainder were subsequently loaned to Mr. Sutton, and others to Dr. Dublin, for comparison with their work on the spermatogenesis of other forms. Certain inconsistencies in the literature relating to the accessory chromosome and the microchromosomes or ‘‘chromatin-nucleoli ” led me to re-examine the preparations of Anasa and some of the other genera, which yielded some new and interesting conclusions in the case of Anasa, and also of Alydus, Lygeus and Conus. Dr. Paulmier being preoccupied with other lines of work did not find it practicable again to take up his cytological studies, and he was generous enough to give me, for the laboratory, his entire set of preparations, com- prising, in addition to the slides already given or loaned, serial sections of more than a hundred testes representing upward of twenty genera of Hemiptera and other insects. A typical series of the Hemip- tera from which these testes were taken had been identified by the eminent specialist, Mr. P. R. Uhler. Much of this material is admirably fixed, sectioned and stained, and the best preparations are a model of technical excellence, showing especially the chromosomes of the spermatogonial and spermatocyte divisions with a clearness and brilliancy comparable with that of the best Ascaris preparations. The 372 Edmund B. Wilson. was described as showing but seven chromosomes) or might remain separate during this division (in which case eight separate chro- mosomes appear). I soon found, however, that in Lygzeus and Coenus whole cysts differed in this respect, all of the cells of a given cyst constantly showing one number or the other. With this is correlated the fact that in the anaphases of the second division no accessory chromosome in the usual sense of the term 1s present, all the spermatid-nuclei receiving the same number of chromo- somes, namely, seven, which is-half the spermatogonial number in both species. Further study conclusively showed that in both of the species the cells with eight chromosomes were primary sper- matocytes undergoing the first maturation-division, while those with seven were the secondary spermatocytes undergoing the second division. Of this fact no doubt can exist, since the second- ary spermatocytes are much smaller than the primary ones, the spindles are shorter, the chromosomes only half as large, the meta- phase-figures are often found in the same cysts with the character- istic late anaphases and telophases, and all the stages of both divisions were found in abundance. ‘Though a great number of division-hgures have been examined, I have never seen seven chromosomes in the first division in any of the forms examined; and though I will not deny that Montgomery may be correct in the statement that such forms occur, I believe he was misled on most successful preparations are from material fixed with strong Flemming’s fluid, and stained with iron hematoxylin followed by long extraction, in some cases followed also by counter staining with Congo red or orange G. ‘These show the cytoplasm completely decolorized, the chromosomes intensely black, and with outlines of such regularity and sharpness that the most careful camera drawings give the appear- ance of being schematized. A few very fine series were stained with Zwaardemaker’s saffranin (which gives a splendid transparent stain). Many others were fastened to the slide unstained, and some of these I have stained with saffranin and gentian violet (the method recommended by Montgomery) which have given very valuable control results, especially in regard to the accessory chromosome and plasmosome which in the earlier growth stages are not well differentiated by the hematoxylin. I am much indebted to Dr. Paulmier’s generosity in placing at my disposal this valuable material, to which I have since added many new preparations of my own. In a subsequent paper I shall describe the results of a re-examination of some of the maturation phenomena in Anasa and Alydus, in both of which there is demonstrative evidence that the accessory chromosome is not the small central chromosome or microchromosome (‘‘chromatin-nucleolus” of Mont- gomery), as Paulmier supposed in the case of Anasa, but the odd or peripheral one, precisely as Gross (’04) has recently described in Syromastes. While looking over some of the other species for the sake of comparison my attention was directed, first in the spermatogenesis of Lygus turcicus and Coenus delius and afterward in that of Euschistus, Podisus and other forms, to the phenomena which form the subject of the present paper. ail Studies on Chromosomes. 373 this point by failing in some cases to distinguish between the two divisions. On tracing out the history of the two divisions step by step, decisive proof was obtained that the apparent reduction in number is brought about in the period immediately following the final anaphase of the first division (which coincides with the earliest prophase of the second division) by a conjugation of two unequal chromosomes that occupy the center of the equatorial plate in the first division and evidently correspond to some of the forms designated by Montgomery as “chromatin-nucleoli.”” ‘This pro- cess can be determined with certainty, owing to the fact that in all of the species, with a single exception, one of the two conjugating chromosomes is much smaller than the others, while in Lygzeus both are much smaller, and they are very unequal in size. ‘The central dyad of the second division is therefore asymmetrical, one of its constituents being in Lygzeus not less than five or six, and in Coenus not less than two or three, times the bulk of the other. The two unequal constituents of this dyad are then immediately separated again in the ensuing division in such a manner that in both species one half the spermatids receive the smaller, one half the larger motety of the central chromosome (or dyad) of the second division. ee essentially similar process was ultimately found to occur in“Euschistus fissilis, in another undetermined species of the same genus, in Brochymena, Nezara, Podisus and Tricho- pepla. The first four of these show the same chromosome- numbers as in Lygzeus and Coenus. In Podisus the number is in each division one more than in the corresponding divisions of the other genera (1. e., respectively g and 8 instead of 8 and 7, while the spermatogonial number is 16 instead of 14).1_ Nezara differs from the other genera in the fact, which is of importance for a comparison with such forms as Anasa or Alydus, that the two chromosomes which undergo conjugation after the first division are of equal size; so that in this form the two classes of spermatids are indistinguishable by the eye. Since the eight species | have 1TIn several of these cases the numbers do not agree with those given by Montgomery (’o1,1). I believe this observer to have been misled by the fact, which he also observed in some cases, that the first division shows one more than half the spermatogonial number of chromosomes; and it is easy to mistake the latter number owing to the fact that the larger spermatogonial chromosomes often show a more or less marked constriction in the middle. Slightly oblique views of the late metaphase, when the chromo- somes are double, may also readily give an erroneous result. 374 Edmund B. Wilson. examined represent two different families of Hemiptera (Penta- tomidze and Lygzidz) the roca: will probably be found to be of wide occurrence in the group.! “The only other case known to me in any higher plant or animal of the unequal division of a chromosome (or chromathe -body) in karyokinesis occurs in Tingis clavata, regarding which Montgomery states that one of the chromosomes of the first division “very frequently is seen to be characterized in having its two components of very unequal vol- ume” (’O1, 2, p. 262). ‘This author also observed a considerable number of cases in which the “chromatin nucleoli’’ are unequal in the rest stage of the spermatogonia, and he describes some forms in which 4 similar condition appears in the growth-period of the spermatocytes (e. g., in ‘Trichopepla, Peribalus and Euschistus tristigmus). In the last-named species he found that a separation of the two unequal “chromatin nucleoli” takes place in the second mitosis (’O1, 1 » Pp. 161, 162), but expressly states that they are not joined ugh | in the apne oe plate (opscit:, p. £02)5 7 Teas evr dent from Montgomery’s brief description ao this phenomenon is similar to, and probably identical with, the one that forms the subject of this paper. TERMINOLOGY. Since confusion may readily arise in the terminology, I wish to define clearly the terms that will be employed throughout. this paper and its successors. I shall apply the term “chromosome”’ to each coherent chromatin-mass, whatever be its form, mode of origin or valence, which as such enters the equatorial plate. In the case of compound or plurivalent chromosomes (“tetrads”’ or “dyads”) McClung’s term “chromatid” may conveniently be applied to each of een univalent constituents. | may call atten- tion, in connection with this, to the fact that the valence of chro- mosomes cannot be determined by mere inspection of their form. In many Hemiptera, for example, the chromosomes of the first maturation-division frequently show a dyad-like or dumb-bell shape (typically the case, for example, in Euschistus, Lygzeus or Coenus) even though in earlier stages they are plainly quadripar- ‘Since writing the above I have found the idiochromosomes in several additional genera. In Mineus they are only slightly unequal, in Murgantia nearly as unequal as in Lygeus. Nezara, Mineus, Brochymena, Euschistus, Murgantia and Lygeus thus show a progressively graded series of stages in the size-differentiation of this peculiar pair of chromosomes. Studies on Chromosomes. 275 tite; and such dyad-lke forms, agreeing both in mode of origin and in fate with actual tetrads, may occur in the same equatorial plate with obviously quadripartite forms (c7. Fig. 2e). Conversely it will be shown beyond that bivalent and univalent chromosomes occurring in the same equatorial plate may exactly agree in form, though having a wholly different mode of origin. The purely descriptive term “Fale elisa gOS” ’ (peculiar or distinctive chromosomes) will be applied to the two chromo- somes, usually unequal 1 in size, which, as stated above, undergo a very late conjugation and subsequent asymmetrical deen to the spermatid-nuclei. These bodies, as already stated, are iden- tical with some of those to which Montgomery (’o1, ’04) has applied the term “chromatin nucleoli.” This use of the latter term 1s, however, undesirable, since the accessory chromosome also appears in the growth-period (of Orthoptera and some Hemiptera) in the form of a chromatin-nucleolus. I shall, there- fore, employ the latter term in a broader’sense to designate any compact deeply staining chromatin-mass, present in the resting nucleus, which afterward contributes to the formation of the chromosomes. When, as in case of the accessory chromosome, or the 1diochromosomes, such a chromatin-nucleolus represents a single chromosome or pair of chromosomes it may conveniently be called a ‘‘chromosome-nucleolus”’; but I think this term should be restricted to the resting nuclei and cannot appropriately be applied to the corresponding chromosome of the division-stage. Especially large or small chromosomes may be designated as ‘““macrochromosomes” or “microchromosomes,”’ irrespective of their behavior. DESCRIPTIVE. In the following account Lygzeus and Coenus will be taken as types, a brief comparison of the other forms being added. Some of the latter—especially Brochymena and Nezara—present features of peculiar interest which I hope to make the subject of a special study hereafter. t. Ihe Maturation Divisions. Lyezus and Coenus show an extremely close agreement in the y Vi g general history of the chromosome-group, and especially in the behavior of the idiochromosomes; though the earlier history of 376 Edmund B. Wilson. these bodies shows a more primitive condition in the former genus. I have followed their behavior in the early stages less completely in Euschistus and Podisus, but their behavior during the matura- tion-divisions in these forms is closely similar to that of the others and leads to an exactly similar result. Ly gzeus Is in some respects the most favorable of all these species owing to the remarkable disparity in size between the idiochromosomes; and to the fact that both are so much smaller than the other chromosomes as to admit of their immediate identification at every period. In all the species, the chromosomes show distinct and constant size-differences. A largest chromosome or macrochromosome may be distinguished in all, and in most cases a second largest; and in all, the small idiochromosome is the smallest of the group and typically lies near the center of the equatorial plate. (Figs. 1b, 2c, 2d, 3a, d, f, etc.) It is difficult to be sure of the size-differ- ences in case of the other chromosomes, owing to variations in form and position,which produce various degrees of foreshortening. In all the forms, with the exception of Nezara, the larger chromo- somes of the first division are typically arranged in an irregular ring within which lie the two idiochromosomes, side by side, but always quite separate (Figs. 1b, 2a, 3a, d, j, etc.). ‘This grouping is apparently invariable in Lygzeus, but in Coenus and Euschistus the larger idiochromosome frequently lies in the outer ring (Figs. 2b, 3c). In Lygzeus the two idiochromosomes, within the ring, are always much smaller than any of the outer ones, and the smaller is so minute that at first sight I mistook it for a centro- some. In Ccenus both the idiochromosomes are relatively larger than in Lygzus, and their inequality of size is less striking (Fig. 2, a, b, gb). The larger one is about equal in size to the smallest of the peripheral chromosomes and hence cannot be certainly distinguished when it lies in the outer ring (Fig. 2). In both species an equatorial plate occasionally occurs in which nine chromosomes clearly appear (Figs. td, 2c), but this is excep- tional, and I have never found a spindle showing this body in division. ‘The presence of this additional chromosome is probably due to a failure of synapsis between two of the spermatogonial chromosomes which normally conjugate to form a bivalent body, and it is evidently to be regarded as an abnormal condition. 1Cf. Montgomery’s Fig. 105, of Corizus, or, I. W ~I “SI Studies on Chromosomes. Ud G a ae tl HM ose ose | r) @ P / 11 0 Fic. 1.1 Lygeus turcicus. a, e, metaphase of first division—in the first two figures several of the chromo- somes are represented out of their natural positions at one side; b, c, normal metaphase-groups in polar view, first division; d, abnormal form with nine chromosomes; f, the idiochromosomes and two others, in early anaphase, first division; g, h, daughter-groups, late anaphase first division, from the same spindle; 7, j, equatorial plates of second division, at the metaphase, in polar view; k, prophase of second division, showing all the chromosomes just before taking up their definitive positions; /, metaphase of second division—three of the chromosomes drawn out of position at one side; m, separation of the idio- chromosomes, second division; 7, 0, daughter-groups, late anaphase of second division, from the same spindle. All of the figures were drawn as carefully as possible with the camera, a iz oil immersion, and compensation ocular 12 (Zeiss), enlarged 24 diameters with a drawing camera, carefully corrected by renewed comparison with the objects and then reduced in the engraving to one-half. At such an enlargement some error is unavoidable, but great care has been taken to represent the chromosomes 378 Edmund B. Wilson. In side views of the spindles both species usually show the chro- mosomes of a symmetrical dumb-bell shape (Figs, 1a, eb 2d), though one or more of them may appear quadripartite, as is espe- atv common in case of the largest one or macrochromosome (Fig. 2b, e). In both forms all of the eight chromosomes are symmetrically divided in the first mitosis (Figs. If, 2e, 7), giving rise to two exactly similar daughter-groups of eight chromosomes each (Figs. 1g, b, 2g, b). The rate at which the daughter- chromosomes separate varies widely in different cases. Fre- quently the idiochromosomes lead the way in the march toward the poles and may be widely separated at a time when one or more of the larger chromosomes are only just separating (Fig. If), while the macrochromosome often lags behind the others (Fig. 2¢); but now and then a spindle shows the reverse condition, the small idiochromosome being the slowest of the group. In the end the daughter-chromosomes come to lie at the same level, and in the final anaphase are drawn more closely together. At this period the grouping of the chromosomes is exactly the same in the two species, the two idiochromosomes lying close together and closely surrounded by a ring formed by the six larger chromosomes. In spindles that lie vertically or slightly obliquely the two daughter- groups may in both species be seen, with the greatest. clearness, to, be exact duplicates of each other, proving beyond doubt the equal division of all of the eight chromosomes of the first mitosis (Figs. g, b, 2g, bh), and the size-relations of the chromosomes persist without noticeable change. In Lygzeus at this period the two idiochromosomes are still as a rule clearly separated; in Coenus this may be the case, but they sometimes lie in close contact, already forming a dyad almost identical in appearance with the central upeuuel dyad of the second mitosis (Fig. 2/). as Fe as possible, and none of the figures are schematized in this respect, except that in a few cases one or more of the chromosomes have been drawn out of their natural positions in order to avoid confusion in the figures. It should be noted that in the division-stages there is some variation in the actual size of the chromosomes, and this is more or less exaggerated in the figures owing partly to slight differences in position, which cause foreshortening in various degrees, and partly to differences in form (different degrees of elongation of the chromosomes cause corresponding variations in thickness as seen in polar view). No attempt has been made to represent the minuter details of the spindle- fibers or asters, though the figures are but slightly schematized in this respect. Some of the stages @f the growth-period (especially stages b, d and f) are difficult to represent adequately in pen drawings; though I have attempted to show them as‘accurately as possible. In all the figures the idiochromosomes are marked 7, the plasmosome p. Studies on Chromosomes. 379 In the stage that immediately follows, the chromosomes for a brief period become so crowded that their exact changes cannot be followed. In slightly later prophases of the second division, which follows without a pause, the chromosomes again spread apart, @*. e ee @e,0 ee. @ee, ®,e° ENGe 2: Coenus delius. a,b, normal equatorial plates, metaphase of first division; c, abnormal form with nine chromosomes; d, the entire chromosome-group, first division metaphase, in side view, the upper four in their natural position; e, f, anaphases in side view showing in each case the idiochromosomes and three others; g, 4, daughter-groups, late anaphase of first division, from the same spindle; 7, 7, equatorial plates, metaphase of second division, polar view; k, metaphase of second division; /, later metaphase, separation of the idiochromosomes; m, —o, p, two pairs of daughter-groups, late anaphase, second division, in each case from the same spindle. 380 Edmund B. Wilson. and it may now be seen, even before the equatorial plate is formed, that the two idiochromosomes, retaining their characteristic size- relations, have conjugated to form an asymmetrical dyad, which shows the most striking contrast to the six other dyads, all of which have a symmetrical dumb-bell shape (Fig. 1k). “The seven dyads are now drawn into the equatorial plate, the asymmetrical one invariably lying at the center of a ring formed by the six symmetrical ones (Figs. 1/, m, 2k, 3b). “These dyads place them- selves with their long axes parallel to that of the spindle, so that when seen in polar view they present a circular or more or less ovoidal outline; if they lie in a slightly oblique position, as is fre- quently the case, they may give a bipartite appearance. Since in polar view the small idiochromosome lies above or below the large one it is usually invisible, and hence only the larger one appears at the center of the equatorial plate (Figs. 17, h 20,4). Un thereanby, metaphase the chromosomes, especially in Ccoenus, are often rather widely separated, so that the equatorial plate may be nearly or quite as wide as in the first division (cj. Figs. 2a, b, 21, 7). Such figures might at first sight readily be mistaken for those of the first division, but without exception, in my material, both the chromosomes and the cell-bodies of the 7-chromosome cells are much smaller than those of the 8-chromosome ones; and the com- pleteness of the series and the great number of division- -figures that I have had under observation precludes, I think, the possi- bility of error on this point. In the ensuing division each of the dyads draws apart into two spheroidal single chromosomes, the peripheral ones dividing equally, while the idiochromosome-dyad separates into its two unequal constituents—invariably, I believe, leading the way in the division (Figs. 17m, 2/, 3c, gk). Owing to this fact the unequal division of the central dyad may be seen with unmistakable clear- ness. In polar or slightly oblique view of the late anaphases, when both daughter-groups are visible, the asymmetrical result may plainly be seen. In such figures the two daughter-groups show a most striking contrast to those of the first division, being no longer duplicates of each other, and both showing seven instead of eight chromosomes. Each daughter-group shows, as in the metaphase-group, a ring of six larger chromosomes (now single spheroidal bodies) within which lies at one pole the smaller, at the other pole the larger, of the idiochromosomes (Figs. In, 0, 2m, Studies on Chromosomes. 381 n, 0, p). Each spermatid-nucleus thus receives seven chromo- somes, one-half the spermatogonial number, and no accessory chromosome, in the usual sense of the word, 1s present; but the spermatids nevertheless consist of two groups, equal in number, one of which contains the smaller, the other the larger of the 1diochro- mosomes. In the mature spermatozoa I have not been able to detect any corresponding difference. Euschistus fissilis, Euschistus sp., Podisus spinosus. a-c, Euschistus fissilis. a, metaphase- group, first division; b, c, metaphase-figure, and early anaphase in side view, second division. d-1, Euschistus sp.; d, e, metaphase groups, first division; f, metaphase-group, second division; g, second division, side view; 4, 7, daughter-groups, late anaphase of second division, from the same spindle; j-m, Podisus spinosus; j, metaphase-group, first. division; k, late anaphase, second division; /, m, daughter-groups from the same spindle, late anaphase, second division. In Euschistus, Brochymena, Podisus and Trichopepla the facts are, with a few variations of detail, essentially similar. In both species of Euschistus and in Brochymena the number of chromo- 382 Edn:und B. Wilson. somes agrees with that of Lygzeus, being r4 in the spermatogonia, eight in ‘the first spermatocyte- divistene sa seven in the second, and their grouping Is similar (Figs. 3, 7), save that in Brochymena, alone among all the forms, TS cea idiochromosome frequently lies in the outer: ring (Fig. 7&). Podisus differs only " the fact that the numbers are respectively 9: and 8 (Pies-275 a), while the spermatogonial number is 16 instead of 14 (Fig. 5). Trichopepla 1s a puzzling case which I have not yet fully cleared up. [he daughter-groups of the second division show 7 chromo- /) Fic. 4. Nezara. a,b, metaphase-groups, first division; c, metaphase-group, second division; d, entire chro- mosome-group, second division, in side view, showing equal division (three chromosome-pairs from a lower level of the spindle shown at the right); e, {, duplicate sister chromosome-groups, anaphase of second division, from the same spindle; g, #, spermatogonial metaphase-groups. somes each (Fig. 7g, r), the idiochromosomes being well differ- entiated. ‘The ee division, however, agrees with Montgomery’ S description in showing either g (Fig. 70) or 8, the emolles: chro- mosome being in the latter case wanting. The conditions in Nezara are of particular interest from a comparative point of view in that the tdiochromosomes are of equal Studies on Chromosomes. 383 size. The first spermatocyte- -division shows 8 chromosomes, which differ in grouping from that of the other forms in that all usually le in a ring without a chromosome at its center (Fig. 4 a,b). For this reason no clue to the identification of the idiochro- mosomes is given by their position. “Iwo of the chromosomes are, however, distinctly smaller than the others; and the relations in the spermatogonia leave little doubt that it 1s these two that corre- spond to the idiochromosomes. ‘They may lie side by side (Fig. 4a) or more or less widely separated (4)). All these chromosomes are, in side view, seen to have a symmetrical dumb-bell shape, and all are equally halved in the first division. None of the prepara- tions show the stage immediately following; but there can be no doubt that a conjugation of the two small chromosomes takes place at this time, since the second division (of which I have a large number) invariably shows in polar view but 7 chromosomes, which have now assumed the usual arrangement, with one in the center of a ring formed by the 6 others (Fig. 4c). Nezara differs again, Hoerer. from all the other forms in the fact that the small chro- mosome (7. ¢., the idiochromosome-dyad) lies in the outer ring in many if not in ail cases, while one of the larger nisin lies at the center of the ring. Lateral views of the spindle show all of the chromosomes as quite symmetrical dyads; and in the ensuing division all divide equally (Fig. 4d). In such views the iachrunoeonee -dyad, which is readily recognizable by its size, may be clearly seen to divide equally; and in this respect Nezara differs from all the others. The anaphase sister-groups of the same spindle, each containing 7 chromosomes (Fig. 42, f) are, accordingly, exact duplicates, the idiochromosome retaining its position in the outer ring. The symmetrical a eicion of the idiochromosome-dyad in Nez- ara 1s a fact of importance for the comparison of the idiochromo- somes with the microchromosomes of such forms as Anasa or Alydus. Were it not for their failure to unite to form a bivalent body until the end of the first mitosis we should find no ground in this case for designating these chromosomes by a special name. 2 T he S permatogonial Chromosomes. We have now to examine the relation of the chromosomes of the first maturation to those of the spermatogonia. The material 384 Edmund B. Wilson. for the spermatogonial division is most abundant in the case of Lygzus, which is much the most favorable form for an accurate count, the chromosomes being well separated and showing with almost schematic clearness. In the preparations of this form numerous spermatogonial plates appear, showing, whenever an accurate count can be made, without exception 14 chromosomes (Fig. 5g, b). Nezara, of which numerous spermatogonial divisions are also available, shows the same number and with almost equal clearness, though the chromosomes are in this form more crowded. In the other forms the material is less abundant, but the relations are clearly shown in most of them. Ccenus (Fig. 57) Euschistus (Fig. 57), and Brochymena (Fig. 77) also show 14 spermatogonial chromosomes, while in Podisus (Fig. 5k) the number is 16.1. In all these cases, therefore, the spermat- ogonial number is double that of the chromosomes in the second spermatocyte-division, and two less than double the number in the first division. The most striking fact is that in all these forms, with the exception of Nezara, the spermatogonial groups show but one microchromosome (marked 1 in Figs. 4, 5, 7); in striking contrast to the fact first determined by Paula. in Anasa and afterward by Montgomery in many other Hemiptera, that two such bodies (“chromatin-nucleoli” of Montgomery) equal in size, are often present. Did this observation rest only on the examination of a few division-figures (as in the case of Coenus, Euschistus, Podisus and Brochymena) I should hardly trust in its general applicability to the species; but in Lygazus numerous demonstrative cases remove every doubt regarding this point, and the agreement of the other forms in their later history makes it nearly certain that my observation is not at fault with them. Distinct, though not very great, size-differences may be observed in the larger spermatogonial chromosomes, as has been indicated by Montgomery in several other genera of Hemiptera. Though these differences are not nearly as marked as those recognized by Sutton in Brachystola, it is nevertheless pretty clearly evident ‘Montgomery (’o1, 1) gives the numbers as follows: Euschistus variolarius 16, E. tristigmus 14 Nezara 16, Coenus 14, Brochymena 16, Podisus 16. This point is emphasized since Montgomery describes and figures two spermatogonial microchro- mosomes (‘‘chromatin-nucleoli’’) in Euschistus variolarius (’o1, 1, Figs. 2, 3), E. tristigmus (op. cit., Fig. 20), Coenus delius (Fig. 55), and Brochymena (Fig. 47). One of the figures of the first-named species shows them equal, the other unequal, in size. Studies On Chromosomes. 385 that the larger chromosomes may be grouped in six pairs; and in Figs. 5), 1,9, 42, 7x01 have attempted to indicate these by corre- sponding numbers; though no pretense to complete accuracy of identification can be aide! since the chromosomes vary somewhat in form and their apparent sizes vary somewhat with their posi- tion, owing to foreshortening. Allowing for all errors of identifica- tion it is obvious that in all but Nezara 12 of the larger chromo- Fic. 5. Ceenus (a-f, 7), Lygeus (g, 4), Euschistus (j), Podisus (k}. a, Ccenus, contraction-phase, showing both idiochromosomes in the form of chromosome-nucleoli; b, prophase, corresponding to stage / in Lygeus, the two idiochromosomes and four of the others; c-f, chromosome-nucleoli of Coenus from a stage corresponding to stage f; c, d, both idiochromosomes present, c, f, single chromosome-nucleoli; g, h, spermatogonial metaphase-groups, Lygeeus; 7, spermatogonial group, Ccenus; /, spermatogonial group, Euschistus, sp.; k, spermatogonial group, Podisus. somes may be symmetrically paired off, two by two, while a thirteenth (unnumbered in the figures) 1s left without a fellow of like size. “The conclusion is, I think, irresistible that in synapsis 4In the last figure the chromosomes have been by inadvertence wrongly lettered. 386 Edmund B. Wilson. twelve of the larger chromosomes unite to form the six peripheral bivalents of the first division, that the thirteenth is the larger idiochromosome and the fourteenth (the microchromosome) the small idiochromosome. ‘These two alone remain separate in the first division as univalent chromosomes, thus giving a group of eight instead of seven. ‘This conclusion is in accordance with Montgomery’s interpretation of the facts observed by him in Euchistus tristigmus, as pointed out beyond, except that he believed that in this form the first division might in many cases show only seven chromosomes, the “chromatin nucleoli”’ having, like the other chromosomes, already conjugated to form a bivalent body. My interpretation is strikingly confirmed by the facts observed in Nezara; for in this case, where the first spermatocyte- division shows two chromosomes of equal size and the smallest of the group, the spermatogonia correspondingly show two equal muicrochromosomes, as in Anasa, Alydus, or Protenor (Fig. 4g, /). Since these two are represented 1 in the second division by a single symmetrical dyad, which is again the smallest of the group (Fig. 4 g-f) it is evident that the two equal microchromosomes must conjugate after the first division. ‘They therefore agree in behavior with the unequal idiochromosomes present in the other forms, and differ from those of the Anasa type, in remaining separate during the first maturation-mitosis. The Growth-Period of the Primary Spermatocytes. Seo (a) General History. It is not my purpose to describe 1n detail at this time the general history of the chromatin during the growth-period, but it ail be convenient to outline the stages in order to trace the history of the idiochromosomes. In Lygzeus ten well marked stages may be distinguished from the early synapsis (a) to the metaphase of the frst division (7), inclusive. Though some of these stages are best characterized by the condition of the idiochromosomes, an account of the latter can more readily be given after considering the history of the larger chromosomes. Ge stage a (early sy napsis), which shortly follows the final anaphase of the last spermatogonial division, the chromosomes have the form of rather ragged, longi- tudinally split loops, the free ends of which converge wane Studies on Chromosomes. 387 one pole of the nucleus. In stage ) (contraction-phase, Fig. 62) they become closely massed in a spheroidal aggregation at the center or toward one side of the nucleus, and surrounded by a large clear space. At this time they stain so deeply that their Fic. 6. Lygeus turcicus. a, contraction-phase of synaptic period (stage b) showing large idiochromosome; b, early post-synapsis (stage c), both idiochromosomes shown; c, stage d, a plasmosome connected with the large idiochromosome; d-f, chromosome-nucleoli (with plasmosome) from stage e; d and e single, f, showing the two idiochromosomes; g, stage f, with plasmosome at its maximum size, and both idio- chromosomes; h-n, chromosome-nucleoli from stage f (represented side by side near the plasmosome, out of their natural position); h-k, four cases in which but one is present, /-n showing both idiochromo- somes; 0, stage g, with both idiochromosomes; Pp, q, stage /, two sections of the same nucleus showing all of the eight chromosomes and a small plasmosome; r, stage 7, showing the six larger chromosomes and the two idiochromosomes. 388 Edmund B. Wilson. outlines can only with difficulty be made out, and then only after long extraction. In stage c (early post- synapsis) they again spread through the nuclear cavity, giving the appearance off an interrupted, contorted and more or less confused spireme, com- posed of delicate and somewhat varicose threads (Fig. 6b). In stage d (Fig. 6c) the threads become somewhat shorter and thicker and have a more open arrangement, but still show a very marked spireme-like arrangement. I believe the threads to be at this period longitudinally split, though this can only be made out with difficulty. ‘This stage is well characterized by the condition of the large idiochromosome, as described beyond, and by the appear- ance of a pale plasmosome. In stage e the threads become mark- edly shorter and thicker, ragged in outlines, often faintly show a longitudinal split, and diminich somewhat in staining-capacity. ane chromosomes now undergo a great change, becoming in stage } very loose in texture, showing only vague “boundaries, and Biase completely losing their staining-capacity, so that it is difficult to represent ei appearance in a black and white figure (Fig. 6g). As may be seen from the figure the chromosomes now give the appearance of a rather vague, pale, finely granular Benwire. in which traces of a spireme-like arrangement can usually be seen, but they cannot be clearly made out as individual bodies. Stage g again “shows a great change (Fig. 60) the chro- mosomes having peed het staining capacity and definite boundaries, shale now appearing as long, coarse, winding threads, often showing rather ragged outlines and a more or less distinct longitudinal split, as in stage e; the two stages may, however, at once be distinguished both by the position of the cells in the testis and by the different relation of the plasmosome and the chromo- some-nucleoli, as described below. The condensation of the chromosomes now takes place, the succeeding three stages follow- ing in rapid succession. In stage / the long split rods shorten and thicken, stain much more deeply, and assume a great variety of forms—curved double rods, dumb-bell shaped figures (some- times longitudinally split), closed rings, and peculiar cross-forms. (Fig. 6p, g, which show all the chromosomes from a_ single nucleus.) In stage 7 (early prophase) all these forms condense into quadripartite tetrads or dyad-like bodies, the latter consisting of two symmetrical halves closely joined together and frequently showing no trace of a second division, though i in the same nucleus Studies on Chromosomes. 389 may occur one or more distinctly quadripartite forms (Fig. 6r). The history of these dyad-like bodies clearly shows, I think, that with the exception of the idiochromosomes they are derived from bivalent longitudinally split rods, and they have therefore the same valence as the actual tetrads with which they are associated. The nuclear membrane now disappears and the chromosomes are drawn into the equatorial plate of the first mitosis. ‘The general history of the growth-period in Ccenus is similar to that of Lygzeus, but is in some respects abbreviated; and stage f is much less marked, the chromosomes not losing this Boundaries or their staining-capacity in so great a degree, and still presenting the appearance of a ragged interrupted spireme. (b) The Idiochromosomes during the Growth-period. The foregoing description applies only to the larger or ordinary chromosomes. ‘Throughout the whole of the growth-period in Coenus and from stage e onward in Lygzus, at least one of the idiochromosomes can alw ays be distinguished as a compact, spheroidal, intensely staining Shiro mioseeie nucleolus, and fre- quently both idiochromosomes are distinguishable in this form in all of these stages. The early stages of Lygzeus (b to d) are of especial interest in that the condensation of the idiochromosomes is delayed, and at least the larger one still has the form of an elon- gate, longitudinally split rod or thread. Even at this time, however, it is immediately distinguishable from the others by its greater thickness and greater staining capacity. It is clear beyond all question, therefore, that at least the large idiochromosome may retain its identity throughout the whole orowth- period. With the small idiochromosome the case is not so strong, as will be seen from the following account. It is convenient to trace the history of the idiochromosomes in the reverse order from stage 1 backward, again taking Lygzeus as the type. In this stage (late prophase), when the six larger chromosomes are in the form of condensed tetrads or dyad-like bodies, both the idiochromosomes have very distinctly the form of dyads. The nucleus now contains therefore eight separate chromosomes, among which the idiochromosomes are at once recognizable by their small size (6r). In stage / the idiochromo- somes have the same general appearance, though their bipartite 390 Edmund B. Wilson. form is often less distinct (67). Up to this point both idiochro- mosomes are apparently always present. In the stages now to be considered both are often plainly distinguishable, but quite as frequently this is not the case, the nucleus showing but a single chromosome-nucleolus the size of which proves it to be either the large idiochromosome or the large and small one united. Between these two possibilities | have not been able to decide in Lygzeus and Coenus; but decisive evidence is given in the case of Brochy- mena, as described beyond. In stage g (60) the idiochromosomes (or the single chromosome-nucleolus) appear as spheroidal com- pact bodies, usually not showing a bipartite structure, and in addition one or more pale rounded plasmosomes are often present. In‘stage 7, owing to the loss of staining capacity by the larger chromosomes, the idiochromosomes show with brilliant clearness, since they are still stained intensely black, and may very readily be studied with care (6g- n). When both are present they appear slightly larger than in the later stages, and often the larger one is plainly seen to be hollow (which probably accounts for its larger size) though this is shown still more clearly 1 in Brochymena, as described beyond. A small plasmosome is sometimes attached to it at one side, but in addition to this there is always present a very large pale plasmosome quite free from both idiochromosomes, and free also from the single chromosome-nucleolus when but one of these bodies is present. In stage e the idiochromosomes present the same appearance, but the /arger one is now always attached to the large plasmosome, and often more or less flattened against it (as is also the single chromosome-nucleolus when but one appears) while the small one is almost always free from it (Fig. 6d, e, 7). The larger body is as before often evidently hollow. Up to this point Lygzeus and Ccenus agree almost exactly. In the earlier stages they differ in that Coenus still shows the idio- chromosomes in the form of compact chromosome-nucleoli, while in Lygzeus the larger one certainly, and I believe the smaller one also, assumes the form of a longitudinally split elongate chro- mosome. In stage d in Lygzus (Fig. 6c) the large idiochromo- some is a rather short, deeply staining rod, longitudinally split, and still attached (usually toward one end) to the plasmosome, which is now considerably smaller. ‘The small idiochromosome is now also more or less elongate, but I cannot be sure whether it Studies on Chromosomes. 391 is longitudinally split. All intermediate stages may readily be observed between this stage and the next earlier one (c) in which the large ciigchromoconne is an elongate split thread that may extend through more than half the diameter of the nucleus (Fig. Op): Lt 1s, however, at once distinguishable from the other chro- mosomes by its straighter course, greater thickness and deeper staining capacity,’ which renders it very conspicuous among the thread-like chromosomes. No plasmosome can now be seen. The small idiochromosome can still clearly be distinguished in many of the nuclei as a short rod staining like the larger one. Finally, in the contraction-phase (stage b) when all the chromo- somes are massed together, the large idiochromosome still unmis- takably appears as a very distinct deeply staining rod, sometimes ° nearly straight, more usually curved, and frequently horse-shoe shaped, at one side of the chromosome-mass (6a). Neither plasmosome nor small idiochromosome can now be made out. In Ccenus at this period (Fig. 5a) both idiochromosomes (or the single chromosome-nucleolus) still appear as compact, deeply staining spheroidal bodies, the larger one typically having a small plasmosome attached to it at one side. The early history of the large idiochromosome proves most clearly that the chromosome-nucleolus into which it afterward condenses is a modified chromosome (as Montgomery first showed in Euschistus variolarius) and one that forms a connecting link between the ordinary chromosomes and the more usual forms of “chromatin-nucleoli” in Hemiptera, described by Montgomery, or the accessory chromosome of Orthoptera; for in none a these latter is the condensation to a compact body so long delayed. Paulmier (99) showed, however, that in Anasa the compact chro- mosome-nucleolus of the synaptic period, afterward elongates considerably and appears as a rather short longitudinally “split rod, similar to that of Lygzeus at stage d (Paulmier, Fig. 22) afterward again condensing into a compact tetrad. In Ly gaeus there can be little doubt that the central cavity of the spheroidal chromosome-nucleolus, often visible in stages e—g, represents the original longitudinal split. It is therefore hardly open to doubt that the division of the large idiochromosome in the first mitosis 1s an equation-division, and the same is probably true of the small 'This has been somewhat exaggerated in the engraving. 392 Edmund B. Wilson. idiochromosome. It is, on the other hand, quite certain that the division of the idiochromosome-dyad in the second mitosis is a reduction-division. [he order of the divisions in case of the idiochromosomes is thus the reverse of that which occurs in the other chromosomes, according to Paulmier’s and Montgomery’s accounts; and as pointed out beyond, it 1s also the reverse of that which takes place in the division of the small central chromosome in Anasa and Alydus. As already stated, I did not in Lygzeus and Ccenus, succeed in finding any certain explanation of the fact that the nuclei of the growth-period may show either one or two chromosome-nucleoli. In Brochymena, however, there 1s very clear evidence on this point. Here, too, the nuclei of the middle growth period show either one or two spheroidal chromosome-nucleoli, the former con- dition being much the more frequent. When both are present they may be widely separated or close together, and both very clearly show a central cavity, which 1s rendered very conspicuous by the fact that the chromatin is frequently concentrated in a dark zone immediately around it (Fig. 7--g). When but one is present it 1s, as a rule, perfectly spherical, hollow, and shows no evidence of a double nature (Fig. 77, g). In the early growth- period, however, the single chromosome-nucleolus almost always appears bipartite, being composed of two unequal halves, forming an asymmetrical dyad (Fig. 7a, b) very similar to that seen in the second maturation-division (Fig. 77m). At a later period both of the constituents become hollow (and hence appear somewhat larger) and stain less deeply; and all gradations may be observed in the fusion of the two bodies to form a single hollow body (Fig. 7¢, d, f) which is plainly as large as the two separate chromosome- nucleoli (such as may be seen in cells of the same cyst) taken together. In Brochymena, therefore, there can be no doubt that when only one chromosome-nucleolus 1s present it 1s to be considered as a bivalent body arising by the fusion or synapsis of the two idiochromosomes. Thus far the facts confirm the interpretation given by Mont- gomery (OI, 1) who observed in Coenus, Euschistus tristigmus and some other forms that the cells of the growth-period may ahars either a single “chromatin-nucleolus” or (in Euschistus “ appar- ently more frequently’ ) two such bodies that are unequal in size; and this fact he interpreted to mean that the two corresponding Studies on Chromosomes. 393 spermatogonial “ chromatin-nucleoli” may either conjugate at the period of general synapsis to form a bivalent body or may remain separate as univalent bodies. As already pointed out, it was probably this interpretation that led him to conclude that the first division in these forms might show either seven or eight chromosomes. But the later stages observed in Brochymena give conclusive evidence that even though such a primary synapsis EG: 7: Brochymena, Trichopepla. a-n, Brochymena, o-r, Trichopepla; a, b, idiochromosomes and plas- mosomes, from early growth-period; c-g, condition of the idiochromosomes in middle and late growth- periods; h, prophase of first division, showing idiochromosome-tetrad; 7, j, two stages, one following and one preceding the last, in the division of the bivalent chromosome-nucleolus; k, metaphase-group, first division; /, metaphase-group, second division; m, side view, second division, showing idiochromo- some-dyad and two other chromosomes; ”, spermatogonial metaphase-group; 0, metaphase-group, first division, Trichopepla; p, spindle of second division in lateral view; q,r, sister-groups, late anaphase of second division. 394 Edmund B. Wilson. of the idiochromosomes takes place the bivalent chromosome- nucleolus again separates into its univalent constituents in the early prophases of the first maturation-division. ‘This process, which at first greatly puzzled me, occurs at the time just preceding the concentration of the larger chromosomes into their final condensed form (corresponding to stage hin Lygzus). In cysts of this period every stage may be seen in the transformation of the single chro- mosome- Futleolis into an asymmetrical tetrad, consisting of two symmetrical dumb-bell shaped bodies of unequal size (Fig. eae and the separation of these unequal dyads to form the idinchrore: somes of the first division (cf. Figs. 7h, 1 i 1): It 1s evident that in these cases the final reunion or conjugation at the end of the first division is not a primary ‘synapsis, but a secondary process.! ‘The facts observed in Brochymena make it very probable that when only a single chromosome-nucleolus is present in Lygzus, Coenus and the other forms, it is there also a bivalent body as Montgomery assumed; but the uniform separation of the 1dio- chromosomes in the first division of all the eight species I have examined 1s almost a demonstration that in all the forms a division of the bivalent body must occur. On the other hand it seems equally certain that in many of these forms the idiochromosomes may fail to unite at the period of general synapsis, and may remain separate through the whole growth- period; and in Brochymena the same cysts Pehab show the division of the single 1diochromo- some-tetrad may also contain nuclei in which the dumb-bell shaped idiochromosomes are widely separated. In such cases it seems probable that the conjugation of the idiochromosomes at the close of the first spermatocyte-division must be regarded as a true or primary synapsis that has been deferred to this late period. DISCUSSION OF RESULTS. ‘The most essential fact brought out by a study of the idiochro- mosomes is that in Lygzeus, Coenus, Podisus, Euschistus, Brochy- mena and Trichopepla a dimorphism of the spermatozoa exists, there being two groups equal in number, both of which contain 'The division of the bivalent chromosome-nucleolus is similar to the process described by Gross (04) in Syromastes; though it occurs at a much later period. For reasons that will appear in a subsequent paper, I am, however, skeptical in regard to Gross’s conclusion which is based on a study not of the idiochromosomes but of paired microchromosomes similar to those of Anasa or Alydus. Studies on Chromosomes. 395 the same number of chromosomes, but differ in respect to one of them. In this respect these genera differ from all those that possess an accessory chromosome (Pyrrochoris, Anasa, Alydus, etc.), since in the latter case one-half of the spermatozoa receive one chromosome fewer than the other half. It is remarkable that two types of dimorphism apparently so different should coexist within the limits of a single order of insects. We are thus led to inquire into the relation between the idiochromosomes and the accessory; and this inquiry must also include the “small chromosomes” of Paulmier and the “chromatin-nucleoli” of Montgomery. It will conduce to clearness if the second part of this question be considered first. lt as evident from the figures and descriptions of Montgomery (O82 OI, 1, OL, 2; 04) that the bodies I have called idiochromo- somes are idendeat with some of those that this author has de- scribed under the name of “chromatin-nucleoli” (which are usually also small chromosomes or microchromosomes); but it is now manifest that the bodies described under the latter name are not all of the same nature. It is evident that two types of these bodies may be distinguished that differ markedly in their behavior during the maturation-mitosis. One of these, typically repre- Fonted in Anasa, Alydus and Protenor appear in the spermat- ogonia in the form of two equal microchromosomes (‘‘chromatin- nucleoli’ e) which like the other chromosomes sooner or later unite in synapsis to form a bivalent body that les at the center of the equatorial plate of the first mitosis. Both divisions accordingly show exactly one half the spermatogonial number of ehromouunice but it is a very noteworthy fact that the final conjugation of the two microchromosomes is long deferred, taking place inthe propha- ses of the first division (as was first observed by ; Montgomery i in Pro- tenor and some other forms, more recently by Gross in Syromastes and by myself in Anasa and Alydus). In this case there seems to be no doubt that the first division of the bivalent body thus formed is a reducing division. It appears to be further characteristic of this type—at least in all the forms mentioned—that a true acces- sory chromosome is associated with the microchromosomes, and that only one-half of the spermatozoa receive one-half the somatic number of chromosomes, the other spermatozoa receiving one less than this. The distinction between the accessory and the microchromosomes or ‘‘chromatin-nucleoli”’ first demonstrated 396 Edmund B. Wilson. by Montgomery (or) in Protenor, has been more recently shown by Gross ((04) to exist in Syromastes; and I have also been able to demonstrate the same fact in Alydus and in Anasa (Paulmier having been in error in his identification of the accessory with the small chromosome in the last-named form). The second type includes the idiochromosomes, which in the forms I have studied differ from the Anasa type in four respects (Nezara being an exception in regard to the first of these), namely, (1) in their unequal size, with which is correlated the fact that only a single microchromosome appears in the spermatogonia; (2) in the fact that the final conjugation or synapsis of these bodies is deferred until the prophases of the second division, a result of which is that the first division shows one more than half the sper- matogonial number of chromosomes while the -second division shows exactly half the spermatogonial number; (3) in the fact that in case of the idiochromosomes It is manifestly the second division that is the reducing one, while my observations on Lygzus render it practically certain that the first is an equation-division; (4) in the fact that no accessory chromosome in the usual sense is present and all the spermatozoa receive the same number of chromosomes. In view of these differences it seems expedient for the present to place these two types in different categories. It is evident from Montgomery’s figures and descriptions that he observed many of the details of the phenomena described in the present paper; but it is equally clear from the varying 1 interpreta- tions that he adopted that he failed to reach any consistent general result regarding the behavior of the idiochromosomes, or to recog- nize the dimorphism of the spermatozoa. For example, it is evident, I think, from his descriptions of Euschistus variolarius, E. tristigmus, Cocenus delius, Oncopeltus fasciatus, and Lygus pratensis, that the essential facts in these forms agree with those I have described, the idiochromosomes being in the last named two species of equal size, as in Nezara; eS Montgomery offers for each of these cases a different interpretation. In the first named species the idiochromosomes are clearly figured in his first paper (98, Figs. 171, 188, 189, etc.), and in Fig. 214 they are shown separating “a quite typical fashion in the pad division (the smaller one designated as a “chromatin-nucleolus”); but it is evident from the descriptions given in both this and the following paper (‘o1, 1) that he did not reach a correct interpretation of the Studies on Chromosomes. 397 facts. In Euschistus tristigmus and Coenus delius the first divi- sion is stated to show either seven or eight chromosomes (the spermatogonial number being 14), but quite different interpreta- tions are given of this in the two species, the conditions in Euschis- tus being assumed to be due to the frequent failure of the two “chromatin nucleoli” to unite in synapsis, while mn the case of Coenus seven of the chromosomes (including the large “ chromatin- nucleolus”) are assumed to be bivalent, while the eighth is an additional small “chromatin-nucleolus”’ not distinguishable in the spermatogonia (’o1, I, p. 166). In Euschistus tristigmus the “chromatin-nucleoli” are stated to be of unequal size and to be separated from each other without divison in the second mitosis. ‘This is evidently the same phenomenon that I have described; though Montgomery overlooked the conjugation of the two facqual: ‘chromatin-nucleoli”’ at the end of the first division, and expressly states that they are not joined together 1 in the second division. In Oncopeltus, likewise, the first division shows one more than half the spermatogonial number, 16 (7. ¢., nine instead of eight, precisely as I have described in Podisus), and this is stated to result from the persistence of the two “ chromatin-nucle- oli” throughout the whole growth period without union; but an interpretation differing from both the foregoing is here sought in the assumption that each of the two “chromatin-nucleoli”’ is bivalent, even in the spermatogonia (oI, 1, p. 186). In Lygus pratensis, finally, the first division shows 18 ics and the second 17, the still different explanation being here offered that the two “chromatin-nucleoli” pass undivided one to each pole of the first spindle (o1, 1). Of these various interpretations only the one given in the case of Euschistus tristigmus, I believe, con- 1The first mitosis is here clearly shown to have eight chromosomes, grouped in the same way as in my ‘‘Euschistus sp.” and the anaphase daughter-plate of the second division is shown with seven (Fig. 220), precisely as in the two species I have studied. Montgomery gave the spermatogonial number, correctly I believe, as 14. He nevertheless concluded that all of the eight chromosomes (seven chromo- somes + 1 ‘‘chromatin nucleolus”’) divide separately in both divisions, apparently overlooking the fact that this would give the spermatozoa one chromosome too many (since he himself demonstrated that the “‘chromatin-nucleolus” is a modified chromosome). This account of the divisions is not modified in the paper of 1901 except in the statement that ‘in the second maturation-division the chromatin-nucleo- lus is not always divided” (p. 161), while the spermatogonial number is now given as 16. Since the figures of the earlier paper show that the divisions in E. variolarius are evidently the same as in the species I have examined, I think that on both these points the first account was probably more accurate than the later one. 398 Edmund B. Wilson. forms to the true one, and it is probable that all of these cases will be found to agree in the essential phenomena with those I have determined in Lygzeus, Coenus, Nezara and the other forms. We may now inquire what 1s the relation of the idiochromo- somes to the accessory chromosome. ‘The observations suggest so obvious an answer to this question that I wish to indicate not only the evidence in its favor, but more especially the difhculties it has to encounter. In forms possessing an accessory chromo- some the spermatozoa fall into two equal groups that differ only in respect to one chromosome. ‘The same is true of Lygzeus and other forms that lack the accessory but possess the idionhmomes somes, with the difference that in the former case the distinctive chromosome is present in but one-half the spermatozoa, while in the latter case two such distinctive chromosomes are present, one of which is present in one-half, the other in the other half, of the spermatozoa. It is impossible to overlook the evident analogy between the two cases; and the idiochromosomes may in one sense be considered as two accessory chromosomes that are never allotted to the same spermatozoon since each fails to divide in the second mitosis (precisely as 1s the case with the single accessory in other Hemiptera)./ The difference between Lygzeus and Ccenus in the size-ratio of the idiochromosomes obviously suggests the view thatthe single accessory of other forms may have arisen by the disappearance of one of the idiochromosomes; and in Lygzeus the smaller one is already so minute as distinctly to suggest a vestigial structure. / We might accordingly assume that in a more primitive type the two idiochromosomes were of equal size (as in Nezara), undergoing synapsis and subsequent reduction in the same way as Bie. Bees chromosomes; that Coenus and Lygzeus represent successive stages in the reduction of one of these chromosomes, and that by the final disappearance of the smaller one in such forms as Anasa or Pyrrochoris a single accessory chromosome remains. This hypothesis at first sight seems to give a clear and intelli- gible view of the origin of the accessory chromosome, and to recon- eile the remarkable mode of spermatogenesis occurring in the insects with forms in which no accessory seems to appear. But further reflection shows that it has to encounter a formidable if not insuperable difficulty in the fact that in some of the forms possessing an accessory chromosome the number of spermato- Studies on Chromosomes. 399 e wr. gonial chromosomes is an even one (as in Anasa and Syromastes); and there seems to be no escape from the conclusion that the acces- sory 1s here a bivalent body arising by the synapsis of two equal spermatogonial chromosomes. Even in cases showing an odd number of spermatogonial chromosomes (as in many Orthoptera and some Hemiptera—for example Alydus or Protenor) it has been assumed, and with good reason, that one of the chromosomes (probably the accessory ) is already bivalent,t and Montgomery has shown G or, 1) that in Protenor the large accessory Gh chrome: some x’’) is sometimes transversely constricted into two equal halves in the spermatogonia. A similar fact was subsequently shown in Harmostes (’01, 2) which also has normally an odd sper- matogonial number. ‘To this should be added the fact that these forms possess the small bivalent central chromosome (which arises by the synapsis of two equal microchromosomes) in addition to the accessory. The difficulty pointed out above cannot be escaped by supposing that the disappearance of one of the idio- chromosomes has been effected by its gradual absorption by the other; for this assumption, too, fails to explain the even number of spermatogonial chromosomes. Apparently therefore the hypo- thesis | have suggested, must in the present state of our knowl- edge be considered untenable.” _ It appears more probable that the idiochromosomes are com- parable to the two equal microchromosomes or “chromatin- 1Cf. Montgomery, ’04. Since this paper was sent to press I have determined beyond the possibility of doubt, I think, that the number of spermatogonial chromosomes in Anasa tristis is 21, not 22 as given by both Paul- mier and Montgomery. ‘This result is based on the study of a large number of preparations, and careful camera drawings of more than twenty perfectly clear metaphase figures have been made. All without exception show 21 chromosomes, and I have sought in vain for even a single cell that shows 22. (Paulmier’s original slides were used.) If corroboratory evidence be needed it is given by the fact that there are always three macrochromosomes, one of which is obviously without a mate of like size, and is probably the accessory. I have, also, positively determined the spermatogonial number to be 21 in a form included in Paulmier’s material and labeled “ Chariesterus antennator,’’ (since this number disagrees with Montgomery’s count of the spermatocytes there may be an error of iden- tification; but the form is certainly different from Anasa) and 15 in Archimerus calcarator (from my own material, identified by Mr. Uhler), both members of the same family as Anasa. This wholly unexpected result perhaps justifies a certain skepticism in my mind in regard to the accounts of other observers, who give an even spermatogonial number for forms possessing an accessory chromosome; and if this be well founded the objection urged above disappears. I shall return to this subject hereafter. It is needless to say that had I been acquainted with these facts, the discussion that follows would have been different. 400 Edmund B. Wilson. nucleoli” which in such forms as Anasa or Alydus conjugate to form the small central chromosome of the first mitosis. “The dif- ferences between the two forms have already been pointed out. ‘Their resemblances are, however, no less obvious, namely, their usual central position in the equatorial plate, small size, occasional persistence as chromosome-nucleoli in the growth-period, and their late conjugation. ‘his comparison finds very definite support in the conditions | have described in Nezara, where the 1dio- chromosomes are of equal size and appear as two equal micro- chromosomes in the spermatogonia. From the analogy of other forms it is very probable that the more primitive and typical form of synapsis is that between chromosomes of like size. It 1s there- fore probable that such a condition as that observed in Nezara is a less modified one than that in which the 1diochromosomes are unequal; and that the latter condition has arisen through a second- ary morphological differentiation of two chremasomies that were originally of equal size, and perhaps are represented by the two equal microchromosomes that appear in the spermatogonia of such Hemiptera as Anasa, Alydus, Sy romastes or Protenor. This comparison involves two assumptions, namely, first that in case of the idiochromosomes the final conjugation of the micro- chromosomes has been postponed from the prophases of the first division to those of the second; and secondly that a reversal in the order of the reduction- and equation-divisions has taken place in case of these particular chromosomes, the first division being in case of the Anasa- -type the reduction- and in case of the idiochro- mosomes the equation-division. The difficulty apparently involved by the second assumption is less serious than may appear. All the facts at our command indicate that a reduction-division is the necessary, or at least invariable, sequel to a foregoing conjugation; and if, as in the case of the idiochromosomes, the final conjugation is deferred to the second division, the reduction- division must also be deferred. “The univalent idiochromosomes— as is shown with certainty in case of the larger one in Lygeus— undergo longitudinal division at the same stage of the growth- period as their bivalent companions and are already double at the time of the first mitosis. ‘There is, therefore, no difficulty in the way of assuming—indeed, the facts seem to admit of no other conclusion—that this is the equation-division. It must be recognized, however, that the foregoing comparison Studies on Chromosomes. 401 wholly fails to explain the origin or meaning: of the accessory chromosome, nor does it account for the surprising fact (of which the phenomena in Brochymena seem to leave no doubt) that two chromosomes may unite 1n synapsis, subsequently part company so as to divide as univalents in the first mitosis, but again con- jugate to form a bivalent in the second mitosis. It seems likely that further comparative study of this phenomenon may throw important light on the general mechanism of karyokinesis and reduction. | The history of the idiochromosomes possesses a more general interest in the strong support that it lends to the general theory of the individuality of chromosomes, to the specific conclusions of Montgomery and Sutton in regard to synapsis, and especially to the correlation of the phenomena of reduction with those of Men- delian inheritance attempted by the last-named author (’02, ’03). It has been assumed by some authors, including some of those who have accepted Montgomery’s remarkable Careleeion Gere » 04) that corresponding paternal and maternal chromosomes nets in synapsis, that in this process the individuality of the conjugating chromosomes is completely lost—‘‘Sie vereinigen sich zu einem einzigen Zygosom, aus dem erst wieder zwei neue Chromosomen hervorgehen.”* It 1s undoubtedly true that frequently all visible traces BE the duality of the bivalents that emerge from the synapsis stage are for a time lost; and as Sutton suggested COR, pe: 243); such cases as those of first crosses that Greed true—and I may add, perhaps also those in which blended inheritance or weakening of dominance occurs—may be taken to indicate that a permanent fusion, or intermixture of the chromosome-substances, may really take place. But, on the other hand, the history of the idiochro- mosomes in cases where they remain separate through the whole growth-period leaves not the least doubt that as far as these particular chromosomes are concerned the same two that unite in synapsis persist as distinct individuals to be afterward separated by the reducing division and assigned to different germ-cells. ‘This preliminary conjugation and subsequent separation ensures that the germ-cells shall be “pure” in respect to these particular ; chromosomes—1. e., that both shall not enter the same spermat- | ozoon—and if this be true of one pair of the conjugating chromo- | 1Strasburger, ’04, p. 26; cf. also Bonnevie, ’05. 402 Edmund B. Wilson. somes we have good reason to conclude that it may be true of all, as Montgomery has urged and as Sutton has so cogently argued, from a study of the size- Tenens Tt is a fair working hy pothesis that the idiochromosomes represent a pair of corresponding or allelomorphic qualities, or group of qualities, that are respectively maternal and paternal, as Sutton, building on the basis laid by Montgomery and himself, has argued for the chromosome-pairs in general. The argument of Montgomery and Sutton is based, it is true, on the fact that chromosomes of different sizes in the spermatocytes are represented by symmetrical chromosome-pairs of corresponding sizes in the spermatogonia; and to this the idio- chromosomes in most of the cases described form an exception in being unequal. If this appears to be a difficulty it is removed by the case of Nezara, where the idiochromosomes are of equal size. Even in the more usual case, where they are unequal, symmetrical synapsis takes place between all the other chromosome-pairs. ‘If the theory of the individuality of chromosomes be granted no other conclusion seems possible, accordingly, than that the remain- "ing two, despite their size-difference, are respectively the paternal and maternal elements of the remaining pair;/and if Sutton’s general hypothesis be well founded, these elements may be peeumned to be physiological correlates or allelomorphs. Their marked difference in size suggests a corresponding qualitative differentiation, and this inevitably suggests a possible connection between them and the sexual differentiation. The visible dimor- phism of the spermatid-nuclei in such forms as Lygzus, Coenus or Podisus shows too obvious a parallel to the sexual dimorphism of the germ-cells, indicated by so much of the recent work on sex- determination, to be ignored; while in Nezara, where no visible dimorphism exists, the spermatozoa nevertheless fall into two equal groups in respect to the previous behavior of one of the chromosomes. But such a suggestion as to the possible signif- cance of the idiochromosomes immediately encounters the difh- culty that both idiochromosomes are present in the male cells (spermatogonia, and spermatocytes), just as McClung’s similar hypothesis regarding the accessory chromosome is confronted with the fact, determined by Montgomery and Gross, that in the Hem- iptera both sexes show the same number of chromosomes. Whether these difficulties can be met by assumptions of dominance and the like remains to be seen; but the fact should be recognized Studies on Chromosomes. 403 that as far as the Hemiptera are concerned neither the suggestion I have made, nor the hypothesis of McClung has at present any direct support in observed fact.! The practical interest of the idiochromosomes lies in the very definite basis that they give for an examination of the question by the study of fertilization, for their disparity in size gives us the hope of determining their history by direct observation. ‘There is good reason to believe that such a study will yield interesting results. SUMMARY OF OBSERVATIONS. 1. In Lygzus turcicus, Coenus delius, Euschistus fissilis, Euschistus sp., Brochymena, Nezara, Trichopepla and Podisus spinosus all of the spermatids receive the same number of chromo- somes (half the spermatogonial number), and no accessory chro- mosome is present; but the spermatozoa nevertheless consist of two groups, equal in number, which differ in respect to one of the chromosomes, which may conveniently be called the “idiochromo- 5h) some. 2. In all of the forms named, excepting Nezara, half the spermatozoa receive a larger, and half a smaller, idiochromosome. In Nezara the Hiachmomiosormes are of equal size, but agree in behavior with the unequal forms. 3. In all of the forms the idiochromosomes remain separate and univalent in the first maturation-division, while the other chromosomes are bivalent; this division accordingly shows one more than half the spermatogonial number of chromosomes. They divide separately in the first mitosis, but at the close of this division their products conjugate to form a dyad, which in all the forms save Nezara is asymmetrical. ‘The number of separate 1The oe referred to in a preceding foot-note, that the spermatogonial number in Anasa is 21 instead of 22, again goes far to set aside the difficulties here urged. Since this paper was sent to press I have also learned that Dr. N. M. Stevens (by whose kind permission I am able to refer to her results) has independently discovered in a beetle, Tenebrio, a pair of unequal chromosomes that are somewhat similar to the idiochromosomes in Hemiptera and undergo a corresponding distribution to the spermatozoa. She was able to determine, further, the significant fact that the small chromosome is present in the somatic cells of the male only, while in those of the female it is represented by a larger chromosome. These very interesting discoveries, now in course of publication, afford, I think, a strong support to the suggestion made above; and when considered in connection with the com- parison I have drawn between the idiochromosomes and the accessory show that McClung’s hypo- thesis may, in the end, prove to be well founded. 404 Edmund B. Wilson. chromatin elements 1s thus reduced to one half the spermatogonial number. In the second maturation-division the asymmetrical dyad separates into its two unequal constituents, the larger one passing to one pole and the smaller one to the other pole of the spindle, while the other dyads divide equally. 4. In all the forms excepting Nezara the spermatogonia possess but one microchromosome (the small idiochromosome), while in Nezara two equal microchromosomes are present as in forms like Anasa which possess an accessory chromosome. 5. Inthe primary synapsis the idiochromosomes may unite to form a bivalent body or may remain separate. In the former case the bivalent body condenses to form a single chromosome-nucleolus that persists throughout the whole growth-period, but again separates into its univalent constituents before the first mitosis (directly proved in Brochymena, inferred in the other forms). If the idiochromosomes fail to unite in the primary synapsls, they remain separate through the growth-period in the form of chromosome-nucleoli. In either case the idiochromosomes divide separately in the first mitosis. : 6. In Lygeus the large idiochromosome has in the synaptic and early post-synaptic periods the form of a long longitudinally split thread which afterward condenses into a Rolle: spheroidal chromosome-nucleolus. Zodlogical Laboratory, Columbia University, May sth, 1905. WORKS CITED. Bonneviz, K., ’05.—Das Verhalten des Chromatins in den Keimzellen ente- roxenos Ostergreni. Anat. anz., XXV1, 13, 14, I5. Gross, J., 04.—Die Spermatogenese von Syromastes marginatus; Zool. Jahrb., Anat. u. Ontog., xx, 3. Montcomery, T. H., ’98——The Spermatogenesis in Pentatoma, etc. Zool. Jahrb., Anat. u. Ontog., xi. ‘o1, 1.—A Study of the Chromosomes of the Germ-cells of Metazoa. Trans. Amer. Phil. Soc., xx. ‘or, 2.—Further Studies on the Chromosomes of the Hemiptera heterop- tera. Proc. Acad. Nat. Sci., Phil., March, 1gor. ’°04.—Some Observations and Considerations upon the Maturation Phenomena of the Germ-cells. Biol. Bull., vi, 3, Feb. Studies on Chromosomes. 405 PautmigR, F. C., ’99.—The Spermatogenesis of Anasa tristis. Jour. Morph., xv, supplement. STRASBURGER, E., ’04.—Ueber Reduktionsteilung. Sitzber. Kon. Preuss. Akad. Wiss., xvill, 24 Marz, 1904. Sutton, W. S., ’02—On the Morphology of the Chromosome Group in Brachy- stola magna. Biol. Bull., iv, 1. ’03.—The Chromosomes in Heredity. Biol. Bull., iv, 5. Witson, E. B., ’05.—Observations on the Chromosomes in Hemiptera. Rept. N. Y. Academy of Sciences, May 8th, 1905; Science, xxi, 548, June 30. CONTRIBUTIONS FROM THE _ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Directror.—No. 169. THE MOVEMENTS OF THE SWIMMING-PLATES IN CTENOPHORES, WITH REBERENCE TO THE THEORIES OF CILIARY METACHRONISM. BY Ga He eA RNs WitH 2 Ficures. I. INTRODUCTION. Since the publication in 1880 of Chun’s elaborate monograph on the ctenophores, it has been generally admitted, contrary to the opinion of many of the older investigators, that the swimming- plates of these animals are their principal organs of locomotion. Moreover, the ciliary nature of these organs may now be regarded as well established, and their relativ ely ¢ enormous size has already made them favored objects with investigators of ciliary phenom- ena. As is well known, these swimming-plates are arranged in rows and the members of each row, like ordinary cilia, beat metachronally, not synchronally. ‘The explanation of this pecu- larity has called forth two somewhat opposing views. According to the first of these, which has been developed chiefly by Engel- mann (68, p. 475; °79, p. 388), it is maintained that one element beats immediately after its next neighbor in a given order because of a nerve-like impulse that is supposed to pass from cell to cell and thus to bring into action in regular sequence the overlying elements. ‘his may be called the neuroid theory of ciliary action. According to the second view, advanced in the main by Verworn (90, p. 175), the cause of metachronal action is not to be sought for in the cell-body proper, but rather in the mechanical effect of one cilium on another, in that the action of one cilium mechanically stimulates the next one to action. This may be called the me- chanical theory of ciliary action. Because of the minute size of ordinary cilia, experimental tests of these two theories are not easily carried out; hence the anatomical conditions presented in ctenophores are of unusual importance. It is the principal object 408 G. H. Parker. of this paper to discuss the cause of metachronism in ciliary action as exemplified in the swimming-plates of these animals. The material upon which I worked consisted almost entirely of the common summer ctenophore of the New England coast, Mnemuopsis leidyi A. Agassiz, though I also made some observa- tions on the winter species Pleurobrachia rhododactyla L. Agassiz. ‘The work was done for the most part during the last few summers at the Wood’s Hole Laboratory of the United States Bureau of Fisheries, to the officers of which I am under obligations for many kindnesses shown me. II. OBSERVATIONS. Anatomical. Mnemuopsis leidyi is a lobate ctenophore measuring often as much as seven or eight centi- metres in length. Its external form is shown in Fig. 1, which is a view of the animal so placed that its sagittal plane corresponds to the plane of the paper. The mouth is directed downward and the two large lobes that charac- terize this group of ctenophores are seen at the right and left of it. [he aboral pole is pointed upward and four of the eight rows of swimming-plates are shown converging toward it. Their relation to the sense body at the aboral pole can be seen Fic. 1. clearly in Fig. 2, where it will . . . . . . 5 Side view of Mnemiopsis leidyi. The sagit- be observed that from the most tal plane corresponds to that of the paper and the aboral pole is uppermost. Two short sub- aboral plate of each row a nar- transverse rows of swimming plates and two row band extends to the sense long subsagittal ones are seen converging to- > ward the aboral pole. The subsagittal rows body. These bands, before they extend as vibratile lines far over the surface of reach the sense body, unite in the lobes. E i pairs and enter that organ as four bands. As will be seen by comparing Figs. 1 and 2, the rows of swimming-plates are either long or short and the pairs The Movements of the Swimming-Plates in Ctenophores. 409 formed by the unions into the bands consist always of a long row combined with a short one. Since the long rows lie near the sagittal plane and the short ones near the transverse, they have been called, respectively, subsagittal and subtransverse. “The combination of a long subsagittal row with a short subtransverse one to form a pair has long been known to be one of the structural characteristics of the lobate ctenophores, and, as will be shown later, this feature is not without its physiological significance. Each such pair, as can be seen in Fig. 2, is restricted to a quad- rant of the animal’s body. The number of swimming-plates in the subsagittal and the sub- transverse rows varies more or less with the size of the animals. Thus in a small specimen eight millimeters long the subsagittal rows contained each about 1g plates, the subtransverse ones about 13; while in a large individual sixty millimeters long, there were about 73 plates in each subsagittal row and about 39 in the Pbemievere ones. In the speci- men from which Figs. 1 and 2 were drawn, there were about 29 plates in each subsagittal row and about 17 in each subtransverse one. In Mnemuopsis the bands that lead from the sense body to the swimming- nt ne ae : Aboral view of Mnemiopsis leidyi. plates are ciliated, as in other cteno- The four subsagittal rows of swimming- phores, and, as Samassa (o2; p- 229) plates, two from each lobe, and the has shown for other lobate forms, a °° percent on eae tne sense body at the aboral pole. band of cilia connects plate with plate. In this species, however, the spaces between the plates seem to be much more sparsely provided with cilia than in other lobate ctenophores, if, in fact, cilia are not sometimes entirely absent from these regions. The second species upon which [ worked, Pleurobrachia thododactyla, was of simpler structure than Mnemiopsis. It belongs to the Cydippidz and has the typical form of an oblong spheroid. Its eight rows of swimming-plates are of about equal length and can be readily distinguished as subsagittal or sub- transverse only by their relations to other parts in the animal’s Fic. 2. 410 G, Ho Parker body. Ina specimen of average length, about sixteen millimetres, there were approximately 40 plates in each row. Physiological. The resting position of the swimming-plates in both Mnemiop- sis and Plewrebrachias is one in which the individual plate is turned close to the body of the animal and with its tip directed orally. In action the plate makes a vigorous stroke aborally and then returns to its resting position. In consequence of such movements carried out more or less simultaneously by certain plates in each row, the animal’s body 1s moved through the water with the mouth forward. ‘The plates in any one row strike one after another beginning at the aboral end, 7. ¢., to use the term proposed by Verworn (’90, p. 152), they beat metachronally. Ordinarily the first plate to strike is the most aboral one and the others follow in sequence giving rise, by the order of their beat, to a wave-like appearance which progresses, of course, in an oral direction. Chun (80, p. 172) has shown that when in a normal animal a wave Starts over one row, a like wave also starts over the other row of the same quadrant, 7. ¢., the two rows of any quadrant act in uni on. ‘[his relation was observed by Verworn (’91, p. 456) in all the ctenophores that he studied, and it is certainly an invariable occurrence in Mnemiopsis, but not in Pleurobrachia. In Pleuro- brachia, though the rows of plates on the same quadrant are often seen to beat in unison, they also frequently beat independ- ently. ‘That their beating in unison is not a mere matter of accident 1s seen from the fact that, whereas rows on the same quadrant often beat in unison, adjacent ones belonging to different quadrants do not beat in this manner. ‘There can be no question, I believe, that the rule laid down by Chun, to the effect that rows on the same quadrant always beat in unison, has its exceptions, for in Pleurobrachia the two rows of any quadrant may beat independently. As might be. expected from the researches of Chun (’80, p. 172), the removal of the sense body from Mnemiop- sis or from Pleurobrachia is invariably followed by a complete loss of the partial or perfect unison of action between rows of plates. Since there is an agreement in the metachronism of the two rows of plates on any quadrant in Mnemuopsis, there should The Movements of the Swimming-Plates in Ctenophores. 411 be a synchronism in the action of the corresponding plates in these two rows, and such proves to be the case. ‘This condition is very noticeable when at the beginning of a series of swimming- plate movements, the waves run at varying rates, for when a wave passes rapidly or slowly over one row, it passes at the same rate over the other row of the same quadrant. Similar conditions were observed in Pleurobrachia when its swimming-plates were acting In unison. The reversed action of the swimming-plates in ctenophores has been stated to occur by numerous observers, but the expression reversed action in this connection is undoubtedly somewhat ambiguous. In the so-called reversal of cilia and other like organs at least two kinds of reversal are possible: a reversal of the propagation wave, “ Reizwelle”’ of Engelmann, and a reversal of the effective stroke of the cilia (Parker, ’05, p. 9). In the first instance the question turns on the sequence in which the cilia beat; thus in the normal action-of a series of cilia, element a may beat first and z last, the propagation wave passing from a to 2; while in reversed action z would beat first and a last, the wave passing in the reverse direction. In the second instance only the effective stroke of the cilium is concerned; this may be normally toward z or reversed toward a irrespective Ae the sequence in w hich the cilia of the series act. In ctenophores a reversal of the direction of the propagation wave has often been observed. ‘This was early noticed on frag- ments of Beroé by Eimer (’80, p. 226), an observation confirmed on this and other species by Chun (80, p. 182) and by Verworn (90, p. 167; 91, p. 459), though the latter is misquoted in this respect by Putter (’03, p. 35). Reversal of the propagation wave occurs occasionally in Pleurobrachia. When a rapid wave from the aboral end of the animal reaches the oral limit of a row of plates, it may be reflected aborally over the row again, but it seldom retraces its course for more than one-third the whole length of the row. As Verworn (’90, p. 167; 91, p. 440) observed, these reversed waves can often be induced by stimulating mechani- cally the oral end of a row of swimming-plates. In Mnemuiopsis I have never observed unquestionably reversed waves, nor have I been able to induce them by special stimulation. Some slight evidence of reversal has been seen when a relatively slowly moving wave near the oral end of its course is overtaken 412 G. H. Parker. by a more rapid one. This is seen to sweep over the slower wave and, as it does so, what seems to be a reversed wave starts from the point of collision and runs aborally over not more than six or eight plates at most. ‘This short wave is the only evidence of reversal that I have found in Mnemiopsis; it is my _ belief that this reversal of the swimming-plate action, so common in many ctenophores, is almost entirely absent from this species. According to previous investigators, ctenophores can reverse - the effective stroke of the swimming-plates as well as change the direction of the propagation wave. Under ordinary conditions, the effective stroke carries the animal with the oral end forward; when this is reversed, the animal moves with the aboral end ahead. Chun .(’80, p. 181) mentions that this reversed form of locomotion is a regular though rare occurrence with all cteno- phores, especially - when by normal locomotion their oral ends collide with some fixed body. Verworn (’g1, p. 432) states that he has on rare occasions observed this reversed swimming in Eucharis and Callianira, but not in other species of ctenophores. ] have never seen any evidence of the reversal of the effective stroke in either Pleurobrachia or Mnemuopsis and I am inclined to believe that Chun’s statement that the effective stroke can be reversed in all ctenophores, may be a mistake based upon a con- fusion of this form of reversal with the reversal of the propagation wave. In Pleurobrachia it can be easily shown that when the propagation wave reverses from an oral to an aboral direction the swimming-plates continue their effective stroke in an aboral direction as before. When a row of swimming- plates in Pleurobrachia or Mnemiop- sis 1s cut through so as to divide it into oral and aboral portions, the plates in both parts cease to move for a short time and when they resume their activity, the two parts are found to beat differ- ently, 7. e., their propagation waves are found to be independent. In this respect the American species agrees with the European forms experimented upon by Eimer (’80, p. 227), Verworn (’90 p. 167), and others. If a row in Mnemiopsis is cut with care, the aboral part almost immediately begins to beat metachronally with reference to its fellow of the same quadrant, and the oral part reéstablishes independent movements in a few minutes or even seconds. In the quickness of recovery of the oral part Mnemiop- sis is in strong contrast with Beroé and Eucharis, in which, accord- The Movements of the Swimmuing-Plates in Ctenophores. 413 ing to Krukenberg (’80, p. 2) and Verworn (’90, p. 156), the activ- ity of the oral part may not return for an hour or so after the row is cut. The waves in the oral part of Mnemiopsis always proceed from near the cut end of the row orally; the most aboral plate to show motion, however, 1s not the one next the wound but usually ~ the third or fourth from it. ‘The oral portion will thus move its swimming-plates for hours without relation to the movements of the aboral part. I have never seen any evidence of the reéstablishment of harmony in the two parts of a severed row such as has been described by Eimer (’80, p. 229) and Verworn (’go, p. 167; 91, p. 463). When a swimming-plate band is cut through, not where ce are swimming-plates but between the most bor plate and the sense body (compare Fig. 2), the whole row in its movements becomes independent of the sense body and if the sense body 1s destroyed, the codrdination of the four pairs of rows entirely disappears, as has already been shown by Verworn (’9gI, pp. 457-459) and others for several European species. ena Mnemiopsis 1 is cut in two transversely, the parts of rows on the aboral portion retain their coérdination as in a normal animal; those on the oral part, as might be expected, lose all signs of such relations. It is clear from this and the preceding experi- ments that the codrdinating influences proceed from the aboral pole, and, when this is lost, codrdination disappears. In this respect my observations confirm those of Krukenberg (’80, p. 2) on Beroé and are opposed to those of Eimer (’80, p. 231), who stated that the oral half of Beroé is indistinguishable in the move- ments of its plates from a whole animal. When a Mnemiopsis is shaken in sea-water, it can be broken easily into fragments and the plates attached to these pieces will continue to beat rhythmically and metachronally for from one to two days. As Verworn (’90, p. 157) has shown for Cestus, so also in Mnemiopsis, even a single plate with a small basal piece of protoplasm will beat rhythmically for a long time. This condi- tion led Verworn to believe that each plate possessed a certain degree of autonomy, which was seen in the continued activity of the isolated plates and must be imagined to be counteracted by some influence when the plate as a member of a row was quiescent. But in my opinion the swimming-plate, when it beats, does so because it is stimulated, and its quiescence is evidence of the absence of appropriate stimuli. When it beats normally on \ 414 G. HiPare a whole animal, it does so in response to a wave of stimulation from the aboral pole, the cessation of which is followed by the cessation of the movement in the plate. When one plate with a small amount of protoplasm attached continues to beat, as it often will for hours, it does so because the fragmentary condition of its base exposes this part to continual stimulation. I see no reason to assume that the plates possess an autonomy that 1s inhibited much of the time by the animal. A fragment of a swimming-plate of Mnemiopsis made by splitting the plate lengthwise will continue to beat if a small basal mass of protoplasm is still attached to it. Whole swimming- plates or fragments of plates cease to beat when the base is trimmed off to such an extent that only the swimming-plate proper is left. In this respect the plates of Mnemiopsis resemble those of the ctenophores on which Verworn (’90, pp. 158, 161) experimented. This failure of the isolated plates to vibrate has generally been attributed to the loss of a stimulus normally received from the basal protoplasm, but Pitter (03, p. 42) has suggested that it may be due to the rapid death of aie plates. after isolation from the livi ing substance of the animal. ‘That this is not so in Mnemi- opsis is seen from the fact that a fragment of a plate cut off from its basal protoplasm and kept in sea water half an hour trembles and curves when a little picric acid is applied to it just as the living plates do on a whole animal when this reagent is poured on them. I therefore believe that the quiescence of “isolated plates is due to the absence of a stimulus to contraction and not to early death. It is evident from what has preceded that the rows of swimming+ plates of ctenophores ordinarily beat in pairs corresponding to the quadrants of the animal’s body and that the plates of any row beat metachronally beginning ordinarily at the aboral end. As Chun (80, p. 172) long ago pointed out, that which regulates their beat proceeds usually trom the region of the aboral pole and here four centers must be assumed, one for each quadrant of the ani- mal’s body. It is also evident that the regulating influence in its passage from the aboral pole is strictly limited to the bands leading from the sense body to the rows of plates, and to the rows of plates themselves, and that, though the waves usually start from the aboral end and progress toward the oral one, they may in some species reverse and run some distance aborally. “All these The Movements of the Swimming-Plates in Ctenophores. 415 facts are explainable on either the theory of neuroid transmission as advocated by Engelmann, or on that of mechanical transmis- sion as put forward by Verworn; for on the former assumption the band of epithelium leading from the sense body to the oral end of a row of swimming-plates may serve as a transmitting tract, and on the latter the ciliated bands leading from the sense body to the rows of plates may serve to transmit the mechanical disturbance from the center to the plates. I propose now to turn to certain observations that are, in my opinion, inconsistent with one or other of these theories. Since in accordance with the idea of mechanical transmission the mechanical activity of the vibratile elements is a necessary accompaniment of transmission, it follows that any means of bringing this activity to a standstill ought to check transmission. It might be supposed that the cutting “of of one or more plates would produce such an effect. When this is done in Cestus, according to Verworn (’go, p. 173), and in Mnemiopsis, according to my own observations, the waves still pass regularly over the whole row of plates and are not interrupted by the interval from which the plates have been removed. Since, however, the spaces between the plates in Cestus, as well as in the lobate ctenophores, have been shown by Samassa (’92, p. 229) to be ciliated, it might be assumed that these cilia in. their vibrations transmit the mechanical disturbance over the whole row. The assumption that in the absence of plates the cilia may transmit the disturbance 1s, however, in my opinion improbable, for the space made by the removal of a plate is so considerable in comparison with the length of the cilia that, unless we assume as Verworn (’g0, p. 173) does that the whole base of the plate is surrounded by cilia, I see no way by which the mechanical disturbance made by the cilia on one side of the root of the plate could influence those on the other side and thus effect transmission. As I have never seen in Mnem- lopsis any reason to believe that the plates are surrounded at their bases by cilia, I do not believe that transmission can be accounted for in the present experiments by the mechanical theory, even admitting the presence of cilia between the plates. A modification of the experiment just described has been employed by Verworn (’90, p. 171) with the view of testing further the nature of transmission. ‘This experiment consisted in restrain- ing a plate from beating instead of cutting it off and then ascer- 416 G. H. Parker. taining whether waves passed beyond it. When a plate in Beroé is turned aborally by a lancet point, waves from the aboral end fail to pass this plate. If only the tip of the plate is held and the base is allowed to move, the wave passes onward to the oral portion of the row. ‘These observations led Verworn (’90, p. 171) to conclude that the mechanical vibrations of the plates were neces- sary for transmission, and he drew this conclusion notwithstanding the fact that in Cestus he (’90, p. 172) found that the holding or even the pulling out of a plate did not interfere with transmission. Verworn confessed to have been astonished at the conditions found in this species, but, as already stated, he believed that they might be explained on the assumption that the base of each plate is more or less surrounded by cilia which after the removal of the plate continue to transmit mechanically. Unfortunately I have been unable to try the experiment of restraining plates in Mnem- iopsis, for the rows of plates in this species, like those in Beroé, as pointed out by Krukenberg (’80, p. 10), are so sensitive to mechani- cal stimulation that the moment they are touched they are drawn down into the animal’s body to such an extent as to make expert- ments of this kind very unsatisfactory, if not impossible. Although the great sensitiveness of the rows of plates in Mnem- lopsis prevented. me from trying the experiment of holding plates individually, it afforded a very natural means of checking their action. As Verworn (90, p. 170) has shown, when the middle of a row of plates is touched, the row in that region becomes depressed and the edges of the depression fold over and cover the plates. Thus in Mnemuiopsis half a dozen plates may become so much restrained that they will not show the least motion and yet waves that arrive at the aboral entrance to this depression emerge from its oral end with the greatest regularity. “his may happen while the covered region is under close inspection through a lens and gives not the least sign of plate or ciliary movement. I am, therefore, forced to conclude, that, contrary to the statement made by Verworn (’90, p, 170), such restrained tracts transmit with perfect regularity even in the absence of observable ciliary and plate motion. Kraft (’g0, p. 223) in his study of the ciliated epithelia of verte- brates, Seen that, though low temperature may bring cilia to a standstill, it does not oreatly check the transmitting power of the tissue. It ought, therefore, to be possible to Beck the action of The Movements of the Swimming-Plates in Ctenophores. 417 plates in a ctenophore by cold and yet leave the transmitting power of the row unimpaired. ‘To test this proposition, I passed a small curved metal tube through the substance of a Mnemuopsis directly under one of its rows of swimming-plates and at right angles to the direction of the row. ‘The animal was anchored by being pinned in a small aquarium of sea water whose temperature was 21°C. Normal waves of action were seen to course over the row of swimming-plates under which the metal tube had been placed. I now passed through the tube water of a temperature between 4° and 5° C. A steady flow was kept up to insure as complete a chilling as possible of that portion of the row under which the tube went. The chilled plates soon ceased to move and the waves appeared to jump from the aboral side from which they approached the chilled region to the oral one beyond it. Sometimes half a dozen waves in rapid succession appeared thus to jump this chilled region. But the best evidence was obtained when the waves ran at considerable intervals, at which times the correspond- ence between the parts of the wave in front of and behind the chilled region was most striking. ‘To be certain that there was no movement of cilia or plates in the chilled region, a small amount of powdered carmine in sea water was placed on the plates of that portion. ‘The carmine remained motionless while wave after wave ran over the aboral and the oral parts of the row. At the close of the experiment the chilled region was allowed to regain its normal temperature, whereupon its plates became vibratile again and the waves passed without interruption. ‘This experiment was repeated on six different individuals and with constant results. In one instance the temperature of the water used for chilling the tissue was 8.5° C. and under this condition the cessation of movement was only petit but in all other experi- ments the temperature was kept at 5° C. or lower with the result that complete cessation of movement invariably followed. Hence it is fair to conclude that in Mnemiopsis a temperature of 5° C. or less will check the movement of the swimming-plates without essentially altering the transmitting power of the row. In handling ctenophores in the experiments last described, | noticed that when the row of plates under which the metal tube passed was subjected to a little local stretching by the awkward manipulation of the tube, the plates often ceased to vibrate in the stretched region. On repeating this operation I found that as a 418 G. H. Parker. rule the slight stretching of a region would bring the plates of that part toa standstill, though it did not interfere seriously with trans- mission. But it must be noted that in such an operation much care must be used not to overstrain the tissue, for otherwise a permanent cessation of action will follow. Avoiding this difheulty, however, mechanical strain, like low temperature, may be made to check motion without interfering with transmission. III. THEORETIC CONSIDERATIONS. The results of the experiments just described, in which the swimming-plates of ctenophores were removed or restrained, or the row chilled or stretched locally, afford good grounds for deny- ing to the mechanical theory any essential part in the explanation of ciliary metachronism. If ordinary transmission is really depen- dent upon the mechanical action of one element on the next, it is inconceivable how such a process can be accomplished when these elements for any reason cease to move. ‘That transmission does take place after the swimming-plates have been brought to a stand- still by physical restraint, cold, etc., is unquestionable. Verworn (go, p. 172) admitted surprise when he found that in Cestus Boe occurred even after the removal of a plate and he was led to assume a continuous band of cilia to account for this condition. In Mnemiopsis no such band is present and yet transmission takes place even after the removal of a plate. The failure of a wave to pass when the plates in Beroé are restrained from moving is not, as Verworn believed, a satisfactory test of the nature of transmission, for, notwithstanding the care used in restraining the plate, the operation may influence the deeper parts of the tissue and thus check transmission as well as plate movement. The fact that transmission does occur in Mnemiopsis when the plates are restrained, shows how treacher- ous such negative evidence is. These facts, together with the © evidence from chilled and stretched rows, show, I believe, that the mechanical theory is not a necessary part of the explanation of ciliary metachronism. Although the mechanical theory may not be the correct explana- tion of transmission, its rejection does not imply a rejection of the idea that the plates are open to mechanical stimulation. Every- one who has worked with ctenophores knows how sensitive the The Movements of the Swimming-Plates in Ctenophores. 419 plates are in this respect. The slightest touch will often cause them to vibrate and will even give rise to a wave which, beginning with the plate stimulated, runs orally over the row. This condition is undoubtedly suggestive of such a view as that advanced by Verworn; his (’90, p. 168) ingenious experiment of attaching plate to plate by cotton filaments and thus obtaining a form of mechanical transmission shows how this idea may find applica- tion. When, however, it is remembered that in rest the plates point orally, that the propagation wave ordinarily proceeds from the aboral end of the row, and that the effective stroke of the plate is made in the aboral direction, it is clear that each plate as it goes into action does not strike toward the next plate to act but away from it and hence in a direction unfavorable for mechanical stimu- lation. When the propagation wave is reversed, as happens in Pleurobrachia and probably in many other ctenophores, the action of the plates is such that an oral one may well stimulate mechani- cally the next in turn, and, while I believe that the normal wave depends for its propagation upon a neuroid transmission, | am inclined to the opinion that the reversed waves may depend largely on mechanical transmission. As is well known, these reversed waves seldom extend far and are always insignificant as compared with the normal ones. Hence I do not elie that mechanical stimulation plays any really important part in transmitting the normal wave. Direct stimulation seems to be a possible means of transmission over a cut in a rowof plates. Since both Eimer (80, p. 229) and Verworn (’90, p. 167) have recorded the occurrence of this form of transmission in European species, it might be looked for in other forms, though I have beenunable to find any evidence of it in Mnem- lopsis or Pleurobrachia. However, I see no reason why the vibra- tion of a plate on the aboral side of a cut may not stimulate to action a plate on the oral side of the same cut provided the two plates are brought close enough together. “The subject is worthy of further investigation. Most of the observations that have been brought forward against the mechanical theory might now be urged in favor of the neuroid theory, for transmission without ciliary or plate motion is what is implied by this view. ‘The idea that the movement of the swimming-plates is controlled by nerves was held by some of the older investigators such as Eimer (’73, p. 45) and Krukenberg 420 1G) pee (80, p. 5), though on insufficient grounds, for no one has ever demonstrated that nerves are connected with these plates. Engel- mann. (’87, p. 442) has used the expression “innervated” 1 reference to the rows of swimming-plates, but it is perfectly evident from other statements in his account (p- 440) that this term is used in a physiological sense and not in an anatomical one, and that he consistently adheres to his original idea (’79, p- 395) of epithelial transmission. Chun (’80, p. 173) made perhaps the best brief statement of the mechanism of transmission in ctenophores when he declared that the rows of epithelial cells served as nerves. It is in my opinion an open question whether in any instance cilia are really controlled by nerves. Such a con- trol is denied by Verworn (’95, p. 251), though Pitter (’03, p. 98) in his recent survey of the whole subject of ciliary activity states that in the larve of certain annelids such control occurs. It seems to me that Putter’s grounds are insufficient for such a conclusion; but, however this question may stand for annelids, in the cteno- phores not the least histological evidence has ever been advanced to show that their rows of plates are accompanied by nerves. Samassa (’92, p. 226), who has studied this matter with care, denies that ctenophores have any nervous system properly so called and points (p. 230) to the epithelial bands in Beroé as the transmitting organs. ‘There thus seems to be good reason for believing that the epithelial cells on the rows of swimming-plates in ctenophores transmit impulses that control the metachronism of these plates; in other words the neuroid theory, contrary to the statement made by Verworn (’go, p. 175), is tenable. Such a conclusion is entirely consistent ae the results of Gruetzner (82) and of Kraft (’90) in their experiments on transmission in the ciliated epithelia of the higher animals, for both investigators found it necessary to assume a deep-seated cellular transmission to explain the spread of ciliary disturbances in active and in quies- cent fields of cilia. Although the results of my experiments make me confident that the metachronism of the swimming-plates of ctenophores is due to neuroid transmission, I do not believe that the facts warrant the extreme position taken by Engelmann (87, p. 440) that no form of mechanical transmission obtains. It seems to me much more likely that, as Chun (’80, p. 174) has declared, mechanical action is a subordinate though real factor in transmission. In my The Movements of the Swimming-Plates in Ctenophores. 421 opinion, this factor would never of itself result in giving rise even to a single full wave, though it might, if vigorously started, carry a wave over a small number of plates. Its influence at most would be only of a subordinate character. Chun’s view that both neu- roid and mechanical factors take part in transmission has been adopted by Pitter (’03, p. 98). While mechanical transmission may be of only subordinate importance, mechanical stimulation must be regarded as of no small significance. It has already been pointed out that a plate, if mechanically stimulated, may become the point of origin of a wave which will be transmitted over the row of plates in all respects normally. Hence mechanical stimulation will not only bring a plate into action but will induce the formation of a normal neuroid propagation wave. If the ciliated epithelia of the higher animals and such special- ized structures as the swimming-plates of the ctenophores are con- trolled by impulses that are passed in a definite direction and within circumscribed limits from cell to cell, it seems highly probable that many of the codrdinated responses of the lower metazoans and of the early larval stages of the higher forms may depend upon this form of mechanism rather than on any kind of true nervous organization. ‘Thus it may well be that the slow but uniform responses of sponges to local stimulation may be due to neuroid transmission through their epithelial layers and not through true nervous tissue, which, as is well known, has been sought for in vain in these animals. ‘The exact orientation to light of larval sea urchins at the blastula or gastrula stage involves a certain coordinated beat of the cilia which, in the absence of ner- vous elements, may well be due to neuroid transmission. Thus animals in such early stagess of growth may carry out by means of their epithelia reactions which in later stages would be performed by a true nervous mechanism. Conditions of this kind lead me to believe that before primitive metazoans possessed any nervous organs whatever, they probably had in their epithelia organs which exhibited the most fundamental property of nervous tissue, namely, a capacity to transmit in a prescribed direction impulses to motion. From epithelia of this kind sense organs and central nervous organs were probably evolved, and yet this evolution did not bring about the entire suppression of these primitive pre- nervous mechanisms; for the ciliated epitheila of the highest 422 G. H. Parker. animals, as well as the swimming-plates of the ctenophores, still possess the power of neuroid transmission. IV. SUMMARY. 1. In Mnemiopsis and Pleurobrachia the swimming-plates normally beat metachronally beginning at the aboral ends of the rOWS. 2. In Mnemuiopsis the two rows of plates belonging to the same quadrant of the animal’s body beat in unison. In Pleurobrachia this is also often true, but all eight rows in this ctenophore may beat independently. 3. The propagation wave (“‘Reizwelle” of Engelmann) shows scarcely any evidence of reversal in Mnemuopsis, but often reverses in Pleurobrachia. 4. Reversal of the effective stroke of the plates was never observed in Mnemiopsis or in Pleurobrachia. 5. On cutting a row of plates in Mnemuiopsis transversely, the oral part quickly recovers and begins beating, but not in unison with any other part; the aboral part also recovers soon and beats in unison with the other row of its quadrant. 6. A single isolated plate will beat if it retains a small amount of basal protoplasm. 3 7. Plates without basal protoplasm will not beat, though they are not dead. 8. Loss of a plate in a row does not prevent the passage of a wave even in Mnemiopsis where the cilia on the rows do not always form continuous bands. 9. When the plates on part of a row in Mnemiopsis are re- strained from moving, an impulse to plate movement may still be transmitted. 10. Cooling a part of a row with water at 5° C. will bring the movement of the plates to a standstill, but not interrupt trans- mission. It. Stretching part of a row will cause local cessation of move- ment, but will not interrupt transmission. 12. [he metachronism of the plates in ctenophores cannot be explained as a result of the mechanical influence of one plate on its neighbor, but the facts observed necessitate the assumption of a deep-seated transmission from cell to cell, nerve-like in character. The Movements of the Swimming-Plates in Ctenophores. 423 13. This neuroid transmission 1s probably supplemented by mechanical transmission, which of itself is insufhcient to carry forward a normal wave. 14. Phylogenetically an epithelium with neuroid transmission probably preceded true nervous structures and such an epithelium is in all likelihood the only means of transmission in many animals at their earliest larval stages (blastula, gastrula, etc.) and in such primitive forms as sponges. BIBLIOGRAPHY. Cuun, C., ’80.—Die Ctenophoren des Golfes von Neapel. Fauna und Flora des Golfes von Neapel. I. Monographie. xviii, 313 pp., 18 Taf. Eimer, T., ’73.—Ueber Beroé ovatus. Zoologische Studien auf Capri. I. 92 pp., orlat: ’80.—Versuche tber kiinstliche Theilbarkeit von Beroé ovatus. Arch. f. mikr. Anat., Bd. 17, pp. 213-240. ENGELMANN, IT. W., ’68.—Ueber die Flimmerbewegung. Jena Zeitschr., Bd. 4, PP: 321-478. ’79.—Physiologie der Protoplasma- und Flimmerbewegung. Hermann, L., Handbuch der Physiologie, Bd. 1, Theil 1, pp. 343-408. °87.—Ueber die Function der Otolithen. Zool. Anz., Jahrg. 10, No. 258, ; PP- 439--444- GruETZNER, P., ’82.—Zur Physiologie des Flimmerepithels. Physiol. Studien, pp- 1-32. Krart, H., ’90.—Zur Physiologie des Flimmerepithels bei Wirbelthieren. Arch. f. ges. Physiol., Bd. 47, Hefte 4, 5, pp. 196-235. KRUKENBERG, C. F. W., ’80.—Der Schlag der Schwingplaettchen bei Beroé ovatus. Vergleichend-physiologische Studien zu Tunis, Mentone und Palermo, Abt. 3, pp. I-23. Parker, G. H., ’05.—The Reversal of Ciliary Movements in Metazoans. Amer. Jour. Physiol., vol. 12, No. 1, pp. 1-16. ay Putter, A., ’03.—Die Flimmerbewegung. Ergeb. Physiol., Jahrg. 2, Abt. 2, pp. I-102. Samassa, P., ’92.—Zur Histologie der Ctenophoren. Arch. f. mikr. Anat., Bd. 40, pp- 157-243, Taf. 8-12. Verworn, M., ’90.—Studien zur Physiologie der Flimmerbewegung. Arch. f. ges. Physiol., Bd. 48, Hefte 3, 4, pp. 149-180. ’91.—Gleichgewicht und Otolithenorgan. Arch. f. ges. Physiol., Bd. 50, Hefte 9, 10, pp. 423-472. ’95.—Allgemeine Physiologie. Jena, 8vo, xii+, 584 pp. ONTA GENERAL THEORY “OF ADAPTATION. AND SELECTION. BY HENRY EDWARD CRAMPTON, Pu.D. It is the purpose of the present brief article to state in general and non-mathematical form some of the results of statistical and experimental studies, dealing with lepidoptera, that have been in progress for nearly six years, and in the second place to develop on the foundation of these results a generalized conception of natural selection and its actual basis during the segregation of the fit or adapted and the unfit or unadapted individuals of a species. I am led to offer this statement in this form and at this time because many months must elapse before the statistical results of recent studies may be put in final form for publication, though the more general conclusions to be drawn from them may be stated in simple 1 terms; and it is my desire, furthermore, to present the theory so that it may bear the scrutiny of the numerous iny esti- gators now at work upon the problems of variation and selection, in order that its validity may be tested in connection with different kinds of biological material. In a word, this paper is an outline. of a fuller discussion that must be deferred until the complete statistical results of my own investigations may be brought into relation with those of other i investigators of the problem of “natural selection and of its actual basis. le In 1899 a brief preliminary investigation was begun upon the pupe of a saturnid moth, Philosamia cynthia, in order to ascertain whether there was a definite relation between the variability of various pupal structures and the elimination that took place after the larvze spun their cocoons in the fall of the year and pupated. The same question was also examined with reference to the reduction in numbers that occurred when the pupe emerged in 426 Henry Edward Crampton. the following spring, when many individuals proved to be unable to form perfect moths, and thus offered a comparison with the pupz that metamorphosed successfully and_ perfectly. The material possessed a peculiar 1 interest for the reason that the pupa does not “‘use”’ many of its structures at all during | its long period of quiescence but remains practically inactive until the time for emergence approaches. ‘To state the results of this investigation in briefest form, it was found in many instances that the pupe which died before metamorphosis were of somewhat different types and were more variable than the surviving individuals; and the same relation between elimination at the time of metamor- phosis and structural variation appeared on the basis of a statis- tical examination of the two groups of pupz distinguished at that time.! Since 1900, the same species has been much more extensively examined to see if similar phenomena were exhibited in other years, and other forms of related moths have also been investi- gated—1. e., cecropia, promethea, ruber, jorulla, etc. ‘The results have been confirmatory, in that in every series where successful pupze have been compared with unsuccessful pupz some of the characters exhibit the stated relation between variation and elimination. Nevertheless, the conclusion that this relation was a general and a final one did not appear to me to be justified, for there were many cases where the reverse was true, where, that is, the survivors were of the same type as the others but were more variable, or they were of the same type and variability. In addi- tion, the characters that exhibited “selection,” where it was indi- cated, were of such a nature that they could hardly have served the pupa either advantageously or disadvantageously. The pupa does not “use” its antenna, and yet the pup that succeeded in producing perfect moths often possessed antennz that were cer- tainly selectively different from those of the contrasted unsuc- cessful group. It was inferred, therefore, that selection was “indirect, and that the real basis for selection was not to be sought in the series of individual characters themselves but in the condition of correlation between and among these char- acters. [he conclusion of the preliminary study reads as follows: 1See Biometrika, vol. iii. On a General T heory of Adaptation and Selection. 427 ce . the test of fitness or unfitness has reference to the physio- logical and morphological co-ordination or correlation among the constituents of the whole organism, and . . . any relaxation in either series, in a fonnaative sense or otherwise, results in an instability which may culminate in death, and which expresses itself in structural deviation as well as ina higher degree of vari- ability. i It is implied in the foregoing that a distinction may be made between formative Eeerelation and functional correlation. In a later instalment! the subject is discussed at length, and it is con- tended that the condition of correlation exhibited by the structures of the pupa is dependent upon the correlation of the formative factors or agencies which control the manufacture of the pupa by the larva, while the immediate functional elements are concerned scarcely if at all. ‘The case is therefore quite different from that of the moth, where indeed formative factors of the general condi- tion of correlation must be operative, but where the physiological co-ordination of the imaginal structures has a large share in deter- mining the “fitness” of the organism. But entirely aside from the relative values to be assigned to these two classes of factors, the point is that the separate “characters” do not serve directly as adaptive or unadaptive elements of the organism, but they do so only in so far as they exist in close or loase correlation with other structural and functional characteristics. bE: The truth of the conclusion stated was next put to the test of quantitative determination. The co-efhcients of correlation were determined, according to the familiar methods, in the case of the characters that had been previously treated individually in their relation to elimination, and the co-efficients of correlation of the two groups were compared, with positive results. While it 1s true that the general condition of correlation, regarded as the basis for elimination, is only imperfectly indicated by the degree of correla- tion between any two characters and that the co-efficients of multi- ple correlation involving three or more characters would be more reliable as indices of this condition, yet if the principle be true 'Now in press, Biometrika. 428 Henry Edward Crampton. the advantage in favor of the surviving or more successful group of pupz should appear more clearly where the co-efficients of _ correlation are used than where the comparison with the eliminated group is based upon the types and variabilities of the individual characters concerned. Such is, indeed, the case. While the former group is not invariably the superior in correlation, there is a smaller proportion of negative cases than where the individual characters are taken singly; so that definite confirmation is found for the conclusion reached at first entirely by inference. |e It now becomes the task to develop the principle of “the corre- lative basis for selection” so as to cover the wider range, over which, I believe, it extends. And I may state at the outset that statistical results have already been obtained proving in part that the wider range 1s indeed covered, though in the nature of the case, as will appear, complete mathematical demonstration is 1mpos- sible. So far, the general condition of correlation, which it is contended serves as the basis for elimination, has been regarded as deter- mined by the whole series of internal or organismal characters taken together. We may next attempt to bring the series of en- vironmental conditions or influences into the case by taking as an illustration the correlation between a single internal character as representing the whole series of internal characters and a single external character as a representative of that series. ‘he first 1S “length of pupal period” in days, and the second is the “time of the year.’ Neither of these varieties is simple, it is true. “The time of emergence will depend upon a number of things, upon the time of pupation, upon the weight of the whole organism, which, ees ks found, is indeed eoeeeired with the type cHarasian while in the second place the time of emergence is dependent also upon the time of the year, as increase of temperature hastens metamorphosis. But the point is rather, that when a given series of pupz is kept under natural conditions of temperature, their times of emergence, even when they are members of a single family, will form a curve of error, like that of structural character such as antenna length, weight, etc. Likewise, the time of the year reckoned as so many days from an arbitrary date such as January first, will form a On a General Theory of Adaptation and Selection. 429 curve; and this external “character” too, is compound or at least representative of a series of external influences that affect lep1- doptera, for not only will temperature conditions agree with calen- dar time, but food-supply and many other things will follow in a general way the temporal curve. It is clear, [ think, that a certain degree of correlation between time of the year and metamorphosis will be adaptive, while a low degree will be unadaptive. Those individuals that mature too early will, even if they find mates, produce eggs and larve that will find poor food- -supply, while those that emerge too late, supposing that they too find similar mates, will produce larve that will not have time to become full fed before cold weather will kill them and cut off their food-supply. Facts might be cited, showing still further that those that differ most from the average as regards the time of metamorphosis, vary also in unadaptive dircermns. in internal characters, produc- ing few eggs, possessing imperfect wings, and in other ways. It is needless to amplify the disadvantages that a lack of correla- tion with external influences or conditions would entail. In brief, then, we find that the principle of correlative basis for selection involves not only the whole constitution of the organism itself, but the whole series of graded external influences as well, be these inorganic or organic, homogeneric or heterogeneric. IV. A few words are necessary with regard to the relations of the conception presented above. In the first place, it differs from the general theories hitherto brought forward in having a concrete basis in the results of statistical investigations of correlation and variation, and secondly, in that it places the series of external con- ditions on the same plane as the series of internal conditions, in their relation to the final welfare of the organism, regarding them also as varying according to the familiar laws of error. How far it may be justifiable to extend this principle over the external world, remains for future investigation; but it will be possible, as I belive, to utilize statistical methods: i in such inves- tigations. Selection is not regarded as in any way originative but only as judicial, so to speak. As the members of any species present themselves at the bar, “selection”’ decides the question of survival 430 Henry Edward Crampton. or destruction on the basis of the condition of correlation that 1s exhibited. Adaptation receives a precise definition: it means a degree of correlation, capable of numerical determination; and the question as to the utility of any given character becomes sub- ordinate to the question of the effectiveness of any given combina- tion of unit-characters, working in a functional of formative complex. Finally, to possess an evolutionary value, this conception must be taken in connection with the view that the heritable characters of an organism are congenitally determined. ‘The heritable nature of fluctuations as contrasted with mutations, however, is not a matter that is necessarily brought into court. Barnard College, Columbia University, June 10, 1905. EXPERIMENTAL STUDIESZON DHE DEVELOPMENT OF THESE YE IN ANMPEIBIA: i ON THE CORNEA. BY WARREN HARMON LEWIS. + Associate Professor of Anatomy, fohns Hopkins University. WitH 2 Ptiares. INTRODUCTION. With the introduction of the binocular dissecting microscope the possibilities of investigating the subject of correlative embry- ology have been greatly enhanced. With its aid, as I have already pointed out in my paper on the origin of the lens in Rana palus- tris, one can make with very diene instruments exceedingly minute dissections of the living amphibian embryo, and by trans- plantation and extirpation OF organs and tissues can gain an insight into the influences of intra-organic environment in dev elop- ment. Spemann’s experiments on Rana fusca and my own on Rana palustris establish without doubt the correlative character of the origin of the lens in these two species and my unpublished work on Rana sylvatica and Amblystoma punctatum show that in these species likewise the lens is dependent for its origin on the influence exerted by the optic vesicle on the overlying ectoderm. _The cornea 1s likewise a correlative product, but of a quite dif- ferent nature from the lens as will appear in the following pages. ~ While the lens is apparently dependent upon specific influences from the optic cup, the cornea or rather corneal changes of the ectoderm may be brought about by such different structures as the lens alone or of the optic cup alone. Spemann! concludes that the clearing of the corneal ectoderm is dependent in Rana fusca on the presence beneath the skin of the eye with its lens. In my own experiments on Rana palustris? it was noted that in 1 Verhand. d. Anat. Gesel., 1901. 2 Am. Jour. of Anat., vol. iii, Fig. 9, p. 512. 432 Warren Harmon Lewis. this species likewise corneal clearing of the ectoderm fails when the eye is wanting. In this paper it will be clearly shown, how- ever, that corneal clearing of the ectoderm in Amblystoma will occur over a naked lens or over the optic cup without the lens, provided the lens or cup are close to the overlying ectoderm. If this be true for Amblystoma it probably holds also for other amphibia and consequently Spemann’s conclusion for Rana fusca should be modified to this extent. In a preliminary communication before the Association of American Anatomists, at Philadelphia, 1903, concerning my experimental studies on the development of the eye in amphibia, the following conclusions were given for the cornea: “(1) The cornea fails to develop when the optic vesicle is entirely removed. (2) Over the regenerated eye with lens a cornea develops, nor- mally, except for size, which is small to correspond to the small regenerated eye. (3) If the optic cup is torn out after the lens has separated from the skin, a small area of clear epithelium will develop immediately over the undisturbed lens. (4) Such clear- ing for the cornea will also develop over an optic cup, from which the lens has been extracted, but not in all cases. These conclusions were based mainly upon experiments on Amblystoma punctatum and Rana palustris. More recent experi- ments on Rana sylvatica show that in this species also the cornea fails to develop when the eye is wanting. The present paper is concerned more especially with the conditions in Amblystoma, which is a more favorable form for the study of corneal forma- tion than Rana. The experiments enable me to consider only the early stages of corneal formation, namely: (1) The thinning of the ectoderm of the corneal area; (2) the clearing of this ectoderm and loss of its pigment; (3) the formation of the endo- thelial layer of the cornea, which 1s in reality the anterior wall of the anterior chamber of the eye. In addition to the above conclusions some of my more recent experiments show that the cornea will form from ectoderm other than that which normally gives rise to the cornea. This spring I have repeated most of the experiments on Ambly- stoma and find that it is easy to confirm all of the conclusions stated above. Some new experiments show that even after the 1 Am. Jour. of Anat., vol. ii. Experimental Studies on the Development of the Eye. 433 cornea is formed it will disappear almost completely if the optic cup and lens are entirely removed, without injury to the over- lying cornea. The experiments in this paper with the sole exception of MD, have been selected each from a series of several similar ones. METHODS. Embryos of various ages were operated upon under the binocu- lar microscope. Either ordinary tap water or a 0.2 per cent salt solution was used. [he older embryos were first anes- thetized with acetone-chloroform. [he embryos were held with a pair of fine forceps, and the incisions were made with a very small pair of scissors, the points of which were ground with great care. Ordinary needles complete the instruments needed. ‘The manipulation requires considerable practice, but one soon finds experiments possible, which at the beginning seemed beyond such methods. ‘The method possesses great advantages over the use of the hot needle or electric cautery. A new sill very wide field of work is opened by means of this dissection method and it will undoubtedly throw much light upon developmental processes. The embryos were killed in Zenker’s fluid, cut into serial sec- tions. 5 # or 10 » in thickness and stained in hematoxylin and Congo red. The operations were all performed on the right side. The figures are all from photo-micrographs of transverse sections through the region of the right eye. EXPERIMENTS. A. Non-development of the Cornea after Total Extirpation of the Eye. ‘The numerous experiments on Rana palustris where the optic vesicle was completely removed at an early stage before lens for- mation and consequently long before there are any indications of corneal formation, have all failed to show corneal changes in the ectoderm which under normal conditions would have farina corneas. Even days after the operation and long after the cornea on the normal side of the head was well developed all traces of 434 Warren Harmon Lewts. corneal clearing of the ectoderm were wanting in the skin cover- ing the region from which the eye had been taken. I have already given an experiment of this nature in my article on the ori- gin of the lens in Rana palustris.!_ In this embryo a skin-flap was turned forward from over the optic vesicle, the latter cut away and the flap returned to its original position without injury to the corneal area of the ectoderm. ‘The embryo was killed eleven days after the operation but showed no signs of corneal changes on the side operated on, while upon the normal side there was a well-developed cornea. In other experiments similarly performed only a portion of the optic vesicle was cut away and over the regenerated eye corneal formation took place provided the regenerated eye was of sufficient size to come into contact with the skin. It is evident then that the lack of corneal formation after complete extirpation of the eye was not due to the turning forward of the skin-flap. If a skin- flap 1 is turned forward from over the eye and then replaced without injury to. either it or the eye, perfectly normal develop- ment of the eye, lens and cornea will ensue. A deeply situated regenerated eye separated from the ectoderm by mesenchyme will not cause corneal formation. In similar experiments on Rana sylvatica like results follow total or partial extirpation of the eye. In Amblystoma punctatum the corneal changes are much easier to follow than in Rana as the ordinary ectoderm is of con- siderable thickness and the contrast between it and the cornea much greater than in the frog. In Amblystoma as in frog larve the early total extirpation of the optic vesicle, before the period of lens formation, results in the failure of corneal development. For example, if in an embryo at this stage a flap of skin is carefully turned forward from over the eye, the optic vesicle completely cut away, and the uninjured skin-flap returned to its original position, it readily heals in place but no traces of corneal formation are to be observed even sixteen days after the operation. Fig. 1 gives an accurate idea of the con- ditions in such experiments. ‘The lens also is entirely wanting while on the normal unoperated side optic cup, lens and cornea are present. The endothelial layer of the cornea likewise fails to develop without the presence of the optic cup. 1 Am. Jour. of Anat., vol. 1, p. 512, Fig. 9. Experimental Studies on the Development of the Eye. 435 If at a much later stage, namely, shortly after separation of the lens from the ectoderm, both optic cup and lens are taken out in a manner similar to that just described, corneal changes fail .to develop, even if the embryos are allowed to live from twelve to eighteen days after the operation. (See Fig. 1, from Experiment XTVE.:) The end result as regards non-development of the cornea is the same in each embryo, whether the eye is taken out before the lens begins to form or shortly after its separation from the skin. In Amblystoma, as in Rana, however, corneal formation occurs after partial extirpation of the eye, whether this is done before the lens has formed or shortly after its separation from the ecto- derm, provided the regenerated eye comes into contact with the ectoderm. ‘The mere lifting of the skin-flap here as in Rana does not interfere with corneal formation, so that the lack of corneal development after complete extirpation of the eye is not due to the lifting of the skin-flap but must be in some manner associated with the absence of the eye. It is evident that the cornea is not a self-differentiating structure. iB: Rudimentary Corneal Area after Late Extirpation of the Eye. If-in Amblystoma the optic cup and lens are taken out some- time after the separation of the lens from the ectoderm but before corneal changes are visible on the surface of the embryo a small clear corneal area will develop in the region where the large normal cornea would have formed. An examination of a normal embryo of the same stage at which the operation was performed shows that corneal changes have begun and consist in a slight thinning of the ectoderm over the eye. The endothelial layer is also in the process of formation. ‘The operations consist in making an incision about the caudal side of the eye and then carefully turning forward the skin-flap from over it without injury to the corneal region. ‘The eye and lens were then cut out and the skin-flap turned back into place where it readily healed. A few days after the operation a small clear corneal area appears in the ectoderm of the corneal region. An examination of the sections shows that this clear area differs from the ordinary ectoderm surrounding it in that it is much thinner, the pigment is wanting and the ectodermal cells have lost or not acquired the 436 Warren Harmon Lewts. usual vacuolization. It is in general like the normal corneal ectoderm except in being somewhat thicker. ‘The rudimentary corneal area, however, never seems to become much larger than that pictured in Fig. 2, even twenty days after the operation. This experiment (Mn,) is only one of several in which almost exactly similar results were obtained as regards the development of the small corneal area. In most of these experiments the rudimentary cornea is at the bottom of a depression in the ectoderm as in Experiment Mn, (Fig. 2). Such depressed areas occur, however, without corneal changes. (See Fig. 3, Experiment ME,.) In this experiment (ME.,) the optic cup was removed some time before the separa- tion of the lens from the ectoderm. ‘The lens has been pinched off from the skin and has become separated from the latter by mesenchyme. ‘The lens is, however, very much smaller than the normal one on the other side of the head. ‘The embryo was killed 16 days after the operation and there is no trace of corneal formation even at the bottom of the depressed ectodermal area. The depression of the ectoderm then can scarcely be looked upon as a factor in its clearing. In explanation of these rudimentary corneal areas it may be that the influences causing corneal formation had, at the time of the removal of the eye, not been acting long enough on the ecto- derm to enable the clearing process to go on independently and form the normal sized cornea. A longer continued influence of the eye is evidently necessary for normal corneal formation and as will be shown later on it is necessary for the eye to be present even after the cornea is well formed if the latter is to continue its existence. It may be that the cornea can never maintain itself independently of the eye; however, farther experimentation is necessary to determine this point. Here it again becomes apparent that the cornea of normal size is not a self-differentiating structure. C. The Size of the Cornea is Dependent upon the Size of the Eye. In the various experiments where portions of the eye have been cut away the regenerated eyes even thirty days after the operation fail to reach the size of the one on the normal side, unless the amount cut away is very small. ‘The size of the regenerated eye is somewhat in proportion to the amount of eye stuff left Experimental Studies on the Development of the Eye. 437 behind so that the new eye is to be viewed more as a re-formation than a regeneration. Such a regenerated eye coming into con- tact with the epidermis causes corneal clearing, the area of which varies in size with that of the eye. If but a call portion of the optic vesicle is cut away the regenerated eye will be of nearly normal size, with a cornea in all respects normal except of slightly less diameter to correspond to the smaller diameter of the eye. If more of the optic vesicle 1s cut away a still smaller regenerated eye and cornea will result. In Experiment VII, (Fig. 4), the somewhat irregular optic cup is about three-quarters of the diam- eter of the normal one and the cornea about two-thirds of the diameter of the normal cornea. In other respects the cornea is like the normal one. The lens which is nearly as large as the normal one is still adherent to the retina and fills the entire posterior cham- ber of the eye. This is not an uncommon condition of the lens in the regenerated eyes even as late as eighteen days after the operation. If a still greater portion of the optic cup is cut away a regenerated eye less than one-half the diameter of the normal one may develop with a correspondingly small cornea. In Experiment VII,, (Fig. 5), the small irregular optic cup contains a large lens which fills completely the cup cavity. The endothelial layer stretches over the lens and is for the most part in contact with it. The cornea although but one-half the normal diameter is in other respects like the normal one on the opposite side. Even more of the optic vesicle may be cut away than in the preceding experiments, yet if the small regenerated eye remains in contact with the skin a very small corneal area will develop. In the above experiments an incision was made around the caudal part of the eye and the skin- -flap with lens attached turned forward. Varying amounts of the optic cup were cut away and the skin-flap with the lens attached turned back into the original position where it readily healed. In another series of experiments both lens and optic cup were turned forward with the skin-flap and then varying amounts of the deep portion of the eye cut away. ‘The skin-flap with the lens and remainder of the optic cup were then turned back into position. ‘The re-formed eyes vary in size according to the amount left attached to the skin-flap, and the cornea in each embryo also varies in size with the size of the re-formed eye. 438 Warren Harmon Lewis. In still another series of experiments a portion of the optic cup together with the lens and all of the epidermis over the eye were cut away. In these experiments new epidermis soon covers the remainder of the eye and from it the cornea develops, and here, too, the size of the cornea varies with the size of the eye. This formation of corneal from strange ectoderm will be treated more fully in another section. The area of the corneal clearing of the epidermis over the optic cup alone or over the naked lens is likewise in proportion to the size of the area of contact of these organs with the skin. A large optic cup may be so situated that only one small corner of it is superficial and in contact with the epidermis. ‘The size of the corneal clearing is in proportion to this area of contact and not in proportion to the size of the eye. D. Corneal Formation with the O ptic Cup Alone and Without the Lens. In order to analyze more completely the influence of the eye on corneal formation I have in the following series of experiments excluded the possible influence of the lens and find that the early stages of corneal formation will develop without the presence of a lens or without a lens ever having formed from the skin. This last point is illustrated by a single fortunate experiment (MD,) on Amblystoma. A skin-flap was turned forward from over the eye in the usual manner at a time when in normal embryos of the same stage the skin is just beginning to show signs of thick- ening for lens formation. A portion of the shallow optic cup was cut out and the skin-flap replaced. ‘The sections show that for some reason the regenerated eye failed to cause lens forma- tion, but nevertheless corneal formation is present (Fig. 6). The optic cup is contracted and the cavity much reduced in size, the pupil is small and the endothelial layer of the anterior chamber reduced in area. The transparent corneal area is smaller than normal and slightly thicker. If at a somewhat later stage after the lens has formed and - separated from the skin, but before there is any corneal clearing, a skin-flap is turned forward and the lens with part of the cup cut out, a small cornea will form over the small optic cup provided the latter is close under the skin. (See Fig. 7, from Experiment va Experimental Studies on the Development of the Eye. 439 MF,.) The contracted cup with small cavity and pupil pre- sents a similar appearance to the condition seen in Experiment MD,. The extent of the endothelium and of the cornea cor- respond in size with the cup. The cornea is somewhat thicker than the normal one on the opposite side of the head, but other- wise similar to it. I have numerous other similar experiments g giving like results. In these experiments the optic cup or its E dothelial membrane lie close to the corneal clearing, which corresponds 1 in size with the area of contact. If, hewegecn, the optic cup and its endo- thelial membrane lie somewhat deeply buried and separated from the ectoderm by mesenchyme the corneal clearing fails to develop. The formation of the cornea is then neither dependent upon the formation of a lens nor upon the presence of the lens. E. Small Corneal Clearing over the Superficial Naked Lens. If in Amblystoma the optic cup is taken out about the time of, or shortly after, the separation of the lens from the skin and the lens left in position close against the skin, a small clear corneal area will develop immediately over the naked lens. In such experiments there was at the time of the operation no trace of cornea and if both optic cup and lens are taken out the small area of corneal clearing does not appear. An incision was made about the caudal two-thirds of the eye and the whole eye and skin-flap turned forward together.. The optic cup was then carefully removed, leaving the lens im situ and the skin-flap with the lens attached turned back into its normal position. At about the time when corneal clearing appears on the normal side the skin over the lens clears also, but is limited to the area immediately over the lens. As both epidermis and lens become perfectly transparent one can look down into the depths of the head in the living animal. The lens in most of the embryos 1s considerably smaller than the one on the normal side and often shows degeneration changes. ‘The endothelial layer does not develop about the naked lens. The corneal area does not seem to spread beyond the extent of the contact area of the lens, nor 440 Warren Harmon Lewts. does the skin become as thin as that of the normal cornea. “The pigment disappears and the large vacuolated cells which are present in the surrounding ectoderm are absent. (See Fig. 8, Experiment MG,.) If, however, the lens is disturbed by the operation so that mesenchyme grows in between it and the skin, the corneal changes do not occur. (See Fig. 9, from Experiment Ma,, and Fig. 3, from Experiment ME.) If after the optic cup is taken out the lens is transplanted a short distance from the normal position, the mesenchyme often separates the lens from ectoderm and in such experiments the corneal clearing likewise fails to develop, and a condition similar to that seen in Fig. g is present. F. Corneal Formation jrom Strange Ectoderm. If the ectoderm covering the optic cup and lens is completely torn away at a stage shortly after the separation of the lens from the ectoderm but before there are any visible corneal changes, the wound thus formed will heal by the ingrowth of ectoderm from the sides of the denuded area. In many of the experiments there was more or less disintegration of the optic cup before the wound healed. In some almost the entire optic vesicle disappears; in others but little of it is lost. In the experiments where the optic vesicle remains of sufficient size to come into contact with the new ectoderm true corneal formation follows, the size of the cornea varying with the size of the eye. In Experiment XVoe2 (Fig. 10), there was very little loss of optic vesicle substance and the new ectoderm soon covered the entire denuded area. The cornea with its endothelial membrane is apparently normal except in size being of less extent than the normal in proportion as the eye 1s smaller than normal, The new cornea was not well developed until about four days after the normal one on the left side had become perfectly clear. The difference in time in the formation of the corneas on the normal and operated sides may be even much greater than this, as when there is considerable disintegration of the eye, after the skin is torn off from over it, healing may be delayed a day or so and the eye much reduced in size. In some of these experiments the corneal clearing may be delayed from four to eight days after the one on the normal side is Experimental Studies on the Development of the Eye. 441 perfectly clear. And more than eight days may elapse before all of the pigment cells are gone. In another experiment performed in a similar way (X Voge) there was considerable disintegration of the eye before the new skin completely covered over the denuded area. ‘The eye is about one-half the diameter of the normal one and the clear corneal area even smaller in size. [he various layers of the retina are irregular and the lens also. ‘The latter fills the pos- terior chamber of the eye and has a process projecting into the pupil. (Fig. 11.) In these experiments as in those in which a portion of the optic cup was cut away without injury to the overlying skin, the size of the corneal area is in direct proportion to lie area of contact of the underlying eye. In these experiments the skin that grows over the eye is at first opaque and shows no signs of clearing. Pigment cells are scattered through it as in the ordinary epidermis. This con- dition often remains until long after the cornea on the normal side is well formed and clear. The clearing of the new epidermis which has grown over the eye is usually a slow process, and a few pigment cells are especially prone to remain even for a long time after the skin has cleared. If not only the skin from over the eye is cut away but with it 1s taken the lens and even the lens and part of the optic cup the adjoining skin will slowly cover the optic cup and after consider- able delay a cornea will form over this optic cup, the lens being absent. The size of the cornea varies with the size of the re-formed eye. Here, too, the corneal formation is much retarded and only appears days after the one on the normal side is well formed. If too much of the optic cup is taken away with the lens, what remains may be so deeply buried that it does not come into con- tact with the skin. In such embryos the cornea does not develop. (See Fig. 12, from Experiment Seva) If skin, lens, and optic cup are completely removed, new epidermis will cover over the large wound but corneal changes fail to appear even four weeks after the cornea has developed on the normal side. So we can scarcely look upon the cornea in the above experiments as a product of regeneration, but must con- sider it as a new product from skin other than that which normally gives rise to a cornea. 442 Warren Harmon Lewts. G. Degeneration of the Cornea after Extir pation of the O ptic Cup and Lens. If after the cornea is well formed an incision is made dorsal to the eye and the optic cup with the lens taken out, care being taken not to injure the cornea, the large cornea will oradually disappear. At first instead of forming a bulge on the surface of the head there results a depressed area owing to the absence of the optic cup. At the bottom of this area is the corneal clearing. This corneal area gradually contracts and pigment cells invade it from the adjoining skin, and in some of the experiments there is scarcely a trace of the cornea left 30 days after the operation. CONCLUSION. It seems very probable that the optic vesicle brings about lens formation through a specific influence. The cornea, however, in so far as its early stages are concerned, namely, the thinning and clearing of the skin and loss of pigment can hardly be ascribed to a specific influence. ‘That the mechanical pressure of the eye, or cup or lens may in some way be accountable for the corneal changes is a possibility. I am more inclined to the view, however, that the changes are due to another and quite different reason. The contact of either the eye or cup or lens with the epidermal cells must of necessity alter the environment of the overlying cells as regards their relation to the mesenchyme. It is possible that this exclusion from contact with the mesenchyme cells may be responsible for changes in the metabolism of the epidermal cells and cause them thereby to alter their mode of development, such alteration leading to corneal changes. ‘That such apparently slight alterations in environment are responsible for other important changes in the history of embryonic ecto- dermal cells I think quite probable. From some experiments already completed it seems highly probable that the central nervous system is in part at least dependent for its origin and differentiation on the difference of environment of cells w al ah at one time possessed the possibilities of producing either ordinary epidermal cells or of producing nerve cells. Experimental Studies on the Development of the Eye. 443 SUMMARY (AMBLYSTOMA.) 1. A normal cornea will not develop without the eye. 2. The size of the cornea varies with the size of the eye, with the area of contact between it and the skin. 3. Contact between eye and skin is a necessary factor, as an eye separated from the skin by mesenchyme will not cause corneal formation. 4. [he optic cup alone (without the lens) can cause corneal formation. 5. ‘The lens alone (without the optic cup) can cause corneal formation, provided, as is the case with the optic cup, it is in contact with the skin. 6. The size of the corneal area over the optic cup or lens is dependent upon the area of contact between these structures and the ectoderm. 7. It is not necessary that the lens should be first formed from the skin in order to have corneal formation. 8. The cornea will develop from strange epidermis other than that which normally forms the cornea. g. After the cornea is once formed it degenerates and disap- pears after extirpation of the rest of the eye. 10. The cornea is neither predetermined nor self-differ- entiating. 11. The cornea is dependent upon the correlation between the eye and the overlying ectoderm for its origin. A44 Warren Harmon Lewts. EXPLANATION OF PLATES. Prann el. Fig. 1. Experiment XIVg5¢. Right optic cup and lens taken out shortly after separation of the latter from the skin. Embryo killed 11 days after the operation. Figure from section through right eye region. A bit of the optic cup with nerve is deeply buried near optic foramen. No traces of corneal formation. ‘The normal left side has a well developed cornea. % 80 diameters. Fig. 2. Experiment Mn. Right optic cup and lens taken out at a somewhat later period than the above, but before there were visible corneal changes. Embryo killed 12 days after the operation. Figure from section through the right eye region. A small depressed area of corneal clearing is seen, which is scarcely 1, the diameter of the normal cornea on the left side. < 80 diameters. Fig. 3. Experiment ME». Right optic cup removed shortly before the separation of the lens from the skin. Embryo killed 16 days after the operation. Figure from section through the right eye region. The lens, much smaller than normal, is separated from the ectoderm by mesenchyme. There are no corneal changes in the epidermis. On the left side the cornea is large and well formed. X 80 diameters. Fig. 4. Experiment VIIz¢1. A portion of the right optic cup cut away before corneal formation. Embryo killed 9 days after the operation. Figure shows irregular eye smaller than normal with cor- respondingly small cornea. X 80 diameters. Fig. 5. Experiment VIIz3. Operation as above except that more of the optic vesicle was cut away. Figure shows small eye and cornea. % 80 diameters. Fig. 6. Experiment MD4. Portion of eye cut away before lens formation. Embryo killed 12 days after the operation. Figure shows absence of lens, small eye with small cavity and pupil, and corneal formation over the optic cup. % 80 diameters. EXPERIMENTAL STUDIES ON THE DEVELOPMENT OF THE EYE. W. H. Lewis. REAR aT: Tue JourNAL OF ExPERIMENTAL ZOOLOGY, vol. ii. 446 Warren Harmon Lewts. Prate II. Fig. 7. Experiment MF2. Lens and part of optic cup removed shortly after the separation of the lens from the skin. Embryo killed 14 days after the operation. Figure from section through right eye region, shows the regular reformed optic cup with small cavity and pupil, and overlying cornea with its endothelium. X 80 diameters. Fig. 8. Experiment MG,. Right optic cup removed shortly after separation of lens from skin, but before corneal formation. Embryo killed 13 days after the operation. Figure from section through the right eye region, shows a lens smaller than normal pressed close against the skin, where there is distinct corneal clearing, but no endothelial formation. 80 diameters. Fig. 9. Experiment Ma). Operation as above except that the lens was disturbed. Embryo killed 13 days after the operation. Figure shows the small lens separated from the skin by mesenchyme. There are no corneal changes in this region nor endothelial formation. A small mass of eye-cells lies medial to thelens. X 80 diameters. Fig. 10. Experiment XVzg2. Epidermis from over the entire right eye cut off before traces of corneal formation present. Embryo killed 11 days after the operation. Figure from a transverse section of the right eye region shows how new skin has completely covered the eye and become trans- formed into cornea. 80 diameters. Fig. 11. Experiment XV3¢¢. All of the skin over the eye and a portion of the optic cup removed. Embryo killed 11 days after the operation. Figure shows small eye with irregular lens and corneal formation from the new skin. X 8o diameters. Fig. 12. Experiment XVIIzig. All of the skin over the eye, the lens and part of the optic cup cut away before corneal formation. Embryo killed 11 days after the operation. Figure shows deep optic cup without pupil or cavity separated from skin by mesenchyme. There are no traces of corneal formation in the new ectoderm. X 80 diameters. EXPERIMENTAL STUDIES ON THE DEVELOPMENT OF THE EYE. W. H. Lewrs. PEATE: Tue JourNat or ExperiMENTAL Zo6LoGy, vol. ii. 0 a) . 1 ih Lge "; > MODIFIABILITY IN BEHAVIOR. I. BEHAVIOR OF SEA ANEMONES. BY H. S. JENNINGS. A thorough study of the modifiability of reactions to external stimuli in lower organisms seems at present one of the great desiderata in the study of animal behavior. Recent asthe has been devoted largely to the study of sharply defined forms of reaction and to the discovery of conditions under which these forms appear in the typical way. As a result there is a wide- spread impression that the behavior of lower organisms 1s com- posed of invariable reflexes, occurring always in the same way under the same external circumstances. This is far from the truth and leads, as it seems to the writer, to a fundamentally false conception of the nature of animal behavior. Inner states and changes are fully as important in determining behavior as are external stimuli, modifying fundamentally the reactions which the latter produce. ‘The present studies are devoted to an analysis of some of these modifying factors; in other words to some of the inner factors in behavior. The study of the behavior of sea anemones herewith presented was made possible by a stay at the Carnegie Research Laboratory at the Tortugas. I am under great obligations to the Carnegie Institution and to the director of the laboratory, Dr. A. G. Mayer, for opportunity to carry on the work, and for supplying every facility that could assist it. The Tortugas laboratory furnishes an ideal situation for carrying on such investigations. An indef- nite number of species of sea anemones and corals can be procured at a few moments notice, and they live as well in the laboratory as in the sea, since the water becomes cooler instead of warmer when brought into the house. 448 Hi. S. Fennings. I. .CHANGES IN BEHAVIOR DUE TO VARYING STATES OF METABOLISM. Nagel (’92) and Parker (’96) have shown that the food reaction of actinians toward weak stimuli becomes changed on repetition of the stimulation. A Metridium or an Adamsia at first readily takes filter paper soaked in dilute juice of crab meat. But after this has been fed several times in alternation with pieces of meat, the reaction to the filter paper becomes slower, and finally ceases, while the meat is taken as readily as before. “Torrey (04) shows that in Sagartia the state of hunger or satiety determines largely the reaction to small solid bodies. A very hungry Sagartia readily swallows inert bodies, such as filter paper and sand grains, while a fairly well fed one rejects these, though it takes meat. Has this effect of hunger and satiety any connection with the changes observed by Nagel and Parker, or are these of a different character? What relation have they to the changes due to experience in higher animals? The whole problem of the changes induced in behavior by changing metabolic states is one of the greatest importance for an understanding of the adjust- ment or regulation produced in behavior. I have attempted to study this matter carefully in a number of sea anemones, and to distinguish modifications due to this cause from those which result from other factors. Stoichactis Helianthus. This large sea anemone has often a disk 10 to 15 cm. in diameter. This is covered closely with short tentacles of uniform size, about 8 mm. in length.t | Stoichactis is voracious; it is usually when captured ready to take large quantities of crushed crab appen- dages. ‘To three specimens I fed piece by piece nearly all of three good sized ghost crabs (Ocypode). ‘The food reaction depends on contact with the meat itself—that is, on chemical stimuli in combination with contact. Hard parts of the crab, or other indifferent objects, are usually not taken, though in rare cases even filter paper is swallowed. Food is taken in the following way: If a piece of crab’s leg, with some of the flesh exposed, is placed on the disk of a hungry 1For photographs of the disk of Stoichactis, see Duerden, 1902, Pl. 1. Modifiability in Behavior. 449 specimen, the tentacles immediately surrounding it (including many not in contact with it) begin suddenly to wave back and forth. After an instant this usually ceases, and all is absolutely quiet for a few seconds. Then the movement begins again. All the tentacles that in their waving motion come in contact with the food, bend over against it and shrink, in such a way as to hold it down against the disk. Now that portion of the disk bearing the food begins to sink inward, by a folding of the surface. The mouth, which may be 4 or 5 cm. distant, begins to open, and the walls of the esophagus protrude from the mouth as large bladdery lobes. The region between the mouth and the food body begins to contract, the tentacles borne here collapsing and almost completely effacing themselves. By this contraction the mouth and food approach each other, the intervening region disappearing. Meanwhile other parts of the disk swell and their tentacles become plump and enlarged; this appears to be a secondary phenomenon due to the squeezing of the internal fluid from the contracted region to other parts. “The esophageal lobes increase in size, becoming 2 to 4 cm. long, and half as thick; they extend toward the food, finally reaching it. By the contractions and expansions already mentioned the mouth may be moved from the center of a disk 10 cm. in diameter to within 1 cm. of the edge. By this time mouth and food may be hidden beneath the surface of the contracted disk, though in other cases they lie on the surface in plain view. Now the esophageal lobes extend over and around the food, while the tentacles progressively withdraw from it until the food body is lying on the contracted portion of the disk, completely covered by the esophageal lobes. Next that part of the disk beneath the food withdraws, involving an enlargement and further displacement of the mouth, till there is nothing beneath the food body, and it is pressed by the esophageal lobes into the internal cavity. “The whole reaction 1s thus very complex. Twenty or more pieces of crab, including entire large append- ages, may thus be successively taken, till the body of the anemone has become a mere stretched sack full of crab appendages. But in the later reactions of a series the process of food-taking becomes much slower, the animal seeming to become gradually satiated. The food may be taken by the tentacles and held for a long time before it is finally moved to the mouth. In other cases the ten- 450 Hi. S. fennings. tacles do not react for some minutes, the food lying on the disk undisturbed, until finally it is slowly taken. Sometimes there is an interesting combination of the positive food reaction and the negative reaction (to be described later). ‘The food is taken by the tentacles and carried very slowly to the mouth, in the way above described, while the mouth opens and the esophageal lobes are protruded. But when the food body reaches the lobes, or sometimes before, the process stops. “The food is released by the tentacles, and is finally carried away and rejected, in the way to be described. Finally, when the animal seems fully satiated, the piece of crab meat may be rejected as soon as it comes in contact with the disk. But after one or more pieces have been rejected one may sometimes see another piece accepted. The internal state is in a condition of most unstable equilibrium, and may easily incline toward the positive or the negative reaction. Thus it is clear that in Stoichactis the reaction to a given stimulus is by no means a set, invariable property of the organism, but depends on the state of the internal processes. ‘To the same stimulus we may get a quick positive reaction or a quick negative reaction; a slow and deferred positive reaction or a combination of the positive and negative reactions. Peculiar effects are observed when several pieces of meat are placed at the same time on different parts of the disk. If the animal is hungry all are carried to the mouth; the entire disk folds inward and the pieces are swallowed simultancously or successively. I have seen six pieces, placed as far apart on the large disk as possible, thus ingested. When the animal is less hungry the results are different. In some cases, when two pieces of meat are placed on the disk, one is swallowed while the other is rejected. If the rejected piece is again placed on the disk after the first piece has been disposed of, it will sometimes be swallowed. Adding new pieces while swallowing is in progress often pro- duces interference. Thus, 1 in one case two pieces of meat, a and 4, were placed near opposite edges of the disk. Both began to approach the mouth in the usual food reaction. Now two new pieces, c and d, were placed near the edge midway between a and b. Thereupon the reaction to a and } cone while d was transported to the edge. of the disk (about 2 cm.) and dropped off. Now the food reaction was resumed, a, } and c traveling toward the mouth. Modifiability in Behavior. 451 Piece d was now replaced on the disk. ‘The reaction to the other pieces was suspended, and d was carried to the mouth. Here it came against the middle of the esophageal lobe that was extend- ing toward a,—in such a way that d could not well be ingested without a rearrangement of the lobes. ‘Thereupon d was again carried away from the mouth and once more dropped over the edge of the disk. ‘The other pieces were now successively swal- lowed. Piece d was readily swallowed when given to another specimen. The Rejecting Reaction.—After Stoichactis has become satiated, it rejects food, as we have seen. ‘The rejecting reaction presents a number of points of much interest. By this same reaction the disk is kept clean when débris falls upon it. If a mass of waste matter of any sort (as a mass of dead plankton or a quantity of sand) is placed on the disk of Stoichactis, measures are set in operation which result, within ten or fifteen minutes, in removing this material and leaving the disk free. ‘The behavior in bring- ing about this result is complex and the operation may be accom- plished in more than one way. The tentacles bearing the débris or the rejected food body collapse, becoming thin and slender, and lying flat against the disk. At the same time the disk surface in this region begins to stretch, separating the collapsed tentacles widely. As a result the waste mass 1s left on a smooth, exposed surface, the tentacles here having practically disappeared—though under usual conditions they form a close investment almost completely hiding the surface of the disk. ‘Thus the waste mass 1s fully exposed to the action of waves or currents, and the slightest disturbance in the water washes it off. Under natural conditions this must usually result in an immediate removal of the débris. If this does not occur at once, often the region on which the débris is resting begins to swell, and becomes a strongly convex, smooth elevation, thus rendering the washing away of the mass still easier. But the process may go much farther. If the débris is not removed in the way just ‘deccnnen, new reactions set in. If the mass is nearer one edge of the Ae this edge usually begins to sink, while at the same time the tentacles between the edge and the waste object collapse and practically efface themselves. ‘Thus a smooth, sloping surface is produced and the waste mass slides off the aisle: If this does not occur at once, after a little time the 452 H1. S. “fennings. region lying behind the mass (between it and the center of the disk) begins to swell, producing a high, rounded elevation, with tentacles plump and swollen. ‘The waste mass is now on a steep slope, and is bound soon to slide down and over the edge. Some- times by a continuation of this process the entire disk comes to take a strongly inclined position, with the side bearing the débris below. Often one portion of the edge of the disk after another is lowered in this way, till all the waste matter has been removed. The disk then resumes its horizontal position, with nearly flat or slightly concave surface. Sometimes the edge bearing the débris cannot be lowered, owing to the fact that it is almost against an elevation in the irregular rock to which the anemone 1s attached. In this case, after perhaps an attempt to bend the edge downward, the part between the edge and the waste body swells and rises, rolling the mass toward the center, while at the same time the region between it and the center sinks down. ‘The sinking continues till it reaches the opposite edge, so that the mass is rolled across the disk to the opposite side and there dropped off the disk. ‘The process is slow, often taking fifteen minutes to half an hour. ‘The rejecting reaction is characterized by great flexibility and variability. The débris or refused food sets in operation cer- tain activities; if these do not remove the source of stimulation, other activities are induced until one is successful. Thus in Stoichactis the same stimulus—crab’s meat—may in the same individual produce sometimes the long train of activities resulting in the ingestion of food; in other cases the complicated and variable behavior resulting in rejection, in still others a com- bination of the two. The deciding factor is internal—the con- dition of the metabolic processes. A 1ptasta. Two species of Aiptasia were studied. One was Aiptasia annulata Les.; the other a smaller and darker species, with shorter tentacles, which I have been unable to identify with certainty. I shall call it Aiptasia No. 2. Both came from the moat surround- ing Fort Jefferson. Rather small specimens, with columns 4 to 10 cm. in length, were used in most of the work. Modifiability in Behavior. 453 The species of Aiptasia are relatively active and quick-moving anemones. Especially is this true of Aiptasia annulata. If the tip of one of the long tentacles is touched, the whole disk and column shrinks with a sudden quick contraction, reminding one of the rapid contraction of a medusa. ‘To the eye all parts of the body appear to contract at once. Often the disk and column have contracted strongly before the actual contraction wave has made any apparent progress from the tip of the long tentacle to the disk. Certainly in this animal the general contraction does not appear to be due to a spreading of an actual contraction wave from one part of the animal to another, through the actual pulling of one region upon the neighboring one, as it Wioce; in Hydra, and according to Torrey (04), in Sagartia. On the contrary, there seems certainly to exist some rapid method of conduction, suggest- ing nervous action. In Aiptasia annulata the use of India ink indicates the presence of cilia driving a current away from the mouth and toward the tip of the tentacles, as in Metridium. Aiptasia annulata usually takes crab meat or filter paper soaked in the juices of such meat, but refuses neutral bodies, such as plain filter paper or sand. Aijptasia No. 2, on the other hand, is usually prepared to swallow readily balls of plain filter paper and other small neutral bodies, as well as crab meat. ‘This furnishes opportunity for some interesting comparative experiments. Food is taken in the following way: If a small object comes in contact with a tentacle it adheres to the surface, and the tentacle contracts strongly, the whole animal usually contracting at the same time. ‘Then the tentacle bends over and places the food with considerable precision on the mouth. ‘The tentacles near by likewise bend over and are applied to the food body, holding it down against the mouth. ‘This happens even when the body is quite neutral, as plain filter paper, so that the bending of the neighboring tentacles is clearly due to some influence transmitted from the one tentacle in contact with the body. The mouth now opens, the lips protruding a little and seizing the food, while the ten- tacles may release it and bend away. But sometimes the tentacles follow the food into the mouth and their tips remain enclosed for some time. The actual swallowing of the food is mainly due to the activities of the lips and esophagus; it may occur without an intervention of the tentacles, when the food is placed directly on 454 HI. S. Fennings. the mouth. A piece of meat or filter paper may be completely enclosed by either species within ten seconds of the time it comes in contact with a tentacle. With these two species of Aiptasia the experiments of Nagel and Parker, mentioned on page 448, were repeated and varied, with somewhat peculiar results. Pieces of crab meat and of filter paper (plain or soaked in juice of crab meat) were given alternately to the individual under experimentation. In Metri- dium and Adamsia, as we have noted, the animal soon comes to reject the filter paper, while still accepting the meat. In Aiptasia annulata a typical experiment is as follows: ‘The animal is fed alternately filter paper soaked in crab juice and crab meat. Both are taken readily till four pieces of each have been ingested. At the fifth piece of paper—the ninth piece of the whole series—the animal balks and rejects it. But it likewise rejects the immediately following fifth piece of meat! It has evidently lost its hunger, and refuses to take anything. ‘This is the usual result with Aiptasia annulata. In Aiptasia No. 2 plain filter paper (not soaked in crab juice) was given alternately with pieces of crab meat. In a typical experiment six pieces of filter paper and six of meat were taken in regular alternation. But the seventh piece of paper and the immediately following seventh piece of meat were rejected. The results above given are the usual ones. But sometimes, though rarely, eeniesn are reached which are analogous to those ied in Metridium by Parker. Thus, 1 in one case a specimen of Aiptasia annulata accepted the first piece of plain paper, but thereafter refused paper consistently, while accepting meat offered in regular alternation with it. For all these results the following explanation suggests itself: The animals when hungry take both meat and filter paper; when satiated they take neither. Usually the tendency to take both ceases at the same point, but sometimes the reaction to the weaker stimulus (filter paper) cease before that to the stronger stimulus— as a higher animal that is not hungry may refuse most things, while accepting peculiarly tempting morsels. If the degree of hunger is thus the determining factor, then it should be possible to produce the rejection of the filter paper by feeding meat alone. This turns out to be the case. Indeed, usually the rejection of filter paper may be induced more readily Modipftability in Behavior. 455 by feeding meat alone than by feeding the two alternately, or than even by feeding filter paper alone. ‘Thus, two specimens of Aiptasia No. 2, which we will call A and B, living side by side, were both found to take plain filter paper readily. “Then A was fed alternately meat and filter paper, while B was fed successive pieces of meat. After eight pieces had thus been fed to each, A still took filter paper (though slowly), while B refused it abso- lutely—though B would still slowly take a piece of meat. ‘Thus B, through satisfying its hunger with meat, had come to reject filter paper, while A still accepted it after devouring several pieces. Apparently meat is more satisfying to sea anemones than is filter paper! In another case a specimen of the same species was fed filter paper alone. It swallowed ten pieces in succession, till the body was puffed out with them, meanwhile ejecting some of the pieces already swallowed, in the intervals between the taking of new ones. In Aiptasia annulata similar relations were found. ‘The animal could be caused to reject filter paper soaked in crab juice much more readily by feeding it meat alone than by feeding soaked paper alone, or by feeding the two in alternation. A large number of comparative experiments were tried, showing this rentils to be general. It is therefore clear that the state of hunger or satiety is the essential factor in this behavior, in Aiptasia. The experiments showed further that it is not the mere mechani- cal fulness of the digestive cavity that determines acceptance or rejection, but some change in the metabolic processes themselves. Filling the digestive cavity with filter paper does not have the same effect in producing rejection as does filling it with meat. Even when the cavity is so filled that pieces of paper are repeatedly disgorged, new pieces are readily taken. In Aiptasia No. 2, a piece of paper that has been disgorged after remaining some time in the cavity, is usually swallowed again immediately, if it 1s returned to the disk. As the animal becomes less hungry the details of the behavior toward food bodies change greatly. In a hungry specimen, as we have seen, the food reaction is rapid, often requiring but ten to fifteen seconds. After several pieces of meat have been ingested the reaction of all parts becomes much slower and less precise. The tentacles touched by the food may not react at all for several seconds; then they bend in a rather languid way toward the 456 Hi. 8. fennings. mouth, while the surrounding tentacles may quite omit their reaction. ‘The food body is not placed so accurately upon the mouth asin the hungry individual. Ata further stage toward satia- tion, a piece of crab meat applied to the tentacles induces either no reaction at all or a straight withdrawal—a negative reaction; they may then bend back from the disk along the column. If the meat is placed directly on the disk, in contact with the mouth, the latter may very slowly open and in a languid way partly or entirely enclose the food, even when there is no reaction of the tentacles. The mouth is thus usually readier to give the food reaction than are the tentacles. In this condition of approaching satiation some peculiar com- binations and alternations of positive and negative reactions may be observed. In a specimen of Aiptasia No. 2 after five pieces of alternate meat and paper had been taken, another piece of paper was swallowed, then after one and one-half minutes this was disgorged. ‘The disgorged piece lay on the disk for a few seconds, then the mouth opened and began swallowing it again. But after it was about half enclosed, it was again rejected. Now it was grasped again and partly re-swallowed, then again rejected. ‘This performance was repeated once more before sae piece of paper was definitely rejected. A fresh piece of paper presented 1 imme- diately after was slowly swallowed, then in two minutes disgorged. The anemone presented exactly the spectacle which we ‘should interpret in a higher organism as a struggle between desire and repugnance for the available food. In another case a piece of meat was presented after six pieces had been swallowed. ‘The tentacles reacted only very slowly, but finally deposited the piece of meat on the disk, and withdrew. The mouth opened part way, then closed again without ingesting the food. Later it opened again a very little and enclosed a minute shred of the meat between its lips. “Uhe piece was thus quietly held for ten minutes, when it was seen to be sinking imper- ceptibly. Fifteen minutes after it was given it was completely enclosed. Many other cases were seen of partial rejection and acceptance of the same piece of meat. At times after one piece has been rejected, another is accepted. In Adamsia and Metridium, according to Nagel (’92) and Parker (’96), after the tentacles of a certain region of the disk have through repeated trials come to reject soaked filter paper, Modifiability in Behavior. 457 those of another part of the same disk will still carry it to the mouth. This shows clearly that a general lack of hunger on the part of the organism as a whole cannot be the only factor involved. In Aiptasia No. 2 I tried experiments to determine whether there was the same independence in the tentacles of different regions. Crab meat was given to the tentacles of the left side; these carried it to the mouth, where it was swallowed, the tentacles of the right side playing no part in the reaction. After the tentacles of the left side had taken five pieces they reacted very slowly, a piece of meat resting against them for several seconds before it was seized. When it was finally carried to the mouth, however, it was swallowed readily. ‘The next piece of meat, not being seized at once by the left tentacles, was trans- ferred to hones of the right side. They seized it instantly and quickly carried it to the mouth. ‘Thus it is clear that the experi- ence of the individual tentacles plays some part in the behavior; either from fatigue or some other cause, tentacles frequently stimulated gradually lose the tendency to respond. ‘The fact that this result is produced by meat, the purest form of food, seems to indicate that fatigue may be the cause. But the rest of the experiment indicates that this plays only a minor part in the change of behavior. After a short rest the giving of food to the tentacles of the left side was resumed. ‘They continued to carry it slowly and with much delay to the mouth, where it was very slowly swallowed. After taking four more pieces, the tentacles of the left side absolutely refused to carry any more food to the mouth. ‘The mouth had now almost ceased taking food when directly applied to it, though after some minutes the food was finally ingested. Now a piece of meat was given to the tentacles of the right side, which had only reacted once, and that more than fifteen minutes ago. Yet they behaved in exactly the same way as did the others, refusing to react at all, save by hanging back from the disk along the column. Thus it is clear that the animal is a unit so far as hunger and satiety are concerned. If the satiety has arisen through the activ- ity of the tentacles of one side, the tentacles of the other side are equally affected by it. It is the general progress of metabolism that is the chief factor in determining the reactions to food. As Torrey ('04) has already noted for Sagartia, the reactions of satiated sea anemones differ in many other ways from those of 458 1. S. fennings. hungry specimens. ‘he well fed animal reacts much less readily and strongly to simple mechanical shock. If touched with a needle the well fed individual of Aiptasia either does not react at all, or contracts very slightly, while the hungry specimen reacts suddenly and powerfully. A slight disturbance in the water has no effect on the well fed individual, while the hungry one contracts strongly. To chemical stimuli the same relations apply. A much stronger solution of any given chemical is required in order to produce contraction in the well fed individual, as compared with the hungry one. The bearing of such facts on quantitative determinations in reaction work is evident. If we should attempt to determine the strength of a given chemical which causes con- traction in Aiptasia, we should obtain totally different results, according as we used specimens that were very hungry, moderately hungry, or thoroughly satiated. No “normal” concentration for causing reaction could be determined for even a single given specimen, for the state of metabolism, and with it the tendency to react, is continually changing. It is, of course, clear that the change due to varying metabolic states cannot be interpreted alone as a general increase or decrease of sensitiveness. Much more significant 1 is the complete qualita- tive change in the nature of the reaction to a certain stimulus, due to this cause, which we have seen both in Stoichactis and in Aiptasia. 2. ACCLIMATIZATION TO STIMULI. Sea anemones show acclimatization to stimuli in the same way as do the protozoan Stentor and many other low organisms. A light stimulus that is not injurious may cause at first a strong reaction, then on repetition produce no reaction at all, or a very slight one. ‘This is easily shown with Aiptasia annulata in the following way: oP Modifiability in Behavior. 459 reaction of a different sort, that often comes on later, will be mentioned in the next section. Experiment shows that the failure to respond is practically universal if the drops fall three minutes or less apart. With drops five minutes apart there is still marked evidence of acclimat- ization, though irregularities appear. With drops falling at intervals of more than five minutes I was unable to satisfy myself with certainty that acclimatization occurs. Related to the present subject are changes in the reaction to light. Aiptasia annulata is very sensitive to light, expanding in darkness, but contracting after a few seconds when exposed to strong light. In ordinary daylight the animal remains contracted for some hours, but after such a period most specimens extend in spite of the light. In comparative darkness the animals direct the disk toward the source of light, through a contraction on the side of the column exposed to the light. After remaining undis- turbed for a long time in an aquarium that is fairly well lighted, the animals give up their orientation with respect to the strongest source of light; with less light they retain it. 3. REACTIONS MODIFIED AS A RESULT OF THE PAST EXPERIENCES OF THE ORGANISM. Under this head will be considered all positive changes in reaction, due to former stimuli or former reactions of the organism, aside from those due to changes in metabolism. We have already described certain cases belonging here. In the reaction by which the disk is kept clean in Stoichactis we find that a mass of débris on the disk causes first one reaction, then another, till one of these or a combination of several rids the animal of the stimulating agent (see p. 451). In this case either the continua- tion of the same stimulus, or the fact that a certain reaction has been given, induces a new reaction, without change in the external _ conditions. A similar phenomenon is often seen in the experiments with falling drops of water, described above. ‘To the first drop the animal responds by a sudden sharp contraction, then to a consider- able number of drops there is no response. Now if the drops con- tinue, the animal usually begins to shrink slowly away from the region where the drops are falling, so that in the course of time the 460 Hi. S. Fennings. disk has been withdrawn some distance below the surface, though no decided reaction has occurred to any one stimulus. ‘These facts are precisely parallel to those which I have described in a previous paper (1902, p. 50) for the infusorian Stentor. More marked changes result when the animal is stimulated by light strokes of a rod. At the first stroke on the disk Aiptasia contracts strongly. It then extends in the same direction as before. When it is fully extended the stimulus is repeated. ‘The animal responds in the same way as at first. ‘This is usually con- tinued for about ten or fifteen stimulations, the animal each time extending in the same direction as at first. But at length, when stimulated anew, the animal contracts, bends over to one side, and extends in a new direction. Under natural conditions, where stimulation at every extension would usually be due to some fixed object, this would of course put an end to the series of stimuli. If, however, the stimuli are still continued after each extension, the animal repeats for a number of times the extension in the new direction, then finally turns again and tries a new position. This may be repeated many times. But in the course of time the reaction becomes changed in a still different manner. ‘The anemone releases its foothold and moves to a new region. ‘This result [ have not succeeded in attaining by striking the animal with a rod each time it extends; the time required is evidently to be measured in hours. But obstructions may be so placed that every time the animal extends, the disk strikes against a solid body. In such a case it is usually found after a few hours that the animal has moved to a new region. Thus to the same stimulus when repeated many times the anemone reacts first by contraction, then by turning repeatedly into new positions, then by moving away. ‘The phenomena are parallel to those described by the present author (’02) for the infusorian Stentor, and by Wagner (’05) for Hydra. Beyond doubt other stimuli would here, as in Hydra and Stentor, produce the same series of reactions. In the behavior just described there are at times certain phe- nomena which bear a striking resemblance to the formation of new habits. Aiptasia annulata frequently extends its body in most awkward turns, the column retaining an irregular and crooked form. ‘This is evidently due to its meth’ Ne life. “The animal lives in irregular crevices and crannies beneath stones or in the Modifiability in Behavior. 461 hollows of the coral reefs. In order that its disk may protrude into the free water, it is often compelled to extend in the irregular way mentioned, and to retain the crooked forms thus reached. W hen removed from the natural habitat it still retains these irregularities of form and action. ‘The lower part of the column may Seen at right angles to the upper part, or there may be permanent S-shaped bends, or still more irregular forms. It would appear that these must have arisen as a result of the way in which it extends in its natural habitat. The peculiar methods of extension found in given individuals could then hardly be characterized otherwise than as habits, the peculiarities of form being the structural cor- relates of the habits. In searching for experiments that would test the possibility of the formation of new habits in sea anemones, the following sug- gested itself. It should be possible to produce new habits in Aiptasia by so arranging the surroundings as to compel the animal to extend in a new way whenever it extends, and to retain the new form thus induced. If the animal when thus compelled by obstacles to extend in a new direction, still extends in the same direction after the obstacles are removed, one would be inclined to hold that a new habit had been formed. I supposed that this result would require a long period of time. But some preliminary experiments showed it to be attained, in some cases, with such absolute ease as to raise the doubt whether we have here anything that can be called habit formation. ‘Thus an individual attached to a plane horizontal glass surface was bent in extension far over to the left. Stimulating 1 it repeatedly , It con- tracted at each stimulation, then bent, in extending, again to the left. This continued for fifteen stimulations, one succeeding another as soon as the animal had become fully extended. At the next contraction the animal turned and bent over to the right. Now when stimulated it contracted as before, then bent regularly, in extending, over to the right. It seemed to have acquired a new habit—bending to the right instead of to the left. Attentive examination showed that when the animal contracted in response to stimulation, the concave side of the column con- tracted a little more than the rest, so that that side remained a little shorter. In other words, the animal did not take on an entirely symmetrical structure, but the region which was most con- tracted in extension remained most contracted also in the con- 462 1. S. fennings. tracted animal. Now on expanding, all parts extended more or less proportionately to their extension in the contracted animal, so that the original curved form was regained. In other words, the structural conditions resulting in the curved form were not really given up even in contraction, and were only made evident when extension occurred. If the animal was compelled by repeated strong stimulation to contract maximally in Ill parts, then in extension there was no greater tendency to bend in the direction previously occupied than in any other. And in about half the individuals this result followed (after once the first habitual position found in nature had been given up) even after a single stimulation, so that there was no indication of anything like the formation of a new habit. What is the interpretation to be given to the numerous cases in which bending in a certain direction when extended does induce, in the way set forth above, bending in the same direction on a new extension? Is this the formation of a habit? It is certainly a condition of affairs that gives the same result as habit formation. The anemone might indeed be looked upon as a sort of structural model, illustrating the principles on which habit formation might occur. A certain action (extension in a certain direction) leaves structural peculiarities, persisting even in the intervals of action (in the contracted state), which result in a repetition of the same action. Is not this the picture that we commonly make for our- selves of the real nature of habit formation? In the sea anemone this seems to occur in a relatively gross way, but it appears difficult to point out any difference in principle between this and habit formation. If the persisting structural peculiarities were of such a nature as to be hidden from observation, there would be no ground for hesitation in calling these phenomena the formation of habits. There can hardly be doubt that the striking individual peculiarities of action and structure, described above, ‘have arisen in precisely this way, so that it plays the part taken by habit formation in higher animals. It would be well if the study of this matter could be extended to the same individual for a long time, beginning with a young, still regular, specimen, compelling i it to live in a position where it would have to extend in a definite irregular way. In this way the development of the structural correlates of the habits (?) could doubtless be observed. Modifiability in Behavior. 463 The facts may be summed up for the anemone as follows: Performance of a certain action involves the assumption of certain structural conditions. ‘These conditions persist in a slight degree even in the intervals between the actions. At a new action they show their influence by causing it to take place in the same way as the former one. ‘This gives the same results as what we are accus- tomed to call habit. . 4. GENERAL AND COMPARATIVE. The sea anemones are among the lowest of the Metazoa, and their behavior, when compared with that of most other animals, is of a very simple character. Yet it is evident that even in these low organisms the reaction to a given external stimulus depends upon many things beside the nature of the stimulus itself. Vary- ing states of metabolism induce totally different reactions to the same stimulus, one state producing the long train of actions look- ing toward the ingestion of food, another inducing the equally long and variable chain of activities resulting in rejection. The same factors cause marked changes in reaction to other stimuli than possible food. Past stimuli received and past reactions per- formed likewise determine the reaction to a given external con- dition, resulting sometimes in a cessation of reaction, in other cases in a complete change in its character. ‘Certain simple con- ditions produce a tendency in the organism to perform more readily an act previously performed (bending, on extension, in a certain direction). Examination of the conditions under which the animals live shows clearly that all the usual reactions and modifications of the reactions are such as to assist in adapting the organism to its environment. In other words, they aid the physiological processes of which the organism is the seat. Aiptasia annulata, for example, lives in crevices beneath and among stones or coral rocks. It 1s, of course, evident that its food reactions maintain its metabolic processes, which would necessarily cease in their absence, that the rejecting reaction keeps the surface clean, so that respiration may take place uninterruptedly, and obstacles or injurious substances be avoided. ‘The transformation of the food reaction into the rejecting reaction after the animal is satiated with food 1s of course as much to the interest of the sea anemone as it is to that of higher 464 1. 8. fennings. animals. If the food reaction were an invariable reflex, occurring whenever food is present, without regard to internal conditions, the results would be disastrous. ‘The fact that the very hungry animal will take indifferent bodies that would otherwise be rejected is of course likewise adaptive; as Torrey (’04) remarks “substances with a very small food value must be of some impor- tance to a starving polyp although they would not be desirable as food to a well penrished animal.” The tendency of Aiptasia to remain in the dark and to contract when strongly lighted keeps it in the crevices where it finds pro- tection for its soft body. ‘The fact that it faces and bends toward the lighted side keeps its tentacles and disk directed toward the entrance to the crevice, where food may be captured; if they were directed toward the darkest part of the crevice little or no food would be obtained. While the contraction under light is protec- tive, it would result, if continued indefinitely by a lighted polyp, in starvation; we find that after a considerable period of light the animal extends. In correlation with its life in irregular crevices or under stones we find that Aiptasia does not take any definite position with reference to gravity, as some other anemones do. Such a reaction would render its usual habitat impossible. The tendency to react by a quick contraction when there is a slight dis- turbance in the water is undoubtedly protective. Yet such a dis- turbance when not?followed by an attack from its author is not harmful and the animal under such circumstances quickly resumes its usual behavior, even though the disturbance continues. But such a disturbance maintained indefinitely would result in loss of opportunity for obtaining food, and the animal after a time shrinks gradually away from such a disturbed region. Injurious stimuli, interfering with the natural physiological processes of the polyp, cause contraction—the animal withdrawing from the field of action for a time. But this continued indefinitely would result in a loss of food and doubtless other injurious effects. We find that the animal has recourse then to extension in another direction, and finally to creeping away and establishing itself elsewhere. Located in an irregular crevice, we find that the polyp extends in various directions, Sunitil it finds a direction in which its disk and tentacles are unimpeded in their spreading to form a trap for prey. It then continues to extend in this manner, even though this may require the body to bend at right angles or to take other irregular * Modifiability in Behavior. 465 forms. It continues to extend in this manner even when removed from its irregular crevice, and the body is found to have become structurally modified, so that a collection of Aiptasias shows many crooked and zigzag shapes, each being an adaptation to the crevice in which the animal lived. The formation of such habitual methods of extension can be imitated and modified in the labora- tory. All together, the activities and their modifications are clearly such as to directly adjust the organism to its environment, enabling the physiological processes to continue under all sorts of conditions. It has become the fashion to neglect such facts, but they fairly force themselves on the attention of the careful student of the behavior, and their existence can hardly be held to be accidental. To remove such an organism to the artificial conditions of the laboratory and then endeavor to understand its behavior is like dissecting an internal organ out of the body and trying to under- stand its functions when thus separated from the other structures with which it interacts. Almost everything the animal does has a direct relation to something in its usual environment, and when cut off from this environment, its activities are likely to become unintelligible. One can hardly resist the belief that the fact that these activities do assist the physiological processes of the organ- isms has determined their selection and retention from among other possible activities. ’ This adaptation and adaptive modifiability of behavior in sea anemones and their relatives has not been explicitly set forth in most works dealing with their reactions. Yet when other careful accounts of behavior in such organisms are analyzed we can dis- cover such relations as clearly as in Aiptasia. Let us look for example at the cases of Hydra, studied by Wagner (’05), and of Cerianthus, as described in the classical papers of Loeb (’91). It will be found instructive to consider the conditions on which the retention of a certain position depends. Hydra and the sea anemones tend as a rule to retain a position at rest, with the foot attached and head free. ‘This usual position is often said to be due to a reaction to gravity, or to contact, or to some other simple stimulus. But when we examine into the matter closely, we find that it is not an entirely simple one. Let us take first the case of Hydta. Suppose the animal is placed on a horizontal surface with head downward and foot upward. It does’ not retain this 466 AH. S. fFennings. position, but bends the body, placing the foot against the bottom, releases its head, and straightens upward. Aiptasia shows the same reaction. In neither of these animals is the reaction due to a tendency to keep the body in a certain position with reference to gravity, for both keep the body indifferently in any position with reference to the pull of gravity, provided that the foot is attached and the disk and tentacles can be spread freely. To what then 1s the reaction due? Evidently there is a tendency to keep the foot in contact with a surface, for the body of the inverted Hydra is bent till the foot comes in contact. There is likewise a tendency to keep the head free, for it is released. But this is not all, for now the body is straightened, then the tentacles are spread out symmetrically in all directions. It is clear that the reaction 1s directed toward getting the organism into a position that may be called “normal,” and this normal position has various factors— attachment of foot, freedom of head, comparative straightness of body, and tentacles outspread. Suppose now that our Hydra has reached this position, and all the conditions remain constant; is this sufficient? We find that it is not. If the conditions remain so constant that no food 1s obtained, the Hydra becomes restless and changes the position of its body repeatedly, though still retaining its attachment by the foot. Later even this is given up, and the animal, of its own internal impulse, quite reverses the position attained through the “righting reaction.” It now bends the body, attaches the head, and releases its foot, thus bringing it back into the inverted position. Is this because the irritability of head and foot have become reversed, so that the head now tends to remain attached, the foot free? Apparently not, for no sooner has the animal taken the inverted position than it draws its foot forward and now performs the “righting reaction”’ again, so that it stands once more on its foot. ‘These alternations of behavior are repeated, and we find that by this means the animal is moving from place to place (see Wagner, 1905, Fig. 3). It seems clearly impossible to refer each of these acts or the whole behavior to any particular present external stimulus. An internal state—hunger—drives the Hydra to move to another region, and these different opposite acts are the means by which another region is reached. Each phase of the locomotion is Modifiability in Behavior. 467 evidently partly determined by the fact that a certain other phase has just been performed, partly by the general state of hunger. ‘The same behavior is shown by Hydra under continued injurious stimuli of different sorts. In speaking of righting reactions, it is often said that the organism is forced by the different irritabilities of diverse parts of the body to take a certain orientation with reference to gravity or | to the surface of contact (see for example Loeb, 1900, p. 184). The facts just brought out (taken from Wagner) show that we cannot in Hydra consider this orientation forced, save in the general sense that all things which occur may be considered forced— including of course the behavior of man. Man takes sometimes a sitting position, sometimes a standing one, sometimes a reclining one, depending upon his “physiological state” and past history, and the facts are quite parallel for Hydra. So far as objective evidence shows, the behavior is not forced in Hydra in any other sense than it isin man. Both organisms take that position which seems best adapted to the requirements of their physiological processes; these requirements vary from time to time. In the sea anemone Cerianthus the conditions for staying in a certain position are somewhat more complex than in Hydra, accord- ing to the account given by Loeb (1891). Cerianthus is usually found in an upright position, inhabiting a tube made of mucus and imbedded in the sand. If placed head downward in a test tube, it rights itself in the same way as Hydra and Aiptasia, freeing the head, bringing the foot into contact, and straightening the body. But in Cerianthus Loeb showed clearly that gravity plays a part in the behavior. If the animal is placed on its side on a wire screen of large mesh, it bends its foot down through the meshes, lifts up its head, and takes its usual position with reference to gravity. If now the screen is turned over, the animal again directs its head upward, its foot downward—as a human being under similar circumstances would do if possible. It may thus weave itself in and out through the meshes. But to be in line with gravity, with head above and free, is not the only requirement for Cerianthus. Loeb found that it would not remain indefinitely in this position on the wire screen, as it does inthe sand. After a day or so it pulls its foot out of the wire and seeks a new abode. Only when it can get the surface of the body in contact with something, as is the case when it is imbedded 468 Hi. S. fennings. in the sand—uin its natural habitat—is it at rest. If this condition is fulfilled, the requirement of the usual position in line with gravity may be neglected. Loeb found that when the animal is placed in a test tube, so that its body is in contact with the sides, it remains here indefinitely, even though the tube 1s placed in a horizontal position (Loeb, 1891, p. 54). The head is bent upward, but the body remains transverse to the direction of gravity. Examples of the fact that a certain orientation with reference to gravity is not a rigid requirement even in animals that usually or at times react to this agent, are common among sea anemones and other lower organisms. Thus, Torrey (04) shows that Sagartia, though it usually maintains an upright position, may ofttimes take | position on the surface film, with head downward. In the rejecting reaction of Stoichactis, described on p. 451, we have clearly a reaction with reference to gravity, though one which even the most sanguine could hardly denominate a fixed tropism. The situation “waste - matter - on - the - disk - not - removed - by - the - first - (usual) - reaction” is responded to by taking such a position with reference to gravity as results in removing the waste; then the reaction to gravity ceases. ‘I’his is somewhat analogous to the reaction to gravity described by Bohn (1903) in the hermit crab. While investigating a shell which it may adopt as a home if fitting, this animal takes up a certain position with reference to gravity— namely, with the body on the steepest slope of the shell, and head downward; it then turns the shell over and ceases to react with reference to gravity. Of a different but equally significant char- acter are the variations shown in the reactions to gravity by the low acelous flatworm Convoluta, as described by Bohn (’03b) and Gamble and Keeble (’03). Under conditions that are favor- able Convoluta remains on the surface of the sand. But when the sun becomes hot, or when the tide rises, so that the animal is likely to be washed away, it becomes “ positively geotropic,” going downward in the sand, where it is protected. When the tide: fale again Convoluta becomes “ negatively geotropic,” thus reaching the surface of the sand, where it obtains food and carries on its usual activities. [hese alternations of reaction become a fixed habit with Convoluta, so that when removed to an aquarium it still goes downward at high tide, upward at low tide, though the con- ditions surrounding it remain constant; it may thus bet used fora time as an in- aioor tide indicator. Gradually, however, when Modifiability in Behavior. 469 removed for a long time from the influence of the tides, this alter- nation of reactions to gravity ceases, showing it to be a true habit, resulting from individual experience. Many other instances of reactions to gravity, of the most diverse sorts and variable charac- ter, could be given. Gravity affects organisms in many diverse ways—determining the distribution of internal substances of dif- fering specific gravity, causing differences in the ease of move- ments in diverse directions, inducing strains or pressure in unac- customed parts of the body when an unusual position is taken— indeed, influencing the life processes in almost every detail. Any of the points at which it comes in contact with the life processes may serve as the basis for a reaction, so that we find behavior induced by relations to gravity in different organisms to be of the most diverse character. We have been assured by various writers that the reaction to gravity must be explained in the same way in all cases, but this is evidently said rather in the capacity of a seer or prophet, than in the capacity of a man of science whose con- clusions are inductions from observation and experiment. Returning to Cerianthus, we find, according to Loeb, that even the usual position in line with gravity and with sides in contact, does not satisfy the animal indefinitely, if left quite undisturbed. If it secures no food it again leaves its place and seeks another region. Thus in order that Cerianthus may remain quiet in a given position, a considerable number of conditions should be fulfilled, constituting the usual, and perhaps what we may call the “normal” state of affairs for this animal. ‘Uhese conditions are the following: (1) The foot should be in contact; (2) the head should be free; (3) the body should be straight; (4) the axis of the body should be in line with gravity, with the head above; (5) the general body surface should be in contact; (6) food should be eee at inter- vals. If these conditions are largely unfulfilled, the animal becomes restless, moves about, and finds a new position. But no one of these conditions is an absolute requirement at all times, unless it be that of having the head free. In the wire screen the animal remains for a day or two in the required position with reference to gravity, even though foot and body surface are not in contact. In the horizontal tube it remains with foot and surface in contact, though the body 1s not straight nor in line with gravity. If all conditions are fulfilled save that of food, the animal remains for a time, then moves away. 470 A. S. Fennings. Clearly, the holding of any given position depends, not on the relation of the body to any one or two sources of stimulation, but on the proper maintenance of the natural physiological processes of the organism. ‘The actinian does not always maintain a certain position with relation to gravity, nor does it always keep its body straight, nor its foot in contact, nor its body surface in contact. It does not at all times receive food. It may remain quiet for considerable periods with one or more conditions lacking. ‘The organism tends on the whole to take such a position as is most favorable to the unimpeded course of its natural physiological processes. Certain usually required conditions may be dispensed with provided other favorable ones are present. ‘The behavior, like that of higher animals, represents a compromise of the various needs imposed upon the animal by its physiological processes. Examination of the literature shows that throughout the Ccelen- terates there is a similar dependence of behavior on the progress of the internal physiological processes, particularly those of metab- olism. ‘The state of metabolism decides whether Hydra shall creep upward tothe surface or shall sink to the bottom (Wilson 91), how it shail react to chemical and to solid objects (Wagner ’05), whether it shall remain quiet in a certain position, or shall reverse this position and undertake a laborious tour of exploration. In the sea anemones it determines, as we have seen, even the details of long trains of reaction. ‘The state of the metabolic processes appears to be the most important determining factor in the behavior of Coelenterates. The same dependence of behavior on the internal physiological processes is found in other groups, even in those much lower than the Ccelenterates—the Protozoa, and particularly the Bacteria. This is brought out especially in some of the work of Engelmann. A number of examples of this relation will be given in the paper which follows the present one, so that they may be omitted here. The fact that in higher animals behavior depends largely on hunger and satiety is, of course, so well known that it need not detain us. The relation of behavior to the internal physiological processes, of which we have given some examples in the foregoing pages, 1s manifestly of the greatest significance for the understanding of behavior. he facie Adameed show directly that in many cases the determining factor in reactions to stimuli is not the anatomical configuration of the body, taken in connection with simple laws Modipfiability in Behavior. 471 of conduction, but is the relation of the action of the external agent to the internal processes. “The problem presented by the fact that the same stimulus, in the same intensity, applied to the same part of the body, produces qualitatively different and even opposite results, depending on the inner metabolic states, seems not to have received the attention it deserves. It evidently places marked difficulties in the way of a simple mechanical conception of the reflex process, based merely on the anatomical structure of the organism. ‘The internal physiological state determines in some way which of various courses within the body the transmitted stimulus shall follow and what organs it shall arouse to activity. The organism cannot be looked upon as a static structure, on which external agents must act in a simple invariable way. The organism 1s a process, and some of the chief determining factors in behavior are given by the relation of the internal to the external processes. As the internal processes change, the reaction to external agents changes correspondingly. We find that reactions which assist the existing internal processes are continued or repeated, while those which oppose them are changed. ‘This gives one of the chief bases for the regulatory character of behavior, as I shall attempt to set forth in farther detail in the paper which follows the present one. ‘The metabolic processes, while the most striking of those taking place i in the lower organisms, are of course not the only ones occurring in animals. Aue immense number of other processes are in progress, and the relation of external agents to these processes may and does equally determine behavior. This gives the phenomena of behavior their complexity, prevent- ing them from being in relations of simple dependence on external agents, as they are often represented of late. Such a view quite underestimates the difhculty of the problem of behavior. ‘Lhe dependence on external agents exists, but is complex, and can usually not be predicted “without a knowledge of the present internal state of the organism—this depending on its past history and the course of its various internal processes. It would of course be more convenient if the problems of behavior were as simple as they are often proclaimed to be. Work revealing their complexity is naturally not received witn the acclaim that greets the announcement that all these things ‘are simple and easy. But if our object is really to obtain concrol of the vital processes, then we must face them in all their com- 472 Hi. 8S. fFennings. plexity. To control animal behavior it is necessary to study animal nature, in much the same way that it is necessary to study human nature in order to control human behavior. It is neces- sary to know the past history of the organisms, and what is going on within them, in order to predict what they will do. He who expects even the lower animals to behave always in certain simple invariable ways when acted upon by the various forces of nature has many disappointments in store, when he comes to make a thorough study of the matter. ‘The internal modifying conditions must be made the object of deliberate and extended investigation in lower animals as well as in higher ones, before the study of behavior can be placed on a really scientific basis. LITERATURE, CElED: Boun, G., ’03.—De l’évolution des connaissances chez les animaux marins littoraux. Bull. Institut Général Psychologique, No. 6; 67 pages. ’03.—b. Les Convoluta roscoffensis et la théorie des causes actuelles. Bull. Mus. d’Histoire Naturelle, pp. 352-364. DuerpDEN, J. E., ’02.—Report on the Actinians of Porto Rico. Bull. U.S. Fish Com., vol. xx, second part, pp. 323-374, 12 pl. GamBLE, F. W.,AND KEEBLE, F., ’03.—The Bionomics of Convoluta roscoffensis, with Special Reference to Its Green Cells. Quart. Journ. Micr. Sci., vol. xlii, pp. 363-431. Jennincs, H. S., ’02.—Studies on Reactions to Stimuli in Unicellular Organisms. IX. On the Behavior of Fixed Infusoria (Stentor and Vorticella), with Special Reference to the Modifiability of Protozoan Reactions. Amer. Journ. Physiol., vol. vi, pp. 23-60. Logs, J., ’91.—Untersuchungen zur physiologischen Morphologie der Thiere. I. Ueber Heteromorphose, pp. 48-59. ‘00.—Comparative Physiology of the Brain and Comparative Psychology. 309 pages. New York. Nace, W., ’92——Das Geschmacksinn der Actinien. Zool. Anz., Bd. xv, S. 334-338. Parker, G.H., ’96.—The Reactions of Metridium to Food and Other Substances. Bull. Mus. Comp. Zool. at Harvard Col., vol. xxix, pp- 107-119. Torrey, H. B., ’04.—On the Habits and Reactions of Sagartia davisi. Biol. Bull., vol. vi, pp. 203-216. Wacne_er, G., ’05.—On Some Movements and Reactions of Hydra. Quart. Journ. Micr. Sci., vol. xlviii, pp. 585-622. Witson, E. B., ’91.—The Heliotropism of Hydra. Amer. Natural., vol. xxv, pp. 413-433. THE METHOD OF REGULATION IN BEHAVIOR AND iN OTHER FIELDS: BY H. S. JENNINGS. The results set forth in the preceding paper, together with certain other relations found in the behavior of lower organisms, that have been detailed in previous papers by the present writer, suggest a certain point of view in regard to the general method of regulation or adjustment in organisms. Everywhere in the study of life processes we meet the puzzle of regulation. Organ- isms do those things which advance their mole There are some exceptions, but this is certainly true in a general survey. If the environment changes, the organism changes to meet the new conditions. If the mammal is heated from without, it cools from within; if it is cooled from without it heats from within, maintaining the temperature that is to its advantage. The dog which is fed a starchy diet produces digestive juices that are rich in the enzyms which digest starch, while under a diet of meat it produces juices rich in proteid digesting substances. When a poison is injected into a mouse, the mouse produces substances which neutralize this poison. If a part of the organism is injured, a rearrangement of material follows till the injury is repaired. If a part is removed, it is restored, or the wound 1s at least closed up and healed, so that the life processes may continue without disturbance. Regulation constitutes perhaps the greatest problem of biology. How can the organism thus provide for its own needs? To put the question crudely, how does-it know what to do when a difficulty arises, so as to overcome this difficulty? It seems to work toward a definite purpose. In other words, the final result of its action seems to be present in some way at the beginning, determining what the action shall be. In this the action of living things appears to contrast with that of things inorganic. It 1s regulation of this character that has given us the theories of vitalism. By these theories the principles controlling the life 474 Hi. S. fennings. processes are held to be of a character essentially different from anything found in the inorganic world. Nowhere is regulation more striking than in the behavior of organisms. Indeed, the processes in this field have long served as the prototype for regulatory action. ‘The organism moves and reacts, on the whole, in ways that are advantageous tOmit.. Lf at gets into hot water, it takes measures to get out again, and the same is true if it gets into excessively cold water. If it enters an injuri- ous chemical substance, it at once changes its behavior and escapes. If it lacks material for its metabolic processes, it sets in operation movements which secure such material, suspending these movements when the lack is fully supplied. If it lacks oxygen for respiration, it moves to a region where oxygen 1s found. If injured it flees to safer regions. In innumerable details it does those things which are good for it, and this is as true of the Protozoan as of the Metazoan. It is plain that behavior depends largely on the needs of the organism, and 1s of such a character as to satisfy these needs. In other words, behavior is adjustment or regulation. ‘There seems no reason to think that regulation in behavior is of a different character from that found elsewhere. But nowhere else 1s it possible to perceive so clearly how regulation occurs. In the behavior of the lowest organisms we can see not only what the animal does, but precisely how this happens to be regulatory. The method of regulation lies open before us. This method is of such a character as to suggest the possibility of its application to other fields; in other words, it suggests a possible general explanation of the method of regulation. This suggestion the present paper attempts to develop. In the lower, unicellular, organisms where we can see just how regulation occurs, the process is as follows. Anything injurious to the organism causes changes in behavior. These changes subject the organism to new Pendicions! As long as the injurious condition continues, the changes in behavior continue. The first change in behavior may not ie regulatory, nor the second, nor the seal nor the tenth. But if the changes continue, subjecting the organism successively to all possible different conditions, a Gocidinicn will finally be reached that relieves the organism of the injurious action, provided -such a condition exists. Thereupon the changes in behavior cease, and the organism remains in the Method of Regulation in Behavior and in Other Fields. 4.75 favorable condition. The movements of the organism when stimulated are such as to subject it to various conditions, one of which is selected. This method of regulation is found in its purest form in unicellular organisms, such as Paramcecium and Stentor. Yet it occurs also in higher organisms, and indeed is found in a less primitive form throughout the animal series, up to and including man. When we ourselves, or other animals, are confronted with a difhculty for which neither experience nor inherited tendency has furnished us with a direct method of relief, the only recourse is to this same method of regulation. We perform movements which subject us to various conditions, till one is found that relieves the difficulty. We call the process searching, testing, trial, and the like. In the lowest and highest organisms the injurious con- dition acts as a stimulus to produce many movements, subjecting the organism to various conditions, one of which 1s selected. In connection with this method of behavior three questions arise, which are fundamental for the theory of regulation. First, How is it determined what shall cause the changes in behavior that result in new conditions? Or why does the organism change its behavior under certain conditions, not under others? Second, How does it happen that such movements are produced as result in more favorable conditions? “Third, How is the more favorable condition selected; what is this selection and what does it imply? Our first and third questions may indeed be condensed into one, which involves the essence of the regulatory process: Why does the organism choose certain conditions and reject others? ‘This selection of the favorable conditions and rejection of the unfavor- able ones presented by the movements is perhaps the fundamental point in regulation. It is often maintained that this selection is precisely personal or conscious choice, and that behavior cannot be explained without this factor. Personal choice it evidently is, and in man it is often conscious choice; whether it is conscious 1n other animals we do not know. But in any case this does not remove it from the necessity for analysis. Whether conscious or unconscious, choice must be determined in some way, and it is the province of science to inquire as to how this determination occurs. To say that rejection is due to pain, acceptance to pleasure, or to other con- scious states, does not help us, for we are then forced to inquire 476 Hi. S. fennings. why pain occurs under certain circumstances, pleasure under others. Surely this is not a haphazard matter! ‘There must be some difference in the conditions to induce these differences in conscious states (if they exist) and at the same time to determine the differences in behavior. We are therefore thrown back upon the objective processes occurring. Why are certain conditions accepted, others rejected? ‘This is essentially what has often been called the pleasure- -pain problem. Such facts as are set forth in the preceding paper give us a basis for an objective answer to this question. Organisms are not static structures; processes of complicated character are in continual progress within them. Among these the processes of metabolism are most prominent. Ostwald (’02) has emphasized the point that one of the chief characteristics of living matter is the fact that processes are occurring with much energy within it. The organ- ism is a complex of processes. In the preceding paper we have seen that the reactions of organisms to external agents depend largely on the relation of the action of these agents to the internal processes. Let us examine certain cases of this dependence in the simplest organisms—bacteria and protozoa. The green Parameecium bur- saria requires oxygen in its metabolic processes. While swim- ming about it comes to a region where oxygen is lacking. It reacts by turning away and going in some other direction. ‘The white Paramcecium caudatum does the same, and so also do many bacteria. All require oxygen in their metabolic processes; lack of oxygen interferes with these processes, and they react to such a lack by changing their movement and going elsewhere. But there are some bacteria that do not require oxygen in their meta- bolic processes. When these come to a region lacking oxygen they do not react, but keep on and enter this region. In many of these anaérobic bacteria oxygen is known actually to interfere with the physiological processes. When these bacteria come to a region containing oxygen, they change their movement and go elsewhere. In Paramoecia and the bacteria that require oxygen, this does not occur (unless the amount of oxygen rises above the optimum). In all these cases, whenever there 1s interference with the metabolic processes, the organism reacts by turning away, otherwise it does not. In the reactions of these creatures with reference to light and darkness we see the same thing. In the Method of Regulation in Behavior and in Other Fields. 477 reen Paramcecium bursaria, in Euglena, and many other green infusoria, light assists the metabolic processes, while lack of | light interferes with them, and the same is true of the so-called purple bacteria. All of these organisms react on coming to a region of darkness by turning away Sand going elsewhere. if the colorless Paramoecium caudatum and in the colorless bacteria ordinary light does not affect the metabolic processes, so that there is no interference with these processes in darkness, and we find that they do not react on reaching a dark region, but enter it readily. For all these organisms, colored and uncolored, light may be made so intense that it does interfere with the phy siological pro- cesses, as 1s shown by the fact that the processes stop, the organ- isms dying. ‘To such light all react by turning away—aincluding even the colorless Paraiiee em caudatum. We find in all of these cases that the animal reacts by turning away when there is interference with the physiological processes, and does not so react unless there is such interference. In some cases the relation between behavior and the effect of an agent on the physiological processes is marvelously precise. Thus, Engelmann (’82) proved that in Bacterium (Chromatium) photometricum the ultra red and the yellow-orange rays are those which most favor metabolism (assimilation of carbon dioxid, etc.). When a microspectrum is thrown on a preparation of these bacteria, they are found to react in such a way as to collect in precisely the ultra red and the yellow-orange. The reaction consists in a change of behavior—a reversal of movement—at the moment of passing from the ultra red or the yellow-orange to other parts of the spectrum, while passing in the opposite direc- tion produces no such effect. Bacteria are not in nature sub- jected to spectral colors in bands, so that there has been no oppor- tunity for the production of this correspondence between behavior and favoring conditions through the selection of varying indi- viduals. What is the explanation of these facts? Why does the infu- sorian or the bacterium shrink back from darkness or the region containing no oxygen? As a matter of fact, it requires the light or the oxygen for the continuance of its metabolic processes, and it does not shrink back from a region lacking them unless it does need them. But we have no reason to attribute to the bacterium any knowledge or idea of that relation. We do not need any 478 H. S. fennings. purpose or idea in the mind of the organism, or any “psychoid”’ or entelechy to account for the change of behavior, for an adequate objective cause exists. We know experimentally that the dark- ness or the lack of oxygen interferes with the metabolic processes. This very interference is then evidently the cause of the change of behavior. When anything interferes with the internal pro- cesses, running with much energy, the energy overflows in other directions, resulting i in changes in behavior. ‘This statement is a mere generalized formulation of the facts determined by observa- tion and experiment in the most diverse organisms. Internal as well as external interference may cause the changes of behavior. If oxygen or other material for metabolism 1s lacking to such an extent as to interfere with the metabolic pro- cesses, the organism changes its behavior. In the sea anemones, as we have seen in the preceding paper, this condition induces the animal to change its position and start off on a laborious tour of exploration. The initiation of changes in movement through internal conditions gives the basis for the reactions which we call positive, as we shall see. The answer to our first question is then as follows: The organism changes its behavior as a result of interference with its physiological processes. Our second question was: How does it happen that such movements are produced as bring about more favorable con- ditions? This question we have already answered, so far as many lower organisms are concerned, in our general statement on page 474. The organism does not go straight for a final end. t merely acts—in all sorts of ways possible to it—resulting in repeated changes in the conditions. In this way a condition is after a time edi that relieves the interference with the internal processes. The nature of the changes in behavior produced—the move- ments that occur in any given organism—depend on what may be called the ‘“‘action system” of the organism. ‘The animal, in other words, performs the movements that it is accustomed to perform, as determined by its structure and its past history. The essential fact is that interference with the internal processes causes a change in behavior. ‘The mere fact of a change under these conditions tends in itself to be regulatory. The original behavior has brought on the interfering conditions, hence the Method of Regulation in Behavior and in Other Fields. 479 best thing to do is to change this behavior. If the unfavorable condition still continues the behavior is changed again; this being continued, the organism is bound to escape from the interfering condition if it is possible to do so. In some cases the move- ments produced are, when considered by themselves, of a rather uniform character, yet are of such a nature as to subject the animal - to many changes of the environmental conditions. ‘This is the case for example in the reactions of such infusoria as Paramoecium, where the character of the movement is determined partly by structure, yet involves a continued change of relation to the outer conditions. In other cases the movements themselves are varied in character, the organism first reacts in one way, then in another, running through a whole series of activities, till one results in ridding the organism of the stimulating condition. This is the method of behavior seen in Stentor and in most higher organisms. Our third question was: How does the organism select the more favorable condition thus reached? ‘This question now answers itself. It was interference with the physiological pro- cesses that caused the changes in behavior. As soon, therefore, as this interference ceases, there is no further cause for change. The organism selects and retains the favorable condition reached merely by ceasing to change its behavior when interference ceases. This process is seen clearly in the behavior of such infusoria as Paramcecium. It is perhaps fairly evident how reaction on the plan just described may result in the avoidance or rejection of sources of interfering stimuli; in other words, in the production of negative reactions. ‘The matter of positive reactions should perhaps receive further elucidation. In conditions that are completely favorable—so that all the life processes are taking place without lack or hindrance—there is no need, from the standpoint of regulation, for a change in behavior— for definite reactions of any sort ‘The most natural behavior on reaching such conditions, and that which is actually found as a rule among lower organisms, is a continuation of the activities already in progress. ‘These activities have resulted in the favor- able conditions, and there is no cause for a change. ‘This we find exemplified in infusoria, bacteria, rotifers, and many other organisms, under most classes of conditions. A change in behavior takes place only when the activities tend to remove the 480 FI. S. “fennings. organism from the favorable conditions; in other words, to pro- duce interference with the life processes. Unfavorable stimuli, in these organisms, cause a change in behavior; favorable stimuli cause none. It is perhaps a general rule in organisms, high or low, that continued completely favorable conditions do not lead to definite reactions. Of course while the external conditions remain the same, the internal processes may change in such a way that these conditions are no longer favorable, and now the behavior may change. ‘This frequently happens. When the organism is not completely enveloped by favorable conditions, but is on the boundary, if we may so express it, be- tween favorable and unfavorable ones, there is often a definite change in the behavior leading toward the favorable conditions— a positive reaction. ‘To understand such reactions, we may start from the fact, already mentioned, that unfavorable internal con- ditions (as well as external ones) cause a change of behavior. It is a general fact, for example, that the animal whose metabolic processes suffer interference from lack of material—the hungry animal—sets in operation trains of activity differing from the usual ones. Interference with respiration, or an increase in temperature above that favorable for the physiological processes, has similar effects. ‘This is indeed a general rule for all internal changes interfering with the usual physiological processes. But the activities thus induced are in themselves undirected, save by structural conditions. There is nothing in the cause producing them to direct them with reference to external things. Let us suppose, however, that certain of these movements lead to a condition which relieves the interference with the internal processes. ‘The cause for a change of behavior is now removed, hence the organism continues its present movement. But perhaps later—sometimes at the very next instant—this same movement may tend to remove the organism from the favorable condition— as when a Parameecium in a heated preparation passes across a small area of water cooled to the optimum, and reaches the oppo- site side, or when a hungry organism comes in contact with food, which will be lost if there is further movement. ‘Thereupon the cause for a change—interference with the life processes—is again set in operation, and the present movement is changed. ‘Thus the animal changes all behavior that leads away from the favorable condition, and continues that which tends to retain it, so that we Method of Regulation in Behavior and in Other Fields. 481 get what we call a positive reaction. The change of behavior is due in each case primarily to the unfavorable stimulation, internal or external. ‘This style of behavior is seen with diagrammatic clearness in the free swimming infusoria. “These animals con- tinue their movements as long as they lead to favorable conditions, changing at once such movements as lead away. ‘They thus retain favorable conditions by avoiding unfavorable ones; the positive reaction is seen to be, in a sense, a secondary result of negative ones. We have a similar condition of affairs in the taking of food by Ameeba. ‘The animal moves forward with broad front, and comes in contact at a certain point on this front with a food body. Part of its movement is taking it away from the food, part toward the food. On coming in contact, all movement which takes it away is changed, only that being continued which keeps the animal in contact with the food. We have here then the same condition of affairs as in the infusoria—the selection of certain conditions through the rejection of all others. This is perhaps the fundamental condition of affairs for organ- isms in general. In higher animals the positive, as well as the negative, reactions, have become complicated through the influences to be brought out later, so that this primitive con- dition is not evident. But the essential point is that unfavorable conditions are rejected as a result of the fact that they produce changes in behavior, and this results in the attainment and reten- tion of favorable conditions. In negative reactions it is the new unfavorable external condition that 1s rejected, retaining the old favorable internal condition. In positive reactions it is the old unfavorable internal conditions that are rejected, retaining the new favorable external conditions. In both cases the impulse to change of movement comes from interference with the physiological processes—external interference in negative reactions, internal interference in positive reactions. To sum up, in the lowest organisms we find individual adjust- ment or regulation on the basis of the three following facts: 1. Definite internal processes are occurring in organisms. 2. Interference with these processes causes a change of behavior and varied movements, subjecting the organism to many different conditions. 3. One of these conditions relieves the interference with the 482 Hi. 8. Fennings. internal processes, so that the changes in behavior cease, and the relieving condition is thus retained. It is clear that regulation taking place in this way does not require that the.end or purpose of the action shall function in any way as part of its cause, as some vitalistic theories hold. ‘There is no objective evidence that a final aim is guiding the organism. None of the factors above mentioned appear to include anything differing 1 in essential principle from such laws of causality as we find in the inorganic world. Now an additional factor enters the problem. By the process which we have just considered, the organism reaches in time a movement that brings relief from the interfering conditions. This relieving response becomes fixed through the operation of a certain law which appears to hold throughout organic activities. This law may be stated as follows: An action performed or a physiological state reached, is performed or reached more readily after one or more repetitions, so that in time it becomes “habitual.” The statement of this law just given is in reality not adequate, and it may be well to dwell upon it a moment, developing 1 it farther, and pointing out some of the phenomena in which it is expressed. In previous papers, including the one immediately preceding the present, I have pointed out the fact that the behavior and reac- tions of an organism depend largely on “physiological states;” the same point has recently been emphasized by Bohn (’05). We may distinguish at least two great classes of physiological states—those depending on the metabolic processes of the organ- ism, treated in detail in the preceding paper, and those otherwise determined. The physiological states of organisms change in accordance with certain laws. The changes in the metabolic states of course depend on the laws of metabolism. In the physio- logical states not directly dependent upon metabolism, but rather upon stimulation and upon the activity of the organism, such as are found in Stentor and Planaria (see Jennings, ’04), we find certain fairly well definéd laws of change that are of a peculiar character. In the organisms just mentioned, and in many others, the fol- lowing phenomena have been found. Under certain external conditions the organism reacts in a certain way. ‘These con- ditions continuing, the organism changes its first reaction for a second, and then perhaps for a fied and fourth. Later the same external conditions recur, and now the organism at once Method of Regulation in Behavior and in Other Fields. 483 responds, not by its first reaction, but by the final one. ‘This is illustrated for unicellular organisms by the case of Stentor, for higher metazoa by the behavior of certain crustacea, as described by Yerkes (’02) and Spaulding (04). There are certain differ- ences between the two cases, but they are not essential for our present purpose. How does this condition of affairs come about? As we have set forth in previous papers (’04), the different methods of reaction under the same external conditions must be due to different physiological states of the organism. ‘The “physiological state” is evidently to be looked upon as a dynamic condition, not as a Static one; It is a certain way in which the bodily processes are occurring; it tends directly to the production of some change. In this respect the “law of dynamogenesis,”’ propounded for ideas of movement in man, applies to it (see Baldwin, ’97, p. 167); ideas must indeed be considered, so far as their objective accom- paniments are concerned, as certain physiological states in higher organisms. ‘The changes toward which physiological states tend are of two kinds. First, the physiological state, like the idea, tends to produce movement. ‘This movement often results in such a change of condition as destroys the physiological state producing it. But in case it does not, then the second tendency of the physiological state shows itself. It tends to resolve itself into another and different state. State I passes to state 2, and this again to state 3. [his tendency shows itself even when the een conditions remain uniform. In this second tendency there manifests itself the important law of which we have spoken above. When a certain physiolog- ical state has been resolved, through the action of an external agent, or otherwise, into a second physiological state, this resolu- tion becomes easier, so that in the course of time it takes place more quickly, and even spontaneously. This may be illustrated from the behavior of the unicellular organism Stentor, as described in previous papers by the present writer (’02 and ’o4), as follows: When the animal is stimulated by the flood of carmin grains (or in any other way), this produces immediately a certain physiological state corresponding to that accompanying a sensation in ourselves. ‘This state we may designate A. It at first produces no reaction. As the carmin continues or is repeated, this state A passes to a second state 484. HA. 8. fennings. B, producing a bending to one side. After several repetitions of the stimulus, the state B passes to the state C, producing a reversal of the cilia, and this finally passes to D, resulting in a contraction of the body. Each state must of course be different from the preceding one, because it produces a different result. The course of the changes in physiological states may then be represented as follows: A—— B—— C—— D Now we find that after many repetitions of the stimulation the animal contracts at once as soon as the carmine comes in contact with it. In other words, the first condition A (direct result of contact) passes at once to the state D, and this results in imme- diate contraction: A—D It seems probable that the same series occurs as before, save that B and C are now passed rapidly and in a modified way, so that they do not result in a reaction, but are resolved directly into D. The process would then be represented as follows: A—— b/ — C’—— D But whatever the intermediate conditions, it is clear that after the state A has become resolved, through pressure of external conditions, into the state D, this resolution takes place more readily, occurring at once after state A is reached. The same law is illustrated in the experiments of Yerkes and Spaulding on association in crabs. In the experiments of Spauld- ing (704) with hermit crabs, the introduction of the dark screen into the aquarium, and the diffusion of the juices of the fish, cause the animals to move about. In so doing they reach the dark screen, which induces, let us say, the physiological condition A. ‘This leads to no special reaction. But this is followed regularly by contact with food, inducing the physiological state B, which is concomitant with a positive reaction. ‘The physiological state A is thus regularly resolved into the state B. In the course of time this resolution becomes automatic, so that as soon as the state A is reached, it passes to B. ‘The positive reaction concomitant with B is therefore given even though the original cause of B is absent. The actual number of physiological states which could be dis- Method of Regulation in Behavior and in Other Fields. 485 tinguished is of course greater than what we have set forth, but this does not alter the principle involved. The law which we have just brought out may then be summed up as follows: The resolution of one physiological state into another becomes easier and more rapid after it has taken place one or more times. Hence the behavior primarily characteristic for the second state comes to follow immediately upon the first. The operations of this law are seen on a vast scale in higher organisms, where they constitute what we commonly call memory, association, habit, and the basis of intelligence. It has been shown to hold in a number of lower organisms, though in these the mani- festations of this law are comparatively little known. Yerkes and Spaulding have demonstrated its applicability to Crustacea. ‘The low acelous flatworm Convoluta evidently shows it clearly, since as we have seen in the preceding paper, it forms definite habits. It has even been demonstrated, as we have seen, in the protozoa, particularly Stentor and Vorticella. According to Hodge and Aikins (’95) a method of reacting thus developed lasted in Vor- ticella as long as five hours. In view of these facts, it is probable that the law is a general one and that it will be demonstrated in some form for other lower organisms. There seems to be no theoretical reason for supposing it to be limited to higher animals. The paucity of experiments fitted to test it is amply sufficient to account for the very slight knowledge we have of it in lower organisms. To return then to the thread of our discussion: In virtue of this law of the readier resolution of physiological states after repetition, the final reaction of a trial series, relieving the organism of the interference with its physiological processes, 1s later reached more readily than at first, and in time becomes the immediate reaction to the interfering condition. ‘Thus the change of behavior induced by interference of a certain sort has come to be of a perfectly definite character, and all trial movements are omitted. It is inthis second stage of the process, when the relieving response has become set through the law above discussed, that an end or purpose seems to dominate the behavior. ‘This end or purpose of course actually exists, as a subjective state called an idea, in man. Whether any such subjective state exists in the 486 Hi. 8. Fennings. lower organism that has gone through the process just sketched we of course do not know. But some objective phenomenon, as a transient physiological state, corresponding to the objective physiological accompaniment of the idea in man, would seem to be required in the lower organism. ‘The behavior in this stage is that which, in its higher reaches at least, has been called intelligent. But so far as the objective occurrences are concerned there would seem to be nothing in even this later stage of behavior involving anything different in principle from what we find in the inorganic world. ‘The only additional feature is this law of the readier attainment of a certain state or action after repetition. We have not attempted to state this law in an entirely adequate manner, but there would seem to be nothing implied by it that is specifically vital, in the sense that it differs in essential principle from what we find in the laws of causality as applied to the inor- ganic world. It certainly by no means requires in itself the action of any “final cause’’—that is, of an entity that is at the same time purpose and cause. On the other hand, it undoubtedly does produce that type of behavior which has given rise to the con- ception of the purpose acting as cause. ‘This conception is in itself of course a correct one, so far as we mean by a purpose an actual physiological state of the organism, determining behavior in the same manner as other factors determine it. That regulation takes place in the behavior of many animals in the manner above sketched seems to the writer an established fact, and it appears to be perhaps the only clearly intelligible way in which regulatory behavior could be developed in a given individual. But we are of course confronted with the fact that many indi- viduals are provided at birth with definitely regulatory methods of reaction to certain stimuli. In these cases the animal is not compelled to go through the process of performing trial move- ments, with subsequent fixation of the successful movement. How are such cases to be accounted for? If the regulatory methods of reaction acquired through the process sketched in the preceding paragraphs could be inherited, there would of course be no difficulty in accounting for such con- genital regulatory reactions, or habits. It is perhaps not going too far to say that this possibility i is not yet out of court, though Method of Regulation in Behavior and in Other Fields. 487 opinion at present seems to be generally against it. Yet Semon (04) in his recent valuable monograph on the phenomena allied to memory and habit, maintains the afhrmative view, and pre- sents evidence in favor of it. We are in the beginning of the study of such problems, and it can hardly be said chit experiments of sufhcient duration and precision have yet been tried to really test the matter. If the inheritance of regulatory reactions acquired after trial should be demonstrated, the process sketched above would give us a satisfactory general method for the develop- ment of regulatory behavior, in the race as well as in the individual. In the protozoa this difhculty of course does not exist; the acquire- ments of individuals may remain as acquirements of the race. If such inheritance does not occur, then the existence of con- genital definite regulatory reactions would seem to be explicable only on the basis of the selection of individuals having varying methods of reaction, unless we are to adopt the theories of vitalism. In the method we have sketched above, a certain reaction that is regulatory is selected, through the operation of physiological laws, from among many performed by the same individual. In what is called natural selection, the same reaction is selected from among many performed by dzfferent individuals—in both cases because it is regulatory—because it assists the physiological processes of the organism. ‘The two factors must work in the same direction. “Intelligence” and natural selection are two analogous methods of selecting the adaptive reactions from among the possible ones; they must then work together, as Baldwin (’02) has so well pointed out. Which of the two factors is the essential one in producing congenital adaptive reactions we shall of course not attempt to decide, since no one knows. We often find in organisms behavior that is not regulatory, or that is regulatory only in a very imperfect way. How are we to account for this? Without going into details, it is evident that there exist at least three general conditions that may result in non-regulatory behavior. First, the organism is formed of sub- stance that is subject to the ordinary laws of physics and chem- istry. Various physical and chemical- agents may act directly upon this substance, producing results that are not regulatory. The fact that the relation of external processes to internal ones is one of the chief determining factors in producing reactions, of 488 A. S. fFennings. course does not exclude the possibility of the direct action of agents on the body substance. ‘The operation of intense physical and chemical agents may injure or destroy the substance of which the organism is composed, and with it the organism, in spite of any reaction the organism can give. Second, the organism can perform only those movements which its structure permits. Often none of these movements can relieve the existing inter- ference with the physiological processes. ‘Then the organism can only try them, without regulatory results, and die. Examples of this are seen in the behavior of Paramcecium, or of Planaria when placed in heated water. Both animals perform practically all the reactions of which they are capable, before they succumb. Third, certain responses may become fixed, in the way sketched above, because under usual conditions they relieve the organism. Now if the conditions change, the organism can respond at first only by this fixed reaction, and if this does not relieve, the animal may be® destroyed before a new regulatory reaction can be developed. ‘This condition of affairs is widespread among animals. All together, the regulatory character of behavior as found in many animals seems perhaps intelligible in a perfectly natural, directly causal way, on the basis of the principles brought out above. We may summarize these principles as (1) the selection by varied movements of conditions not interfering with the physio- logical processes of the organism; (2) the fixation of the move- ments by which the selected conditions were reached, by the law of the readier resolution of physiological states after repetition. Neither of these principles seem to contain anything specifically vitalistic, or opposed in principle to what we find in the inorganic world Is it possible that regulation is based on similar principles in other fields than behavior? Bodily movement is only one of the many kinds of activity that may vary, and variations of any of the organic activities may impede or assist the physiological processes of the organism. Is it possible that interference with the physio- logical processes may induce changes in other activities—in chemical processes, in growth, and the like—and that one of these activities is selected, as in behavior, through the fact that it relieves the interference that caused the changes? There is some evidence for this possibility. Let us look for Method of Regulation in Behavior and in Other Fields. 489 example at regulative changes in the chemical activity of the organism, such as we see in acclimatization to poisons, in responses to changes in temperature, or in the adaptation of the digestive juices to the food. What is the material from which the regulative changes may be selected? One of the general results of modern physical chemistry is expressed by Ostwald (02, p. 366) as follows: “In a given chemical structure all processes that are possible, are really taking place, and they lead to the for- mation of all substances that can occur at all.’’ Some of these processes are taking place so slowly that they escape usual obser- vation; we notice only those that are conspicuous. But in its enzyms the body possesses the means of hastening any of these processes and delaying others, so that the general character of the action shall be determined by the more rapid process. Such enzyms are usually present in the body in inactive forms (zymo- gens), which may be transformed into active enzyms by slight chemical changes, thus altering fundamentally the course of the chemical processes in the organism. It is evident that the organism has presented to it, by the con- dition just sketched, unlimited possibilities for the selection of different chemical processes. ‘The body is a great mass of the most varied chemicals, and in this mass thousands of chemical processes in every direction—all those indeed that are possible— are occurring at all times. ‘There is then no difficulty as to the sufficiency of the material presented for selection, if some means may be found for selecting it. The process which will relieve any unfavorable condition, if any such process is possible, is actually occurring in an infinitesimal way, and needs only to be hastened. Further, it is known that interference with the physiological processes does result in many changes in the internal activities of the organism, as well as in its external movements. Intense injurious stimulation causes not only “excess”? movements of the body as a whole, but induces marked changes in circulation, in respiration, in temperature, in digestive processes, in excretion, and in other ways. Such marked internal changes involve, and indeed are constituted by, alterations of profound character in the chemical processes of the organism. ‘These chemical changes are sometimes demonstrated by the production of new chemical substances under such circumstances. Furthermore, it is clear that the internal changes due to interference with the 490 Hi. S. Fennings. physiological processes are not stereotyped in character, but varied. Under violent injurious stimulation respiration may become for a time rapid, then is almost suspended. ‘The heart for a time beats furiously, then feebly, and there is similar varia- tion in other internal symptoms. Thus it seems clear that interference with the life processes does induce varied activities in other ways than in bodily move- ments, and that among these are varied chemical processes. There is then presented. opportunity for regulation to occur in the same way as in behavior. Certain of the processes occurring relieve the disturbance of the physiological functions. ‘There results then a cessation of the changes. In other words, a certain process or condition is selected through the fact that it does relieve. ‘There is much evidence that the law of the readier resolution of physiological states after repetition applies to other bodily pro- cesses as well as to behavior. “The much readier induction of digestive trouble by a small quantity of a certain food, after a large quantity has once induced it is perhaps an example; many better ones are given by Semon (’05). If the analogy with behavior holds in this respect, there will be present at a later period certain fixed methods of chemical response, by which the organism reacts to certain sorts of stimulation—as by the pro- duction of a definite antitoxin when a certain poison is introduced. Definite organs or organisms will have left open to them only certain limited possibilities of variation—due to the development of something corresponding to the “action system” in behavior. Thus, in the pancreas there will not exist unlimited possibilities as to the chemical changes that may easily occur. Its “action system’ will be limited perhaps to the production of varied quantities of certain enzyms—amylopsin, trypsin, etc. ‘The proper selection of these few possibilities will then occur by the general method sketched. When digestion is disturbed by food that is not well digested, variations in the production of the different enzyms will be set in train, and one of these will in time relieve the difhculty, through the more complete digestion of the food. ‘Thereupon the variations will cease, since their cause has disappeared. By still more complete fixation of the chemical response, through the law of the readier resolution of physiological states after repetition, an organ or organism may largely lose its power of varying its chemical behavior, and thus be unable to Method of Regulation in Behavior and in Other Fields. 491 meet new conditions in a regulative way. A condition compar- able to the establishment of a fixed reflex in behavior will result. It is perhaps more difficult to apply the method of regulation above set forth to processes of growth and regeneration. Yet there is no logical difficulty in the way. ‘The only question would be that of fact—whether the varied growth processes necessary do primitively occur, under conditions that interfere with the physiolog- ical processes. When a wound is made or an organ removed, is the growth process which follows always of a certain stereotyped character, or are there variations? It is, of course, well known that the latter is often the case. In the regeneration of the earth- worm, Morgan (’97) finds great variation; he says that in trying many experiments, one finds that what ninety-nine worms cannot do in the way of regeneration, the one hundredth can. ‘The very great variations in the results of operations on eggs and young stages of animals is well known. Removal of an organ is known to produce great disturbance of most of the processes fi the organism, and among others, in the process of growth. It appears then not impossible that in growth processes regula- tion may be brought about in accordance with the same principles as in behavior. A disturbance of the physiological processes results in varied growth activities. Some of these relieve the disturbance; the variations then cease, and these processes are continued. In any given highly organized animal or plant the different possibilities of growth will have become practically much limited, and it is only from this limited number of possibilities that selections can be made. In some cases, by the fixation of certain processes through the analogue of the law of the readier resolution of physiological states, the organism or a certain part thereof will have lost the power of responding to injury save in one definite way. Under new conditions this one way may not be regulatory, yet it may be the only response possible. ‘This may result in the formation under certain conditions of such things as heteromorphic structures—a tail in place of a head, or the like, from a part of the body that is accustomed (in normal develop- ment) to produce such an organ. ‘This would again correspond to the production of a fixed reflex action in behavior, even under circumstances where this action is not regulatory. It appears to the writer that the method of form regulation recently developed in a most suggestive paper by Holmes (’04) 492 HI. S. Fennings. is in essential agreement with the general method of regulation here set forth, and may be considered a working out of the details of the way in which growth regulation would probably take place along such lines. It may be noted that regulation in the manner we have set forth is what, in the behavior of higher organisms, at least, is called intelligence. If the same method of regulation is found in other fields, there is no reason for refusing to compare the action to intelligence. Comparison of the regulatory processes that are shown in internal physiological changes and in regeneration to intelligence seems to be looked upon sometimes as heretical and unscientific. Yet intelligence is a name applied to processes that actually exist in the regulation of movements, and there 1 iS, a priori, no reason why similar processes should not occur in regulation in other fields. When we analyze regulation objec- tively, there seems indeed reason to think that the processes are of the same character in behavior as elsewhere. If the term intelligence be reserved for the subjective accompaniments of such regulation, then of course we have no direct knowledge of its existence in any of the fields of regulation outside of the self, and in the self perhaps only in behavior. But in a purely objective consideration there seems no reason to suppose that regulation in behavior (intelligence) is of a fundamentally different character from regulation elsewhere. It is perhaps hardly necessary to point out the relation of the method of regulation in behavior here discussed to the process of “selection of overproduced movements,” so ably set forth in Baldwin’s well-known works (’97, 02). The account here given is based on this same process, but differs in a number of points which seem to the writer of fundamental significance for a proper understanding of the method of regulation. Baldwin has like- wise made some suggestion as to the possibility of extending this point of view to other fields (Baldwin ’o2). We may make a general statement of the features in the method of regulation set forth in this paper, as follows: The organism 1s primarily activity. It is the seat of many processes, of chemical change, movement, and growth; these are proceeding with a certain amount of energy. ‘These processes depend for their unimpeded course on one another and on the relations to the environment which the processes themselves largely bring about. Method of Regulation in Behavior and in Other Fields. 493 When any of the processes are blocked the energy overflows in other directions, producing varied changes in chemical processes, movement and growth. Some of the conditions reached through these changes relieve the interference that was the cause of the changes. Thereupon the changes cease, since their cause has disappeared; the relieving condition is therefore maintained. After repetition of this course of events, the change which leads to relief is reached more directly, as a result of the law of the readier resolution of physiological states after repetition. ‘Thus are pro- duced finally the stereotyped and under certain conditions non- regulatory changes sometimes resulting from stimulation. This method of regulation is clearly seen in behavior, where its operation is, in the later stages, what is called intelligence. Its application to chemical and form regulation is at present hypo- thetical, but appears probable. LITERATURE CITED. Batpwin, J. Mark, ’97.—Mental Development in the Child and in the Race. Methods and Processes. Second ed., 496 pages. New York. ’02.—Development and Evolution. 395 pages. New York. Boun, G., ’05.—Attractions et Oscillations des animaux marins sous |influence de la lumiére. Institut Général Psychologique, Mémoires, i, 110 pages. ENGELMANN, TH. W., ’82.—Bacterium photometricum. Ein Beitrag zur vergleichenden Physiologie des Licht- und Farbensinnes. Arch. f. d. ges. Physiol., Bd. xxx, S. 95-124. Hopcg, C. F., anp Atkins, H. A., ’95.—The Daily Life of a Protozoan: a Study in Comparative Psycho-physiology. Amer. Journ. Psychol., vol. vi, pp. 524-533. Hormes, S. J., ’04.—The Problem of Form Regulation. Arch. f. Entw.-mech., Bd. xviii, S. 265-305. Jennincs, H. S., ’02.—Studies on Reactions to Stimuli in Unicellular Organisms. IX. On the Behavior of Fixed Infusoria (Stentor and Vorticella), with Special Reference to the Modifiability of Protozoan Reactions. Amer. Journ. Physiol., vol. vii, pp. 23-60. ’04.—Physiological States as Determining Factors in the Behavior of Lower Organisms. Contributions to the Study of the Behavior of the Lower Organisms, fifth paper, pp. 109-127. Carnegie Institution of Washington, Publ. 16. 494 H. S. fennings. Morean, T. H., ’97.—Regeneration in Allolobophora fcetida. Arch. f. Entw.- mech., Bd. v, S. 570-586. Ostwa.p, W., ’02.—Vorlesungen iiber Naturphilosophie. 457 pages. Leipzig. Semon, R., ’04.—Die Mneme als erhaltendes Prinzip im Wechsel des organischen Geschehens. 353 pages. Leipzig. Spau.pinc, E. G., ’04.—An Establishment of Association in Hermit Crabs, Eupagurus longicarpus. Journ. Comp. Neurol. and Psychol., vol. xiv, pp. 49-61. YERKES, R. M., ’02.—Habit Formation in the Green Crab, Carcinus granulatus. Biol. Bull., vol. ii, pp. 241-244. HPOUARITY~ CONSIDERED AS A°SPHENOMENON OF GRADATION OF MATERIALS. BY T. H. MORGAN. In my paper’ entitled “An Attempt to Analyze the Phenomena of ‘Polarity’ in Tubularia” I offered an interpretation of certain experiments carried out by Stevens and myself.? In another paper,’ “An Analysis of the Phenomena of Organic Polarity,” written at about this time the same interpretation was applied to the general question of polarity. ‘hese attempts to analyze the problem suggested a number of new experiments in which the conclusions could be tested further, and I shall give in the following pages the results of some new work on ‘baler. I may recall my hypotheses that the phenomenon of polarity in Tubularia depends on the graded distribution of materials from the hydranth to the stolon. ‘This gradation is the basis in which the formative action takes place and produces the new hydranth. The stimulus calling forth the reaction is the presence of a free end. From the previous experiments and from those to be described here the following conclusions may be drawn: (1) That a gradation of hydranth-forming substances 1s present in the stem of Tubularia, and that the amount present at any level determines the rate at which both the oral and the basal hydranth develop. (2) That in addition to the quantitative factor the direction of the gradation or the polarity 1s a qualitative factor in the result. Journ. Exp. Zodl., i, 1905. ?Journ. Exp. Zodl., i, 1905. 3Science, xx, Dec., 1904. ‘Following Driesch I have sometimes stated that the stimulus comes from the action of the sea water on the free end, but it is difficult to distinguish between the action of the sea water and the simple exposure of the free end (an internal factor). Since the normal regeneration of Tubularia takes place in sea water we can not distinguish between these two possibilities. If regeneration could be made to take place in moist air the sea water, as a factor, would be eliminated. 496 T. H. Morgan. (3) That it is probable that the materials of the oral end set free in the circulation may influence, under certain conditions, the rate of regeneration of the aboral hydranth. ‘The experiments that bear on these questions may now be given under their respec- tive heads. EXPERIMENTS. The Rate of Development at Different Levels. Long pieces of unbranched stems were used.1_ In one set the cut was made just below the hydranth; in the second set about the middle of the length of the stem; and in the third set near the base (leaving the piece still so long that the primordium of the hydranth would not be shortened). ‘The long pieces produced their oral hydranths first; those cut through the middle next; and the short pieces last. Driesch obtained similar results. At the same time experiments were made to determine the rate of development of aboral hydranths at different levels. Long stems were again selected and all tied near the oral end. ‘The aboral ends were then cut off at different levels. “Chose in which the aboral end was near the ligature developed first; those whose aboral ends were near the middle of the piece developed exes and those whose aboral ends were near the base of the piece developed last. ‘The results show that the rate of both oral and aboral development 1s determined by the level at which the end lies. The most probable interpretation of these facts is that the amount of hydranth-forming substances decreases from the free end to the base, and that their amount determines the rate of regeneration. Influence of the Direction of the Gradation of the H ydranth- Forming Materials. If a stem is tied at its two ends and then cut in two in its middle the cut ends will be at exactly the same level and the neighboring parts are nearly alike. ‘The anterior piece has, in fact, the advan- tage in the amount of hydranth-forming material near the cut end, although its gradation is in the reverse direction from that at the oral end of the posterior piece. If the direction of the grada- 1In all cases where comparisons are made the pieces came from the same colony. “Polarity.” 497 tion is a factor in the rate of development the posterior piece might develop a hydranth at its anterior end before the anterior piece makes a hydranth at its posterior end, despite the slight advantage of the material out of which the aboral hydranth develops. ‘This is, in fact, what occurs, although the difference in rate is not very great and might be overlooked were not a close watch kept on the pieces. Halves of the same piece must of course always be compared rather than different pieces. An experiment of this kind gave the following results: Long pieces were tied at the oral and basal ends and were then cut in two in the middle. After twenty-four hours five primordia were present at the oral cut ends of the posterior pieces, and none at the basal cut ends of the anterior pieces. Six hours later the former had primordia in five pieces, the latter in three (but less advanced). After forty-eight hours one of the posterior pieces . had produced an oral hydranth, and eight hours later four hydranths were out on these pieces but no aboral hydranths as yet on the anterior pieces. “These results show that the hydranth at the oral end of a piece (closed at the basal end) develops a little sooner than does the hydranth at the basal end of a piece (closed at its oral end). ‘This difference can be safely attributed, I think, to the difference in the direction of the gradation of the material near the two cut ends. The results confirm an experiment of King.? The Probable Influence of the Materials in the Circulating Fluids on the Rate of Development. It has been shown by Driesch, Morgan, and Loeb that by closing the oral end of a piece by a ligature the development of the aboral hydranth is greatly hastened; so much so, in fact, that it develops nearly as soon as an oral hydranth at the same level, as described in the last section. What factors cause this accelera- tion? It is clear that the suppression of the oral hydranth is in some way connected with the result, and it seems not unreason- able to suppose that when the oral hydranth develops it draws on the food supply and thus holds in check the aboral hydranth. The problem is, however, complicated in several ways. In the 1Biol. Bull., vi, 1904. 498 7 ae Morgan. first place the ligature does not in some cases entirely prevent the beginning of the development of the oral end and occasionally I have seen, as Stevens and I have previously observed, that the primordium of the oral hydranth may be laid down. Further- more, if the material in the circulation is derived mainly from the broken-down ridges, etc., of the oral end it is not clear why a similar breaking down might not also occur at the aboral end and in this way by doubling the total amount present make possible the simultaneous development of both hydranths. Other diffi- culties are also present that may be spoken of later. In the hope of gaining some further insight into these questions the following experiments were carried out: The hydranths were removed from a number of pieces, the oral ends of some of these pieces were tied at once (A); others were tied at the end of six (B); or of twelve hours (C). Now during the first six to twelve hours after cutting, the endodermal ridges of the oral ends (in the pieces not yet tied) begin to break down and their material is thrown into the circulation. If the presence of this material in the circulation has any influence on the rate of development of a hydranth (oral or aboral) it might accelerate the aboral development if the oral end is now tied. A number of experiments of this sort show that the aboral develop- ment often takes place sooner in pieces whose oral end 1s tied after six hours or often even after twelve hours than in check pieces tied at once. Unfortunately the material began to “go bad” before a sufficient number of results could be obtained to place the con- clusion entirely beyond doubt, but there seemed to be evidence in all cases of some acceleration in the development of the aboral hydranth in pieces tied later than in those tied at once, and in most cases the acceleration was so great that the former developed even before the latter. Some of the more satisfactory experiments may now be described. In the first experiment some pieces were tied at once (A); others after six hours (B). Forty-seven hours later the A-pieces showed nothing, while four of the seven B-pieces had produced primordia. After seventy-two hours two of the A-pieces had primordia, while four of the B-pieces had primordia, one a hydranth, and two nothing. In another series, in which the B-pieces were tied after fourteen hours, the aboral hydranths of the A-series developed first. “The “Polarity.” 499 start of fourteen hours was so great that even the acceleration of the B-pieces did not suffice to make them develop first, and this would hardly be expected since the whole development often occurred in less than forty-eight hours, but the B-pieces were not fourteen hours behind the A-pieces. In another series tied at once (A); after six hours (B), and after eighteen hours (C), it was found after forty-eight hours that five of the A-pieces barely showed primordia and five others nothing; that four of the B-pieces showed primordia further advanced than the primordia of the A-pieces, and six nothing; that one of the C-pieces showed the barest beginning of a primor- dium and nine nothing (in poor condition). After seventy-two hours all of the A-pieces had primordia; four of the B-pieces had hydranths and the remaining six primordia; all ten of the C- pieces had young primordia. In an experiment of this kind different pieces are necessarily compared, and, since no two pieces can be assumed to be cut at exactly equivalent levels, it is, perhaps, unsafe to draw conclu- sions from so small a number of observations. If, as seems to be the case, pieces tied after six hours develop as soon as, or sooner than, those tied at once (1. ¢., six hours earlier), the result is probably due to the presence of material in the circulation derived from the oral end before tying. It may be asked, why may not the reserve material of the wall throughout the piece be used rather than that thrown into the circulation by the breaking down of the ridges? If this were the case the total amount of material would be much more than neces- sary to supply both ends of a long piece at once, as shown by the fact that a long piece cut into shorter ones will produce as many oral hydranths as there are pieces, in the same time that long pieces cut at equivalent levels produce oral hydranths. ‘Therefore if an appeal is made to the amount of food material to explain the acceleration of the aboral hydranth, it must be the material of the circulation postulated rather than that of the wall. In still another way I have tried to find out what part, if any, of the materials of the circulation influence the rate of develop- ment. ‘The hydranths were cut off from a number of pieces and then after six hours (or more) ligatures were tied around the pieces at different levels, in some pieces (A) near the oral ends; in others (B) near the middle; and in others (C) quite near the basal end. 500 T. H. Morgan. If the rate of development of the aboral hydranths depends, to any extent, on the amount of fluid in the circulation, the A-pieces should develop first, then the B-pieces, and lastly the C-pieces; for the amount of gastro-vascular fluid, with its contained material shut off in the basal end is greater in (A) than in (B), and greater in (B) than in (C). ‘The results show that in most cases the order is that just given. In the first set tied after six hours, the rate in (A), (B) and (C) seemed to be about the same. In the second set, tied after twelve hours, the (A) and (B) appeared at nearly the same time, but the C-pieces were distinctly delayed. In the third set, tied after six hours, the A-pieces developed ahead of the (C’s). ‘There were no B-pieces. In the fourth set, tied after six hours, the (A’s) developed before the (B’s), and the latter sooner than the (C’s). (The difference between the (B’s) and the (C’s) was not marked.) In the fifth set, tied after six hours, the (A’s) averaged better than the (B’s), and the (B’s) better than the (C’s), although the difference was more apparent at first than later. In the sixth set, tied after six hours, the (A’s) began to develop before the (C’s). There were no B-pieces. ‘The same difference could be seen throughout the later development. The results from these experiments all point in the same direc- tion. ‘The nearer the ligature to the basal end, after the oral end had been allowed to develop for six hours, the later the develop- ment of the aboral hydranth. Nevertheless without further experiments I feel it unsafe to rely too much on these data, because here also different pieces have to be compared, but if the results are established by further work they indicate that the materials of the circulation are a factor in the rate of aboral development. It should be clearly understood that whether this is true or not the general theory of polarity here proposed 1s little affected, for the question of the rate of aboral development in a piece tied at the oral end has only an indirect bearing on the prob- lem of polarity. Only the rate is affected. ‘The heteromorphosis is due to the totipotence of the stem and the stimulus of the free end. “Polarity.” 501 GENERAL DISCUSSION OF RESULTS. From the data furnished by these and by previous experiments we may, I think, formulate a statement in regard to the phenomena of polarity in Tubularia. Several factors enter into the result: (1) The material of the stem is totipotent, and may produce a hydranth at any level, but more quickly at an oral end of a piece than at an aboral end. ‘The quicker response at the oral end is due, on my view, to the gradation of the material in the direction of more to less. (2) Ihe gradation 1s the polarity, and on this as a basis the formative changes take place. Whether these formative changes involve only known physical elements need not be discussed here, but whatever the kind of process the gradation gives the basis for its directive action—the presence of a free end calling forth the for- mative changes. (3) The development of the aboral hydranth may appear, on first thought, to contradict this idea of polarity. In reality it does not do so, for the gradation is only one of a number of factors that may possibly determine the result. A stronger influence of another sort may call forth, in the totipotent material, the hydranth- forming action. In fact, all the phenomena of axial heteromor- phosis show that the polarity may be overcome; sometimes one condition, sometimes another causing this result. “Thus when the totipotence is lost and the material can only produce one kind of structure, if it produces anything, as in the tail of the tadpole, the polar influence—the gradation of the material—is overcome in heteromorphic regeneration from the anterior end and here a tail and not a head develops. That even in Tubularia there is a conflict between opposing factors when an aboral hydranth forms (in a piece tied at the oral end) is shown by the delay in the development compared with the oral development at the same level of the basal piece. LOCALIZATION IN EGG AND ADULT AND ITS BEARING ON DEVELOP- MENT AND REGENERATION. A number of recent results in experimental embryology indicate that the protoplasm of the egg 1s composed of a number of mate- rials—quantitatively or qualitatively different—that go to dif- ferent parts of the embryo, and become later the basis of the parts 502 T. H. Morgan. of the body. ‘That the early development depends on the proto- plasm, and not on the nucleus, as previous theories had assumed, was first demonstrated by Driesch and myself by means of an experiment on the unsegmented ctenophore- egg, from which a part of the protoplasm was removed but the entire nucleus left. Defects appeared in the embryo. Later observers have con- firmed the conclusions that we drew from our experiment and have greatly extended the results. ‘The observations of Wilson and of Conklin have been especially interesting in showing that extensive processes of protoplasmic migration may occur. Fur- thermore the results of Wilson and of Yatsu have shown that the localization of the materials takes place only after the germinal nucleus of the egg breaks down. Even in such eggs as the sea urchin there is evidence of a similar localization.t. Although in this egg and in some other eggs the totipotence of the different regions often so overbalances the difference of the parts that isolated portions of the egg do not show strikingly the evidence of the specification of their materials. Since the different regions of the adult animal are formed out of the different materials of the egg, which must be assumed to increase enormously in volume as the animal grows larger, we must sup- pose that these different materials furnish the basis for the regen- eration of the same organs. In many animals the gradation in the amount of each kind of substance may be very gradual and extend throughout almost the entire length of the body, e. g., in Lumbni- culus, as shown in that a head or a tail may regenerate from any level. If on the other hand sharp regional differences exist, such as that between a leg and the body of an animal, we may expect to find a corresponding limitation in the regenerative capacity, so that the leg is no longer capable of making a body, etc. Even where the material is proliferated at the cut surface to make the new part, as happens in many cases of regeneration, the gradation of the material of the old part still maintains— that first produced coming from the more distal end and that produced later coming from further in, from the more proximal parts—but the formative action must be supposed to take place not only under the influence of the new material, but of the neighboring parts as well. IDriesch (’00); Boveri ("or); Morgan (’o1). “Polarity.” 503 It must be assumed, of course, that while some materials grade off from head to tail others grade off from tail to head, etc. “Thus in Lumbriculus, the head-end material grades from the anterior end backward, while the tail-forming materials decrease from behind forward. ‘There must often be regions of considerable overlapping of these materials as shown again in Lumbriculus and Tubularia and less so in Lumbricus. Furthermore certain regions may consist so largely, or even exclusively, of certain kinds of substances that despite the postulated polar gradation these regions are capable of Tegenerating only one anal of structure. Thus as I have shown in the posterior regions of the earthworm, and in the tail of the tadpole, only one kind of structure, e. g., tail, can develop. I have tried also to show that the heteromorphosis of very short cross-pieces of Planarians finds its best explanation on the assumption that by the partial removal of the polar influences in such short pieces, this influence no longer dominates the develop- ment, and the centripetal influences determine that a new head will develop—the head-forming substances being in excess. In other Planarians the tail-forming substances seem to be dominant in certain parts and a heteromorphic tail develops at the anterior end when the polar influence is lessened. The most striking example of the influence of the gradation of the material on the formative action is shown by lateral pieces of Planarians. If cross-pieces are first cut from the anterior, middle, and posterior regions of a Planarian, and if the sides are then cut from these pieces, so that none of the median organs are left, it will be found that the position of the pharynx in the new worms that regenerate will depend on the region of the original worm from which the pieces were cut. In the new worms from lateral pieces from the anterior part of the original worm the pharynx lies near the posterior end, in the worms from the middle pieces the new pharynx lies in the middle of the length; and in the worms from posterior pieces the new pharynx lies nearer the anterior end of the new worm. ‘Thus, although in all these pieces having the same shape (and open at both ends) the possibility would seem to exist for the pharynx to lie in the same place in the new worm, in reality its position is different, and appears to be determined by the gradation of the material. ‘hus in the anterior pieces there is more of the pharynx material in the part of the piece nearer to the old pharynx, 7. ¢., at the posterior end. 504. T. H. Morgan. In the middle pieces its amount is greatest at the middle. In the posterior pieces the amount is greatest nearer the anterior end. These relations throw more light on the problem of localiza- tion in regeneration than any others that I know of, and the con- clusions to be drawn from them are so obvious that they can scarcely fail to carry conviction. ‘The reader may probably have observed that in the preceding attempt to account for the phenomena of polarity I have referred to the protoplasm in the sense of cytoplasm rather than nucleus. Yet on the current view of embryologists every nucleus contains the sum total of all the hereditary qualities and may transfer, in some unknown way, these qualities to the protoplasm. Every part, therefore, is looked upon as potentially totipotent, and its only limitations are those due to its protoplasmic differentiation. Even this is supposed to be capable of being worked over under the influence of the nucleus so that it may at times return to its “embryonic condition” of indifference and may then under the influence of the nucleus again be differentiated in new ways. If this belief represents the actual conditions in the tissues then the remarkable limitations of regenerative power in some instances can only be explained by assuming that the protoplasm when once differentiated can in these cases no longer return to the so-called “embryonic condition.” It is not apparent, if this be the case, why nuclei should always be present in somatic cells unless they have some other important function to perform than that of transmitters of hereditary qualities. ‘There is, of course, much evidence to show that the nuclei have important physiological functions to perform, v7z., in connection with the metabolism of the cell. “This admission at once raises the question as to whether the main function of the nucleus may not be connected with metabolism and have nothing to do with hereditary transmission, unless indirectly. If we inquire on what evidence the accepted view rests that the nucleus is the transmitter of the hereditary qualities we shall find the evidence not entirely conclusive. ‘The principal argument in favor of this doctrine is that the spermatozoon brings into the egg only, or mainly, the nucleus of the male germ-cell, and thus the paternal qualities must become transmitted to the offspring by means of the nucleus. It is pertinent to ask what becomes of the cytoplasm of the male germ-cell? Is the nucleus simply “Polarity.” 505 ejected from the cell, leaving the cytoplasm behind? Assuredly not! A small part of the cytoplasm goes into the tail of the sper- matozoon, and since, in some cases, the tail is said not to enter the egg that part of the cytoplasm is lost. ‘The rest of the cytoplaam— by far the largest amount in many cases'—concentrates around the nucleus, enters the egg with it, and, no doubt, mingles with the cytoplasm of the egg. It is a matter of common observation that the chromatin of the nucleus must also greatly contract to be stowed away in the minute sperm-head. If we compare the size of the nucleus of the sperm mother-cell with the size of the head of the spermatozo6n the enormous difference in volume between the two becomes apparent. It is true that most of the volume of the nucleus consists of nuclear sap rather than chromatin, but there can be no doubt that the chromatin itself may expand and shrink within very wide limits. Have we not laid too much em- phasis on the nucleus of the sperm-head because we can trace its history with great clearness, and have we not ignored the cytoplasm that is carried in, because becoming at once commingled with that of the egg it is lost to sight? May it not be true that the paternal cytoplasm becomes incorporated in the fertilized egg as a part of the cytoplasm and as it increases in volume comes to play its part in the differentiation of the cell. “The accumulation of cytoplasm around the male-nucleus? which accompanies the latter as it moves toward the female nucleus, its division and its distribution to the daughter-cells suggests how the mechanism of transmission may be accomplished. From this point of view the protoplasm and not the nucleus might transmit the hereditary qualities of the male as well as of the female. “The nucleus would be concerned with the metabolism of the cell. “The more difh- cult question still remains, of course, as to how far the metabolic influence of the nucleus might influence the cytoplasm and affect its hereditary properties, but a discussion of this possibility in the absence of data would be too speculative to be profitable. In conclusion we find that the localization in the cytoplasm of the egg is directly comparable to the localization of the materials of the adult animal. ‘The “polarity” in both cases is an expression of the gradation in the material. “The phenomena of regeneration Its more watery parts are thrown off. *Generally supposed to come mainly from the egg, but which may also in part come from the sperm. 506 6. Vek Morgan. are, in part, the outcome of this gradation; in part also of the kind of substances in a given region and also of a formative action using the preceding condita as a basis, as well as taking into account the amount of material present. ‘The formative influence acting in a centripetal direction always gives precedence, as it were, to the terminal organs. In regard to the specification of the cytoplasm, as the basis of regeneration, versus the assumed toti- potence of the nuclei, we have at present a choice of three views, no one of which can be said to be satisfactorily established: (1) The cytoplasm alone furnishes the basis for the action of the formative changes without regard to whether the nuclei are storehouses of hereditary qualities or whether they have to do only with the feeding (and respiration?) of the cell—not directly with its growth and specification. (2) The nuclei are reserve storehouses of all of the hereditary elements and may be called upon to supply whatever is needed for the formation of the new part, the cytoplasm returning to an “embryonic condition” to be worked over under nuclear control. ‘This view is the logical outcome, it seems to me, of the current view in regard to the relation of nucleus and cytoplasm. I have tried to show by a brief examination of the evidence on which this view rests that it is not established beyond doubt. (3) Both nuclei and cytoplasm may be progressively specialized, hence it may not be profitable to make any distinction between the part they play in regeneration. The gradation of the material—the polarity—is, on this view, expressed as much by one as by the other, and by both alike. STUDIES ON CHROMOSOMES. II. THE PAIRED MICROCHROMOSOMES, IDIOCHRO- MOSOMES AND HETEROTROPIC CHRO- MOSOMES IN HEMIPTERA.' EDMUND B. WILSON. With 4 Ficures. In investigating the physiological significance of the chro- mosomes and their individual values in heredity, it is important to determine as accurately as possible how far they are differen- tiated in respect to individual behavior, and to ascertain by the comparative study of different forms to what extent the chro- mosomes can be grouped in well-defined classes. “The work of Henking, Paulmier, Montgomery, Gross and Stevens on the Hemip- tera has shown that this group is peculiarly favorable for such a study; and I believe from my own observation that no group of animals has thus far been examined that offers greater advan- tages in this direction.? But although the general results obtained by the above-mentioned observers are of great value and interest they nevertheless show many discordances of detail that stand in the way of a consistent general interpretation of the phenomena, while some of Gross’s conclusions are a stumbling block in the way of the whole theory of the individuality of the chromosomes. For this reason I propose in this paper to record a series of obser- vations that I hope may serve to clear away some of the con- fusion that now exists in the accounts of the subject, and that open the way, I believe, to a true interpretation of the “accessory chromosome” and its relation to the determination of sex. In a series of suggestive papers (oI, ‘04, 05) Montgomery ‘Attention is called to the Appendix in which are briefly recorded facts, determined by later observations, that exactly realize the theoretic expectation regarding the sexual differences of the chromosome-groups, stated at p. 539. An abstract of these observations was published in the issue of Science for Oct. 20, 1905. ?T am much indebted to Mr. Uhler’s kindness in identifying many of the species examined. 508 ~ Edmund B. Wilson. has endeavored to bring together under the name of “hetero- chromosomes” two classes of chromosomes in these insects, namely, the “unpaired heterochromosome”’ (“accessory chro- mosome”’ of McClung)! and the “paired heterochromosomes” (or “chromatin nucleoli”), which differ markedly in behavior from the other chromosomes during the maturation process. Montgomery gives as the most essential characteristic of these chromosomes “their difference in behavior from the other chro- mosomes in the growth period of the spermatocytes and ovocytes, as sometimes during the rest period of the spermatogonia, a dif- ference which appears usually to consist in the maintenance of their compact structure and deep-staining intensity, so that while the other chromosomes become long loops or even compose a reticulum, these do not undergo any such changes or only to slight extent” ('05, p. 1gt). “Thanks to this peculiarity they can be followed with extreme certainty from generation to genera- tion, even during rest stages; and so are splendid evidence for the thesis of the individuality of the chromosomes” (’04, p. 146). The study of these chromosomes has led Montgomery to some very important conclusions regarding synapsis and reduction with which, as far as their more general features are concerned, | am glad to find my own results in substantial agreement. Considered more in detail, however, there are many points regarding which 1 think Montgomery’s general treatment of the “heterochro- mosomes”’ requires emendation. Ina preceding paper (Wilson,’05) the fact was indicated that two types of “paired heterochromosomes”’ or “chromatin nucleoli” occur in Hemiptera. ‘The first, including what I have called the 1Since there is no reason for considering the ‘accessory chromosome” as in any sense accessory to the others, it appears to me that McClung’s term might well be abandoned in favor of a less com- promising one. I suggest that until their physiological significance is positively determined chro- mosomes of this type may provisionally be called heterotropic chromosomes (in allusion to the fact that they pass to one pole only of the spindle in one of the maturation-divisions) in contradistinction to am phitro pic chromosomes, the products of which pass to both poles in both divisions. ‘There are several objections to this term, one of which is that the ‘‘accessory’’? chromosome behaves as a heterotropic body in only one of the divisions (and probably in one sex only). Another is the fact that the members (“chromatids”) of every chromosome-pair are heterotropic in the reducing division, since this only separates univalent chromosomes that were previously in synapsis; but if, as in these studies, the term **chromosome” be consistently applied to each coherent chromatin-element of the equatorial plate, whatever be its valence or mode of origin, this objection is perhaps not serious enough to weigh against the convenience of the term. . i Studies on Chromosomes. 509 “idiochromosomes” (which occur in such forms as Lygzus, Euschistus, Coenus, Brochymena, etc.) are typically unequal in size, and differ from all other known forms of chromosomes in the fact that their union in synapsis gives rise to an unequal or asymmetrical bivalent. ‘The spermatogonial groups correspond- ingly show but one small chromosome, since the larger idio- chromosome is not noticeably smaller than the ordinary chromo- somes. The second type includes the equal paired “chromatin nucleoli” of such forms as Anasa, Alydus, Syromastes. or Archimerus. Since the latter are almost always markedly smaller than the others they may conveniently be called the paired microchromosomes, or better, in order to avoid all ambiguity, simply the m-chromosomes; and these are distin- guishable in the spermatogonial groups as an equal pair of especially small chromosomes. The most obvious difference of behavior between these two types, so far as is now known, 1s that the idiochromosomes divide as separate univalents in the first maturation-mitosis, which accordingly always shows one more than half the spermatogonial number of separate chromatin elements, while the m-chromosomes, like the other chromosomes, always unite to form a bivalent before the first mitosis—which therefore shows the same number as in the second division. Other no less characteristic differences are described beyond. These two forms are not yet known to coexist in the same species; and, as a rule, forms that possess the idiochromosomes do not have an “accessory” or heterotropic chromosome, while as far as now known such a chromosome is always associated with the m-chromosomes. The confusion that has grown out of the failure to observe these differences arose in the first instance from two conclusions—both of which I shall show to be untenable—reached by Paulmier in his valuable, and, as far as the general history of the maturation- process is concerned, very accurate, study of the spermatogenesis of Anasa tristis (’99), and was increased by the subsequent efforts of Montgomery (’or, ’04, ’05) to reduce the behavior of the “chromatin nucleoli” to a uniform scheme. Paulmier, who was the first to reéxamine the history of the “accessory’’ chromosome since its discovery by Henking, was also the first to describe the m-chromosomes (in Anasa) as two very small chromosomes of equal size in the spermatogonial metaphase-groups. “These two 510 Edmund B. Wilson. small chromosomes, he believed, united in synapsis to form a single condensed bivalent chromosome-nucleolus which persisted throughout the growth-period of the spermatocytes and later gave rise to the small central “tetrad” of the first maturation-mitosis. He believed, further, that after an equal division of this small “tetrad” in the first mitosis each of its products passed undivided to one pole of the second spermatocyte-spindle. He therefore compared the “small tetrad” (microchromosome- -bivalent) of Anasa to the body, first discovered by Henking in Pyrrochoris, and afterward found in the Orthoptera and some other insects by McClung and others, to which the last-named author gave the name of “accessory chromosome.” In identifying the’ chro- mosome-nucleolus of the growth-period as the microchromosome- bivalent Paulmier has been followed by Montgomery in all of his papers and with some modifications by Gross (04) in his recent study of Syromastes. Paulmier’s conclusion on this point cannot, however, be sustained, as I shall try to show; and the same is true of his identification of the microchromosome-bivalent as the “accessory”’ or heterotropic chromosome. « I. GENERAL HISTORY OF THE M-CHROMOSOMES AND THE HET- EROTROPIC CHROMOSOME DURING THE GROWTH-PERIOD AND IN THE MATURATION-DIVISIONS. ‘The behavior of the m-chromosomes in the maturation-divisions may conveniently be considered first. Paulmier’s original preparations,' as well as my own more recent ones, give demonstrative evidence of the equal division of the small central chromosome in both maturation-mitoses, and the same appears no less clearly in Alydus and in Archimerus, pre- cisely as has been shown by Montgomery (’o!) in Protenor and by Gross (’04) in Syromastes. I was long since led to suspect an error in Paulmier’s conclusion in regard to this point from the fact, which clearly appears in his own figures, that the “‘accessory ” is nearly or quite as large as the ofier chromosomes, and much larger than the products of the first division of the small bivalent. 1] have in the previous paper acknowledged my indebtedness to Dr. Paulmier’s generosity in placing at my disposal his entire series of preparations of Anasa and other insects. He has since added to this indebtedness by sending me from time to time a large amount of valuable living material. Studies on Chromosomes. 511 (C7. Paulmier’s Figs. 28, 34-36, and my Fig. 2, k-n.) Both in Anasa and in Alydus careful search among longitudinal sections of the second division shows in fact in the clearest manner, that the “small dyad” divides into equal halves, so that each of the spermatids received one of its products (Figs. 1, 1-7; 2, m, n). ‘The heterotropic chromosome is a much larger body, as shown by the figures, in Anasa fully equal in size to some of the larger single chromosomes of the anaphases of the second division. Paulmier’s failure to observe the second division of the small bivalent is easily explained by the difhculty of observing this body owing to its usually central or subcentral position, and the mistake was a very natural one at the time his paper was written. Had he examined Alydus where there are but seven chromosomes, which show marked and constant size-differences, he could not have failed to observe this division. We have now to examine a second and more difficult point, namely, the nature of the condensed nucleolus-like body (chromosome-nucleolus) of the growth-period, which so closely simulates the heterotropic chromosome of the Orthoptera at the corresponding period. I have always doubted Paulmier’s and Montgomery’s conclusion that this body is the microchromosome- bivalent, from the fact, clearly shown in the figures of both these authors, that the chromosome-nucleolus of the synaptic and growth- periods 1 is always larger, and in some species very much larger (e. g., in Alydus)! than the two spermatogonial micro- chromosomes taken together, or than the small central bivalent to which it was assumed to give rise. (C7. Paulmier’s Figs. 16-21, with 26, 28.) ‘This fact did not escape Montgomery’s attention, but he explained it as due to an increase of volume on the part of the chromatin-nucleolus in the early growth-period and a corresponding decrease in the late growth-period or in the pro- phases of the first division (oI, p. 203). ‘This explanation was, however, not supported by any sufficient evidence;? and the only detailed evidence on this point has been brought forward by Gross (’04) in the case of Syromastes. ‘This observer, however, while apparently confirming Paulmier and Montgomery as to 1Cf. Montgomery, ’or, Figs. 96-98. 2Montgomery’s study of the facts in Euschistus (’98) is not in point, since he was here undoubtedly dealing with the idiochromosomes and not with the m-chromosomes, 512 Edmund B. Wilson. the fate of the chromosome-nucleolus, differs entirely from them in regard to its origin, concluding that it is derived from two of the Jarger spermatogonial chromosomes. In the attempt to reconcile these contradictory results (with both of which my own are in disagreement) he is led to some speculative conclusions that I think must be regarded as highly improbable.’ A careful study of all the intermediate stages, not only in Anasa, but also in Alydus, Archimerus, and Chariesterus gives in point of fact, evidence that I believe is quite decisive, that the small central bivalent 1s not derived from the large chromosome-nucleolus of the growth-period, and that the latter 1s nothing other than the accessory or heterotropic chromosome, precisely as in the Orthoptera. To the differences between the idiochromosomes and the m-chro- mosomes already stated may therefore be added the fact that the former, like the heterotropic chromosome, may form a single chromosome-nucleolus during the growth-period, while this is not the case, in the forms I have studied, with the -chromosomes. It may seem strange that Montgomery, after accurately tracing the history of the heterotropic chromosome (“chromosome x”’ in Protenor and showing its complete independence of the “chro- matin-nucleoli” (s-chromosomes) was not led to suspect a similar relation in the other forms. ‘That he apparently did not do so was doubtless due to his having failed to distinguish between the »-chromosomes and the idiochromosomes, which latter bodies he correctly identified (in Euschistus, etc.) as the bivalent chromo- some-nucleolus (or two separate univalents) of the growth-period. The entire independence of the large chromosome-nucleolus and the m-chromosomes is most obvious in Alydus and Archi- merus, partly because in both these forms the heterotropic chromosome is at every period recognizable by its characteristic size, partly because—in Alydus certainly, and I believe also in Archimerus—the m-chromosomes frequently assume a compact condensed form at a much earlier period than in Anasa; they can therefore be recognized in addition to the heterotropic chromosome, throughout the latter part of the growth-period, at a time when the larger chromosomes are still in the pale, vague condition characteristic of so many of the Hemiptera at this period. In Alydus pilosulus the first mitosis invariably shows seven 1Gross (’04) pp. 481, 482. Studies on Chromosomes. 513 bivalent chromosomes, which show very marked and characteristic size-differences (Fig. 1, c-g, b). ‘There are always (1) a largest chromosome or macrochromosome, which is frequently quad- Tripartite; (2) a second largest; (3) three slightly smaller ones of nearly equal size; (4) a fourth, considerably smaller than the last; and finally (5) the smallest or michrochromosome-bivalent. These show a characteristic grouping, the five larger ones forming an irregular ring with the small biv alee (‘ ronan nucleolus’’) at its center, while the next smallest lies more or less at one side of the ring (Fig. 1, g). In the first division all these chromosomes are equally halved (Fig. 1,7). In the second all are again halved with the exception of the fecond smallest which passes undivided to one pole of the spindle (Fig. 1, :-o). ‘The size-relations leave not the least doubt that this chromosome is derived from the one of corresponding size in the first division—1. e., the odd or eccen- tric one—and the latter accordingly is to be identified as the “accessory”? or heterotropic chromosome. In the first division this chromosome sometimes shows a quadripartite form (as was described by Paulmier in Anasa) sometimes a dumbbell-shaped or dyad-like form. In the second it is usually unconstricted and often curved (Fig. 1, 1, m, ), sometimes into a U-shape so as almost to appear double (Fig. 1, 0). A study of the growth-period shows that the heterotropic chromosome may be traced uninterruptedly backward from the metaphase of the first division to the contraction-phase of the synaptic period, being always in the form of a condensed chro- mosome-nucleolus, which in the early growth-period is attached to a large, pale plasmosome, from which it afterwards separates. It is impossible to mistake this chromosome, ‘owing to the fact that its characteristic size does not noticeably change except that it becomes slightly larger as the growth-period advances (probably owing to the presence of a central cavity), again becoming slightly smaller as the general condensation takes place. (Cf. Fig. 1, a-c.) Inthe contraction-phase (Fig. 1, a) and in the early post- synaptic spireme the m-chromosomes are not visible, but as the larger chromosomes assume the peculiar pale, ragged, clumped condition, characteristic of the middle and late growth-periods, the m-chromosomes frequently come into view, in the form of two compact, intensely-staining bodies, that may occupy any relative position (Fig. 1, 5). The period at which these bodies 514 Edmund B. Wilson. FicureE I. Alydus pilosulus.—a, Contraction-phase of synaptic period, ‘‘accessory”’ (4) in the form of a con- densed chromosome-nucleolus attached to a large plasmosome (fp); 0b, spermatocyte-nucleus, middle growth-period, showing large diffused chromosomes—‘‘accessory”” still attached to the plasmosome— and the two condensed m-chromosomes on opposite sides of the nucleus; c, early prophase of first division, showing all of the chromosomes, the larger ones condensing; d, late prophase, showing “‘accessory” (k) and the two m-chromosomes still separate; e, slightly later prophase, showing all of the chromosomes; f, initial anaphase, first division, the m-chromosomes separating; g, polar view of metaphase-group, first division; h, polar view of metaphase-figure, second division; 7, 7, initial anaphases, second division; k, spermatogonial metaphase-group; /, m, n, 0, anaphases of second division. Studies on Chromosomes. 515 Ficure 1.! 1The figures are all drawn to the same scale as those of the preceding study. 516 Edmund B. Wilson. condense into the compact form appears to vary considerably, for they cannot always be distinguished until the later growth- period, and it should be noted that during the pale period the nuclei often show a variable number of smaller deeply-staining granules. I believe, however, that there can be no doubt as to the nature of the two larger bodies on account of their great con- stancy, their size, and the completeness of the series that connects the earlier with the later conditions (such as is shown in Fig. 1, c), where no doubt of their nature can exist. ‘The persistence of the larger chromosome-nucleolus (“accessory’’) throughout all these stages without any considerable change renders it manifestly impossible that it should give rise to the m-chromosome bivalent, either directly as assumed by Paulmier and Montgomery, or by division into two univalents that subsequently conjugate, as described by Gross in Syromastes. In the early prophases the larger chromosomes resume their staining capacity and condense into characteristic cross-forms (Fig. 1, c), and finally into compact quadripartite tetrads or bipar- tite bodice. At this time the heterotropic chromosome assumes a dumbbell or quadripartite shape, and the m-chromosomes, which are still quite separate and may even lie on opposite sides of the nucleus, also frequently become bipartite. “Che nucleus now contains, accordingly, eight separate chromatin-elements, one more than the number of bivalents in the first mitosis, as is also the case in Archimerus and Anasa, as described beyond. As the spindle forms the two microchromosomes lose their bipartite shape, approach each other, and in the stage just preceding the metaphase finally conjugate to form the small bivalent chromosome at the center of the group. Without fusing, the two halves are then immediately separated, the division always taking place more rapidly than in. the case of the larger chromosomes (Fig. I, /). It is clear to demonstration accordingly, that in Alydus the small central bivalent does not arise from the large chromosome-nucleolus of the growth-period, but is formed by the late conjugation of two separate microchromosomes that have no genetic connection with that body. ‘The same fact is shown no less clearly in Archimerus calcarator (which shows eight chromosomes in the first mitosis), where the m-chromosomes, and the corresponding bivalent, are of extraordinary minuteness and are so much smaller than the acces- sory that they could not possibly be confused with the latter (Fig. 3). Studies on Chromosomes. 517 I believe that in this form, too, the m-chromosomes are fre- quently recognizable as condensed separate bodies in the growth- period; but owing to their minute size it 1s difficult to be sure of this. In any case, in the period just before the disappearance of the nuclear membrane they are quite distinct from the “acces- sory, ” which is, as in Alydus, immediately recognizable by its size (Fig. 3, g). From this period, as in Alydus, the latter body may be traced continuously backward into the growth-period. The foregoing facts, observed in Alydus and Archimerus are in close agreement with Montgomery’s results on Protenor, differing only in that the condensation of the m-chromosomes takes place somewhat later.’ In Anasa the condensation of these chromosomes from the diffused condition takes place still later; and this, combined with the fact that the “accessory” cannot be certainly distinguished from the other larger chromosomes by its size, renders the question more difficult of solution than in Alydus, though I believe the result is equally decisive. In Anasa, as in Alydus or Archimerus, the small central bivalent of the first equatorial plate is formed by a very late conjugation of two separate microchromosomes that only come together as the spindle forms, precisely as Gross describes in Syromastes. Of this fact no doubt is left by the study of a large number of preparations that show every stage of the process, step by step. In the late prophases, just before the nuclear membrane disappears, the nuclei invariably show twelve separate, condensed, intensely-staining chromatin-elements (one more than the number of chromosomes in the first mitosis) in addition to one or more pale rounded plasmosomes with which the chromosomes cannot for a moment be confused. ‘Ten of these are larger bivalents which have the form of quadripartite tetrads or dumbbell shaped bodies. The remaining two are much smaller bodies, irregularly ovoidal or frequently more or less distinctly bipartite (m, Fig. 2, e, 7); they may occupy any relative position. As the spindle forms, the microchromosomes lose their bipartite form, assume an evenly rounded ovoidal shape, and conjugate at the center of the equa- torial plate to form a small dyad-shaped bivalent (Fig. 2, g—). Without fusion the two halves are then immediately drawn apart ‘ 1In Alydus pilosulus this author believed the m-chromosomes, as usual, to be derived from the large chromosome-nucleolus. 518 Edmund B. Wilson. FiGureE 2. Anasa tristis.—a, Contraction-phase of synaptic period, showing ‘‘ accessory” (1) and plasmosome (p); b, spermatocyte-nucleus, late growth-period, beginning of the condensation, showing “accessory” () and the m-chromosomes (mm); c, a slightly later stage than the last; d, later stage, immediately before the final condensation, from a long-extracted preparation; e, f, two sections of one nucleus, show- ing all of the twelve chromosomes immediately before the disappearance of the nuclear membrane; g, view of one pole of the late prophase just after disappearance of the nuclear membrane, the m-chro- mosomes still wide apart; /, early metaphase-group in side view, showing approach of the m-chromo- somes; 7, four chromosomes from the metaphase, conjugation of the m-chromosomes to form the small central bivalent; j, early anaphase, separation of the m-chromosomes, ‘‘accessory”’ at the left; k, polar view of metaphase-group, first division; /, polar view of metaphase-group, second division; m, n, anaphases of second division, showing division of m-chromosomes and the undivided heterotropic chromosome; 0, p, spermatogonial metaphase-groups drawn as carefully as possible to show sizes of the chromosomes. Studies on Chromosomes. 519 Ficure 2. 520 Edmund B. Wilson. in the initial anaphase, always separating in advance of the larger chromosome-halves (Fig. 2, 7). It is not possible in the prophases just described to identify the heterotropic chromosome; but from the analogy of Alydus, Syromastes and Archimerus it may be assumed with great probability that it is the “odd or eccentric chromosome which in the metaphase-group lies outside the principal ring (Fig. 2, &). During the orowth- period, as Paulmier described, the chromo- somes, with the exception of the single conspicuous chromosome- nucleolus, remain in a loose, diffused, lightly-staining condition from the post-synaptic spireme stage until the condensation of the tetrads begins; and until the end of this period the m-chromo- somes cannot be distinguished. “Throughout this whole period the chromosome-nucleolus is distinctly visible; and it may at every period, even in hematoxylin preparations, if long extracted, be at once distinguished from the true nucleolus or plasmosome (as is shown in Paulmier’s figures), since the former stains intensely black, the latter pale blue or in double-stained preparations, pale red or yellow. In the contraction-phase of the synaptic period it is more or less elongated, ovoidal, or sometimes slightly con- stricted in the middle (Fig. 2, a). In the late post-synaptic period, at a time when the other Sheanmagannes are beginning to shorten and to give rise to the characteristic double cross-figures and V-figures it is usually more or less elongated, the transverse constriction is less obvious or disappears from view, and the body often shows faintly but distinctly a longitudinal split. (C7. Paul- mier, Fig. 22.) Slightly later, as the other chromosomes continue to shorten and thicken, the chromosome-nucleolus also shortens and thickens, often assuming a spheroidal form in which a central cavity may sometimes be seen.- As the remaining chromosomes condense to form the tetrads it again alters its shape, often becom- ing bipartite (Fig. 2, b-d), but sometimes showing a more or less distinctly quadripartite form as described by Paulmier (e. g., in his Figs. 23, 24). It now becomes indistinguishable from the other larger chromosomes, since the latter have also condensed into Slee tetrads or dyad-like forms, but the two m-chromosomes are immediately recognizable by their small size. It might therefore be supposed that the chromosome-nucleolus has divided to form the two microchromosomes, as Gross believed to be the case in Syromastes. The stage that immediately precedes this gives, Studies on Chromosomes. 521 however, conclusive evidence that such is not the case. In this stage (corresponding to-Paulmier’s Figs. 22, 23) the chromosome- nucleolus is still unmistakably recognizable by its compact and rounded appearance, while the other chromosomes, including the two microchromosomes are still in the form of paler and more diffuse bodies. The m-chromosomes at this period (one of them is clearly shown in Paulmier’s Fig. 24) appear as short, more or less ragged, paler, irregular rods that give the appearance of being longitudinally split (Fig. 2, b-d). Some of the cysts at this period show every stage in the condensation of these two small diffused chromosomes to form the two small, dyad-like micro- chromosomes that conjugate to form the small central bivalent. I have studied numerous nuclei in these stages with great care, and believe that they remove every doubt that the two micro- chromosomes that conjugate to form the small central bivalent in Anasa arise neither from separate small condensed bodies, as in Protenor or often in Alydus, nor from the single large chromosome- nucleolus as assumed by Paulmier, Montgomery and Gross, but from diffused masses similar to the larger ordinary chromosomes during the greater part of the growth-period. ‘The same fact may be seen in Chariesterus, though I have not in this case so complete a series of stages. [he chromosome-nucleolus must therefore give rise to one of the larger chromosomes; :and the exact agreement of Anasa with Alydus and Archimerus, save in the one point of the later condensation of the microchromosomes in the former form, justifies the confident conclusion that in Anasa the chro- mosome-nucleolus 1s the “accessory” or heterotropic chromosome. Anasa, Alydus, Chariesterus, and Archimerus thus fall in line with the facts observed in the Orthoptera, and I believe the same will prove to be the case with other Hemiptera in which an “odd,” “accessory”? or heterotropic chromosome occurs.! ‘This result, which is wholly at variance with the accounts of previous observers, forms the first step in clearing away the confusion that has hitherto stood in the way of a consistent general inter- pretaticn of the heterotropic chromosome. 1] cannot at present offer a definite explanation of the divergence between this result and that reached by Gross in Syromastes. Without questioning the accuracy of his figures, I feel confident, in view of what I have seen in so many other forms, that further examination of this genus will give a different result, both on this point and on a number of others. 522 Edmund B. Wilson. 2. RELATION OF THE CHROMOSOME-NUCLEOLUS TO THE SPER-= MATOGONIAL CHROMOSOMES. In view of the foregoing conclusion it will readily be admitted that a derivation of the chromosome-nucleolus from the two spermatogonial microchromosomes is a priori highly improbable; and in point of fact, all the actual observations not only of myself, but also, I believe, of Paulmier and Montgomery, are opposed to such a conclusion. This question has been complicated in a most unfortunate way by errors in counting the spermatogonial chromosomes. It was natural that the earlier observers should have expected to find an even number of chromosomes in the spermatogonial divisions; and the number is in point of fact an even one in all the forms that possess the idiochromosomes, as I have shown in the first of these studies. Regarding the forms that possess an accessory or heterotropic chromosome the existing accounts are, however, in conflict in giving sometimes an even number (Anasa, ¢. Paul- mier and Montgomery, Syromastes, t. Gross, Alydus pilosulus, ¢ Montgomery), and sometimes an odd one (Protenor, Harmostes, (Edancala, Alydus eurinus, t. Montgomery). A similar difference occurs in the existing accounts of the spermatogonia in Orthoptera, some of which are described as showing an even number and some an odd. ‘This contradiction has enormously increased the com- plication of the subject; for it has necessarily involved the view that in cases showing an even number the heterotropic chromo- some is a bivalent body, formed by the synapsis of two of the spermatogonial chromosomes; and this, in turn, very naturally led Montgomery (’04, ’05, etc.) to the further conclusion that in cases showing an odd number one of the chromosomes (presum- ably the “accessory’’) is already bivalent in the spermatogonia. I myself had at first no doubt of the correctness of both these interpretations, and my faith was not shaken even after the dis- covery that the number is 13 in Alydus pilosulus (Fig. 1, k), 15 in Archimerus (Fig. 3, 7), and 21 in “Chariesterus.’! When, however, demonstrative evidence was obtained that even in Anasa —in opposition to the concordant results of Paulmier and Mont- gomery on Anasa and those of Gross on the related form Syro- 1The indentification of this form (from Paulmier’s material) is doubtful. Studies on Chromosomes. 523 mastes—the number is 21 instead of 22 (Fig. 2, 0, p) I confess that surprise at this result was followed by ee regarding all of the accounts asserting the occurrence of an even number in other forms. ‘This result, which was totally unexpected to me, rests on the study of a large number of division-figures exactly in the metaphase, many of which are of almost schematic clearness. Of these, twenty-five (selected from six testes from different indi- viduals, including both adults and larval forms) were drawn with the camera, chromosome by chromosome, and subsequently counted. Without one exception these drawings show exactly twenty-one chromosomes; it is therefore out of the question that my result (worked out on Paulmier’s original preparations) can be due to an accidental displacement of one of the chromosomes in the process of sectioning, or to other similar sources of error. I believe the error of previous observers on this point is owing to the fact that one or more of the chromosomes sometimes show a more or less obvious constriction near the middle, and the larger ones are not infrequently curved—sometimes almost into a U-shape—so that one might readily be mistaken for two. Quite in harmony with this result is the fact that in Anasa the metaphase groups always show not two but three chromosomes that are distinctly larger than the others,’ one of these being obviously without a mate of like size, while all the others may be symmetrically paired, two by two, as a study of Fig. 2, 0, p, will show. It is obvious therefore that the heterotropic chromosome, and hence the chromosome-nucleolus of the growth-period, must be compared with one, not two, of the spermatogonial chromo- somes. In Alydus the heterotropic chromosome appears in the con- traction phase of the synaptic period as an ovoidal single body, always attached to one side of a large plasmosome and imme- diately distinguishable from the latter by its different staining- reaction (Fig. 1, a). Comparison of this figure with that of the spermatogonial chromosomes (Fig. 1, &), shows that the hetero- tropic chromosome at this period is much larger than the two spermatogonial microchromosomes united. In the spermato- gonial equatorial plates of Alydus or Archimerus it is not possible 1] regret to find myself here again in disagreement with Montgomery, who finds only two large spermatogonial chromosomes in Anasa (’04, p. 151, Fig. 16). 524 Edmund B. Wilson. positively to identify the heterotropic chromosome by its size; though it is evidently not one of the largest ones, since the latter form a symmetrical pair (Fig. 1, £) which doubtless unite to form the single macrochromosome of the spermatocyte-divisions (in accordance with Montgomery’s account of several other forms). In Anasa, however, it may be regarded as highly probable that the heterotropic chromosome is one of the largest three chro- mosomes, the remaining two of which pair as usual to form the spermatocyte macrochromosome-bivalent (Fig. 2, 0, p). ‘This is confirmed by comparison with the chromosome-nucleolus at the synaptic contraction- -period (lig>'2, a), At this time it vanes considerably in form, but is always more or less elongate, often ovoidal, sometimes almost rod-shaped, and sometimes more or less distinctly constricted in the middle; it rarely appears to be composed of two symmetrical halves (described by Gross as the typical condition in Syromastes.) It is rarely attached to a plasmosome, the latter body, when present, being usually separate (as in Fig. 2, a). The discrepancy in size between the chromosome-nucleolus and the spermatogonial microchromosomes is here still greater than in Alydus. On the other hand, as a comparison of the figures will show, the chromosome-nucleolus of this period is of very nearly the same volume as one of the largest three spermat- ogonial chromosomes. All the facts therefore point to the con- clusion that one of the latter is the heterotropic chromosome, and that it persists throughout the growth-period as the chromo- some-nucleolus, precisely as in Alydus or Protenor. Exactly the same result is indicated in Archimerus, where the discrepancy in size between m-chromosomes and heterotropic chromosome is even greater than in Anasa (Fig. 3, a, 7). 3. BEHAVIOR ,OF THE HETEROTROPIC CHROMOSOME IN THE MATURATION-DIVISIONS OF ARCHIMERUS CALCARATOR. In all the Hemiptera thus far described (Pyrrochoris, Anasa, Alydus, Protenor, Syromastes, Harmostes, C£dancala, Charies- terus), the heterotropic chromosome, when present, divides equally in the first spermatocyte-mitosis, but fails to divide in the second, thus showing a marked contrast to the phenomena in the Orthop- tera where the reverse order occurs. In the present section I wish ‘ Studies on Chromosomes. 525 briefly to record the fact that Archimerus, which agrees so closely with Alydus in most other respects, differs from this and all the above-mentioned forms in that the heterotropic chromosome fails FIGURE 3. Archimerus calcarator.—a, Side-view of first division metaphase showing heterotropic chromosome and m-chromosome bivalent; b, polar view of metaphase-group, first division; c, anaphase group, first division, side view; d, late anaphase, first division; e, f, polar views of metaphase-groups, second division, the former including, the latter lacking, the heterotropic chromosome; g, spermatocyte- nucleus, prophase of first division, showing heterotropic chromosome (/), the two separate m-chromo- somes (m), and five of the six large bivalents; h, views of the chromosome-nucleolus (heterotropic chromo- some) at different periods—1, from the contraction-phase of the synaptic period; 2, middle growth-period; 3, 4, later growth-period (the last three showing central cavity); i, spermatogonial metaphase-group. to divide in the first mitosis, passing over bodily to one pole and dividing equally in the second mitosis, precisely as in the Orthop- tera (Fig. 3, c,d). ‘This fact, which at first I myself hardly found 526 Edmund B. Wilson. credible, is placed beyond doubt by numerous preparations show- ing every stage in the first division, and no less certainly by the occurrence of two forms of the second division, in equal numbers and appearing side by side in the same cyst, one of which shows seven chromosomes, the other eight, the additional chromosome in the latter case being usually recognizable by its size. Fig. 3, c, d, shows two stages in the history of the heterotropic chromo- some in the first division. Fig. 3, e, 7, gives polar views of the two forms of equatorial plates in the second division, one showing seven, the other eight, chromosomes. A large number of sections from different individuals show no exception to this mode of distribution, the two divisions being immediately distinguishable by the size of the cells and by both the size and the form of the chromosomes. A similar case will be described, in Banasa calva, in the following section. 4. THE CHROMOSOME-GROUP IN BANASA CALVA. In this section I shall briefly describe a remarkable form that is unique among the Hemiptera thus far described in that it possesses both the idiochromosomes and a heterotropic chro- mosome; and as a consequence of this it 1s unique among all described animals in possessing not merely two but four visibly different classes of spermatid-nuclet in equal numbers. ‘These four classes are in no visible way distinguishable 1 in the fully formed spermatozoa, but are clearly apparent in the chromosome- groups of the spermatid-nucle1. o spermatogonial metaphase-groups are shown with sufficient clearness to admit of an accurate count, but there are great numbers of dividing spermatocytes which show every stage of both the maturation-divisions. The first division constantly shows, in polar view of the metaphase, fifteen chromosomes, of which two are markedly smaller than the others (Fig. 4, a, 6). As is demonstrated by their later history, one of these smaller chromosomes is the small idiochromosome (7) and one the heter- otropic chromosome (/). One of them frequently, but not invariably, lies at one side of the group, sometimes outside the principal ring of chromosomes (Fig. 4, a); but it Hs, lie inside the ring (Fig. 1,5). One always lies within the ring; and judging by the analogy of such forms as Lygzeus, Ewthistné or Coenus, a Studies on Chromosomes. [eae much larger chromosome beside which. it lies is to be identified as the larger idiochromosome. Besides these fifteen undoubted chromosomes one or more paler rounded bodies are often present, lying outside the chromosome-group, sometimes close to it, that are undoubtedly the remains of the plasmosome of the growth- period. In side views of the metaphase-figure all of these chromosomes, with one exception, have a symmetrical bipartite (rarely a quad- ripartite) shape; and in the ensuing division these are equally divided. One of the small Pico maseiee (heterotropic) never shows a bipartite shape, but is simply elongate and more or less fusiform (Fig. 4, c, d, e). As the division proceeds, this chro- mosome at first remains near the equator of the spindle and then passes over bodily toward one pole where it enters the daughter group (Fig. 4, 7, g), finally shortening again so as to assume a spheroidal form. One of the secondary spermatocytes there- fore receives fifteen chromosomes, the other fourteen. The failure of this small chromosome to divide in the first mitosis at first seemed to me so anomalous (I had not then observed the similar phenomenon in Archimerus, described in the foregoing section) that for a time | thought that this body must be one of the fragments of the plasmosome; and this suspicion was strengthened by the fact that other plasmosome-fragments are often found lying near or in the spindle (Fig. 4, g)- Further study, however, conclusively showed that this suspicion was not well-founded. The plasmosome-fragments are always rounded, paler, wholly inconstant in position and never lie in the equatorial plate. ‘The heterotropic chromosome, on the other hand, is always present (many division-fgures in all stages have been studied) and every stage of its asymmetrical distribution has been repeatedly observed. All doubt is, moreover, removed by a study of the metaphase-figures of the second division. Great numbers of these, showing the relations with schematic clearness, are avail- able for study. In polar view these show either fourteen or thirteen chromosomes (Fig. 4, /, 7), the two classes existing in approximately equal numbers, and side by side in the same cyst. At first sight neither of the small chromosomes of the first division can be distinguished in polar view of the second. This is owing to two causes: First, the small heterotropic chromosome, having failed to divide while all the others are but half as large as before, 528 Edmund B. Wilson. is sometimes hardly distinguishable from the latter—though, as in Fig. 4, 7, it can often be identified on careful scrutiny. Second, the small idiochromosome, now only half as large as in the first division, has conjugated in typical fashion with the larger one, so as to be visible, as a rule, only in side view (Fig. 4, 7), though careful focusing will often reveal it also in polar view, especially when the idiochromosome-dyad lies in a slightly oblique position. In this way the idiochromosome- -dyad has been positively identi- fied in Fig. 4, 6,7. In side-view the second division shows with entire clearness the separation of the idiochromosome-dyad into its two unequal constituents, precisely as in Lygzeus, Euschistus, etc., while all the other dyads, including the small heterotropic, dade equally (Fig. 4, ;-/). From this it follows that four visibly different classes of spermatid chromosome-groups are formed in equal numbers. ‘Two primary classes are formed that possess respectively fourteen and thirteen chromosomes, according to the presence or absence of the heterotropic chromosome; and each of these falls into two secondary classes, one of which contains the large idiochromosome, the other the small. Although this result necessarily follows from the mode of division, it is not a matter merely of inference, but of observed fact; for with a little pains spindles of both classes in the anaphases may readily be found in a vertical position that show both the sister-groups. Such a pair, from the early anaphase of a fourteen-chromosome spermatocyte, are shown in Fig. 4, m, the two groups exactly corresponding, chromosome by chromosome, except in case of the idiochromosomes (which are shown by focusing to be more widely separated than the others). A similar pair from a some- what later anaphase of the thirteen-chromosome class 1s shown in Fig. 4, 0, the relations being as before save that the heterotropic chromosome is lacking. A pair from a later anaphase of the fourteen-chromosome type is shown in Fig. 4, 7, showing a prin- cipal ring of ten ordinary chromosomes within which lie four others. ‘Iwo of these (below) are ordinary chromosomes; the other two are, at one pole the heterotropic and the small idio- chromosome, at the other pole the heterotropic and the large idiochromosome. Studies on Chromosomes. 529 VA 1] J R Ws Vag LR / @ o?, © 0/,@ 2 ec®, oe 08% SER Sey: © 6 Gee e%e e ;~ @ ° m eee va oS FiGure 4. Banasa calva.—a, b, Metaphase-figures, first division, in polar view, showing fifteen chromosomes, including two small ones (h, heterotropic chromosome, /, small idiochromosome—the large idiochro- mosome not distinguishable); c—g, successive stages of first division, in side-view, showing division of the small idiochromosome (7), and the unipolar movement of the heterotropic chromosome (/); #, meta- phase-group of second division, with thirteen chromosomes; 7, metaphase-group of the same division with fourteen chromosomes; j-/, metaphase and early anaphase of second division, showing separation of the idiochromosomes, and equal division of the heterotropic chromosome; m, sister-groups from the same spindle, early anaphase second division, fourteen-chromosome type; 1, similar pair, late anaphase; o, similar pair, middle anaphase, thirteen-chromosome type; /p, 7, entire chromosome-group from a single nucleus at the end of the growth-period, showing idiochromosome-dyad (7) and heterotropic chromosome (h). 530 Edmund B. Wilson. The four classes thus formed may be tabulated as follows: Primary Class A ! (1) 12 ordinary chromosomes, 1 heterotropic, 1 large chromosome. (14 chromosomes) { (2) 12 ordinary chromosomes, 1 heterotropic, 1 small idiochromosome. Primary Class B ; (3) 12 ordinary chromosomes, 1 large idiochromosome. (13 chromosomes) { (4) 12 ordinary chromosomes, 1 small idiochromosome. Restating the facts from the point of view of mere size, it appears that class (3) contains no especially small chromosome, class (2) two small chromosomes, and classes (1) and (4) each one small chromosome, the latter being in one case the heterotropic, in the other the small, idiochtomoceene.: I have not yet studied in sufficient detail the history of this form in the growth-period, which will require additional material; but the main facts are such as might be expected. In the middle growth-period the nuclei show, with great constancy, two unequal chromosome-nucleoli, both of which frequently appear hollow. The larger of these is almost certainly the idiochromosome- bivalent; for in the prophases of the first division it may be seen separating into its two unequal constituents, precisely as I described in Brochymena (Fig.45 P5 q)., eAuthis period the hetero- tropic chromosome 1s unmistakably recognizable by its size and shape, showing no constriction like that of the other chromo- somes. I believe this to be identical with the smaller chromo- some-nucleolus of the earlier period, but cannot offer decisive proof. CRITICAL AND COMPARATIVE. The three well-defined classes of chromosomes that have been described in this and my preceding paper differ from the others, each in its own way, especially in respect to their behavior in the process of synapsis and during the growth-period. The most characteristic common feature of the first two classes is their long delayed synapsis, which in both cases is deferred to the period Jt is probable that additional light will be thrown on this form by further study of the related one, Thyanta custator, which I now have under investigation. The general aspect of the chromosome group in this species is closely similar to that of Banasa, and the first mitosis also shows fifteen chromosomes, of which however three, instead of two, are smaller than the others. The second mitosis differs from that of Banasa insbowing always but thirteen chromosomes, and I have not thus far found a heterotropic chromosome in either division. Though I cannot yet speak positively, these conditions seem only explicable under the assumption that two pairs of idiochromosomes are present. From such a con- dition one nearly similar to that observed in Banasa might readily be derived by the disappearance of one of the small idiochromosomes. Studies on Chromosomes. 531 immediately preceding the reduction-division—z. e., in case of the m-chromosomes to the prophases of the first division, at the very end of the growth-period, and in case of the idiochromosomes to a still later period following the first division (though a temporary or preliminary union frequently occurs at a much earlier period). The “accessory” or heterotropic chromosome, finally, does not undergo synapsis at all, since it is without a mate with which to pair. : As regards their behavior in the growth-period, the idiochro- mosomes and the heterotropic chromosome agree in being “hetero- chromosomes” in Montgomery’s sense—1. e., are distinguished from the other chromosomes by their compact form and deep- staining capacity. The m-chromosomes, on the other hand, may remain in a diffused condition throughout the early and middle growth-periods, only condensing to the compact form at the same time as the ordinary chromosomes (Anasa, “Charies- terus’); their condensation may, however, take place in the middle growth-period (Alydus), or even earlier (Protenor, ac- cording to Montgomery). An analogous difference in the time of condensation exists in case of the idiochromosomes, which in case of Lygazus do not condense as early as in Ccenus or Euschistus. My observations prove definitely in some cases (Alydus, Anasa, Archimerus, “Chariesterus’’), and I think render it prob- able for all cases, that in those Hemiptera that possess an “acces- sory’’ or heterotropic chromosome and two equal spermatogonial microchromosomes (m-chromosomes), the large chromosome- nucleolus of the synaptic and growth-periods is not, as other observers have supposed, the microchromosome-bivalent (“chromatin nucleolus” of Montgomery) but the heterotropic chromosome, precisely as in the Orthoptera. This error of identification has led Montgomery to designate three quite distinct kinds of chromosomes by the same name of “chromatin- nucleoli.” “These are (1) the equal paired spermatogonial micro- " chromosomes and the corresponding bivalent of the first sper- matocyte division; (2) the idiochromosomes, which are typically unequal and do not form a bivalent in the first division; and (3) the heterotropic chromosome as it appears in the growth-period. It is therefore desirable, despite some repetition, to bring together in brief form the principal distinctions between these three. 532 Edmund B. Wilson. 1. The paired microchromosomes—or preferably ‘m-chro- mosomes,” since forms may be found in which they are not smaller than the others—form an equal pair in the spermatogonia, and in most of the forms thus far known are much smaller than the others. These do not, ordinarily conjugate to form a bivalent in the general synaptic period, and may (Alydus, Archimerus) or may not (Anasa, ‘“Chariesterus’”’) condense early in the growth- period to form two small separate chromosome-nucleoli which can be distinguished in addition to the principal one (hetero- tropic chromosome). ‘They undergo a very late synapsis (in the prophases of the first maturation division) to form a small sym- metrical bivalent, typically central in position, that undergoes a reduction-division in the first mitosis and an equation- -division in the second. Each spermatid nucleus therefore receives a single m-chromosome. ‘They are always, as far as known, associated with a heterotropic chromosome, and the number of spermato- gonial chromosomes 1s odd (with the more than doubtful exception of Syromastes). ‘The first maturation-division shows a number of chromosomes which when doubled is one more than the spermato- gonial number (as in Orthoptera). Known to occur in Anasa, “Chariesterus,’’ Syromastes, Protenor, Alydus, Archimerus, Har- mostes, (dancala, and doubtless occur in many others. 2. The idiochromosomes are typically unequal 1 in size (Nezara forms an exception) forming an unequal pair in the spermatogonia (which accordingly show typically but one small chromosome); they may conjugate to form a bivalent at the time of general synapsis, or may remain separate, in either case condensing to form a chromosome-nucleolus (or two separate unequal ones) which persists throughout the greater part or the whole of the growth-period. In either case they are in the Hemiptera always separate univalents at the time of the first maturation- -mitosis, and separately undergo an equation-division in that mitosis. This division accordingly shows one more than half the spermat- ogonial number of separate chromatin-elements, the latter number being in all cases an even one. At the end of the first mitosis their products conjugate to form a bivalent dyad (thus reducing the number of separate chromatin-elements to one-half the spermatogonial number). ‘This dyad, typically unsymmet- rical, undergoes a reduction-division in the second mitosis, and all of the spermatozoa receive the same number of chromosomes, Studies on Chromosomes. 533 one-half receiving the larger and one-half the smaller idiochro- mosome. ‘They are not ordinarily associated with a heterotropic chromosome, the single known exception being Banasa. ‘The idiochromosomes are known to occur in Lygzeus, Ccenus, Podisus, Trichopepla, Mineus, Nezara, Murgantia, Brochymena and Banasa and are doubtless of much wider occurrence. 3. The “accessory” or heterotropic chromosome is certainly in most Hemiptera—and | believe will be found to be in all— unpaired in the spermatogonia, and its behavior is throughout that of a univalent body. It fails to unite in synapsis with any other chromosome, and persists throughout the spermatocytic growth-period as a chromosome-nucleolus. During the earlier part of this period it resembles the idiochromosome bivalent (or the univalent large idiochromosome) in being attached to a large plasmosome from which it afterward separates This chro- mosome divides in only one of the maturation-divisions, passing undivided to one pole of the spindle in the other. ‘The latter division is usually the second (Pyrrochoris, Anasa, Protenor, Alydus, Chariesterus, Syromastes, Harmostes, Ofdancala), but in Archimerus and Banasa it is the first. In either case one-half the spermatozoa receive one more chromosome than the other half. From the foregoing it will be seen that Montgomery correctly identified the chromosome-nucleolus in the growth-period of such forms as Euschistus, Coenus, Podisus, Brochymena, Tricho- pepla or Nezara, which possess the idiochromosomes. He was, however, at fault in the conclusion that it gave rise to a small bivalent in the first division, the small chromosome of this division being always a univalent that is not at this time paired with its (usually) larger fellow; and further, owing to a failure to discrimi- nate between these bodies and the paired microchromosomes of the Anasa or Alydus type, he describes and figures the spermat- ogonial groups in most of these forms as containing a symmetrical pair of “chromatin-nucleoli.”” Owing to his having overlooked the constant separateness of the idiochromosomes as univalents in the first mitosis he has also, I believe, been misled in several 1Tt is doubtless a similar condition that has led Moore and Robinson (’o5) in the case of Periplaneta, to conclude that the ‘‘accessory”? chromosome is nothing but a ‘“‘nucleolus.” These observers have evidently studied the phenomena in a very superficial manner. 534 Edmund B. Wilson. instances in regard to the spermatogonial number (e. g., in Euschistus variolarius, Nezara and Brochymena). ‘The state- ment given in the general summing up of his latest paper (’05) “Whenever the héterochromenames occur in pairs in the sper- matogonia they (7. ¢., the “chromatin nucleoli’) always conjugate to form bivalent ones in the first spermatocytes, and their univalent components become separated in the first maturation mitosis, 1. é., divide prereductionally” (p. 195, and elsewhere), is inapplicable to the idiochromosomes; for even though they conjugate to form a bivalent chromosome-nucleolus in the growth-period they again separate to divide as separate univalents in the first mitosis, asi showed in detail in Brochymena, and as must also occur in the other forms (as is proved by the number of the chromosomes and their later history). The statement cited above applies only to the m-chromosomes of such forms as Anasa, Chariesterus, Alydus, Archimerus or Protenor; but the name “chromatin nucleoli”’ is in these cases not very appropriate in view of the fact that in the very form (Anasa) in which they were first discovered they do not appear as chromatin-nucleoli at any time during the growth- period of the spermatocytes. As to their behavior in the rest- period of the spermatogonia | have at present no opinion to express. It is further probable that the distinction urged by Montgomery between the “odd chromosome” and the accessory (’05, p. 192) is also not valid; for my observations prove that in Alydus and Archimerus the “odd chromosome” (“‘accessory”’) is a typical chromosome-nucleolus (7. e., ““heterochromosome’’) in the growth- period, and it is extremely probable that the same will be found to hold true of the “odd chromosome” of Harmostes and Q(dancala. I think therefore that Montgomery’s general con- clusions regarding the ‘“heterochromosomes”’ require some revision. We may now briefly consider the nature of the “accessory” or heterotropic chromosome. So long as any of the forms possessing such a chromosome were supposed to have an even number of spermatogonial chromosomes the conclusion drawn by Mont- gomery (01, ’04, ’05) that this chromosome is a bivalent seemed an almost necessary one, even in cases where it appears as a single body in the spermatogonia. The observations brought foovard ances paper cast grave doubt, I think, on all of the earlier accounts asserting an even spermatogonial number in Studies on Chromosomes. 535 the Hemiptera that possess a heterotropic chromosome. Of these accounts (in cases positively known to have such a chro- mosome) there are but four, namely, Henking’s original account of Pyrrochoris (90), Paulmier’s (’99), and Montgomery’s (01, ’04) accounts of Anasa, Montgomery’s of Alydus pilosulus (’01) and Gross’s more recent one of Syromastes (’04). Henking states that he counted but four cases, one of which seemed to show twenty-three, the other three twenty-four, and it is evident both from the figures and from the frank statement of this able observer, that he adopted the latter number more on account of theoretical considerations than as a result of any adequate study of the facts. I have shown the counts of Paulmier and Montgomery to be erroneous in the case of Anasa, and also that of Montgomery in the case of Alydus pilosulus. There remains therefore the single case of Syromastes; but perhaps, in view of the results I have reached in other forms, I may be allowed the pre- diction that a reéxamination of this one will lead to a similar conclusion. If this expectation is verified every ground will be removed for considering the heterotropic chromosome as a bivalent body; and I think that until definite evidence to the contrary is forth- coming we are bound to take this chromosome at its face-value, so to speak, as univalent. ‘This conclusion involves a series of other conclusions and possibilities of which I shall here undertake to indicate only the more important. 1. As was indicated by McClung (’02, p. 71), if the “accessory”’ be univalent, its behavior in the maturation-mitoses at once falls into line with that of the other spermatogonial chromosomes; for each of these, too, undergoes but one division in the course of the two maturation-mitoses. One of these divisions (the reduction division) merely separates the univalent chromosomes that have previously paired in synapsis (as is so convincingly shown in case of the idiochromosomes or the m-chromosomes); and only the fact that the “accessory”’ has no mate with which to pair renders its behavior in one of the divisions apparently different from that of the ones that do pair. 2. The objections that I myself urged to the suggestion made in the first of these studies regarding the origin of ans heterotropic chromosome are thus set aside, and my attempt to compare the idiochromosomes with the m-chromosomes was made on incorrect 536 Edmund B. Wilson. premises. My suggestion was that a heterotropic chromosome might arise from a symmetrical bivalent by the gradual reduction and final disappearance of one member of the conjugating pair, conditions corresponding to several of the stages of such a reduc- tion being shown to exist in Nezara, Mineus, Coenus, Euschistus, Murgantia, and Lygzus. All of the facts seem to me to indicate that this interpretation is the true one. Were the small idio- chromosome to disappear in such forms as Lygzeus or Euschistus, the large idiochromosome would be left as a heterotropic chro- mosome agreeing, point by point, with that of such forms as Alydus, Protenor or Anasa, namely, in its persistence as a chro- mosome-nucleolus during the growth-period; its association with the plasmosome in the earlier part of this period and its subse- quent separation from it; its equal bipartition by an equation- division in the first spermatocyte-mitosis, and the failure of the resulting products to divide in the second mitosis; and in corre- lation with the foregoing the existence of an odd number of spermatogonial chromosomes. ‘The exactness of this corre- spondence is such, I think, as to lend a high degree of probability to the interpretation. ‘The only apparent obstacle in its way is the fact that in Banasa a heterotropic chromosome coexists with a typical pair of idio- chromosomes; but this difficulty only exists under the assumption that a heterotropic chromosome has arisen but once in the history of the species, and nothing is known to justify such an assumption. I think, on the contrary, that the facts in Banasa may fairly be taken as evidence that a process is here in progress which if con- tinued would lead to the formation of a second heterotropic chromosome.! 3. The formation of a heterotropic chromosome in the manner indicated involves a reduction of the total number of chromo- somes by one; and it is possible that this may represent one process by which changes from a higher to a lower number or chromo- somes have been brought about. But I doubt whether such a process can have gone very far, since, as pointed out beyond, there is reason to believe that it has occurred in only one sex. 1Should my surmise (stated in the footnote at p. 530) be correct that in the related form Thyanta two pairs of idiochromosomes are present without a heterotropic chromosome, I think additional support will be lent to the above interpretation. Studies on Chromosomes. 537 It seems, on the other hand, probable that the m-chromosomes may be of more general significance in this direction, since the facts distinctly suggest that they are diminishing or disappearing, and perhaps in some cases already vestigial, structures in both sexes. Paulmier was the first, as far as I am aware, to suggest that a reduction in the size of particular chromosomes might fore- shadow their total disappearance; that chromosomes might in this way assume a vestigial character; and further, eg such chromosomes might represent ‘ “somatic characters which belonged to the species in former times, but which characters are disappear- ing” (’99, p. 261). ‘This conception was applied by him to the small m-chromosomes (which he believed to represent the “acces- sory”), but was further supported by his observation of a very small chromatin-body that may divide like a chromosome (Paul- mier, Fig. 28, a) but is only rarely visible.’ Paulmier’s suggestion, which I suspect may prove to embody one of the most important results of his paper, has been further developed by Montgomery. This author first suggested that an uneven number of chro- mosomes “represents a transition stage between a higher number and a lower” (OI, p. 215); and he has more recently assumed that the “unpaired heterochromosomes”’ (“accessory”’ or hetero- tropic chromosomes) have arisen from paired heterochromosomes (“chromatin nucleoli’) or ordinary chromosomes by fusion of the members of a pair to form a bivalent body (’05, p. 197). Both the paired and the unpaired heterochromosomes are con- sidered to be chromosomes on the way to disappearance. ‘Though my conclusion regarding the origin of the unpaired or heterotropic chromosome is an entirely different one, it agrees with that of Montgomery in assuming a reduction in the original number of chromosomes; and it is possible that by a subsequent disappear- ance of the heterotropic chromosome a further reduction may take place, though as indicated above there are difficulties in the way of this assumption. My conclusion is, however, distinctly opposed to the view that heterotropic chromosomes have arisen from “paired heterochromosomes” (m-chromosomes), and although they have some features in common the evidence is opposed to 1Tt seems quite possible that this body may be the last remnant of a small idiochromosome, of which the corresponding larger one has remained as the heterotropic chromosome; but definite evidence of this is lacking. 538 Edmund B. Wilson. any direct relationship between these two classes of chromosomes. Montgomery has called attention to the fact that the m-chro- mosomes vary greatly 1 in size in different species, graduating down to excessively minute forms (such as those occurring in Archi- merus.) It is evident that these chromosomes Ae. undergone a symmetrical reduction which, if continued, might lead to the disappearance of both; and such a process, if repeated, would lead in the history of a species to a progressive and parallel reduction of the number in both sexes. When these facts are compared with those presented by the idiochromosomes the thought can hardly be avoided that the reduction of the m-chro- mosomes may be correlated with a corresponding change that is taking place equally in both sexes; while the reduction of the small idiochromosome may represent a change that is taking place more rapidly in one sex than in the other, or affects one sex only. 4. How the foregoing conclusions and suggestions regarding the idiochromosomes and heterotropic chromosomes will square with McClung’s hypothesis (’02, 2) and my own. similar sug- gestion (’05) that these bodies may be in some way concerned with sex-determination, does not yet clearly appear from the known data; but there are some considerations that are too interesting in this connection to be ignored. If the heterotropic chromosome be a univalent body the conclusion is unavoidable (since the spermatogonial number is odd) that in the production of males, the number of chromosomes contributed by the two germ-cells cannot be the same. ‘To this extent the facts har- monize with the view of McClung; but further consideration gives reason to doubt some of the more specific features of his hypothesis. The presence of the heterotropic chromosome in the male by no means proves that it is of paternal origin in fer- tilization, still less that it is specifically the male sex-determinant— indeed, I believe the facts point in the opposite direction. In Anasa, for example, where the spermatozoa possess either ten or eleven chromosomes, offspring (males) having twenty-one would be produced by the fertilization of an egg having ten chro- mosomes by a spermatozoon having eleven (as McClung would assume); but the same result would follow from the fertilization of an egg having eleven by a spermatozo6n having ten. I believe the second of ‘iene alternatives to be the more probable one for the following reasons: According to my view, the heterotropic Studies on Chromosomes. 539 chromosome has assumed its unpaired character by the reduction and final disappearance of its parental mate or homologue (1. e¢., a small idiochromosome); and it is highly probable that this pro- cess has occurred in one sex only, namely, the male.’ If this be the fact, it 1s evident that the heterotropic chromosome that remains in the male is the maternal mate or homologue of that which has vanished. I think therefore that we may expect to find that the heterotropic chromosome present in the male is derived in fertilization from the maternal group of chromosomes; and also that the female will be found to possess one more chromosome than the male (exactly the opposite of McClung’s assumption), the additional chromosome being the homologue of that which has vanished in the male. If this be the fact, it follows with great probability that in the egg-synapsis this chromosome pairs with its paternal homologue (originally the heterotropic chro- mosome) to form a symmetrical bivalent, and that all the eggs receive eleven chromosomes; while in the male the heterotropic chromosome fails to pair (having no mate) and hence remains univalent. The expectation may therefore be stated as follows: Egg 11 + spermatozoén 10 = 21 (male). Egg 11 + spermatozoén 11 = 22 (female).$ Important direct evidence in favor of this expectation is given by the discovery by Stevens, briefly referred to in my preced- ing paper, that in the beetle Tenebrio a small chromosome, evidently analogous to the small idiochromosome of Hemiptera, is present in the somatic cells of the male only, while in the female 1T will here not go into the somewhat intricate difficulties encountered under the supposition that it has occurred in both sexes, except to point out that if an unpaired heterotropic chromosome be present in the female and is allotted to only half the eggs (as in the male) it is necessary to assume a fertiliza- tion of each form of egg by the opposite form of spermatozoon, since otherwise three forms of offspring would result. Such a mode of fertilization is a priori very improbable. Still greater difficulties stand in the way of assuming that an unpaired heterotropic chromosome, present in the female, is retained in all of the eggs. "Montgomery (’o4) has in fact found in the odgonia and follicle-cells of the female Anasa twenty- two chromosomes, and Gross (’04) reports the same number in those of the female Syromastes. But since the first-named observer is certainly, and I believe the second-named is probably, in error as to the number in the male, both these cases require reéxamination. On the other hand Sutton has found twenty-two in the odgonia and follicle-cells of the Orthoptera (Brachystola) while the spermatogonial groups show twenty-three; but here again I think a result so important should be supported by more adequate evidence than he has brought forward. I now have this subject under investigation. 5For the confirmation of this, see Appendix. 540 Edmund B. Wilson. it is represented by a corresponding larger one (both sexes having the same number of chromosomes). Were the small chromo- some to disappear, the female would show one more chromosome than the male in accordance with my general assumption. We have now therefore good reason to hope that observation will directly determine whether sex is predetermined in the chro- mosome-group; and further, whether the sex-determining func- tion can be localized in a particular chromosome or pair of chromosomes, as McClung suggested. 5. Ihe foregoing offers no specific suggestion as to the mean- ing of the four classes of spermatozoa observed in Banasa. But it may be remarked that the existence of two or four (or more) classes of germ-cells in the same sex is in itself nothing anomalous; for as Sutton has pointed out, under the conception of himself and Montgomery there may be as many classes of spermatozoa as there are combinations of paternal and maternal chromo- somes (in accordance with the Mendelian ratios). Forms which possess idiochromosomes or heterotropic chromosomes differ from the more usual ones only in that two or four of these classes are made visible by a greater or less differentiation of the members of one or two of the chromosome-pairs. It seems admissible to suppose that such a visible differentiation of the members of particular chromosome-pairs may stand for a corresponding differentiation of corresponding or allelomorphic qualities in the adult. I would therefore suggest the possibility that such a visible polymorphism of the male germ-nuclei as exists in Banasa may be accompanied by a visible polymorphism in the adults; and, while I am not aware that such a polymorphism has been observed in the Hemiptera, I believe this subject should be care- fully examined. ’ It is hardly necessary to point out, finally, how strong a support the foregoing observations lend to the general hy pothesis of the individuality of chromosomes, and to the conception of synapsis and reduction first brought forward by Montgomery and developed in so fruitful a way by Suttonand Boveri. I must frankly confess that until I had followed step by step the behavior of the idiochro- mosomes and the m-chromosomes in the Hemiptera I did not appre- ciate how cogent is the argument brought forward in Montgomery’ s paper of ’or in support of his conclusion that synapsis involves an actual conjugation of chromosomes two by two, and that the Studies on Chromosomes. 541 chromosomes thus uniting are the paternal and maternal homo- logues. In the case of the m-chromosomes, no less clearly than in that of the idiochromosomes, the conjugation is not in any way an inference but an easily observed fact; and in both cases it is equally clear that the subsequent reducing division separates, with their individuality unimpaired, the same chromosomes that have previously united in synapsis. I believe that any observer who will take the trouble to study in detail the history of the chromosomes in these insects must sooner or later in his task acquire the firm conviction that he is dealing with definite, well characterized, entities which show the most marked individual characteristics of behavior, which in some manner persist from one cell-generation to another without loss of their specific character, and which unite in synapsis and are distributed in the ensuing maturation-divisions in a_ perfectly definite manner. All the facts indicate that these phenomena are the visible expression of a preliminary association, and subsequent distribution to the germ-cells, of corresponding hereditary char- _ acters. It is evident, therefore, that the time has come when cytologists must seriously set themselves to the task of working out a comparative morphology and physiology of the chromosomes, with the ultimate aim of attempting their specific correlation with the phenomena of heredity and development. SUMMARY. 1. The chromosomes that have been called “heterochromo- somes” in Hemiptera (Montgomery) include three distinct forms that may provisionally be called (a) the paired microchromosomes or m-chromosomes; (b) the idiochromosomes; (c) the “accessory” or heterotropic chromosomes. 2. The m-chromosomes are usually very small, form a sym- metrical pair in the spermatogonia, and do not unite (in the forms I have studied) to form a bivalent chromosome-nucleolus in the growth-period. At an earlier or later period they condense to form two separate chromosomes that finally pair to form the small bivalent central of the first division, but are immediately separated without fusion. Each divides equally in the second division. 3. The idiochromosomes are typically unequal, and hence do not form a symmetrical pair in the spermatogonia. ‘They may 542 Edmund B. Wilson. or may not pair at the time of general synapsis to form a bivalent; in the former case they appear in the growth-period as a single bivalent chromosome-nucleolus, in the latter case as two separate univalent chromosome-nucleoli. In either case they undergo equal division as,separate univalents in the first maturation- mitosis, their products conjugating at the close of this division to form an asymmetrical dyad the two constituents of which are, without fusion, immediately separated in the second division. 4. The heterotropic chromosome is without a mate in the spermatogonia (which accordingly show an odd number of chro- mosomes) and hence fails to undergo synapsis. Its behavior is throughout that of a univalent body. It divides only once in the course of the two maturation mitoses, this division taking place usually in the first, but in some species in the second, mitosis. It has probably arisen by the reduction and final disappearance of one member of a symmetrical chromosome-pair, this process having taken place in the male only. 5. [he m-chromosomes are always associated with a hetero- tropic chromosome, while the idiochromosomes and heterotropic chromosomes are known to coexist in only a single case (Banasa). This case indicates that the formation of heterotropic chromo- somes may have taken place more than once in the history of the species and possibly represents one mode of change from a higher to a lower number of chromosomes. 6. In forms possessing the idiochromosomes two classes of spermatozoa exist in equal numbers, which receive the same number of chromosomes but differ in respect to the idiochro- mosome. In forms possessing a heterotropic chromosome two classes of spermatozoa likewise exist, one of which possesses one more chromosome than the other. When both idiochromosomes and heterotropic chromosomes are present (Banasa) four classes of spermatozoa are formed, two having one more chromosome than the other two, each of these groups again differing in respect to the idiochromosome. 7. The facts support the general theory of the individuality of chromosomes, the theory of Montgomery 1 in regard to synapsis, and that of Sutton and Boveri regarding its application to Men- delian inheritance; and they point toward a definite connection between the chromosome-group and the determination of sex. Zoélogical Laboratory, Columbia University, July 29th, rgos. Studies on Chromosomes. 543 APPENDIX. During the summer, and since the foregoing paper was entirely completed in its present form, I have obtained new material which shows decisively that the theoretic expectation in regard to the relations of the nuclei in the two sexes, stated at p. 539, is realized in the facts. In Anasa, precisely in accordance with the expectation, the odgonial divisions show with great clearness one more chromosome than the spermatogonial, namely, twenty-two in- stead of twenty-one; and the same number occurs in the divisions of the ovarian follicle-cells. Again in accordance with the expec- tation, the odgonial groups show four large chromosomes instead of the three that are present in the spermatogonial groups. In other respects the male and female groups are closely similar. In like manner, the odgonial divisions in Alydus and Protenor show fourteen chromosomes, the spermatogonial but thirteen; and in Protenor the spermatogonial chromosome-groups have but one _large chromosome (unquestionably the heterotropic) while the odgonial groups have two such chromosomes of equal size. The interpretation is unmistakable. Taking Protenor as a type, all of the matured eggs must contain seven chromosomes, of which one, much larger than the others, corresponds to the heterotropic chromosome present in one-half of the spermatozoa. These spermatozoa (seven-chromosome forms) contain a chromo- some-group exactly similar to that of the egg; and fertilization by a spermatozoon of this class produces a female having fourteen chromosomes. ‘The other half of the spermatozoa (six-chromo- some forms) lack the heterotropic chromosome; and fertilization of an egg by aspermatozoon of this class produces a male having but thirteen chromosomes, the unpaired one being derived from the egg and appearing in the maturation of this male as the heterotropic chromosome since it is without a mate. ‘There can, therefore, be no doubt that a definite connection exists between the chromosomes and the sexual characters, and I believe that the conclusion can hardly be escaped that the chromosome- combination, established at the time of fertilization, is, in these insects, the determining cause of sex. The result reached in Anasa is confirmed by a comparison of the male and female chromosome-groups 1 in Lygeus, Coenus and Euschistus, all of which possess in the male a pair of unequal 544 Edmund B. Wilson. idiochromosomes 1n place of an unpaired heterotropic chromosome. In all of these forms, as I showed in my first paper, the spermato- gonial groups show fourteen chromosomes that may be equally paired with the exception of a small and a large idiochromosome. The odgonial groups in these forms also show fourteen chromo- somes, but all may be equally paired, the small idiochromosome being represented by a larger one that has a mate of equal size. In these forms, accordingly, males are produced as a result of fertilization by spermatozoa containing the small idiochromosome, females by fertilization by spermatozoa containing the large idio- chromosome (which accords with Stevens’ result in Tenebrio). This proves the correctness of my conclusion that the size-reduction and final disappearance of the small idiochromosome has taken place in the male sex only, and that the large idiochromosome corresponds to the heterotropic chromosome. Complete disap- pearance of the small idiochromosome in the male has led to each a condition as exists in Anasa and other forms possessing a heterotropic chromosome. ‘These facts will be described and discussed in the third of these studies. October 4, 1905. Studies on Chromosomes. 545 LITERATURE.! BAUMGARTNER, W. J., '04.—Some new Evidences for the Individuality of the Chromosomes. Biol. Bull., viii, 1. Bovert, TH., ’04.—Ergebnisse tiber die Konstitution der Chromatischen Substanz des Zellkerns. Jena, 1904. Gross, J., °04.—Die Spermatogenese von Syromastes marginatus. Zool. Jahrb., Anat. Ontog., xx, 3. Henke, H., ’90.—Ueber Spermatogenese und deren Beziehung zur Entwickelung bei Pyrrochoris apterus. Z. wiss. Zodl., li. McC ung, C. E., ’00.—The Spermatocyte Divisions of the Acrididz. Bull. Univ. Kansas) 1x91, ‘02, 1.—The Spermatocyte Divisions of the Locustide. Jbid., xi, 8. ’02, 2.—The Accessory Chromosome. Sex Determinant? Biol. Bull., ite Te 2p Montcomery, T. H., ’98.—The Spermatogenesis in Pentatoma, etc. Zool. Jahrb., Anat. Ontog., xii. ’o1.—A Study of the Chromosomes of the Germ-cells of Metazoa. ‘Trans. Amer. Phil. Soc., xx. °o4.—Some Observations and Considerations upon the Maturation Phenomena of the Germ-cells. Biol. Bull., vi, 3. ’05.—The Spermatogenesis of Syrbula and Lycosa, etc. Proc. Acad. Nat. Sci. Phil., Feb., 1905. Issued May 18, 1905. Moore AND Rosinson, ’05.—On the Behavior of the Nucleolus in the Spermato- genesis of Periplaneta Americana. Q. J. M.S., xlviii, 4. Pauimier, F. C., ’99.—The Spermatogenesis of Anasa tristis. Jour. Morph., xv, supplement. Stevens., N. M., ’05.—A Study of the Germ-cells of Aphis rosz and Aphis ceno- there. Journ. Exp. Zodl, ii, 3 Sutton, W. S., ’00.—The Spermatogonial Divisions in Brachystola magna. Bull. Univ. Kansas, ix, I. ’02.—On the Morphology of the Chromosome Group in Brachystola magna. Biol. Bull., iv, 1. ’03.—The Chromosomes in Heredity. Biol. Bull., iv, 5. Witson, E. B., ’05.—The Behavior of the Idiochromosomes in Hemiptera. Journ. Exp. Zodl., ii, 3. : 1Including only works directly cited in the text. A full literature-list is given in the works of McClung (02, 2) and Montgomery (’o5). * ae he meee el, » VARIATIONS AMONG SCYPHOMEDUS£:.! BY CHAS. W. HARGELT, Witu I PiLate AND 17 FiGuREsS IN THE TEXT. During the course of a study of variations among Hydrome- dusz in 1901 the present writer became interested in facts of a similar sort which came under observation incidentally as certain specimens of various Scyphomedusze were observed. Further- more, during the course of extended studies in the development of Cyanea additional facts of a very interesting kind were observed. Still later in connection with experiments on regeneration in Rhizostoma pulmo (’04) other facts bearing more or less directly upon the same general problem were accumulated. It is the purpose of the present paper to present a synopsis of the facts and to attempt a statistical exhibit of certain features of the variations observed, as well as an analysis of the data with a view to determine something of their bearings on the general problem of adaptation. MATERIAL. Most of the material was obtained at Woods Hole during the summers of Ig0I, 1902, 1905. Most of the ephyrz were obtained in April and May 1902, and in March 1904. The adults were collected by the writer at various times during these years in part, and in part by Mr. Vinal N. Edwards, collector for the laboratory of the United States Fish Commission, for whose kindness. in turning it over to me for investigation it 1s a pleasure to acknowl- edge my grateful obligations. I was also permitted to examine a collection of about two hundred specimens of Aurelia collected by Mr. Geo. M. Gray, curator of the Marine Biological Labora- tory supply department, for which courtesy my thanks are due. he material was preserved for the most part in 5 per cent formalin. That of my own collecting was preserved in formalin 1Contributions from the Zodlogical Laboratory, Syracuse University. 548 Chas. W. Hargitt. after treatment with the chromic acid method suggested by Browne, to the excellent results of which I am glad to certify. My thanks are due to my son, George T. Hargitt, who has drawn most of the diagram sketches. AURELIA FLAVIDULA. The general facts of variation, or abnormality, as formerly regarded, i in species of Aurelia have long been known. It is no part of the purpose of this paper to review in detail the history of observations along this line, yet it may not be amiss to cite some few of the more noteworthy among them. In a recent paper, “Uber Hypomerie und Hypermerie bei Aurelia aurita Lem.,” Ballowitz has given a brief summary of the more impor- tant literature. Von Baer’ seems to have been among the first to record obser- vations upon the several numerical variations in Aurelia aurita, and to point out certain correlations noticed, as well as their absence in some cases. According to this author the variation was estimated to be about Io per cent. Of more critical character are the observations of Ehrenberg? in 1835. This naturalist in an extended paper “Uber die Acale- phen des rothen Meeres, etc.,” reports with considerable detail upon variations observed in this medusa, and illustrates by numerous figures the more conspicuous aspects of the problem. He was able to confirm the earlier observations of von Baer, and considerably to extend them. Among many thousands of specimens casually noticed, and several hundreds examined with care he records having seen but two specimens of octamerous division of the gonads and comparatively few having a three-, five- and six-merous character. He records a single specimen observed with but one circular gonad about the mouth, which he considered to have been the result of a fusion of three or six single organs, as there were several openings into the stomach. In a case having double gonads he considered the condition to be due to a similar fusion of six organs as there were six openings distinguished. Like von Baer, this observer also recognized a 1Uber Medusa aurita, Meckels Archiv f. Physiol., Bd. viii, 1823. ?Abhandl. d. Kénigl. Akad. Wissenschaften, Berlin, 1835, S. 199-204, 1837. Variations Among Scyphomeduse. 549 more or less perfect correlation among the several variable organs of the medusa, but also cites and figures an exceptional case in Taf. I, Fig. 12, in which in a rare octamerous specimen there were found fourteen rhopalia and twenty-eight principal canals, instead of sixteen and thirty-two respectively, as required to complete the symmetry. According to this author the ratio of variation was estimated as about 10 per cent (though Agassiz in a following quotation seems to have overlooked this point in Ehrenberg’s work). It is not specified as to \ hether this includes the totality of variations, or refers to those of the vegetative organs. If the jormer it was probably too low; if the latter probably too high, as will be seen in the following data: Haeckel! has recorded numerous facts of variation among European species of Aurelia and suggested their significance in relation to problems of evolution, though in his earlier account no details were given. In a later contribution’ he has, however, discussed the problem in much detail, not only in connection with the adults but also in relation to the embryonic development, from segmentation and gastrulation on through the several transi- tional stages—scyphostoma, strobila and ephyra—up to the adult, giving excellent figures and descriptions of typical examples. This author strongly | maintains that numerical variations sus- tain intimate relationships throughout the entire ontogeny, and quotes Claus as holding similar views. “Ich nehme mit Claus an, dass alle Zahlenabnatanititen der reifen Aurelia schon bei ihrer Ephyrula-larve auftreten, und dass diese letztere sie bereits von ihrer Scyphostoma-amme geerbt hat. Wenn also Scypho- stoma nur 2 gegenstandige Tzeniolen besitzt, so zeigt ihre Ephy- rula nur 2 gegenstandige Filamenten und die reife Aurelia spater nur 2 gegenstandige Gonaden.” In connection with the recent interest in the general problems of variation several brief accounts have appeared in reference to this medusa, but only two have gone into any details as to facts, or undertaken any analysis of them, namely, Browne* who in several contributions has discussed with pains and ability a very large number of observations made upon both adult and ephyre; 1Das System der Medusen. Jena, 1879. 2Metagenesis und Hypogenesis von Aurelia aurita,S.26. Jena, 1881. 3Quart. Journ. Mic. Soc., vol. xxxvii, pp. 245. Biometrika, vol.i,p.90. 1901. 550 Chas. W. Hargitt. and Ballowitz,! who has devoted attention chiefly to adults, giving excellent figures of noteworthy variations, and includes also a valuable review of literature. In view of these rather extended observations on the part of European observers and the almost entire lack of similar study of American medusz it has seemed to the writer for several years that a comparison of Aurelia flavidula with Aurelia aurita might afford valuable results. And with this in view the data presented in the following pages have been worked out at such intervals during the past three years as have been available. I regret very much that a larger number of adult specimens have not been available, the hope of securing which has delayed the final appear- ance of the paper. It is believed, however, that sufficient data are presented to show at least something of the extent and sig- nificance of the variations. As already intimated, almost nothing along these lines has been attempted in relation to American Scyphomedusz, while the summary of observations by the present writer is all that has been attempted on the Hydromeduse. L. Agassiz? has left a few very brief and rather indefinite records of variation in Aurelia flavidula. Concerning numerical variation he says: ‘These variations in number arise from the interpolation of similar parts, or from the abortion of some of them. I have observed on our coast specimens with three, five, six and seven crescent-shaped bodies, and the number of indentations along the margin increased correspondingly. ‘These deviations from the normal number are rare with our species, and though Ehrenberg does not allude to their frequency in the European, I should infer that they are more frequent in Aurelia aurita than in Aurelia flavidula, for the simple reason that malformations of the crescent-shaped bodies are rarely met with in our species.” As will be noted, there is apparent in these observations of Agassiz, the same general assumption of a more or less close corre- lation among the paca organic systems of the medusze. I am inclined to regard this as in some measure due to a temperamental predisposition on the part of these earlier observers, perhaps growing out of the peculiar ideas in reference to such matters 1Archiv. f. Entwickelungsmechanik der Organismen, Bd. viii, S.239. 1898. Contr. Nat. Hist. U. S., 1862, vol. iv, p. 51. Variations Among Scyphomeduse. 551 more or less prevalent at that time. Certain it 1s, that either the more recent work on these lines have been more critical and discriminating, or a remarkable change has taken place since the earlier records were made. Possibly something of both may be true, though I incline to regard the former as more probable. My investigations have been directed chiefly to two series of facts, namely: Variations as exhibited in the ephyra, and those found in the adult of Aurelia flavidula. Incidentally I shall direct attention briefly to certain other species which have been studied in connection with those of Aurelia, though of these no details will be undertaken, since in ohly a few cases have sufhicient numbers been examined to warrant any general conclusions. Being strongly convinced of the general correctness of Haeckel’s view as to the relations of variations of the adult to similar con- ditions found in the larva, or ephyra, 1 it seemed desirable to secure collections of specimens from various localities differing more or less in physical conditions of environment in order to estimate the probable influence of such conditions in relation to variation. Accordingly I secured ephyre from three localities adjacent to Woods Hole, in about equal numbers, approximately 500 from each. (Unfortunately I was unable to obtain adults from the same environments, since their locomotor powers, influence of currents, winds, etc., carrying them every whither, made this quite impracticable.) As is very well known, the Discomedusz are characterized in general by the octamerous lobing of the umbrellar margin, cor- related with which are eight rhopalia, or sensory bodies; and by the tetramerous form of the stomach and oral arms. ‘This is more particularly the case with the semostomous group, to which belong most of our larger medusz, of which Aurelia and Cyanea are good examples. As will be seen, therefore, the organization of these medusz, leaving out of account the tentacles, which differ greatly in the several genera, presents two fairly differentiated and independent sets of organs, namely, the marginal or sensory, and the central or vegetative. In many cases these sets are correlated for nutritive purposes through the radial canal system, though of themselves they may for the present discussion be considered as independent systems, distinctively organized and definitely correlated, as 552 Chas. W. Hargitt. indicated above. In keeping with this view we may naturally proceed upon our analysis and comparisons under two heads: (1) The marginal system; and (2) the nutritive and repro- ductive, or vegetative system. Concerning the canal system nothing will be said in connection with the study of the ephyre, since during the early larval history this system is but slightly developed and therefore of but small consequence in relation to variation. Furthermore, since as already suggested the purpose is in part a comparison of the aspects of variation presented by the ephyra and adult, we have again a two-fold division of the subject. And since in the order of nature the ephyra precedes the adult this may as well be taken as the order of research. Variation in the Ephyra. Aside from the investigations of Haeckel (op. cit.), so far as I am aware Browne 1s the only investigator who has taken up this phase of the subject in detail. And furthermore, since hereto- fore investigation has been directed almost wholly to European species it has seemed to me highly desirable that similar observa- tions be made upon those of American waters, in order to have some broader basis of comparison and deduction. Marginal Organs. The ephyre studied are of two series, first those collected in 1901-02, and second, those collected in 1904. I choose to consider them in this detached way chiefly from the fact that the latter were just in process of metamorphosis into young Medusz, and it seemed better to study them with a view to securing possible evidence as to any ratio of selection which might be detected as occurring during this process. ‘The ephyra were all of Aurelia flavidula, except possibly a stray specimen of Cyanea which might have drifted among them. But these were exceedingly rare, if occurring at all, since an examination of several hundred of the series of 1904 in process of metamorphosis failed to reveal the presence of a single specimen. Moreover, since in an earlier study of the development of Cyanea I found abundant evidence of similar variation, the presence of an occasional specimen in the estimation of percentage variation could hardly affect the results. Variations Among Scyphomeduse. 553 Of the first series a total of 1512 specimens were examined. Of this number 398 or 26 per cent showed variations in some one or more of the organs. Of specimens having less than the normal number of marginal organs there were but 19, or 1.25 per cent. Of those having more than the normal number of these organs there were 379, or 25 per cent. Details concerning these data are given in tabulated form in the following tables. Tas_eE I.—Showing Correlations of Marginal Lobes and Rhopalia. RHOPALIA. Male Colette] 8 Que | TO" emerge 2: | Te |e 4: | TorTaLs 5 I | ! I 6 I | | I 7 15 13 28 a 8 Salhi aime Wall Calls | 1121 (Q | | ° 4 | || = g | 7 | 206} 4 | | 217 < | | Z | | || S 10 |} 2/84] 1 | 87 < | = see ee EN(y U II ig cath oh 5 BES 30 12 | | 3 13 | 16 13 I | 8 9 14 | eel 2 Torarsiier | \o a 17 1134 204080) 1.33 | 13° | Siena L512 ‘Table I presents in graphic form the correlations existing between the marginal lobes and the rhopalia. In the vertical column at the left are given the number of marginal lobes rang- ing from 5 to 14, the smallest and largest number respectively found in any specimen. In the horizontal column at the top are 554 Chas. W. Hargitt. given the number of rhopalia, while the number of specimens are arranged in the squares. While in general there is a very close correlation between these organs it will be seen that there are not infrequent departures from this rule, or in other words absence of correlation. For example, two specimens were found having only seven rhopalia while there were eight marginal lobes. Like- wise there will be seen to be five specimens having more thopalia than marginal lobes in normal octamerous individuals. A simi- Tas_e I].—Showing Correlations of Oral and Gastric Lobes. ORAL LOBES. 2 ay | et 5 6 7 |) Tomas | 2 I I wo 3 I 3 - Q Q | | [o) | 7 4 2 474 | 476 1S) | | fe — a 5 I I 9 | 6 3 3 | ae ey I Torars | 1 | 3 Se 477 T 3 is 486 lar condition of variation is shown in specimens having seven, nine, ten, eleven, and twelve marginal lobes and rhopalia. A curve constructed by which to portray even more graphically the facts would involve the following factors :! Marainat Loses. RHOPALIA. Meant vaiiatton sy niy ac snes sicaerensio a erste tee Cision ante SR EEE EIEN 8.396 8.379 Standard/devratiom sta «sm csc eracotoe cone oe sisiete leo ciel erate eee Cerone -896 872 Coethcientiofivariability: Camas shines cia eecletenetstascie oe aoe ia: 1.06 1.04 Probableverronvoksmeant ns tiaeieiciaaiciel eccistacs sore oelerverseciet tee ere eee a5 don'ts + .002 Probable\errorjofistand ard) deviationses 1.9. een ==) .O01 st O11 Average’ deviations -9.\5::cieip crite tiers Sctste wicca soe icinn ie tad Sea ee Teter acre -626 641 1For calculating the factors of this curve I am under obligations to my colleague, Dr. Smallwood. Variations Among Scyphomeduse. 555 A study of the table will naturally give rise to the question as to how the several variations in these marginal organs are to be explained. For example it will be observed that twenty-three specimens had more rhopalia than marginal lobes. A reference to Figs. 1 to 3 will quickly afford an explanation of this phenome- non. The figures also show double and twin rhopalia, several cases of which were found. Similar cases are cited by Browne (op. cit., p. 90), and in connection with their discussion he ven- tures the suggestion that perhaps in later development and during metamorphosis, by a growth of the margin these so-called twin rhopalia may become separated thus giving rise to an independent lobe. This seems to me to be extremely doubtful. As a matter of fact it is well known that during growth following metamor- ‘Maul Fig. 1. - Diagram of marginal lobe showing twin rhopalia. I. Fig. 2. Compound marginal lobe, one of which contains twin rhopalia. Fig. 3. Compound marginal lobes the central one notched at the tip. phosis, increase of the marginal dimension takes place entirely by growth of the inter-rhopalial areas. ‘This is very easily seen by a study of the appearance of new marginal tentacles and by the origin and multiplication of the branching canals. It may therefore be accepted as practically certain Ten the twin rhopalia of the ephyra continue such in the adult medusa. It is also almost certain that a similar condition 1s involved in the case of such double rhopalia as are shown in Figs. 3 and 4. In these respects, as in others, as cited by Haeckel (op. cit.) we may, I believe, regard it as undoubtedly true that the larval variations are carried over into the adult through the several phases of meta- morphosis. Furthermore, this view is confirmed by the facts clearly established, that the ratio of variation found in the adults is essentially the same as that found in the ephyre. 556 Chas. W. Hargitt. As suggested above, it seems reasonably clear that the excess of rhopalia may be accounted for in the manner already proposed. But it remains to consider those cases in which the number of marginal lobes is in excess of the number of rhopalia. Of these there were found seventeen cases, as against twenty-three of the former. It is quite obvious that for these a different explanation must be found. We are here limited to two alternatives, namely, either there are cases in which for some reason there has been a failure of a given lobe to develop its usual organ; or on the other hand there may possibly be a subsequent and independent origin of an extra lobe. While I have found undoubted cases of the occurrence of the former condition, and am constrained to regard it as the more usual and probable explanation, at the same time I have found an occasional case in which a belated lobe appears to arise after the ephyra has become free from the strobila, but at the same time it must be admitted that in these cases there is usually found the accompanying rhopalium, though this is not always true. I am therefore constrained to consider both alter- natives as possible, though giving to the first the larger probability. A few unusual features in the marginal and rhopalial variations call for a merely passing notice. Fig. 1, showing an ordinary twin thopalium, calls for little note aside from the statement of fact that it plainly occupies the position and relation of a typical organ, namely, the terminus of a single canal. And in this connection it may be well to recall that in the early ephyra-life all the canals are simple, that is, unbranched. It is only during the progress of metamorphosis that the complexity of the adult canal system is gradually differentiated. Figs. 2, 3, show a series of extremely interesting variations of graduated complexity. ‘The first shows a trifid condition of the © otherwise normal ephyra lobe, though with the added abnormality of twin rhopalia in one of the notches. In the second there is shown a quadrifid lobe, the lappets of the median pair being small and not particularly remarkable, while in each notch of the outer lappets is a normal sensory body. Several other figures show similar features. Among 486 ephyre taken in the “eel pond,’ Woods Hole, in April, 1902, the variants numbered 144, or 29.6 per cent. Variations Among Scyphomeduse. 557 *Fig. 4. Hexamerous ephyra, one lobe of which is compound. Fig. 5. Hexamerous ephyra, with two-lobed mouth, and two gastric lobes. Fig. 6. Ephyra with small supernumerary rhopalium, several examples of which were found. Fig. 7. Ephyra with an adradial rhopalium, and one of the normal lobes devoid of an organ, perhaps due to injury. 558 Chas. W. Hargttt. TasLe III. No. Specimens. | Gasrric Lopes. | Ora Lopes. RuHopatia. MarGiInat Loses. I 2 | 2 6 6 I 3 3 7 7 I 3 + i i 2 3 4 9 9 I 4 3 IO if) I 4 | 3 12 12 78 4 | 4 9 9 I 4 | + 8 9 35 if vn 10 | IO 2 4 4 9 10 8 4 4 | II II 7 4 4 12 12 I 5 5 IO ie) I 6 | 6 | II II I 6 6 | 12 2 I 6 6 13 13 I 4 4 | 14 14 I 7 7 10 10 | | | Each of these rhopalia occupied a single octant of the ephyra margin, and differed but little in size from the others. It should be stated that each was found on a different specimen. In two specimens were found a very rare feature among these varied rhopalial phenomena, namely, the presence of a rhopalium in an iterobular, or adradial position, as shown in Figs. 6, 7. One of such cases I also discovered in connection with the study of the development of Cyanea. Here we probably have the origin of the condition which eventuates in the equally rare occur- rence of an adradial rhopalium in the adult medusa, cases of which will be considered in a later connection. Oral and Gastric Organs. As compared with the marginal system that of the vegetative shows comparatively little variation, at least in the ephyra stage, though the present data are far from complete. In the first place Variations Among Scyphomeduse. 559 the number of specimens tabulated was less than one-third of the entire number in the preceding series. ‘This is due in part to the poorly differentiated stage of these organs in the early ephyra, the gonads being entirely lacking, and the mouth-lobes being often so coutmieted as) to geile certain determination im- possible. In Table II is shown the range of variation so far as accurate data are at command, including as in Table I divergencies or lack of correlation between the two sets of organs. As will be noted, out of a total of 486 specimens, only 12 or 2.68 per cent vary from the normal. In several figures are shown illustrations of these aspects ofour Fig. 8. Ephyra with seven gastric and oral subject. In Fig. 5 is shown 4q__ lobes, and ten marginal lobes. sketch of one of these, in which there are but two oral divisions and two gastric pouches. One might suppose the directly opposite relations of these organs as figured to be unusual, but when compared with Figs. 6 and 7 it will be seen to be quite in keeping with that found in almost every case, that is, the angles of the mouth correspond with those of the stomach so that the pouches of the latter of course occupy inter- mediate positions. In other words, the angles of the mouth occupy the perradui of the body while the gastric pouches or lobes occupy the interradii. A comparison of figures will readily show the lack of correlation of these organs with those of the marginal system, and at the same time the close correlations between themselves, the latter being likewise evident in the data of the table. ~ Additional data of a similar sort will be presented in connection with the second series, a comparison of which will still further emphasize the small ratio of variation as compared with that of the marginal organs. 560 Chas. W. Hargitt. A Comparison of the Variations Exhibited by Ephyre During Metamorphosis. During the current summer, 1905, I was able to secure a collec- tion of about 1000 ephyre from Waquoit Bay, a body of water some ten miles east of Woods Hole, from which had also been secured a portion of the previous series in 1g01. Among these were found 392 specimens which were just emerging into young medusz. ‘They varied in size from 7 to 14 mm. in diameter, the radial canals were well differentiated, and the gastric pouches easily distinguishable. ‘There were also 218 ephyrae among the number which were entirely devoid of any indications of meta- morphosis, indeed, apparently but recently escaped from the strobila. I was particularly glad to have an opportunity to study a series of this character from a strictly local environment and from the same brood, so to speak, since it afforded an opportunity to test a feature of variation already referred to, namely, whether varietal features existing in one stage are carried over into another, or whether during a period of metamorphism there was at work any selective processes. While the numbers examined in these cases are too small to afford conclusive data on a problem of this character, they may at any rate afford a fair indication as to probabilities, and when taken in comparison with similar series in larger numbers, as in the former case, and also in connection with the observations of Browne (op. cit.), they may become correspondingly more convincing. A comparison of the data presented in Tables IV and V will show, both in relation to the percentage variation and to the question of the persistence of varietal features during the several phases of metamorphism, rather striking points of likeness. So far as the ratio of variation is concerned it will be seen at a glance that it is so nearly the same in the several cases as to pre- clude the probability of anything more than slight and incidental differences. For example, in Table I the total per cent of variation is 26; in Table V it is 22.2; while in Table IV it is 22.9. Compared with the ratio obtained by Browne, which for 359 ephyre taken in 1893 was 22.6, and for 1116 speci mens taken in 1894 was 20.9, the results become still more conclusive. Variations Among Scyphomeduse. 561 OF. 218 ephyre taken at Waquoit Bay in April, 1904, there were 50 variants, or 22.9 per cent. The chief features are tabulated as follows: Tase IV. No. or Specimens.| Gastric Lopes. | Orat Loses. RHOPALIA. | Mareinar Loses. I 3 3 6 | 6 1 3 | 3 7 7 4 | + 6 6 4 | 4 7 | 7 31 4 4 9 | 9 4 4 4 IO fe) I 4 4 II II I 4 4 12 12 I 5 5 II | Il I 5 5 12 | 12 I 6 3 12 12 3 6 6 12 12 I 6 7 12 12 Now if we compare these results with those shown in Tables VI and VII, in which are presented in detail the variations found in 226 specimens of young Aurelia flavidula taken at Waquoit Bay in May, 1905, and in a collection of adults made at New Bedford about the same time it will be seen that they all tend to confirm the general propositions under consideration, namely, that varietal features found in the ephyra persist in the adult, and furthermore, that there is no evidence of any selective process involved during these several changes in ontogeny. Of 392 ephyrz in process of metamorphosis, taken at Waquoit Bay in April, 1904, there were 87 specimens, OL 22.2 (pet cent, which showed various aspects of variation, the principal features of which are classified in the following tabulated form: 562 Chas. W. Hargitt. TABLE V. Soe oe She RHOPALIA. Cana SystTEeM. SPeEcIMENS.| Loses. Loses. if iRermadialis.aee «ae Acree OY aie SI I 2 2 6 ; iI interacial s+. acer Oe. Wea : ; : 5 { Perradial ates eee js Cm a i interradiallee serra Ou 1) eiaenl . , ; . if Ronee Bo esc Gime Tt i interzadialieeee eee Tt. Sie ater ‘ 7 | A | ‘ J Sa Fas oboe. as tren oe ge I Imtexradiallmanmee eee Coyne ily ie f Perradialiee ne eeriee 2 Th TI 45 : 4 ? i) Intermadiall= seer joe in - i | ‘ e J ene BAR SEs ah ee Dy ne \ Imternadia] ere moa oT Fe r r i i Perradiall. serene PAG Gil i Interradialleas sie eee oi : 4 P Ps if iPerradialaem ace yy I \ InteradialiSss--eee 0: 1. We sigur ; a if Pernadialaeeereeerae Dy PD si a) + \ interzadiallcersce Wy yee Vals eel : i i i f iRerradtalleesr cere yy TT I\ intergadialleegsererrr 1: (Te eteas : i Pi 3 f Perradial ico creche yeah SI 3 \ Imtemadialle. see eeeee ace iy : ‘ i ne { Peradial Me Mosc yao a gee) Imteradiall secs Cheat vee era dial eyelet nees ay elaecl 2) : 4 = ‘ Interradiallee reece i ie 2 6 ; Ne f iPernadialweaeieeserere 7 Th iy oi \ Enterradialles-tceeee rote oe or 6 6 6 ee Lf noe seve teers pe ee ‘h Interxvadialleci.-t eee oer t pt mt ot 5 6 6 é IJ parent ae eee Verein i Interradialles . eer farsit WG it ill Permradial er ceesene ee Be BHAA) EN : 3 p "3 Iinterradialle.). eee May evike tie Vil If Rermadiallt sae ete viayiG i ; ° 5 2 i\ Interraditall yee je eee I I 6 6 13 Thid. I 6 8 (double) 15 [Gye Bigs; ; i 8 8 - if FRmEChe Sean Sec ©.) ae een iN nterradialleece eee. Or olen Ge 36 STi eye Shi Variations Among Scyphomeduse. 563 Variations in the Adults. As in the study of the ephyre attention was directed chiefly to the marginal or sensory bodies, and to the central or vegetative, so likewise in the study of the adults the same systems have received primary consideration, though in the latter including also the canal system, as a correlating medium between the others. Attention was also directed to the | problem of the probable influence of local conditions in determining variations. As favorable localities from which to secure specimens more or less subject to a definite environment, New Bedford harbor and Waquoit Bay were selected, the latter serving moreover, the further end of ascertain- ing the variations exhibited by ephyra and adults under the same environment. In addition to authorities cited in a previous part of this paper attention may be directed to the observations of Bateson and Romanes on the variations of Aurelia aurita. ‘The latter has described in some detail variations found in this medusa and has illustrated by diagrams many of the features described. In both the illustrations and the analysis of the facts there is an apparent effort of the author to reduce the variations to as few symmetrical types as possible. As I have pointed out in another connection in reference to the work of Ehrenberg and Agassiz, these attempts to discover a law of symmetry, or perhaps better in modern phrase, a law of regulation, in the diverse variations encountered, have apparently been only partially justified. While it is doubt- less true that in many cases, perhaps in a majority, some form of regulation may be distinguished, there are too many cases in which this is lacking to be considered as merely exceptions to such a law. A study of the following facts and illustrations, will I believe justify this view. As Bateson in commenting upon Romanes’ work has remarked, “Tt is impossible in regular threes, sixes, etc., to say that any particular segment is missing or added rather than another.” And if this be the case with an organism like Aurelia, in which the several organs are so sharply differentiated as to be easily distinguished at a glance, it is much more likely to be true in organisms of more complex structure and less sharpness of differentiation. In an attempt to ascertain the comparative frequency of certain 564. Chas. W. Hargitt. variations in Aurelia, Bateson examined 1763 adult specimens taken on the Northumberland coast in 1892. In the tabulation of his results he presents details of only the gonads and oral lobes. Of these there were but 28 abnormal specimens, or a variation of only 1.6 per cent. Of the 28 abnormal individuals 19 he considers as ““symmetrical varieties,’ and observes that the other 9 speci- mens, or 33 per cent are “irregular varieties” and are seen “for the most part in single specimens only.” Here Bateson apparently falls into the same error which he has criticized in Romanes, namely, the attempt to reduce the variations to ‘symmetrical varieties,’ regarding “irregular varieties” as exceptional. But the presence of 33 per cent of the so-called “irregular varieties” is too large a proportion to be designated as exceptions. As is well known Aurelia is an octamerous medusa, each octo- mere being characterized by a single, more or less dichotomously branched radial canal, at the terminus of the central stem of which is located the sensory body, or rhopalium, and separated from the adjacent octomere by an unbranched canal, as shown in several of the diagrams. In normal individuals this arrangement is very symmetrical, and easily distinguishable. It must not be inferred, however, that the several branching canals are exactly similar, or symmetrical. Indeed it may be safely said that probably no two in a given individual are exactly alike, any more than are two leaves of a given plant. Still, the differences are usually slight, striking variations occurring chiefly in those cases where departures from the typical arrangement are considerable. For convenience in following readily the subsequent discussions, it may be well to briefly remind the reader that for descriptive purposes the several canals have been designated by the special names, perradial, signifying those canals arising between the gastric pouches, or mouth angles; interradtal, indicating those occupying intermediate positions, or emerging from the outer median portion of the gastric pouches; while the term adradzal refers to the unbranched canals alternating with the other two series. Gastric and Reproductive Organs. Among the most conspicuous variations from the typical con- dition just described are those involving a numerical, or meristic departure. This will be readily understood when it is remembered Variations Among Scyphomeduse. 565 that these organs are large and conspicuous, four in number, and usually the first to attract attention. It is doubtless on this account that so many of the earlier observations concerning variation in these medusz dealt almost exclusively with this feature. Among the commonest variation is the hexamerous form, where there are six each of the gastric lobes, gonads, and oral arms. This will be observed at a olance by comparing the several tables, especially Nos. [V and V. Next in frequency is the pentamerous type, where there is a symmetrical arrangement of the organs upon the plan of five. As the several details of these variations are specified so far as their numerical aspects are concerned it is only necessary to refer to the tables already cited. It may be well to notice briefly a few features not capable of tabulation. Among these are the not infrequent occurrence of signs of atrophy, as shown in Figs. 11 and 12. In the former it will be observed that associated with the small size of the pouch and gonad is the entire absence of the interradial canal and its marginal organ. In the latter will be observed the presence of a mere rudiment of a regres- sive gonad in one of the pouches, while in the opposite compound pouch there are two gonads, and in this case the absence of the perradial canal system. Associated with variations 1n the number, is that of variation in the size and relations of the organs, as already pointed out in the figures cited. Attention was directed to the compound character of the organs. ‘This 1s a very common occurrence, and probably is indicative of the manner of the origin of supernumerary organs of this character. However, the pentamerous and hexamerous condition is frequently distinguishable in the ephyra, and seem to be quite distinct from the beginning And I have found in Cyanea that occasionally trimerous polyps occur, and probably give rise to trimerous ephyrz and later trimerous meduse. It may not be improbable that the suggestion of Ehrenberg (op. cit.), that the circular gonads which he observed were the result of fusion of what may have been earlier distinct organs, is quite as likely as that above. It may be suggested in this connection that I have never seen a case such as that cited by Ehrenberg, though its occurrence does not seem improbable, but in every case which has come under my observation of a compound gastric pouch, the gonads have been more or less distinct, as indicated in Fig. 12. 566 Chas. W. Hargitt. Of 226 small adults collected at Waquoit Bay, May, 1905, there were 55 variants, or 24.3 percent. The general features of variation are tabulated as follows: TasB_e VI. OBERT a | eee ESSE LS Cana System. | J Perradial arts Tt gr : 4 3 : | Interradial aoe ee Goi eT J Perradial Saeucrane oy al he a : + | 4 | y | Interradial “at ne Sgr ie J Perradial vets eT Or a 3 4 4 | \Interradial ... Ones ea | J Perradial eae at tte eE a8 | 4 9 | Interradial es OD Se | | J Perradial onat et ea gc 7 4 4 9 \Interradial . eR aes | Perradial 2... 2. Pt LD 5 | 3 2 ua | Interradial ae oN iinet Re | | Perradialis.5-5 2 Pees) Sy 2 4 4 am eae es Pui, Et Penradialccen elie a a : 4 | 4 ex ee aa yer a! 20 | Petradial.. 0: Sp yer ; 3 . | eae Sis | eh a a ie iS Perradraly..2 55 feo Ts) 3 et ; : : + ae Rae a a ee Variations Among Scyphomeduse. 567 Of 129 large adults taken at New Bedford in May, 1905, there were 29 variants, or 22.5 per cent. The chief features of variation are shown in the following table: Tas_e VII. | | No. or | Gastric Orat | RHOPALIA. CANAL SyYsTEM. SpeciMENS.| Lopes. Lozes. | | | Rerradialeeeeees ler I 3 3 6 ; Interradral) 2... - ame ert Penradial peer jinn y eee 2 4 4 7 lbnwerrachiall 5.5.6 - Gb ile je | Petradial =e oe Ah 9 SS cit I 4 4 ; 3 | 7 Interradial 7 -< :. ie Soy Perradial 335-525 Te we Tat ol 2 4 4 9 aetna 1-1) en DT) SOT eae Renradialy == oe IN), TH I 4 4 ie) : ntennadiall see i a a A Perradiall =... ee Age Me il I 4 4 IO Imterradiala see tat a enacts ee i a et I 4 4 IO a Anternadiale eee 2 2 { Perradial Beek os a) i 2 2 4 ATE < : Interadials.. 52. ee iets el Rerradialaee nee 210 2 2 4 4 12 Interradialeee es ij eae wee 81 |Pesaraghiall o 45 oo 2 2g Gal I 4 4 i372 i Intennadiale 5s Seo Ci See (~ . 6 6 Pereachiall 55.2426 Gu inh am ih Ti i II ; Interradial 5545 rgb ble eel enradialaaa eee je OR es hee 2 6 6 12 : | Imterradial +2... . LD) bal le I have frequently been able to confirm the observation of Bateson, (op. cit.), that where there are cases of marked dif- ference in the size of the gonads of a given quadrant or of adjacent quadrants, that there is frequently a noticeable decrease in the size of the corresponding portion of the bell, as shown in Blate aie: ‘5. 568 Chas. W. Hargitt. As compared with the variations occurring in other organs, par- ticularly the marginal organs and canals, the per cent is extremely small, as will be seen at a glance 1 in comparing the several tables. My observations on this point confirm those of both Bateson and Browne. ‘The former found but 1.6 per cent, while the latter found it as large as 2.4 percent. My observations gave the average of 2.75 per cent, as the total variations to be detected in Aurelia flavidula. As already suggested in connection with Ehrenberg’s observa- tions, in which he claimed that variation reached Io per cent, either this must be taken to include the total, in which case it is evidently too low, or if it refer to the vegetative organs alone it is certainly too high, unless indeed it may be possible that the Aurelia aurita of the Red Sea differs very greatly from the species in other waters, or from our own species. Rhopalia and Radial Canals. An examination of the several tables will show that there is a general variation in the direction of an increase in the number of both rhopalia and radial canals. ‘This has been shown to be the case in Aurelia aurita by both Ballowitz (op. cit.), and Browne (op. cit.). While confirming for the most part the results obtained by both these observers, there are points of difference which must be reviewed with some detail, and other points wherein I am unable to accept the conclusions of either in all par- ticulars. Some of these will be considered in their appropriate connections. Concerning the number of rhopalia little need be said further than to direct attention, as above, to the tabulated facts. 1> se 587 Tie Descriptionvof the) Culturesiy oe ye ereleieielel loin ims) oe l= « wieinle =) =1eleia /elaieiel=)/s1e\e/ 1c ile wie w1eiei'e 590 foe Oxytricha fallax, Culture Atwastetetetseeie lee 1 1o(eiele |= «1 lelelefeleleelicin'els = /~/0\=/elsin)sil>\ 2 590 Pee Oxytrichartallaxs| Culture ws sperterewetete es ele tose later =1eleelnieve =fo)ellaletevete)sialelef=\-/sin)> =)=t-1-\=1= 594 3. Pleurotricha lanceolata, Culture A. 2.2.2.2... 2s eee e cece cere eee enter cence 594 4. Pleurotricha lanceolata, Culture Be 22... 2... . 0.52 s ewe nee e cent cee c ences 596 5. Gastrostyla steinii, Culture A. ........-.-.eee eee e eter e rere cette ence eenece 596 VAN Discussion) obsthey Data onthe Cwleiress mjslerley=lelets ler syeial =i) oie eialal)~/= 622 Bape Comparison, Of: RESUltssraeraleie1< esegel ela! «Ieirlsio’ sie <\ele/*1 ele(nsn)s1=lo}eieje\elie|-(n\«ls «1+ leo» «ief=l= 622 VII. Effect of Light on the Rate of Division. ............-...2esee cece cece cee eccececoee 625 \ltl,,. Suimnengs Seeanissadcodbooocodocsancods6o0D50E Pepe ETA eet eres iere steha| sens heraiakeleretare 626 I. INTRODUCTION. The first suggestion of the cyclical character of the life-history of Infusoria was advanced by Dujardin as an argument against Ehrenberg’s theory that the Protozoa, because of their simple organization and method of reproduction, are not subject to natural death. The observations of Biitschli (’76) and Engel- 1§ubmitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University. 586 Lorande Loss Woodruff. mann (’76), that in infusorian cultures after a number of genera- tions the organisms are reduced in size and show other signs of degeneration, were evidence in favor of Dujardin’s theory. As is well known, however, Weismann (’84) greatly elaborated the theory of the potential immortality of unicellular organisms, maintaining, on a priori grounds, that the Protozoa, like the germ-cells of higher forms, are not subject to natural death. Maupas (’88; *89) in his classic researches on the life-history of Infusoria brought forward data which weighed heavily against Weismann’s hypothesis. In his long-continued cultures he found marked evidence of “senile degeneration” and he confirmed the eneral conclusion of earlier workers as to the cyclical character of the life-history of certain species. More recently still Joukow- sky (‘98) and Simpson (or) have investigated the life-histories of various forms, and Calkins (’02, 1, 2, 3; 04, 1) in a series of papers on Parameecium has submitted strong evidence that this species passes through more or less regular periods of vigor and weakness, the periods of weakness invariably ending 1 in the death of the culture unless the organisms are “stimulated” by conjuga- tion or by changed environment. ‘This work, besides throwing light on the 45 of conjugation in the life- -cycle, gave the fee experimental proof that various stimuli will “ ‘rejuvenate ” the lagging functions of exhausted protoplasm and incite the Para- moecia to further periods of reproductive activity. In the light of the previous investigations on the physiology of Infusoria, the following questions seem to be of sufficient impor- tance to warrant still more extensive experimental work on dif- ferent forms, in order to place the problems involved on a broader foundation: 1. Does the life-history of Infusoria, in general, run in cycles? 2. If so, will changes in the environment bring about renewed activity during depression-periods? 3. Will conjugation effect “rejuvenation” ? 4. What are the physiological and morphological changes, if any, characteristic of declining vitality? 5. What effect has initial and daily application of various stimuli on the division-rate, 7. e., on the metabolic activity of protoplasm? 6. Is the division-rate affected by light? The present investigation is an attempt to answer these ques- The Life-History of H ypotrichous Infusoria. 587 tions as far as possible for hypotrichous Infusoria. With this in mind, experiments on five cultures of hypotrichous Ciliata, includ- ing Oxytricha fallax, Pleurotricha lanceolata, and Gastrostyla steinii, have been carried on during the last three years. Antici- pating the conclusions, it may be stated briefly that the experiments offer affirmative evidence upon the first two points and negative evidence upon the last, while owing to failure of the infusorians to conjugate there is no evidence upon the third point. Regarding the fourth and fifth points, it may be said that morphological changes, particularly such as concern the cytoplasmic and macro- nuclear structures, are characteristic of declining vitality, and that initial and daily stimuli have a marked effect upon the metabolic activities of the forms studied. I take pleasure in acknowledging my great indebtedness to Professor Gary N. Calkins, at whose suggestion this investigation was undertaken, for his advice and criticism throughout its prosecution. I also wish to express my thanks to Professor Edmund B. Wilson for many helpful suggestions. II. GENERAL METHODS AND TECHNIQUE. In the experiments on Protozoa here described, which have been followed continuously for the past three years, I have employed, with but slight change, the method used by Calkins (’02, 1) which is itself an improvement on the method of Maupas. As this method is described in detail by Calkins, a brief outline of it with my own modifications will suffice. The organisms were cultivated on slides having a central cir- cular concavity with a capacity of about five drops of water. Cover-glasses, used by Maupas and Calkins, were not employed, as it seemed to me that a more natural condition was obtained without them, and as I found that unless great care was exercised in cleaning the slips they afforded a possible source of contamination. The slides were kept in moist chambers to prevent evaporation of the preparations. ‘These were ordinary stender dishes about ten inches in diameter and three inches deep. In the bottom of the dish was placed about two inches of wet sand. Over the sand was placed a glass plate on which rested four parallel strips of glass and on these the depression slides with the Protozoa were arranged. [he whole was covered with a ground-glass top. 588 Lorande Loss Woodruff. The Infusoria were handled with a pipet drawn out to a fine point. Each pipet was used for one purpose and only one. All of the Infusoria employed are of sufficient size to be seen readily with a lens having a magnification of about ten diameters, and as it is far more easy to operate with this than with a compound micro- scope, it was used almost entirely in transferring specimens with the pipet from one slide to another. At first hay-infusion was employed as a culture-medium, but later it was found that an infusion of fresh grass gave equally good results and had the advantage that one kind of grass could be selected and used to the exclusion of all others, thus securing a more uniform culture-medium. ‘The infusions were prepared as follows: About three grams of grass or hay was washed in tap- water and then placed in a beaker containing about 200 cc. of tap-water; this was boiled for one minute. In most cases this infusion was used shortly after it had cooled but occasionally it was allowed to stand for twenty-four hours. Except at certain periods of physiological depression and during certain experiments, to be described, this type of culture-medium was used throughout the work. As pointed out by Bitschli and Calkins, Maupas’s method was inaccurate in that he assumed the rate of division of all individuals of a culture to be the same and allowed a large number of speci- mens to accumulate before computing the number of bipartitions. Protozoa, like all other animals, have their individual physiolog- ical peculiarities, as is shown by my own and similar experiments. In order to obviate this source of error as far as possible and to exclude the possibility of endogamous conjugation occurring in the direct line of the culture, one individual from each line of the culture was isolated almost every day. In the great majority of cases not more than four individuals, representing two genera- tions, were present at the time of transference. At each icglaen the single infusorian was put in fresh culture medium, the remainder being kept as a reserve, or “stock,” in case, through accident or otherwise, the individual isolated did not live. Following the earlier workers, the maximum and minimum tem- perature of the laboratory in the vicinity of the cultures, as recorded by a registering thermometer, was noted daily. ‘This is, of course, but a rough method as the temperature within the moist chambers is more constant than in the room itself; still, it The Life-History of H ypotrichous Infusorta. 589 gives the greatest variation which could possibly occur, and by averaging the maximum and minimum points of each day for ten- day periods the result is quite satisfactory for comparative work. For the purpose of following as closely as possible the changes in cell structure during the life of the cultures, permanent prepa- rations of individuals 7 different lines were made from time to time. Here again I employed with little change the method used by Calkins, which is briefly as follows: ‘The specimen to be preserved is isolated by means of a fine-pointed pipet on a clean depression slide (which is kept just for this purpose) with as little of the culture-medium as possible. ‘To this is added three or four drops of bichlorid of mercury in saturated solution with 5 per cent of glacial acetic acid. After about five minutes the specimen is transferred to another slide and a few drops of 75 per cent alcohol is added. A slide is now smeared with a trace of egg- albumin and the specimen is taken from the 75 per cent alcohol and gently spurted onto the albumin. After a short time, when the alcohol has coagulated the albumin, the slide with the speci- men adhering to it is transferred to a jar of 75 per cent alcohol and is thereafter treated by the ordinary slide method. For staining, Ranvier’s picrocarmin was used, although Delafield’s hematoxylin gives quite satisfactory preparations. Clearing was done with xylol, and damar was used in mount- ing. For convenience in description the main cultures are designated by letters, and the individual lines (four in number) which make up each of these cultures are designated by figures. ‘Thus, the two cultures of Oxytricha fallax are designated respectively A and B, and the lines under them as A-1, A-2, A-3, A-4, and B-1, B-2, B-3, B-4, In each case the culture was started by isolating one wild individual and when this had divided twice, giving four individuals, these were isolated to start the four lines. These four lines thereafter were kept distinct except in cases where one died out through accident or through the isolation of a weak individual, in which case its place was supplied by a speci- men from one of the three closely related lines. Of course, the more lines of a culture that are carried on, the closer their average rate of division will approach the true one for the culture. I have found that four lines is all that can be reasonably carried without undue labor and the average here is probably near enough to give 590 Lorande Loss Woodruff. the general result. ‘Throughout this work, as in that of my predecessors, the rate of cell-division is taken as the indication of the physiological status of the cultures; it being generally accepted that this is a just criterion of metabolic activity. The experiments were started in the Zoological Laboratory of Columbia University, New York City, and carried on there con- tinuously (except for a short period during the summer months) during the first two years of the work. ‘The last year of the work was done at the Thompson Biological Laboratory of Williams College, Williamstown, Massachusetts. Mc) DESCRIPTION OF THE CULTURES: I. Oxytricha fallax, Culture A. On October 26, 1901, a specimen of Oxytricha fallax was found in an aquarium, in the Columbia laboratory, containing water and superficial slime taken a few weeks before from a stagnant ‘pond at Van Cortlandt Park, New York City. The individual was isolated on a depression slide in a few drops of hay-infusion as previously described. ‘Two days later the infusorian having divided twice, each of the four individuals was transferred to a separate slide, thus starting the four lines of Culture A which are designated respectively A-1, A-2, A-3, and A-4. The accom- panying diagram shows fe. daily record of divisions of all four lines averaged together and this again averaged for each ten-day period of the life of the culture. As is indicated in the diagram, the culture started with an average rate of a little over one division per day for the first ten-day period. ‘This was increased to one and three-quarters divisions for the second ten-day period, after which there were two Scere in which the rate fell each time below that of the first oe e., in period four to exactly one division per day. 1From November 20 to 24, in the third period, A-1 and A-2 were changed from the hay-infusion to a medium of flour and water, prepared by boiling a pinch of flour in about 25 cc. of tap-water for fifteen minutes. This was used about an hour after cooling. This change of medium was made because I became alarmed at the rapid fall in the division-rate—not having become acquainted, as yet, with the general life-cycle of hypotrichous forms. That the use of the flour had no apparent effect was shown by a comparison with the Gee rate of A-3 and A-4 which were continued on the hay-infusion diet. | a7 *pauieje o19M Ady} YOIYA ut sporsed ay} ut paseyd are pue suotyesoued quasaidar “aja fooz ‘oor ‘sandy ayy, *[]9y Aparyd sumysAys oy YOrYA ur syjuOUT ay} ‘Mozeq {poyeotput aie sumMIAYI ayy NTT YDTYA spotsod Aep-ua} ay] Jo sraquinu ay) ‘aaoqy 3 ssuyiky4 snorsea ayy Jo syuny ayy ayeusisap saury uayorq ayy, ‘aINy[No ayy Jo sauT] INoJ ay} Jo UOTSTAIP Jo ayeI Ajtep aderaae ay} Juasaider sajeurpio ayy, ‘spotiad Aep-uay S15 pade19ar UOISIAIp Jo ary ‘woRe1sued yIogg ayy UT (Lobr “br A[n[) UoNIUTVXa 03 (1061 ‘gz 1aq0}90) JAeIs WoAZY aaNQINC ‘xeTTe} eYDIAKO Jo Ar04sIy a397duI0D: uy Ss ‘I WvuovVIG > 1061 ey ‘idy pue £061 Avy pue ZOb1 “AON pure 2 Ajn{ ounf pur key Ie | “qa puecuef ‘20q “AON 290 ‘dag asn3ny Ajnf ounf dy ‘ey pue‘qag ‘uef vq 20 9 098 008 OOL 009 00g OOF 008 006 Oot ars l 7 > eager 0°0 ay | : ! | | | | = pa + + 4 ——— 4 = — 0 a, | - | | = [ aoraes : CPT” | | | ! | | 1 — t ap | 1 U ST 2 | : | | | It : > | ' zy } : ; 0% i | : | ! & eae ae ——_|_ 1s. aia | | eal | 1 ae on ) : ae see ses Ee y | | | I ! | 1 S ; : reed | ene | | mmr Sie | | | | ( \ | ' ao |e eae oe i= a a en eee eee ee { =: == | i OF Ww 19 vg 0g vY Tv 9€ &€ 0€ 16 GG IG LT Or Z 4 ‘Y GANIIAD ‘XvTIvd VHOINLAXO 592 Lorande Loss Woodrujf. During period five there was a marked rise for no apparent cause to over two and one-eighth divisions, and then a fall to about, seven-eighths of a division per day in the seventh period, which was below the lowest rate so far attained by the culture. The next fall, however, was still lower, when seven-tenths of a division per day was recorded. After this there was another rising period extending over about a month and attaining a maximum rate of nearly two divisions. From here there was a gradual decline for four periods when a minimum of six-tenths of a bipartition per day was averaged—the lowest point so far attained. Again a slight rise for twenty days, and then a fall at the twenty- first period (210 days since the culture was started) to one- quarter of a division per day, the lowest point reached in the division-rate, which had been gradually diminishing since the beginning. It was apparent that unless something was done to stay this decline or “rejuvenate” the culture at this point that it would soon die out. Calkins had succeeded in reviving Paramoecium cultures with an extract of beef, and acting on this clue all four lines were transferred to weak beef-extract' for five days and then changed back to the regular hay-infusion diet. As the beef- extract showed no immediate results, flour and water was tried again, apparently to no purpose. I now returned to beef-extract, this time making it stronger and varying the strength from day to day, and this treatment was continued up to June 1. This time a very slight rise in the division-rate for the period occurred (cf. Diagram I, period 22) and during the following ten days it increased to almost one division per day. ‘Then it fell again for two periods, but the slowest rate here attained was considerably faster than the previous low mark of period 21. In period 26 the diagram shows a considerable rise which was brought about by a sudden springing into activity of one line (A-1) of the culture. Instead of dividing at the rate of about once in two days, it started off on July 7 at the rate of three times per day. When I first noted this sudden change I thought that possibly in some way, in spite of all precautions, an adventitious specimen, perhaps as a cyst, had 1The beef-extract was made by boiling for a few minutes a piece of lean beef about the size of a silver half-dollar in 200 cc. of tap-water. This was allowed to settle for a number of hours and then the clear extract was used. The Life-History of Hypotrichous Infusorta. 593 vitiated the culture, and I immediately examined the stock of the line—some of which had not been touched for a number of days. I found that this also had started dividing at the same rapid rate, and as there was apparently no way in which all the preparations could have become contaminated simultaneously, I was convinced that the increase in rate was due to some change in the culture itself—a conviction which was substantiated by a study of the cytological changes in the permanent preparations; but to leave no chance for error I removed the rapid line (A—1) to another moist-chamber and thus isolated it from all the rest. ‘This con- dition of affairs—A-1 dividing about three times each day and the other lines once in two days—continued for just a month when the three other lines sprang into activity. ‘This at once, of course, brought up the average of the four lines as is seen in the twenty-ninth period (Diagram I) when the average rate of multi- plication reached over three and one-half divisions per day. This twenty-ninth period was the high record for the division- rate of this culture. During the next ten days the rate fell to three divisions per day; then occurred a slight rise above this for twenty days, and then another drop to about two and three- quarters divisions per day for the thirty-third period. This rising and falling of the division-rate continued to the very end of the life of the culture, or from August, 1902, to July, 1903, nearly a year. At the fifty-third period it was clear that the culture was again approaching extinction and, accordingly, two lines, A-1 and A-2, were transferred for a day to beef-extract, leaving A-3 and A-4 in the normal hay-infusion. ‘This had no visible effect on the lines treated and both died out at different times, and their places were supplied by individuals from the other two lines. During period 54 the culture medium for all lines was changed from hay-infusion to an infusion made with fresh grass in order to see what effect a change in medium would have on the behavior of the culture. The very slight rise in the division-rate which followed for the next three periods may be due to this change, but I think it is more probable that it is due to a decided rise in temperature which took place at this time (cf. Diagram VII). During the next two periods no attempt was made to revive the culture, and as the fission-rate remained quite uniform I postponed all experi- ments in order to see what would take place if the culture was Dae Lorande Loss Woodrujf. allowed to run its natural course. A very slight falling of the division-rate occurred in the next twenty days; but in the ten-day period after that there was a more decided rise than had taken place for a long while. ‘This proved to be only temporary for the Infusoria suddenly began to die off and within four days only six specimens were left. Efforts to stimulate by artificial means (K,HPO,, return to the usual hay-infusion, etc.) were unavailing, and the last individual died on July 14, in the 860th generation— 626 days after the first isolation. 2. Oxytricha fallax, Culture B. A second culture of Oxytricha fallax was started on December 10, 1902, with an individual found in a hay-infusion, made with boiled water, in the Columbia laboratory. “The method of pro- cedure was the same as that already described for the A-culture. The accompanying diagram shows the history of the fission-rate of all four lines averaged together and this again averaged for each ten-day period of the life of the culture. Culture B was continued for a period of 348 days during which time it attained 429 generations. Its loss was due entirely to an accident resulting in the drying up of the preparations. The general rate of division for the first twenty-three periods averages about one and one-half divisions per day, and compared with the curve of Culture A, the curve of B is considerably more uniform. From period 24 on (August), however, the rate shows a consider- able falling off and it was averaging about one division in two days—the lowest rate in its history—at the time that the culture was lost. 3. Pleurotricha lanceolata, Culture A. A culture of Pleurotricha lanceolata was started November 10, 1902, with an individual from an aquarium in the laboratory of Columbia University which contained material collected during the previous month at Fort Lee, New Jersey. The treatment of the culture was the same as that already described for the Oxytricha cultures. The general trend of the division-rate, as shown by Diagram III, was steadily downward from the beginning to the nineteenth period (May), when the low rate of one division in eight days was reached. During the next three periods a marked eh 5) 1a. “] wesSvIC] JO} se WES ay} are S[lejap JayIO BY, “Papuayxa ainy[nd oY} YIIYM Jado sysuow snolIeA dy} JO SHUT] IY 939 -Iput saut] ueyorq ayy, “(£061 ‘zz Jaquiaaoyny) Ystuy 0} (zo61 for raquisdaq) yeIs Wo; ‘g aINgND ‘xeTLeY eypuyAxO jo Aroysry azayduiog ‘TL WvYOVIG £061 zob1 "AON ‘pO ydag ‘dny Atnf aunf ACW Judy qoie yl qoq -ue[ 29 66P OOP 00€ 006 Oot ; . eee I | ere | | | | | | | | ‘g AUNLIND ‘XVTIvd VHOIYLAXO The Life-History of H ypotrichous Injfusor 596 Lorande Loss Woodruff. rise took place for which there is no apparent cause, but this recovery was not lasting and the rate fell somewhat during the next period, while in the period following this all the infusorians encysted, thus bringing the culture to an end at the two-hundredth generation, and after being under observation for two hundred and thirty-five days. PLEUROTRICHA LANCEOLATA, CuLTuRE A. 100 200 Noy. Dec. Jan. Feb. March April May June 1902 1903 Diacram III. Complete history of Pleurotricha lanceolata, Culture A, from start (November 10, 1902) to finish (July 3, 1903). For method of plotting, see Diagram II. 4. Pleurotricha lanceolata, Culture B. A second culture of Pleurotricha lanceolata was begun Novem- ber 25, 1902, with an individual found in some material in the Columbia laboratory which had been recently collected at Van Cortlandt Park, New York City. ‘This culture was carried on by the method used in all previous cultures for 480 days, and reached during this time the 448th generation, when it was lost by an acci- dent similar to that which terminated the Onn alae The culture-curve plotted in Diagram IV shows that throughout the life of the culture a general average rate of nearly one division per day was maintained. 5. Gastrostyla steini1, Culture A. A culture of Gastrostyla steinii was started on May 28, 1904, with a specimen which was captured in a hay-infusion in the Williams College laboratory. For convenience I have desig- 597 hous Infusorta. 1¢ The Life-History of H ypotr 24} st 3ur330]d Jo poy "AT NYUOVIG *[] WieiseIG 10j sv owes *(vo6r ‘£1 yore) ysruy 03 (zobr ‘Sz raquisAoN)) RIS WO] “g aINI[NZD ‘ej efoaduR] eYSIjOIMe[g Jo A10ysIYy a3a[dwI0D Fo61 fo61 ob ary qaq 0S ‘ue aya, Mae “ABY9) ydag oo -Sny 0S A(n{ ss ounf = Key Soudy Srey‘ ‘uel 1:99q SPP OOF 00€ 006 OOT | T Pog Fae Maar elt mal a 3 ie : | | | \ | | | | ' | | | | Roms | | | _| | | | | | I =e 7 aii syne oa | 1 cal | | [ots | | + | i l T | | at al ae | ; | | ee gaa Sees : eee Ee SSasey feos | eae ] — | | | | “gq aunLing ‘VIVIOGONVI VHOINLONNATY | 0°0 0 OT GT 0% 598 Lorande Loss Woodruff. nated this Culture A, although but one culture of this species has been studied. ‘The culture was put at once on a grass- infusion diet and continued on the same during its life. From Diagram V it will be seen that the rate of division for the first three periods was very close to one division per day. On June 25, which fell just at the end of the third period, I moved the culture from Williamstown, Massachusetts, to New York City. The greatly increased division-rate, which appeared in the follow- ing period and was augmented in the period succeeding that to almost two and one-half divisions per day, is difficult to account for with any certainty. ‘The jolting which the animals received GasTRosTYLa stEInu, Cutture A.—(Ten-day Periods.) June 1904 July Aug. Sept. Oct. Nov. DiacraMm V. Complete history of Gastrostyla steinii, Culture A, from start (May 28, 1904) to its extinction (December 5, 1904) averaged for ten-day periods. Method of plotting the same as in previous diagrams. on the trip to the city, the change to city tap-water, the change of grass with which the infusion was made, and the increased atmospheric pressure are prominent among the factors which may have tended to stimulate the fission-rate. Further, the treatment of the culture was exceedingly uniform beginning with its location in New York as I wished to see if the minor fluctua- tions in the division-rate, so prominent in the earlier cultures, could be modified or entirely eliminated by still more stable conditions. Again, I employed this culture as a “control” for certain experiments on the effects of salts on the division-rate and ao9 The Life-History of H ypotrichous Infusorta. aN wieIseid qi aivdwog AIGUIIAO WT ‘TA WvdOVICG, ‘spotiad Avp-aayf roy padesaar “y ainq[nd ‘ules e[AsoNseg) jo Aroysty ajojdwiog 19q0}99 Jaquajdag qjsnsny Atnf tobr ‘aun{ 006 OOT T alte le | | | | | | | | l ae i ¢0 | | | ! | : a Tt OT ) : sae : whee é | ee | | | é =| —| 0% | | | | = | oS | | | sat fete at ee all 2 oe VIALSOULSYS) (‘spotsag Kvp-aaty)—Y FANLIAD “IINIGLs 600 Lorande Loss Woodruff. for this purpose exactness was most essential.1_ Whatever factor or factors caused the high division-rate of the fifth period, the effect was not lasting for in all of the succeeding periods up to, and including, the thirteenth, the rate steadily decreased and at a remarkably uniform rate. During the twelfth period (end of Sep- tember) I moved the culture back to Williamstown. No effect 1s to be seen in the succeeding period but the rise in the fourteenth period undoubtedly is due to this change. This time the rise was by no means so marked and it was evident only after a latent period of ten days or more. ‘This possibly can be explained by the fact that the “potential of vitality” of the infusorians was considerably less than when the first removal took place? Like the acceleration at the first removal, this second one was not lasting as, during the following ten-day period, the fission-rate settled down to where we should expect to find it if the culture had been carried along without any disturbing influence. Begin- ning with the next period (No. 16) the very exact treatment which I had employed was discontinued and the change of liquid was made only every other day, and then not at exactly forty-eight hour intervals. ‘The effect of this is at once apparent in the con- siderable fluctuations in the fission-rate shown in the culture- curve during the remaining four periods of the life of the culture. At the beginning of period 20, 7. ¢., at the 1g1st day of the life of the series, when the animals were dividing on the average three times in two days, the culture suddenly died out, stock and all, at the 288th generation. I noted that the infusorians were exceptionally active on the slides just previous to their extinction. This sudden death of the culture cannot be attributed to any accidental change in the liquid medium as the stock was affected similarly at the same time.® 1T endeavored to secure this uniformity of treatment and culture medium: (1) By changing the culture medium daily and at the same hour, thus making the daily records of just twenty-four-hour periods. (2) By using the same kind of grass and grass grown in the same place. (3) By washing the grass very thoroughly and boiling it for one minute. This was given as soon as it reached the room temperature. Calkins (’02, 1) found, however, that a journey which he made with his Paramecium cultures when they were on a descending cycle accelerated the fission-rate, while a return journey made when the cul- tures were on the ascending cycle produced a retarding effect. 3Tt will be recalled that the death of Maupas’s culture of Stylonychia pustulata was preceded by a period of more rapid division of almost’ three weeks’ duration. The Life-History of Hypotrichous Infusorta. 601 Vi. DISCUSSION OF THE DATA OF THE CULTURES. I. Rbhythmical and Cyclical Variation in the Rate of Division. One has but to glance at the plotted curves of the various cul- tures (Diagrams I to VI) to see that all the species of Infusoria studied pass through periods of greater and less dividing activity when subjected to a stable environment. ‘These periods, upon analysis, are resolved into two kinds: First, the short, more or less rhythmical fluctuations in the fission-rate which I shall refer to as “rhythms”; and second, the long downward trend of the cultures (especially prominent in the Oxytricha A-culture) from their beginning to end, or, in the case of Oxytricha A, from Its start to its recovery by stimulation at about the 250th genera- tion, and again from this point through the second long downward sweep which ended with its extinction. This second type of change of fission-rate I regard as the * “cycle.” Iam satisfied that these two kinds of variation are due to different causes. I believe the rhythms to be somewhat superficial in character and due in part to slight variations in the environment, the most important of which is change in temperature. ‘This belief is based on the remarkable agreement which obtains between the rhythms and the fluctuations in temperature. In Diagram VII there is plotted a section of the culture-curves of all the four cultures which were carried on simultaneously, and above them the temperature curve. [he agreement is seen to be more marked in the Oxy- tricha cultures; in the Pleurotricha series, the similarity, while not as striking, is too exact to be a mere coincidence, and serves to emphasize the fact that while temperature does influence the rate of multiplication, it is not the most important element among the factors which cause fluctuations in the rate. It is only natural that temperature variations should affect the division-rate, if not directly, at least indirectly through the effect on the multiplica- tion of bacteria and therefore upon the food-supply, and this has been shown to be the case by Maupas and Calkins. I believed there was another and more fundamental factor underlying the rhythms, and with this in mind I took still greater precautions to have the environment as nearly constant as possible in the more recent culture of Gastrostyla stein (see note, p. 600). From the results of this series when plotted in periods of ten days (Diagram V), it would seem that my idea 602 Lorande Loss Woodrujf. was wrong and that with a constant medium all rhythms could be removed. ‘lo test it still further the same results were plotted for five-day periods. ‘This brought the rhythms to view. again (cf. Diagram VI) in such beautiful regularity that it seems to me to show beyond doubt that the rhythmical element of the division- rate cannot be caused entirely by temperature changes or by imperceptible fluctuations in the food supply, but that it is due, in the last analysis, to factors of a more complex character. Varia- tion in the rhythm of division is well known in the development of the metazoodn egg, and it has yet to be satisfactorily explained. ‘Towle (04) in a recent paper on the effects of stimuli on Para- mececium is led to make this interesting statement: “There may even prove to be rhythmical changes in sensitiveness like those described by Lyon (02; ’04) for cleaving eggs, and Scott (’03) for unfertilized eggs. Something of this nature is indicated by the fact that Parameecia from hie same culture vary in sensitiveness from day to day.” In my work on the effects of chemicals on Infusoria I have found that individuals react differently at various times to a given stimulus (cj. p- 616 et Seq. ) and I believe we have the clue to these “changes in sensitiveness” manifested in the rhythms of the fission-rate. A point of some interest in regard to the rhythms i in the Oxy- tricha A-culture is the fact that the slowest fission-rate of each rhythm in the descending cycle is less than that of the slowest rate of the preceding rhythm. In the ascending cycle also, the slowest rate in each rhythm is greater than the slowest rate of the preceding rhythm. So far we have not considered the long trend of the division- rate, which I regard as the cycle as I believe that this is directly comparable with the cycle of Calkins’s Paramcecium cultures. The cycle obviously extends over more generations in Oxytricha than in Parameecium though in both cases it is a variable number both in the same culture ae in different cultures of the same species. Maupas’s rather definite limits to the life-cycle are not substantiated by this work as will be readily seen by comparing the various culture-curves. It must be borne in mind, however, that neither Maupas’s chief cultures nor my own were started with ex-conjugants, and therefore the number of generations does not afford a just basis of comparison, since they indicate merely the number of bipartitions since the culture began and in no sense The Life-History of H ypotrichous Infusorta. 603 | TEMPERATURE OXYTRICHA - A ' | OXYTRICHA — B I PLEUROTRICHA = 8B 1 Dec. Jan. February March April 1902 1903 Diacram VII. Sections of the culture-curves of Oxytricha A, and B, and Pleurotricha A, and B, together with the temperature curve for the same period (December 10, 1902 to May 9, 1903), showing the corre- spondence of the ‘‘rhythms” of the division-rate with the fluctuations in temperature. 604 Lorande Loss Woodruff. the “age” of the culture with reference to the last conjugation period. The number of generations from the recovery of my Oxytricha A-culture to its extinction gives the number of divisions in a cycle of an artificially stimulated line, but it remains to be shown that this is directly comparable to “rejuvenescence”’ by conjugation. Joukowsky carried a series of Pleurotricha lanceolata through 458 generations and found no signs of degeneration, and he sug- gested that degeneration depends not on the number of divisions only, but on the rapidity with which they succeed each other. This conclusion, on a priori grounds, would seem reasonable, but my cultures give no evidence to substantiate it. Calkins, on the other hand, lays more emphasis on the duration in time of the cycles than on the number of generations passed through, and he showed (’04) that about six months is the period of the cycle in Parameecium, but it would seem that about three months was the result reached by other workers on this species. Calkins (02, I, 2, 3) himself, in his earlier studies on Paramcecium, believed that the cycle in this species was approximately of three months’ duration, as he interpreted the smaller trimonthly fluc- tuations as the cycles. I am satisfied that these’ periodic lesser changes in vitality which are so conspicuous in his culture- curves are identical with what I have termed rhythms in my cultures, and in the light of his results with Paramcecium, it is probable that the earlier workers on this species, Joukowsky and Simpson, have been dealing with rhythms rather than cycles. I believe that it is essential to recognize a sharp distinction between “rhythms” and “cycles,” which may be defined as follows: A rhythm 1s a minor periodic rise and fall of the fission-rate, due to some unknown factor in cell-metabolism, from which recovery 1s autonomous. A cycles a periodic rise and fall of the fission-rate, extending over a varying number of rhythms, and ending in the extinction of the race unless it 15 “ rejuvenated” by conjugation or changed environment. The question of the number of generations, as well as the time duration, of a life-cycle, is very uncertain and extremely difficult to determine as it is probably dependent upon more than one factor. My cultures lead me to believe, with Simpson, that the personal equation, if I may use that term, of the individual selected to start a culture has the most influence in determining the number The Life-History of H ypotrichous Infusorta. 605 of generations attained before “the initial potential of vitality” is exhausted. Calkins’s discovery of what he calls “incipient fertilization” in Paramcecium—that is, of two ex-conjugants which continue to live “one is invariably far more vigorous than the other” —would seem to bear out this point and to show that the number of generations or the period over which a cycle extends is not a point of great moment. Taken as a whole my cultures show conclusively that the three species of hypotrichous ciliates studied are subject to periods of greater and less dividing activity, and since the fission-rate is probably a fair criterion of the metabolic activity of the protozoan cell, that ciliates pass through alternating periods of greater and less general vitality. ‘This is also the general conclusion reached by Engelmann, Bitschli, Maupas, Joukowsky, Simpson, and Calkins, and from the range of species investigated it can probably be accepted as of quite general occurrence among the Infusoria.' 2. Artificial Rejuvenescence. Calkins (’02, 1) showed conclusively that Paramcecium cultures when becoming extinct can be revived by the application of various 1Peters (’04) working on Stentor, states that ‘‘neither direct observation nor the experiments made, furnish evidence of any inherent periodicity of division. The present experiments show that, except when some special modification of the medium exists (e. g., presence of potassium chlorid in excess), multiplication runs, in the main, parallel to metabolism.” Peters’s experiments were not planned directly to investigate this point and I fail to see, from his description of the methods employed, how cyclical variation in the fission-rate, unless very pronounced, would be apparent. That “multiplication runs, in the main, parallel to metabolism” is, I take it, not open to question, and is in no way opposed to periodic fluctuations of the fission-rate. Peters says further, in regard to the culture medium employed in determining periodicity of division, that ‘‘such promiscuous mixtures as hay infusion of unknown com position will not suffice. Since frequent chemical analyses are impracticable, it will be necessary to construct by trial artificial media of known composition.” Undoubtedly hay infusion is not an ideal culture-liquid, but when the hay or grass is carefully selected and thoroughly washed and otherwise treated uniformly, and when this is prepared fresh each day and employed as soon as it is has reached the room-temperature, there is little chance for fermentation, and I believe that about as near a perfect medium is obtained as is practicable. Undoubtedly the ideal culture-liquid would be one artificially combined so that its salt content, etc., is accurately known; but as Peters himself says, “‘. . . afood supply must be added to the salt solution, and this requirement has proved to be a difficulty. For the addition of any food that has been found available utterly changes the salt content both qualitatively and in its proportions.” To supply this demand Peters added to the artificial medium which he con- cocted, ‘‘some dry leaves or dead reeds, or both. . . . The final step is to ‘seed’ this culture with a mixture of all sorts of Infusoria, and other living material from thriving cultures.” This done, I do not see how a hay-infusion could be a more promiscuous mixture. 606 Lorande Loss Woodruff. stimuli, and he found that an extract of beef, among others, was most effectual. As previously stated, I employed beef-extract as a stimulant during the first depression period of the Oxytricha A-culture which was at its height in May, 1902, and in July, 1902, after a latent period of about six weeks, one series suddenly sprang into new life. It is certain that something “rejuvenated” the culture at this time and I have every reason to believe that it was brought about by the salts of the beef-extract, and that we have here a case of stimulation analogous to “artificial partheno- genesis’ as Calkins suggests in his Paragnoseiain work. 3. Conjugation. My endeavor to study the effect of conjugation on the life- cycle of Oxytricha fallax, Pleurotricha lanceolata, and Gas- trostyla steinii has been in vain, as at no time during the life of any of the five cultures have I succeeded in getting a single syzygy. Numerous individuals from the A and B cultures were placed together at different times in an endeavor to get exogamous conjugations, but to no purpose. ‘The same is true of endoga- mous conjugations. With Maupas’s conditions of conjugation in mind attention has been paid to the amount of food present but without result. It seems rather remarkable that Oxytricha should pass through 860 generations, Pleurotricha through 448 generations, and Gastro- styla through 288 generations and at no time show any tendency to conjugate. The significance of this is rather difhcult to see. Joukowsky, however, found no conjugations in his long culture of Pleurotricha, and Maupas secured none in his cultures of Stylonychia mytilus or Oxytricha sp. though his other series yielded plenty of syzygies. It is not uncommon to find hypo- trichous forms conjugating in wild cultures in the laboratory, so that it is evident that some condition must prevail there which does not obtain in the experiments, and it is just possible that an excess of carbon dioxid and other noxious gases in these wild cul- tures may be the provoking cause; but it seems more probable, since the physical state of the protoplasm of the infusorian undoubtedly plays an important role in the conjugating process, that the required “ miscible state” is prevented in artificial cultures through the scarcity of certain salts in the liquid medium used. In the The Life-History of H ypotrichous Infusoria. 607 light of Maupas’s results with Oxytricha sp. and Stylonychia mytilis, and of Joukowsky’s with Pleurotricha lanceolata, and also my own on two cultures of Oxytricha fallax, wwo of Pleuro- tricha lanceolata and one of Gastrostyla steinii, it would seem to be questionable whether conjugation is of so frequent occurrence among the Hypotrichida as in some other groups of Ciliata. Vv. PHYSIOLOGICAL AND MORPHOLOGICAL VARIATION DURING THE Et b-CYClEs Maupas emphasized the fact that various changes, cytoplasmic and nuclear, take place in Protozoa as “senile degeneration” advances; and he also found physiological evidence in the form of lessened vitality, increase of endogamous conjugation, and infertile syzygies. Joukowsky found no morphological changes in Paramoecium but observed that the rate of division deed as the cultures advanced and that many of the animals became sluggish. In an eight month culture of Pleurotricha he found no signs of degeneration. Simpson made some observations on three to four month cultures of Stylonychia pustulata, Para- moecium caudatum, and Paramcecium putrinum, and while he did not find degeneration in such specific form as nuclear changes or loss of external appendages, still he was “convinced of a gradual ebbing of vital energy as the series proceeds, which expresses itself in slower motion, in a tendency to inactivity and general listlessness, if the word be admissible in this connection, as also in a certain diminution of size that was not remedied by any amount of food.” Calkins (04), however, found marked cytoplasmic and nuclear changes in his long Paramcecium cultures, and physio- logical degeneration was manifested by irregular and abnormal divisions, decreased division- “rate, tendency to endogamous con- jugations, and above all by the “death of all members of a series fed continually on the same diet of hay-infusion.” I. Physiological Variation. In my cultures, physiological changes have been manifested chiefly in the slowing down of the division-rate after a greater or less number of generations, and coincident with this, in a con- siderable lessening of the general activity of the infusorians. 608 Lorande Loss Woodruff. The general behavior of individuals on the slide is quite different at various periods in the life-cycle, and by it the condition of the culture can be estimated with some degree of accuracy. Although the activity of the animals is considerably lessened during depres- sion periods, I have not found that their power of taking food 1s diminished since the oral cilia vibrate normally and keep a con- tinuous stream of food particles passing into the mouth opening, and this results in a black appearance of the infusorians due to accumulated and unassimilated food. Another indication of physiological disturbances during periods of depression is the greater frequency of pathological divisions at this time, and Calkins found this to be the case in Paramoecium cultures. An interesting specimen which occurred in the A-cul- ture of Oxytricha, when the vitality was extremely low in June, 1902, is shown in Fig. 21. 2. Morphological V artation. For the purpose of determining the morphological changes which occur during the life-history, permanent preparations were made from time to time during the life of each of the cultures.’ The series of preparations is particularly complete for the Oxy- tricha A-culture, from the time that series was approaching its first depression period through its recovery by stimulation, and then through the second cycle which resulted in death. On this account, the following description is based on this series of some two hundred slides, while the lesser series of Oxytricha B, Pleuro- tricha A, and B, and Gastrostyla A are used for confirmation and comparison. The typical cytoplasmic structure of Oxytricha fallax, Pleuro- tricha lanceolata, and Gastrostyla steinii, is practically identical and is best described as alveolar throughout. As in all the hypo- trichida, no distinction is visible between ectoplasm and endo- lasm. The ectoplasmic modifications such as cilia, cirri, and membranelles, of course, vary in a characteristic manner for each species, but it is unnecessary to consider these here. In Oxy- tricha and Pleurotricha, as is well known, there are two ellipsoidal macronuclei situated more or less symmetrically in the cell, while 1For description of technique, see section on General Methods and Technique. The Life-History of H ypotrichous Infusorta. 609 in Gastrostyla there are four macronuclei similarly placed. The macronuclei in all three species consist of at least two elements: First, a substance, undoubtedly chromatin, having a strong afhnity for nuclear dyes; and second, a clear sabes resisting all stains, which may be termed achromatin. ‘The general appear- ance of the nucleus is nearly homogenous though this 1s probably caused by the massing of a granular matrix. A mem- brane surrounds the nucleus and a very delicate commissure apparently connects the macronuclei though it is very difficult to determine. From time to time a Kernspalt is observable. Asso- ciated with each macronucleus is a small spherical micronucleus; in Oxytricha and Pleurotricha there are typically two, and in Gastrostyla four, micronuclei. ‘The staining reaction of the rest- ing micronucleus is the same as that of che macronucleus. A typical specimen of Oxytricha fallax is illustrated in Fig. 15. During the earlier part of the Oxytricha A-culture no prepara- tions were made, so that during the first period of decline up to the sixteenth ten-day period (Diagram I) I am unable to trace the morphological changes. On April 2, 1902, however, two indi- viduals of the 230th generation were preserved. A glance at the photographs of these specimens (Figs. 1 and 2) shows that marked vacuolization of the cytoplasm has occurred in each case. In Fig. 1 the two macronuclei are considerably displaced in the cell, and each shows a peculiar vacuolized condition of the nuclear material, the chromatin being segregated about what appear like bubbles of the achromatic substance. Each macronucleus is sur- rounded by a clear area which separates it sharply from the cytoplasm. I believe that this clear area 1s caused by an accumu- lation of the achromatic substance against the nuclear membrane, which thus produces the appearance of a halo about the nuclear bodies. In this particular specimen there are two micronuclei present, one being nearly invisible in the photograph as it is somewhat below the plane of focus. Fig. 2 shows the same condition of cytoplasm and nuclear material but the two macronu- clei are fused and the whole mass is surrounded by the halo. At least three micronuclei are present in the preparation, two of which are visible in the figure, so that we have a case of micronu- clear reduplication similar to that which Maupas described in his culture of Oxytricha sp. Specimens from A-2 of the 239th genera- tion (Fig. 3) and A-1 of the 241st generation (Fig. 4) show a 610 Lorande Loss Woodruff. fused condition of the macronuclei similar to that in Fig. 2, though the chromatic material appears somewhat more homogenous. Here again the micronuclei, with two exceptions, are out of focus. Other characteristic specimens of this period of declining vitality are shown in Figures 5, 6, 7 and 8, all representing forms from the 243d to the 247th generation (c7. Explanation of Plates). The specimen illustrated in Fig. 9 is of the 250th generation and is the last of the descending cycle of A-1, since on July 7, 1902, this line sprang into renewed dividing activity (cf. p. 592). The marked improvement of the cytoplasmic and nuclear con- dition of the infusorians is shown in an individual of the 256th generation (Fig. 12). Here the macronuclei, in outline and in general appearance, are again approaching the typical condition, and the position of the micronuclei in relation to the macronuclei is also more typical. Lines A-2, A-3, and A-4, however, which remained dividing at the slow rate, show no improvement in their nuclear condition, as is seen in specimens of the 255th generation (Figs. 10 and IT). The cytoplasm of the ‘ ‘rejuvenated ” individual, from line A-1, represented in Fig. 13 1s still somewhat vacuolized, but the macronuclei and micronuclei are nearly typical. The specimen is quite small but this is due to the high rate of division prevailing at this period. This reduction in size is still more apparent in preparations of the 331Ist generation (Fig. 14), but beginning at about the 409th generation (Fig. 15) the size again increases with the slightly decreased fission-rate. ‘This beautifully diagram- matic condition of the nuclear apparatus is the prevailing state in the large majority of specimens at this period of great repro- ductive activity. One most interesting exception, here is that in two lines of the culture, specimens from the 361st to 369th generations lack the posterior micronuclear body. ‘This is but temporary and for almost one-hundred generations after this the normal condition prevails. Preparations of the 458th generation again show evidence of a changed condition of the micronuclei since now they appear pale; the chromatin having but little affinity for the stain. ‘This peculiarity reaches a pie at the 473d generation when the micronuclei appear almost perfectly clear; but from this time on they again resume their normal staining capacity. Starting at about the 542d generation the cytoplasm shows signs T he Life-History of Hypotrichous Infusoria. 611 of vacuolization, and this increases steadily and at approximately the 6ooth generation the nuclear apparatus begins to differ from the normal. An early stage is shown in Fig. 16, and a later stage exhibiting nuclear fragmentation in Fig. 17. The last stage in this cytoplasmic and nuclear degeneration is shown by specimens of the 853d and 854th generations (Figs. 18, 19 and 20) in which the cytoplasm is greatly vacuolated, the ventral cirri reduced, the macronuclei distorted and fragmented, and the micronuclei increased beyond the typical number; a condition closely similar to that which obtained at the 230th generation (Figs. 1 and 2). The series died out at the 860th generation (7. P - 594). An interesting feature is the marked variation in size of the infusorians at different periods of the life-cycle. Previous workers have found that a gradual decrease in size occurred as “old age” ensued. This certainly does not hold for the species in question. Fig. 15 shows about the typical relative size of a normal specimen, and a comparison of this with the figures of the succeeding generations and with Figs. 1 through 8 shows that the size gradually Pleprogh fs the ee of fh eee This, however, is true only up to a certain point for, during the last two days before death, the size decreased quite rapidly, a decrease due to a shrinking of the cytoplasm which pro- duced a more or less abnormal contour of the individuals. This condition is shown somewhat inadequately in the speci- men illustrated in Fig. 9, which is the last of the line before the culture was “rejuvenated ” in July, 1902. After this recupera- tion, however, the size of the infusorians decreased remarkabl (from the normal) with the high rate of division (cf. Figs. 13 and 14). The B-culture of Oxytricha (cf. Diagram II), which was lost by accident at the 429th generation, shows far less fluctua- tion in vitality than does culture A, indicating that the potential of vitality of the B-series was considerably greater. Cytological study of the preparations made from time to time shows that, beginning at about the 140th generation and extending over approximately the ensuing seventy-five generations, the anterior micronucleus was not present. [his was the only morphological change apparent during the life of this culture. A typical speci- men in the 365th generation is shown in Fig. 22. 612 Lorande Loss Woodruff. In the two series of Pleurotricha I have found no nuclear variation at any time. Although neither of these cultures was actually carried to natural death, still from the large number of generations attained it would seem that nuclear changes should have appeared if they occur in this species. Joukowsky’s culture of 458 generations of this species, however, gave the same result and that this has been held unjustly as opposed to Maupas’s conclusions is evident from my cultures. A slight cytoplasmic vacuolization appeared in both of my cultures as the series advanced. A specimen, in a late division-stage, from the 413th generation of culture B is shown in Fig. 23. The Gastrostyla culture showed morphological changes in the form of vacuolized cytoplasm and distortion of the macronuclei during the later generations; but at the time of the sudden death of this series “degeneration” was by no means so marked as in the Oxytricha A-culture long before death ensued. Briefly reviewing the chief morphological changes apparent during the various cultures, we have: Oxytricha A, cytoplasmic vacuolization, disappearance of one of the micronuclei for a period, and later an increase in their number beyond the norm, distortion and fragmentation of the macronuclei, degeneration of part of the ciliary apparatus, and, finally, a gradual increase in the size of the infusorians as degeneration advances; Oxytricha B, one of the micronuclei was not present during a number of generations; Pleurotricha A and B, slightly vacuolized cytoplasm; and Gas- trostyla A, cytoplasmic vacuolization and distortion of the macronuclei. Wallengren (’o1) made a careful study of the morphological changes which occur in starved Paramoecia, and discovered that in the later stages the endoplasm 1 is distorted by huge vacuoles and finally the macronucleus is deformed and broken. The micronucleus, however, remains unscathed throughout the starva- tion changes. Calkins confirmed these starvation observations and also found that quite similar morphological changes occur in degenerating Parameecia cultures, and he believes that the simi- larity of the changes in the two cases indicates that it is the diges- tive function which becomes impaired in the declining series, since when in this condition the organisms still take food but apparently are unable to utilize it. - In my own cultures it has been clear that the power of taking food is not diminished appreciably The Life-History of Hypotrichous Injusorta. 613 during depression periods and the very similar morphological changes which occur in the hypotrichs studied, justifies, I believe, the assumption that the power of assimilation becomes diminished as the culture proceeds and that the effect of the beef-extract is essentially that of concentrated nutrition, resulting in the rapid assimilation of the salts, etc., necessary for the continued life of the animal. It has been customary to regard the macronucleus as relatively vegetative in function and the micronucleus as reproductive; and this accords well with the results of these experiments, in so far as the morphological variation of the macronucleus may be regarded as an indication of the apparent lack of assimilation of the food taken. Throughout the culture no form-changes were apparent in the micronuclei themselves, but they showed a tendency to numerical reduction when the fission-rate was at the highest, and to reduplication when the lowest rate of multiplication ensued. This may be explained by supposing that the exceedingly rapid rate of assimilation, calling for such frequent bipartitions, results in the exhaustion of the micronuclei during these periods; but when assimilation is at a low ebb, the little demand for the dynamic forces of the cell results in the reduplication of the micronuclei beyond the typical number. Thus Maupas’s observations that in certain hypotrichous forms the micronuclei are reduced in number, and later appear again in greater number, is entirely substantiated by these cultures. Variations in the number of micronuclei is not unknown in other forms. Johnson (’93), for instance, working on Stentor, found that from one to eight may be associated with each node of the macronucleus. However, the disappearance of all the micronuclei in certain forms, as described by Maupas, has never occurred in my cultures, and the continuance of his series for many generations without this cell-organ I believe is open to question. Whatever may be the correct interpretation of the nuclear changes taking place in the life-history of the hypotrichida, these cultures strongly suggest that it is customary to regard the struc- ture most frequently observed in “wild” Infusoria as too fixed in character, and to overlook the fact that under varying condi- tions, modifications may occur which are in no way abnormal. Biitschli (83) comments on the frequent presence of a coarsely alveolar or vacuolar structure of the protoplasm of certain ciliates 614 Lorande Loss Woodruff. and believes that this should be sharply distinguished from the fine honey-comb structure which obtains in other forms, such as many of the hypotrichida; and he regards the observations of Sterki (’78), that Stylonychia mytilus has a markedly vacuolized structure, as an indication of abnormality. Simpson (oI, 2) made sections of what he regards as “absolutely normal” Stylonychia, and he states that they “showed the vacuolization fairly well developed. .’ Again, the question of the fixity of form and position of the macronucleus has been variously dis- cussed since Balbiani more than forty years ago observed a shifting in Paramcecium, to its recent consideration by Simpson through observations on various species. My own cultures give conclusive proof that the cytoplasm becomes considerably more vacuolated at certain periods in the life-cycle; but further, daily observation has shown that hardly any two individuals are identical in their cytoplasmic condition, and the same can be said of the position of the macronuclei and the accompanying micronuclei. The fact that subjection to beef-extract gradually revived the cellular activity and caused the resumption of the normal condi- tion of cytoplasm and nuclei, shows that up to the verge of extinc- tion the cell-life can be revivified. I think this indicates that we are hardly justified in assuming that Protozoa, when dividing ata low rate, with nuclei fragmented, etc., are exactly “abnormal.” The fact that it is possible to restore such remarkable types as | have figured to the text-book “normal” condition suggests that we are justified in regarding these changes as phases 1 in the life- history of the susan aha occur Tce certain conditions after a considerable period of vegetative reproduction. VI. EFFECT OF INITIAL AND DAILY STIMULATION WITH SALTS ON THE RATE OF DIVISION. The first essential for experimental work with salts on the fission-rate of Protozoa is to have a constant subject on which the stimuli are to be applied so that the results obtained shall be directly comparable. This condition is admirably fulfilled by cultures of Infusoria fed daily on the same diet and carried on in this way for many weeks; and such cultures probably afford as near a perfect “control” as it is possible to get for work of this kind. The Life-History of H ypotrichous Infusoria. 615 The results obtained with beef-extract as a stimulant for worn- out Protozoa led me to test the effect of some of the more common salts on the fission-rate, since, as Liebig claimed, the stimulating property of beef-tea is probably due to the extractives and not to the small amount of proteid which it contains. For this work potassium phosphate (monobasic and dibasic), potassium chlorid, potassium bromid, and potassium sulphate; sodium chlorid, and magnesium sulphate were chosen. ‘The series of experiments with these seven salts extended from the early part of July to the middle of September, 1904. ‘The work with each salt extended over twenty days. ‘The salts were made up into equivalent normal solutions’ and these were then diluted as indicated in the descriptions of the individual experiments. In each case two solutions of dif- ferent strength were employed, and each of these was applied both as an initial and as a daily stimulus. The culture of Gas- trostyla steinii was used in this work (cj. Diagrams V and VI). I. Experiments with Potassium Phosphate (Monobasic and Dibasic). On July 6, 1904, eight cultures (each consisting of four lines) of Gastrostyla were started with individuals isolated from my culture A of this species, which had been under observation since May 28. Four of these cultures were used for experiments with the monobasic and four with the dibasic salt. Of these cultures, half were used for initial stimulation and half for daily stimulation; ane of each half, one was used for ;%,5 solutions and the other for z75y solutions of the salt in question. The method of applying the salt was, briefly, as follows: In the case of initial stimulation, one individual was placed on a slide with as little of the culture-medium as possible. To this was added the solution of the salt to be tested and this was removed again immediately and fresh salt solution put on. Each trans- ference was performed with a pipet used only for this purpose. The length of each initial stimulus was thirty minutes and when this had expired the specimen was transferred back to the grass- infusion. In the case of daily stimulation the method of procedure The solutions were made according to the definition of ‘normal’? solutions as given in Sutton’s Volumetric Analysis, Eighth edition, 1900. 616 Lorande Loss Woodruff. was identical except that the duration of the stimulation was ten minutes instead of thirty minutes. The immediate effect of immersion in the monobasic salt was to cause the infusorian to rotate rapidly on its short axis for a couple of minutes, after which it began to move slowly about the slide, and by the end of the thirty minutes normal locomotion was entirely resumed. Practically the same behavior was caused by the application of the dibasic salt. I found, however, that this typical reaction varied somewhat with different individuals at various times; sometimes, for instance, the duration of the whirl- ing motion was very much shorter and sometimes it was entirely absent. ‘This is true not only for stimulation with potassium phosphates but also for stimulation with the various other salts tried. Slightly different reactions occurred with some of these other salts, but I have noticed the same variability. I am inclined to believe that ‘he explanation of this variability in the reaction to a given stimulus at different times is in some way correlated with the slight changes in vitality which I have described as rhythms. I found also that when the salts were applied daily they soon ceased to cause any abnormal movements, even when their effect on the vitality of the animals, as determined by the division- rate, was detrimental. Here again there were occasional exceptions which point to periodical fluctuations in sensitiveness. ‘This holds true for all the salts employed. A glance at the diagram shows that the culture stimulated ini- tially with K,HPO, in ;,%5 solution divided more rapidly than the control during three out of the four five-day periods of the experi- ment, and produced a greater effect than any of the three other experiments involving initial stimulation. Culture K,HPO, 74,, initial stimulation, showed the next greatest effect, but this was manifested in a slowing of the rate in three out of four periods. In initial doses, then, the dibasic salt proved to be more effective—the greater dilution producing an accelerating effect and the lesser dilution producing a retarding effect. An examination of the data of the experiments on daily stimulation shows that K,HPO, zssy again produced the greatest change in rate, though this time it had a retarding influence. Summarizing the results of the 1The curve for daily stimulation is not plotted for KH,PO, ;2, and , 745, during three periods 0 because the individuals stimulated were lost accidentally at these times. The Life-History of Hypotrichous Infusoria. 617 twenty-day experiments with the two potassium salts, it is apparent that the dibasic salt had in every case a greater “net effect” than the monobasic salt; and that the more dilute solution of the dibasic salt produced a greater acceleration than the less dilute solution when used as an initial stimulus, and also produced a greater depressing effect when given daily. ‘This is a clear-cut example Potassium Puospuate (Monorasic anp Dirgasic). K,HPO, KH,PO, Diacram VIII. Effect of initial and daily stimulation with KZHPO, (2. and ) and KH2PO,4 (-2_ and _2_)on 100 1350 [50 1250 the division-rate of Gastrostyla. Averages are for five-day periods. Control (Gastrostyla A, on regular medium) is indicated by a continuous line; initial stimulation, by a broken line; and daily stimulation, by a dotted line. The eight experiments plotted in this diagram were carried on simultaneously from July 6 to July 26, 1904. (Cf. text.) of a chemical being beneficial in a single small dose but detrimental when used frequently. In view of the interesting results with the dibasic salt the K,HPO, =2, initial-stimulus culture was continued for some two months after the twenty-day experiment was over. ‘The result of this work is plotted in Diagram IX. From the curve it will be seen that in the sixth period of the experiment the rate fell 618 Lorande Loss Woodruff. below that of the control, and at this point the infusorians were again stimulated, as previously, for thirty minutes. ‘This apparently accelerated the rate (as compared with the control), but only temporarily as in the eighth period it was again below the control. Another stimulation at this time again raised the rate, but as in the previous case the effect was not lasting. After Porasstum PHospuate (Dreasic). 2 oy 14: 3) 6 te SD | 10. iS elas 5 1G ay July 1904 August September Diacram IX. This diagram shows the continuation of the experiment with KzHPO, ;. (cf. Diagram VIII) illustrating the effect of stimulation with this salt at different periods in the life-cycle. Method of plotting, as in Diagram VIII. Control = continuous line; regular K2HPO, series = broken line; new series stimulated = dotted line; time of stimulation = @. ‘The figures above the diagram indicate the five-day periods. (See text.) two more periods had passed, in each of which the stimulated line was dividing at a rate below the control, they were treated still another time with the salt-solution; but this time no acceleration was produced, but instead the rate, as compared with the control (cf. Diagram IX), fell still lower. The behavior of the culture here suggested the possibility that the lack of effect of the salt The Life-History of H ypotrichous Infusorta. 619 was due to the series becoming accustomed to it, and accordingly I started a new series from the control and stimulated both this and the old series at the same time. From the results (see Diagram IX) it was evident that this hypothesis was not sub- stantiated, for the new series showed even a greater drop in the fission-rate than did the old. Instead, it was apparent that the difference in effect of the salt at these later periods must be sought in the change in the general vitality of the culture itself. When the salt was first used the vitality of the series was considerably PotasstumM CHLoRID AND SopiuM CHLOoRID. KCl NaCl Dracram X. Effect of initial and daily stimulation with KCI, . and ~?., and NaCl, }, and ,7., on the division- rate of Gastrostyla. Averages are for five-day periods. The eight experiments plotted in this diagram were carried on simultaneously from July 26, to August 15, 1904. Method of plotting is the same as in Diagram VIII. greater than toward the end of the experiment, as is indicated by the comparative fission-rates of the two times; and the conclusion seems to be justified that a given stimulus produces different effects at different periods in the life-cycle. ‘This result shows how com- plicated is the whole problem of the effect of stimuli on protoplasm, and the great amount of work that will have to be done before it will be possible to attain any satisfactory knowledge of the part played by a particular salt in the economy of the protozo6n. 620 . Lorande Loss Woodruff. 2. Experiments with Potassium Chlorid and Sodium Chlorid. The experiments with KCl and NaCl were conducted precisely the same as those with the phosphates of potassium, except that an fy solution was used in place of the 72% of the phosphates. ‘The accompanying curve (Diagram X) gives the results of the experi- ments. The striking point about the effect of initial stimulation with KCI and NaCl in each dilution used is that they all accelerated the fission-rate during the first part of the experiment, and had a still greater opposite effect during the latter part, so that the “net PorassitumM SULPHATE AND MaGNesiuM SULPHATE. K2SO, MgSO, eestor seeee . ee eee | We kaccied rn vitie@lee | *ieieiciee |) tr te nets msOlelniele 0.5 te eee 0.0 DiacraMm XI. Effect of initial and daily stimulation with K2SO4, ,7, and ,2, and MgSOug, 7, and zup» on the division-rate of Gastrostyla. Averages are for five-day periods. The eight experiments plotted in this diagram were carried on simultaneously from August 15, to September 4, 1904. Method of plotting is the same as in Diagram VIII. effect” for the twenty-day experiment was a marked falling off in the number of divisions. A closer analysis of the results shows that KCl produced a greater variation from the control than did NaCl both when used as an initial and as a daily stimulus. In every case also the greater dilution produced the greater variation. Daily subjection to each of the salts proved to be uniformly T he Life-History of H ypotrichous Infusorta. 621 detrimental,! as was seen to be the case in the work with the two potassium phosphates. As far as these experiments go they would seem to indicate that potassium has more effect than sodium on the metabolic activity of Gastrostyla. 3. Experiments with Potassium Sulphate and Magnesium Sulphate. The third series of experiments was with ;3; and 2, solutions of K,SO, and MgSO,. The results of the eight cultures together Potassium BromipD. KBr Diacram XII. Effect of initial and daily stimulation with KBr, P. and ;%., on the division-rate of Gastrostyla. Averages are for five-day periods. The four experiments plotted in this diagram were carried on simul- taneously from August 30, to September 19, 1904. Method of plotting is the same as in Diagram VIII. with the control are shown in Diagram XI, and they indicate that an initial application of K,SO, in both dilutions used produced a slight acceleration in the division-rate, whereas MgSO, under the same conditions produced a retardation. It is evident here again that the daily use of the salts was invariably detrimental; and also 1The omission of the curve at certain points is due to accidental loss of the specimens under experi- mentation. 622 Lorande Loss Woodruff. that the greater dilution produced the largest variation in the fission-rate, except in the MgSO, daily stimulus experiment. 4. Experiments with Potassium Bromid. The results obtained with potassium bromid in # and +45 solutions, are plotted in Diagram XII. This shows that KBr in both dilutions had on the whole a very slight accelerating effect on the division-rate, and also that the greatest variation from the control was caused by the 73; solution. ‘The chief effect of KBr, however, seems to have been to change the rhythm of division as shown when plotted in periods of five days. ‘The daily applica- tion of this salt also was deleterious, and [ had especial difhculty in maintaining the culture for more than two days when subjected to daily stimulations, which accounts for the omission of the daily curve in three out of the four periods of the experiment. 5. Comparison of Results. Comparing the results of all the experiments with the seven salts when used as initial stimuli, it is clear that K,HPO, 735 caused the greatest acceleration of the division-rate, while NaCl wh produced tbe greatest slowing of the rate. The largest variation from the control, when plotted in five-day periods, was shown by KCI ;%,. All the salts tested agreed in ey a marked deleterious effect when employed daily: K,HPO, 7255 being slightly the most active in this regard. ‘The table on the opposite page gives the actual status of each experiment in relation to the control for each five-day period of the work. Calkins tried stimulating his Parameecium cultures with various salts, among them the dibasic potassium phosphate and found that it not only produced an acceleration of the division-rate, but also that there were less fluctuations in the rate. His results show far more uniformity with this salt than do my own. Greeley ('04) investigated the effects of a number of salts on the physical struc- ture of protoplasm and incidentally on the division-rate of Para- moecium, and he arrived at the general conclusion that “with Paramececia from alkaline Siitures, anions or liquefying agents stimulate cell-division, cathions or coagulating agents inhibit it. Thus I have frequently observed in my experiments that when the liquefying solution is too weak seriously to modify the structure The Life-History of Hypotrichous Infusorta. 623 TABULATED Resutts oF SALT EXPERIMENTS. Five-pay Perriops. Theat Nee rae poets SALT SoLu- Varia- | Errect a AGE AGE UseEp. TION. TION IN |IN Four Vine Net Ist 2d 3d 4th Four | Lines. z Errecr. LINEs. AON: zoo ° mean (enrte’Qy lps ahg 14 + 4 ed ar KH2PO. | ——_——_ |———_ —— 1250 Be cy Ia ol a fa et) ° 5 ° sey -3 —6 + 2 —2 13 = 9) aa —24 KoHPOg ae S| eee aos +3 —2 +15 + 3 23 +19 52 +42 =i +8 | +6 -7 —22 43 —15 10} —3? KCl — —_$ _ Kr gyi ure ——\c—- at +9 +1 + 1 28 34 —12 84 =3 a +1 +4 = 7 —19 31 —21 73 om NaCl | ———_ | | J — | —————_— — x a Wy (lima eb soe cS Sees Be nn +4 =i + 2 =<) 10 an 2 24 ane) KeSO4 sha +8 ° + 4 —2 14 +10 34 +24 shi ° —8 + 1 -— I 10 — 8 24 —2 MgSO, — $$ | |! —____ | —__— ae +9 -3 — 8 — 6 26 -— 8 64 | —2 ae -7 +7 ° aie 18 an a 44 +1 KBr — ——— ———__|—___— Ss ° =] — 5 + 4 16 + 6 4 +14 Record of the variation in the number of divisions of each initial stimulus experiment from the control, during each five-day period; and also the net effect for the whole twenty days of the experiment. For example: the KH2PO,4 ;™., culture, during the first five-day period, divided exactly the same number of times as the control; during the second period, two times less; during the third, nine times more; and during the fourth, three times less than the control. For the four per- iods of the experiment, then, there was a total variation of fourteen divisions, or a “net effect”” of four more divisions than the control 624 Lorande Loss Woodruff. of the protoplasm it will however, greatly increase the motility of the protoplasm and the rate of cell-division.” Among the electrolytes employed by Greeley are three of the salts which I have used: KCl and MgSO, with predominant cathions and NaCl with the anion predominant. He found that KCl # and MgSO, 335 each exerted an inhibiting influence on the fission- rate, through a coagulating of the protoplasm. Referring to these salts he remarks that “the less active solutions, such as KCl and MgSO, do not produce quite so dense a coagulum as the others, and the reaction is considerably slower.”’ As already stated, my work with an initial stimulation of thirty minutes with KCl 3, and MgSO, 335 produced a quickening of the rate of fission for the first five days or more; the total result, however, for the twenty days of the experiment showed an inhibiting influence. With NaCl 2, Greeley found an increase in the rate and this agrees with the first period of my NaCl experiment, but here again I found a slowing of the rate for the total twenty days. It is impossible, though, to make a direct comparison of Greeley’s results with my own, both on account of the great difference in the methods employed and because he gives no details of the individual experiments. Peters (’04) describes some experiments with KCl on Stentor in which he found that initial stimulation for ten minutes produced an increased division-rate for the three days over which the longer experiments extended. ‘This accords with my results for the early periods of stimulation with the zy and with the ;2, solutions of this salt. To draw any general conclusions from my experiments with salts on the division-rate of Gastrostyla, I think, would be hazar- dous. Before this can be safely done it will be necessary to per- form many experiments on different forms. Work on this subject up to the present time, while affording a nucleus of data as a basis for future investigation, is too meagre and the methods employed by different workers too varied to make the results at all compar- able. As Towle (’04) aptly remarks: “the first step toward a clearing of the haze that envelops the subject will be found, I believe, when an effort is made to unify the conditions under which different investigators are working.” From this work on the Protozoa, I am persuaded that the most adequate method of attacking the problem is by breeding long cultures of Infusoria on a fixed diet. While this is a tedious process, it is the only way The Life-History of H ypotrichous Infusorta. 625 in which it is possible to know with any degree of certainty exactly what the pedigree of the subjects of the experimentation is, and unless one has the daily record of the ancestry of each protozo6n and knows its status in the life-cycle, any results obtained lose a large part of their value. Nothing emphasizes this point more forcibly than the record of my experiments with the dibasic potassium phosphate. Vil- SS ERFREECT -OF SLIGHT ON THE RATE OF DIVISION: Maupas (’88) made some interesting experiments on the effect of light on the division-rate of various Infusoria, by keeping cultures for one month in the light and then for one month in the dark and then comparing the rate of division during the two periods. But it would seem that his method is open to criticism for it is clearly impossible to keep the conditions absolutely con- stant during the two months of the experiments, not to mention the fact that, according to Maupas himself, “senescence” is increasing. Consequently it is impossible to say that the dif- ference, or absence of difference, in the rate during two consecu- tive months shows the effect, or non-effect, of light on bipartition. I would call attention to the fact that he found less difference in light and darkness than my records show for any two consecutive months of any of the cultures when light and all other factors have been apparently constant. With this in mind I made an experiment on the effect of light on the division-rate of Oxytricha fallax, and endeavored to elimi- nate the factors which seem to vitiate Maupas’s experiments. This was accomplished by isolating an individual from each line of Oxytricha A-culture, and starting with them a second culture (designated A‘) in absolute darkness.!_ By this method the light and dark series were carried on simultaneously and this ruled out the question of relative “senescence’’; and at the same time varia- tion in the food was reduced to a minimum, since the same infusion was supplied to both cultures simultaneously. “lempera- ture differences were avoided also. It would seem, therefore, that light was the only factor removed in the case of culture At, and 1The culture was necessarily, of course, subjected to light for two or three minutes each day when the record of divisions was being taken. 626 Lorande Loss Woodruff. that this had a very insignificant influence on the fission-rate is shown by the accompanying table. The experiment certainly substantiates Maupas’s result, however obtained, that light is of little or no direct importance in the economy of the ciliate. AKT atyeieec ae 23 divisions = ¢ Gxyteche A (lisht)eio soto ee _ 2 ocoladegs ae =2 cooupgoac 17 INS ee copeerae 20 se | ASNT, tetas eves 22 divisions i= “ Oxytricha A! (darkness) ............. fi eft ette is m 3 sadiereeiatels I Naa Sisto lorets 18 cs Total, 82 divisions. Excess in light, 6 divisions. VIII. SUMMARY. 1. The chief object of the work was to ascertain if the life- history of hypotrichous Infusoria is characterized by “cycles,” and if so, the cytological changes which occur and the effect pro- duced on the cycles by changes in environment. 2. [wo cultures of Oxytricha fallax, two of Pleurotricha lan- ceolata, and one of Gastrostyla stein have been carried on. Oxytricha culture A extended from October 26, 1901, to July 14, 1903, during which time 860 generations were attained. Culture B was started December 10, 1902, and died out through an acci- dent November 22, 1903. Pleurotricha culture A was isolated November 10, 1902, and became extinct July 3, 1903. Culture B was carried continuously from November 25, 1902, to March 13, 1904, when it was lost by an accident. ‘The culture of Gastrostyla was started May 28, 1904, and died out December 5, 1904. The life-history of each culture is represented graphically by a curve which is plotted by averaging the number of divisions per day of the four lines constituting each culture, and then averaging this for five- or ten-day periods. 3. All the cultures give incontestable proof that the species studied pass through periods of greater and less general vitality T he Life-Hstory of H ypotrichous Infusoria. 627 as measured by the rate of division. ‘This cyclical change is most prominent in the Oxytricha A-culture. ‘The periods of depres- sion lead to death if the culture is subjected continuously to the same environment. : 4. Minor fluctuations occur in the division-rate which I have termed “rhythms” and which are to be clearly distinguished from cycles. ‘The rhythms are probably indicative of a rhythmical change in the metabolism of the organism, though they are influenced somewhat by almost imperceptible changes in the environment. The results of the experiments seem to indicate that “rhythms” and “cycles” should be defined as follows: Arhythm is a minor periodic rise and fall of the fission-rate, due to some unknown factor in cell-metabolism, from which recovery is autonomous. A cycle is a periodic rise and fall of the fission-rate, extending over a varying number of rhythms, and ending in the extinction of the race unless it is “rejuvenated” by conjugation or by changed environment. 6. Changes in the environment will revive the lagging func- tions during the descending cycle, as is shown conclusively by the sudden recuperation of Oxytricha A during July, 1902. ‘There is every reason to believe that this “rejyuvenescence” was produced by treatment with extract of beef. 7- Seasonal and temperature changes have no apparent influence on the cyclical fluctuations of vitality. Variation in temperature, however, undoubtedly affects somewhat the daily rate of division, if not directly, at least through the food supply. 8. The number of generations which constitute a cycle is not at all constant; and there is no evidence to show that duration in time is of any significance in the forms studied. g. Periods of extreme depression of vitality are manifested on the physiological side chiefly by a greatly decreased division- rate, and by the comparative frequency of pathological divisions. Morphological changes are apparent chiefly in (1) an increased vacuolization of the cytoplasm; (2) distortion and fragmentation of the macronuclei; (3) numerical increase of the micronuclei; and finally (4) in a reduction of the ciliary apparatus. 10. Variation in the size of the infusorians during the life- cycle is marked; the organisms being very small during periods 628 Lorande Loss Woodruff. of high reproductive activity and progressively increasing in size as “degeneration” advances. In the last couple of generations before death ensues the size is secondarily reduced by a shrinking of the cytoplasm. 11. A disappearance of one of the micronuclei occurred at certain periods of high reproductive activity. 12. These cultures strongly suggest that it is customary to regard the structure most frequently observed in “wild” Infusoria as too constant in character, and to overlook the fact that, under varying conditions, modifications may occur which are in no way abnormal. 13. Throughout the entire period of the cultures no tendency to conjugate was shown in any of the series, and experiments for endogamous and exogamous syzygies failed to produce a single case. 14. Experiments with KH,PO, K,HPO,, KCl, KBr, K,SO,, MegSO,, and NaCl gave evidence of the extreme sensitiveness of Protozoa to solutions of electrolytes. Initial stimulation with KH,PO,, K,SO,, and KBr in ;2, solutions caused in each case a slight acceleration of the division-rate; while initial stimulation with =2, K,HPO,, KCl, NaCl, and MgSO, caused a slowing of the rate. Daily stimulation with the same solutions of each of these salts invariably, caused a marked inhibition of the fission- rate. Initial stimulation with KH,PO, ;.%, showed no change in the rate while K,HPO, 7%, produced a marked increase. K,SO, 32, accelerated division; and KCland NaCl each in 4 solutions, retarded it; while KBr 33, accelerated the fission-rate. Comparison of the effects of the two solutions of each salt shows that, almost without exception, the more dilute solution produced the greater variation in the rate from the control. 15. Stimulation with K,HPO, 75 gave different results at various periods of the life-cycle, which indicates that the state of the general vitality of the culture, and also the rhythms, are factors which must be taken into account in experimental work of this nature. 16. Light has little or no direct effect on the division-rate of Oxytricha fallax. Zoélogical Laboratory, Columbia University, New York, 1905. The Life-History of Hypotrichous Injusorta. 629 LITERATURE. Butscuut, O., ’76.—Studien tiber die ersten Entwickelungsvorgange der Eizelle, der Zelltheilung und der Konjugation der Infusorien. Abh. d. Senckenb. nat. Gesellsch. Frankfurt a. M., x. ’83.—Protozoa. Bronn’s Klassen und Ordnungen der Thierreichs. Catxins, Gary N., ’01.—The Protozoa. Columbia University Press. ’02, 1.—Studies on the Life-history of Protozoa. I. The Life-Cycle of Parameecium caudatum. Archiv fiir Entwickelungsmechanik der Organismen, xv, I. . ’o2, 2. (With C. C. Lieb.)—Studies on the Life-history of Protozoa. II. The Effect of Stimuli on the Life-Cycle of Paramcecium caudatum. Archiv fiir Protistenkunde, i, I. ’02, 3.—Studies on the Life-history of Protozoa. III. The Six Hundred and Twentieth Generation of Paramoecium caudatum. Biol. Bull., iii, 5. ’o4.—Studies on the Life-history of Protozoa. IV. Death of the A- Series of Paramcecium caudatum. Conclusions. Journal of Experimental Zodlogy, 1, 3. Duyarpin, F., ’41.—Histoire naturelle des zodphytes Infusoires, comprenant la physiologie et la classification de ces animaux, etc. ENGELMANN, 1. W., ’76.—Ueber Entwickelung und Fortpflanzung von Infusorien. Morphologisches Jahrbuch, 1. Geppes, P., anp THomson, J. A., ’00.—The Evolution of Sex. Second ed. =: Scribners. GreELEY, A. W., ’04.—Experiments on the Physical Structure of the Protoplasm of Parameecium and its Relation to the Reactions of the Organism to Thermal, Chemical and Electrical Stimuli. Biol. Bull., vii, 1. Hertwic, R., ’03.—Ueber Korrelation von Zell- und Kerngrésse und ihre Bedeu- tung fur die geschlechtliche Differenzierung und die Teilung der Zelle. Biol. Centralblatt, Bd. xxiii. Jounson, H. P., ’93—A Contribution to the Morphology and Biology of the Stentors. Jour. of Morphol., viii, 3. Jouxowsky, D., ’98.—Beitrage zur Frage nach den Bedingungen der Vermehrung und des Eintrittes der Konjugation bei den Ciliaten. Verh. Nat. Med. Ver. Heidelberg, xxvi. Lyon, E. P., ’04.—Rhythms of Susceptibility and of Carbon Dioxide Production in Cleavage. Amer. Journal of Physiology, xi, I. ’92.—Effects of Potassium Cyanide and of Lack of Oxygen upon the Fertilized Eggs and the Embryos of the Sea Urchin (Arbacia punctulata). Amer. Journal of Physiology, vii, 1. 630 Lorande Loss Woodruff. Maupas, E., ’88.—Recherches expérimentales sur la multiplication des Infusoires ciliés. Arch. d. Zod]. exper. et gén., 2me sér., vi. ’89.—Le rejeunissement karyogamique chez les Cilies. Arch. d. zodl. exper. et gén., 2me sér., Vil. Peters, A. W., ’04.—Metabolism and Division in Protozoa. Proc. Amer. Acad. Arts ang@sct., XXxIx, 20. Rywoscu, D., ’00.—Ueber die Bedeutung der Salz fiir das Leben der Organismen. Biol. Centralblatt, xx, 12. Scott, J. W., ’03.—Periods of Susceptibility in the Differentiation of Unfertilized Eggs of Amphitrite. Biol. Bull., v, 1. Smmpson, J. Y., ’o1, 1.—Observations on Binary Fission in the Life-history of Ciliata. Proc. Roy. Soc. Edinb., vol. xxiii. "oI, 2.—Studies in Protozoa. JI. Notes on the Intimate Structure of Protozoa, as Exhibited by Intra-vital Staining. Proc. Scot. Microscopical Soc., ii, 2. STEIN, F., ’83.—Der Organismus der Infusionsthiere. Leipzig. STERKI, V., ’78.—Beitrage zur Morphologie der Oxytrichinen. Z. w. Z., xxxi. Tow es, Exizasetu W., ’04.—A Study of the Effects of Certain Stimuli, Single and Combined, upon Parameecium. Amer. Jour. of Physiol., xa Verworw, M., ’97.—Allegemenine Physiologie. WALLENGREN, H., ’01.—Inanitionserscheinungen der Zelle. Zeit. f. allg. Physiolo- PaGaL. Weismann, A., ’84.—Ueber Leben und Tod. Wiison, E. B., ’oo.—The Cell in Development and Inheritance. Second ed. Columbia University Press. T he Life-History of Hypotrichous Injusorta. 631 EXPLANATION OF PLATES. The photographs were taken by Dr. Edward Leaming, of Columbia University, from permanent preparations stained with picrocarmin. ‘The magnification is the same in every case and the relative sizes, therefore, represent absolute differences. The figures, unless otherwise specified, are of Oxytricha fallax, Culture A. Pirate I. Figs. 1 and 2. Two individuals in the 230th generation, period 16, April 2,1902. (Cf. Diagram I.) The cytoplasm is vacuolated and the macronuclei are vacuolated and displaced in the cell. A charac- teristic “‘halo” is visible about the macronuclei. The individual shown in Fig. 2 has three micronuclei. Figs. 3 and 4. Individuals in the 239th and 241st generation respectively. Period 24, June 1902. The two macronuclei in each are fused and their structure appears somewhat more homogenous than is the case in those illustrated in Figs. 1 and 2. Fig. 5. Specimen in the 243d generation, period 25, June 24, 1902, showing an extreme case of cytoplasmic vacuolization. The nuclei are exceptionally normal for this period of the cycle. Fig. 6. Specimen in the 246th generation, period 25, July 1, 1902. Fig. 7. Individual in the 246th generation (A-2), period 25, July 2, 1902. The macronuclei are surrounded by a “‘halo” (cf. Fig. 1). Fig. 8. Individual in the 247th generation (A-1), period 25, July 2, 1902. Note the condition of the cytoplasm. Prater II. Fig. 9. Specimen in the 25oth generation (A-1), period 26, July 6, 1902. The cell is shrunken and the cytoplasm considerably vacuolated. Note the somewhat reduced size and irregular contour of the cell. This is the last of the line A-1 before it was “‘rejuvenated.” Figs. 10 and 11. Specimens in the 255th generation, period 27, July 21, 1902. These individuals are from line A-2 which remained dividing, at this time, at the slow rate. The specimen photographed in Fig. 10 has ingested a Trachelomonas volvocina. Fig. 12. Specimen in the 256th generation (A-1), period 26, July 8, 1902. This line had divided six times within the past forty-eight hours. Note the normal condition of cytoplasm and nuclei as compared with the preceding specimens. Fig. 13. Specimen in the 287th generation (A-1), period 27, July 20, 1902. Size is reduced. Compare with Fig. 12. Fig. 14. Individual in the 331st generation (A-1), period 29, August 7, 1902. Size is reduced. Nuclei are proportionately large. Fig. 15. Specimen in the 4ogth generation, period 32, September 1, 1902. Apparently a ‘‘normal” individual in every respect. Fig. 16. Specimen in the 542d generation, period 36, October 17, 1902. Cytoplasmic vacuoliza- tion begins to appear. Fig. 17. Individual in the 829th generation, period 56, April 29, 1903. Nuclear fragmentation has begun. Fig. 18. Specimen in the 853d generation, period 62, July 2, 1903. Nuclear and cytoplasmic degeneration is far advanced. The size of the cell is greatly increased. Same as Fig. 18, Plate IT. : Fig. 20. Specimen in the 854th generation, period 62, July 4, 1903. Fig. 21. A double monster from A-1, 238th generation, June 16, oe Fig. 22. Oxytricha fallax, B-culture. Specimen in the 365th generation, August 2 125; 1903. “Con: dition is normal. Fig. 23. Pleurotricha lanceolata, B-culture. Individual i in the 413th generation, January 29,1 Late division-stage, he = - i ay be etek ct he cee ‘ Sa we : SS ane av * _ “THE LIFE-HISTORY, OF HY HYPOTRICHOUS INFUSORIA. L. L. Woovrvrr. ao Ne Bagley ee ue oe PLATE TI. ic Tue Journat or ExprriMENTAL Zo6LoGy, vol. u. THE LIFE-HISTORY OF HYPOTRICHOUS INFUSORIA. L. L. Wooprurr. PLATE II. Tue Journat or ExperimentaL Zooéxoey, vol. ii. ialua i matic ee ‘s ey Spee oe is ae ise yf " ; - . 7 : Pot gta ey aan. ‘ ; te a ; wat 5 ve’, ' J or | oe oe val hy ane | : i , : a a ped y oy peel! - if : , : - : i ay) ( 7 a i , a ’ i 4 ~ We ar a7 7 \ ie)” ( ’ 1 i > * Lye if ih Stu" ' yy ‘ ais @ = 5 - - y 7 THE LIFE-HISTORY OF HYPOTRICHOUS INFUSORIA. L. L. Wooprurr. PAGE) ii: Tue JournaL or ExperiMENTAL Zo6LoGy, vol. il. a ard Dp ae ie ah Lo ha eee ek et ANCIENT Bas Jae ai RS os petra