Please handle with _ EXTREME CARE This volume is Brittle — - and CANNOT be repaired! * Photocapy only if necessary Return to library staff, do not in bookdrop ~ _ “GERSTEIN SCIENCE INFORMATION CENTRE Library staff, please retie with black ribbon and reshelve LINIV. OF ToRONTO LIBRARY ~ Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/journalofexperim0S5broo ¥,) ay Oa ie wi tak JOURNAL OF EXPERIMENTAL ZOOLOGY EDITED BY WILLIAM K. BROOKS Johns Hopkins University WILLIAM E. CASTLE Harvard University EDWIN G. CONKLIN University of Pennsylvania CHARLES B. DAVENPORT Carnegie Institution ROSS G. HARRISON Yale University HERBERT S. JENNINGS Johns Hopkins University FRANK R. LILLIE University of Chicago JACQUES LOEB University of California THOMAS H. MORGAN Columbia University GEORGE H. PARKER Harvard University CHARLES O. WHITMAN University of Chicago EDMUND B. WILSON Columbia University ROSS G. HARRISON, Managing Editor 2 HILLHOUSE AVENUE, NEW HAVEN, CONN. VOLUME V PUBLISHED QUARTERLY BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY 36th STREET AND WOODLAND AVENUE PHILADELPHIA, PA, CONTENTS No. 1—November, 1907 Davin Day WHITNEY Determination of Sex in Hydatina senta ArTHUR B. Lams A New Explanation of the Mechanics of Mitosis. With Two Figures... . HERBERT EUGENE WALTER The Reactions of Planarians to Light. With Fourteen Figures... . No. 2—December, 1907 HERBERT EUGENE WALTER The Reactions of Planarians to Light. With Fourteen Figures (con- chided) pete Sy lsterie ce wo eta MEMES ora CR eae eee Mary ISABELLE STEELE Regeneration in Compound Eyes of Crustacea. With Sixteen Plates and De Mane WOR endian mace edcae bdeauoheb hor sorgoesse onan H. V. WiLson On Some Phenomena of Coalescence and Regeneration in Sponges. ANS NI SOME MN ateS a hace boo manbeu ss sco un bob aw ou oaU pmaOal dick Hans PrzIpRAM Equilibrium of Animal Form. With) Templo reson rerescaheteeretieeeteha (ter CHARLES ZELENY The Effect of Degree of Injury, Successive Injury and Functional Activity upon Regeneration in the Scyphomedusan, Cassiopea xamachana. \WitchvBourphiipuresssteilt qesicteece tty sa te cletmetstteret teeters Oot acy ett ALEXANDER PETRUNKEVITCH Studies in Adaptation. I. The Sense of Sight in Spiders. With Six INANE, Foc pone ee Ree ah: SE Sk 245 259 No. 3—March, 1908 Gruman A. Drew The Physiology of the Nervous System of the Razor-Shell Clam (Ensis directus, Cons)) sWathi@neiPlate see cen: enecace.o = ote rer 311 FLORENCE PEEBLES The Influence of Grafting on the Polarity of Tubularia. With Twenty- Chay writs choeoss Guid bod O An ou 6 DMbore iD ROME ad oso. vac 32 N. M. STEvENS A Study of the Germ Cells of Certain Diptera, with Reference to the Heterochromosomes and the Phenomena of Synapsis. With Four Pl atesi hire cei uakeee iS cphane ence BRS eea: ADE he kee Sus 9, ed Sie ee Re ere 359 Ratpu S. LILiie Momentary Elevation of Temperature as a Means of Producing Artificial Parthenogenesis in Starfish Eggs and the Condition of its Action....... 375 Tuos. H. Monrcomery, JR. The Sex Ratio and Cocooning Habit of an Aranead and the Genesis of Sex Rationse With aewoll i punes mec canioe aii «6 Meee a Ae 429 No. 4—June, 1908 N. M. STEVENS The Chromosomes in Diabrotica vittata, Diabrotica soror and Diabrotica 12-punctata. A Contribution to the Literature on Heterochromosomes and Sex Determination. With Three Plates...................-. 453 Victor. E. EMMEL The Experimenal Control of Asymmetry at Different Stages in the Devel- opmentofithe lobsters.) ome eee Erte one 471 C. M. CuiLtp Physiological Basis of Form-Regulation. With One Figure .......... 485 H. H. Newman The Process of Heredity as Exhibited by the Development of Fundulus Hybrids. With Five Plates and Sixteen Figures in the Text ..... 503 C. C. GUTHRIE Further Results of Transplantation of Ovaries in Chickens. With Three Bigures acct raer siete rapt esi cterers reels, ee ieiateaer eer eS eee 563 H. S. JENNINGS Heredity, Variation and Evolution in Protozoa. With Twenty-two Figures SiS ce hectare ease eerieiee tee ann. Oe eee 577 DETERMINATION OF SEX IN HYDATINA SENTA BY DAVID DAY WHITNEY Mie oIntro duction: cram c yet eeieveieree vein etctetede ol 1eCotelsfoisislatavers:= nvessioLole/eale/ouclerarearfourtaletere: tae e/e}selel I TE Material and methods: «<..2 2.0 cccceec cence creases deeceneensce cee eeseeuseecteee 3) III Influence of temperature 4 1 Maupas’ experiments......-.00-es cece cece ete cee e rere eee t eee sence r een ers ces 4 2 Author’s experiments.........6 02-6 e secre cere eee e teen eee ener teen e eee ees 5 a Temperature 20° to 22°C. .... 1... e cece eee eee eet e eee t tn t eens 5 b Temperature 25° to 29°C... 1. eee eee eee eee tee teen ete e ene ees 8 c Temperature 14° to 15°C... 2. eee eee eee teen eter e teen eens 9 IV The relative number of eggs which a male-laying female and a female-laying female produc: 10 I Temperature 20° to 22°C... 21. cece cece tenet eet eee e cent ence eens II 2 Temperature 24° to 29° C.... 2.222 see eee erent ee tence ete cnt e erect ees II V_ Early production of male-laying females in a family of daughter-females...............--. 13 Villm lin men celo Mt Oo samc isrovarcitereisveleilalalelsvol-porelaye ofosshaxeseps¥elcVerebesepsltaretceseleieds laidtsiel-(o[olaloriaretiere 15 1 Temperature 20° to 22°C... 2... ee eee ee ee eee ete teen entree cent es eees 16 2 Temperature 14° to 15° C.... 6.00 eee eee eee cent eee eee ee tenet ee eee ees 18 3 Temperature 25° to 26° C.... 1... cece eee eter e eect eee erence tees 18 aVI Male and female'strains........0..20.0ccecccs cer ecce cece cece etsccestesteneceerersce 19 VIII Production of fertilized eggs... ... 0... ce cece cece ec eee cere eet t eee eet e teen eee ene 23 YR S we ary ate ea Yay set saket ole vole aisieke orelalsatcla = ehtetaret=te)sfoin in) tove:wielalete\oL-setehegesonel se eisieieielape=;nheyaks 25 I INTRODUCTION On account of the supposed influence of external factors in determining sex in Hydatina senta, this rotifer has attracted much interest in recent years. As is well known Hydatina produces three kinds of eggs, viz: (1) parthenogenetic eggs which develop into females; (2) smaller parthenogenetic eggs which develop into males; and (3) fertilized eggs which develop into females. Each female produces only one of these three kinds of eggs. ‘Thus three types of females may be distinguished, viz: (1) females which produce females parthenogenetically, or female-laying females, 2 2; (2) females which produce males parthenogeneti- cally, or male-laying females, @ 9; and (3) the sexual females that lay fertilized eggs. Tur Journat or ExreriMENTAL ZOOLOGY, VOL. V, NO. f. 2 David Day Whitney Both female-laying females and male-laying females can be impregnated by males, but on the former, impregnation is sup- posed to have no effect. If the male-laying females are impreg- nated by the male in the first few hours after they leave the egg, such females produce fertilized eggs instead of parthenogenetic male eggs, thus showing that male-laying females can develop into sexual females that lay fertilized eggs. The female-laying female can produce a family of daughter- females, some of which may lay female eggs and others may lay male eggs. With the view of finding out the ratio in which these two classes of daughter-females are produced under various conditions I have carried out the experiments to be described. Maupas found that a temperature of 26° to 28° C. would pro- duce as high as 95 per cent of male-laying females while a tem- perature of about 14°C. would produce as low as 5 per cent of male-laying females. Nussbaum, on the contrary, came to the conclusion that nutri- tion and not temperature is the sex controlling factor. He found that by starving the young females for the first few hours after they emerge from the egg they would produce a high percentage of males, but if they were fed at the time they leave the egg they pro- duce a high percentage of females. Punnett has carried out a few experiments along the lines laid down by Maupas and Nussbaum and finds that neither tempera- ture nor nutrition is influential in determining the sex. He finds, on the contrary, that there are definite “‘sex strains.”’ Some strains produce 40 to 50 per cent of males, others produce a very low percentage, 2 to 5 per cent, while others produce no males at all, although reared through as many as seventy-two generations. The greater part of the work of the present paper was planned and begun in the spring of 1906, under the direction of Prof. T. H. Morgan, before the results of Punnett were published. Not knowing how to obtain proper food cultures the rotifers all died in July and the continuation of the experiments was deferred until October, 1906. Determination of Sex in Hydatina senta 3 Il MATERIAL AND METHODS In the latter part of April, 1906, Hydatina senta was discovered in great numbers in a small pool on the Palisades of New Jersey near Grantwood. The pool was fed by a little stream or ditch which carried away the drainage from several cottages. “The ditch was an extremely favorable place for the growth of Euglena viridis which collected in large patches on the sides and bottom. Immense numbers of Euglena floated down into the pool at the end of the ditch and served as food for the rotifers which abounded there in countless thousands. Sometimes as many as 150 to 250 individuals could be drawn up by a pipette in a few cc. of water. About May 15 the pool dried up completely. The ditch still contained water but no rotifers were found in it after May 20. At this time there were innumerable larve of insects in the ditch and perhaps they exterminated the rotifers by feeding upon them. In all experiments each individual female was isolated in a square or round watch glass which contained about 5 cc. of water and fed with Euglena, other protozoa and bacteria. In order to obtain the Euglena and other protozoa a culture of horse manure and water (one to two ounces to a quart) was made, inoculated with Euglena and allowed to stand for two to three weeks at room temperature. At the end of this time the green coating of alg, Euglena, etc., could be removed from the sides of the glass jar and served as an excellent food for the rotifers. Great care was taken to keep these food cultures uncontami- nated by rotifers. All watch glasses were placed in hot water after each experiment in order to destroy all eggs which adhered to the sides, thus preventing contamination of the following experi- ments by eggs of the preceding ones. The experiments at temperature of 24° to 29° C. were con- ducted in an incubator. ‘Those at a temperature of 20° to 22° C. were conducted on the laboratory tables at room temperature, while those at a temperature of 14° to 15° C. were carried on in an ice chest. These rotifers are exceedingly hardy and can be very easily kept in the laboratory throughout the year. In Mayof 1906 a Euglena culture was prepared in a glass jar containing 2000 cc. of water 4 David Day Whitney and a few rotifers put into it. The jar was covered so as to pre- vent evaporation of the water. Rotifers have lived in it to this time, April, 1907, although no more food material has ever been added. It is absolutely necessary that the surface of the water be free from a scum for the rotifers will die within a few hours if it is present. It is safer, in order to keep the surface free, to tie the horse manure in a muslin cloth and place it in a well covered jar nearly filled with water. III INFLUENCE OF TEMPERATURE I Maupas’ Ex periments The experiments of Maupas were so briefly described that it is very difhcult to understand clearly how he obtained his results. Nussbaum and Punnett are inclined to believe he determined that a female was a male-laying or a female-laying individual by the size of the eggs that she produced. Small eggs being assumed always to give rise to males while larger eggs give rise to females. Nussbaum has measured a series of both male and female eggs and found that in some instances the two kinds of eggs over-lap in size. ‘Thus he points out an error through which Maupas’ results might have been obtained. Isolating and counting the eggs of this rotifer would be exceed- ingly tedious and require almost constant attention. As the sexes can be readily distinguished at any period and as it requires only 36 to 48 hours for a female to mature and produce eggs it seems to me extremely probable that Maupas must have allowed some at least of the eggs to hatch before recording his results. As his experiments are so few and briefly described it may be well to present them here in order that they may be compared with and interpreted by my own. Experiment I. Lot A, tempera- ture 26° to 28°C. Five female-laying female sisters produced 104 eggs; 97 per cent developed into male-laying females. Lot B, temperature 14°C. Five other female-laying females, which were sisters of lot A, produced 260 eggs; 5 per cent developed into male-laying females. Determination of Sex in Hydatina senta 5 Experiment II, temperature 14° C. Five female-laying females, kept from the time of hatching at this temperature, produced 110 eges; 24 per cent developed into male-laying females. The same five female-laying females were then placed at a tem- perature of 26° to 28° C. and produced 81 per cent male-laying females. Experiment III, temperature 14° C. Six female-laying females which had been kept at this temperature from the time of hatch- ing produced 34 eggs, of which 12 per cent developed into male- laying females. The same six female-laying females were then placed at a tem- perature of 26° to 28° C. and allowed to produce 44 eggs, of which 95 per cent developed into male-laying females. These six females were alternately placed at 14° C. and 28° C. several times and always gave a high percentage of male-laying females at the higher temperature. 2 Author's Experiments a ‘Temperature 20° to 22°C. Experiment I, October 24, 1906. A female-laying female was isolated from a jar which was stocked with rotifers collected Octo- ber 2, from the same pool in which the animals were found in the preceding spring. This strain was carried through twelve generations and the percentage of male-laying females determined. Each female was supplied with an abundance of food from the time of hatching, isolated in a separate watch-glass, and kept upon the laboratory table at room-temperature. Table I gives the ratio of the mother individuals producing male and female offspring in the 3264 daughter-females of 95 female-laying females in the twelve generations. This experiment was made in order to obtain the percentage of male-laying females produced at room temperature of 20° to 22° C., in order to be able to have some standard percentage of male-laying females with which to compare the results of the experiments conducted at lower and higher temperatures. 6 David Day Whitney TABLE I Record of the production of male-laying and female-laying females among the 3264 daughter- females of 95 female-laying mothers. Temperature 20° to 22° C. No. | Eggs | Offspring | Per | | No. Eges | Offspring | Per Gen. | 22 ale [> ae || cent -||Gen--) 299 oc | po ercireeceneenn ECCOE | mother fe | oe yc Wh ears: mother laid (sional) Louse eine) SS | eta | Se ape = see | | | | I I ar | ets Vere) | 48a 3 32 8 | 24 25 | | 4 15 3 12 20 I I 28. || 0: |) 28 ° | Ce Ne 223 Sie ess 18+ } 2 | 53 | 8 | 45 | ase | 6 48: 9) rte! Rage eae | 3 27) hn: 23.«| «14+ | 7 25 o | 25 ro) | | | 8.938 | 15) 23 tao It I 440 cay | a7 38+ | 9 17 La be 23+ | 2 | 20.0) ara 325) 20h 10 48 15 3300 Stats | | | II 48 | 16 33+ IV Te al ees I so | i+ | 12 43 4 39 | 9+ 2 | 47 15 32 | Bit 13 44 | 10 34. |) 722-5 30 N52 elo Mh iea6 tao 14 49 | +14 35. | 28+ lisa asoonie 71/8 28 ulezo | Sx¢/ 1 Wasa ees 27 40 | 16 16 | 6 10 37+ My I 47 Our 47 ot 17 45 | 4 41 8+ 2 44 2 42 Piaoe Ml 18 50 7 43 14 3 41 40 2+ | 19 | cpa il) A 28 12+ 4 45 ° 45 | | 20 21) |" 0 21 ° 21) 0) 2) See 17 19+ NAD pag ae Mie 12 20 27+ 2220 2 18 10 i) ee 45 4 41 8+ 230+) «925 ° 25 ° Saas o | 36 | © | | eae illsa7 | go. | 14+ ar 49 8 40 16+ 5 | 48 22 26 «| 45+ } | | | XII I 38 3 35 7+ VIE]; 1 30 15 15 | ‘50 2 45 9 36 | 20 | 2 35 I 34 2+ 3 48 4 44 8+ 3 | 27 ° 27 ° 4 31 5 26 16+ 4 24 ° 24 ° 5 shee (aie) 26 | 33+ 5 19 5 14 | 26+ | 6 31 10 21 32+ | latZ 54 To 440) 18+ Vill I 41 16 25 39+ es 45 6 39 13+ Panes 17 I 16 | s+ | 9 35 II 24 31+ 3 47 18 29 38+ | 10 43 5 B85 u) Erte 4 26 ° ye | xr 38 3 35 iia 5 38 | ° 38 ° 12 27 9 18 33+ 13 42 16 | 26 |- 38+ Ix I 24 | I 23 4+ 14 34 ei Og Bae 15 83 15 | 28 | 34+ x I 49 | 13 36 0 | 26+ | 16 37 6 | 31 16 axe 45 10 35 224+ | | 47 31 26 16+ Determination of Sex in Hydatina senta TABLE I—Continued Temperature 20° to 22° C. Now Eeeeet| a Obspaugs |) be Noo ees ||) Otspung, || Se Gen. Se a TERE. || |i ea | ass Gen. 2h) heat Ise — mother | au oon] Lo) {0} ae mother Gs ae? 29 XII 18 26 7 19 | 22+ || XII | 30 16 5 II 19 33s S| 3r 9 oe A 20 4 | 3 41 6+ 32 18 8 | 10 21 20 ° 20 Qi | 33 20 6 14 Bam Aleetsc i ltee 36 | 20 | 34 47 14 33 | | } 31 ut ZO E350) 35 x9) 4 15 24 28 10 i | 35+ 36 26 8 18 25 15 3 2 [20 | 37 II ° II 26 34 I 33 2+ 38 37 II 26 27 15 ° 152) Ce sl 3934 19 15 28 14 3 II 20 40 25 6 19 29 6 I 5 | 16+ | 41 35 4 31 | \ 1 cent 31+ Dict 44+ 3° The nature of the sex-producing power of the daughter-females of each individual mother is given separately in order to show that the ratio between the daughter-females producing male and female offspring varies with different mother-individuals, and also varies as much in the daughter females of sister-mother-individuals. Summary of each generation in Table I and also the final summary of all the generations taken 29 mother TABLE II together. Temperature 20° to 21° C. Offspri Per Werceonl EE eesa | les aR: Sb \inoeher laid 29 29 Sad Gen. Tye 31 15 16 | 48+ | VIZ | I 3 108 12 96 i+ | VOI Ii 2 go 31 59 34+ ee x IV 4 185 39 146 21+ XI Vv 4 177 3 174 I+ xi VAS WG 208 45 163 21+ Eggs Offspring Per aid Ee | ae cent Sa a? _ | a al 135 21) 114 | 15+ | | 169 35 | 134 | 20+ 24 I |} 23 4+ 819 176 643 -| 21+ 49 8 41 16+ 1269 268 | roor | 21+ 3264 654 | 2610 | 204 8 David Day Whitney Table II gives the summary of each generation and the final summary of the twelve generations. In this experiment 95 mother-individuals produced 3264 daugh- ter-females of which 20+ per cent were male-laying females. It will also be noted that the percentage of male-laying females varied in the different generations from I + per cent to 48+ per cent regardless of the number of isolations in each generation. b Temperature 25° to 29° C. Experiment II, October 26. Two female-laying females were TABLE III Record of the production of male-laying and female-laying females among the 208 daughter-females of 26 female-laying mothers. Temperature 25° to 26° C. Temperature 26° to 29° C. | No. Leges| Ofspring| Per No. Eggs Offspring | Per Gen.| 99 | ale __| cent Gen.| 29 laid | — | cent | mother | ] 82/29} oe | mother J 2)9 9 | oS? | | | | | | | StrainIV) I) 1 | 17| o| 17] © Strain TT IX | I | Werey [Naz | ° | | 2 || go" | ral ° | I I (Ts ae Ly | we (or | | | | | | xX 1 |e 31, ONS StrainIII| I I 17 corals 4 | o | 2 Sa sol) 5 ° | {<3} 1h Srh3|) an Fo) IE100 Ul I 13} 4] 9] jot+ AL || <t\feo: | ex ° 5 8| 2 | 6 | 24+ peel I 16 | 2/14] 12+ 6 6) t] 5 | 16+ | oh |W 2 | 6| 24+ Vil | I 16 | Mal 12+ 8 oat foie} ° | | 2 | 10 | 3 7 30 9 | 5 2 | 3) 4° 3 TE | (6:5 Gi Sat Tor | 143) 225i) 450 | 4 25 | 13 | 12 | 52+ are Nie CNAs aac : 725 |/91\) 05203 ae 13 i at I Ce 0c | 1 | 10 3| 7] 30 | ef | © | 100 | 26/208 46 |162 | 22+ isolated from the same stock jar in a similar manner as in Experi- ment I, and placed in an incubator. ‘Two generations from one Determination of Sex in Hydatina senta 9 individual, strain [V, and six generations from the other individual, strain II],were recorded. Six generations were kept at a tempera- ture of 25° to 26° C. and two generations at 26° to 29° C. Table III gives the results of the experiment obtained at this higher temperature. The 26 female-laying mothers from eight generations, and from two strains, produced 208 daughter-females of which 22 + per cent were male-laying females. ‘This percent- age is practically the same as that obtained at room temperature. @ Wemperature 14° to 15°C. Experiment III, October 8. A female-laying female was isolated from stock jar as in Experiment I, and placed in an ice chest. A record of only a few of her offspring was kept and is shown in Table IV. TABLE IV Record of the production of male-laying and female-laying females among the 167 daughter-females of 7 female-laying mothers. Temperature 14° to 15° C. No. of | eo | Eggs ; Ofspring ; Per cent mother | laid | Fed oo | 99 Fe I 48 47 11 36 23+ 2 so «| «28 4 24 14+ 3 30 20 3 17 15 4 | 22 9 2 7 22-+- 5 41 19 5 14 26+ 6 | 36 15 ° 15 ° 7 | 51 2 ro) | 19 34+ 7 | 278 167 35 132 20+ Out of 167 daughter-females from 7 different mothers 20+ per cent were male-laying females. “Uhe mother-individuals were reared at this low temperature as well as the daughter-females. The percentage of male-laying females is about the same as that obtained at temperatures 20° to 22° C. and 25° to 29° C. The foregoing results agree with those obtained by Nussbaum and Punnett but seem contrary to Maupas’ results. 10 David Day Whitney IV THE RELATIVE NUMBER OF EGGS WHICH A MALE-LAYING FEMALE AND A FEMALE-LAYING FEMALE PRODUCE It seems evident from Maupas’ account of his own experiments that he did not isolate each female-laying mother and each one of her daughter-females but kept the female-laying mothers to- gether in one dish and their daughter-females together in another dish. If it is assumed that Maupas made no mistake in determining the sex character of the eggs before they hatched, or even that he allowed all eggs to hatch before he recorded their sex character, his results can be easily explained. TABLE V The number of eggs laid by each of 13 sisters, of which 6 were male-laying and 7 were female-laying, showing that the average number of eggs laid by each of the two kinds of females is very nearly the same. Temperature 20° to 22° C. 13 Sisters F 9 99 | 98 Mother | Eggs | rae Mother | Eggs | ane I 46 I 47 I 50 I 38 I 47 Li 37 I 46 I 43 I 41 I 38 I 31 I 45 I 38 6 261 434 7 268 408 - He gives no results of experiments conducted at a tempera- ture midway between 14° and 28° C. but only results obtained at these two extremes. The results that were obtained at 14° C. may very likely be identical with those that could have been obtained at a room temperature around 20° C, Maupas recognized the fact that male-laying females produce eggs faster than female-laying females but makes no mention of the number of eggs that each kind of female may produce at dif- ferent temperatures. He seems to assume that they always pro- Determination of Sex in Hydatina senta II duce about an equal number, 40 to 50 each, but the following experiments will show the error of this assumption. I Temperature 20° to 22° C. Experiment IV, November 5. Of 13 sister individuals kept at room temperature and with the same amount of food 6 pro- duced male eggs and 7 produced female eggs. ‘The average num- ber of eggs produced by each female was nearly the same. ‘The results are shown in Table V. 2 Temperature 24° to 29° C. Experiment V, November 9. ‘Three lots of sister-individuals from three different mother-individuals were kept in an incubator TABLE VI Record of the number of eggs laid by 11 sisters, of which 6 were male-laying and 5 were female-laying, showing that the average number of eggs produced by the male-laying females is about two times as great as the average number produced by the female-laying females. Temperature 24° to 25° C. II sisters | 7 A =) route) 9 a? | 2 2 | oO z 2 | Av. Mother Eggs we Mother | Eggs | if = —- —_ ls = I 37 I 16 u 35 1 17 I 26 I 16 I 38 I II I 26 I 10 I 22 e 184 305 se a ze 14 and the number and sex character of the eggs that each produced was very carefully noted. The results are shown in Tables VI, VII and VIII. The records of the individuals in Tables VI and VII were taken at the same temperature of 24° to 25° C. while those of Table VIII were taken at a higher temperature of 26° to.29° C. These tables show a decided change in the ratio between the number of eggs produced by a male-laying female and a female- laying female. As the temperature is raised the female-laying 12 David Day Whitney TABLE VII Record of the number of eggs laid by 21 sisters, of which 9 were male-laying and 12 were female- laying, showing the average number of eggs produced by the male-laying females is about two times as great as the average number produced by the female-laying females. Temperature 24° to 25°C. 21 sisters rr Peles se = oe oe | 29 g Mother | Eggs aN | Mother Eggs Ny loa fe ee I 25 «| I 6 I 39 | ee 14 I 34 I 16 I 37 I II I 28 | | I 10 I 25] | I 12 I 29 | st 12 I 23504 | I 9 I 22. 4 | I 17 \fesjeat 14 | | I 16 A Pee se | a | F ae ; 9 261 294 lh 33 158 13 TABLE VUI Record of the number of eggs laid by 14 sisters, of which 2 were male-laying and 12 were female-lay- ing, showing that the average number of eggs produced by the male-laying females is nearly four times as great as the average number produced by the female-laying females. Temperature 26° to 29° C. 14 sisters a5 Se a ey oun) a | ; | eye} ie) Mother | Eggs ox Mother | Eggs ENG | = = — | | I 23 | I 3 | ee | 1 5 | | I I | I 8 I 6 I 8 : 3 | a 5 | 1 4 | I 4 | : Dial Al od ees oe feces 2 | 42 [he oan | 12 66 Ci 53 Determination of Sex in Hydatina senta 13 females produce fewer and fewer eggs while the decrease in the number of eggs produced by male-laying females is not as great. Table LX shows a rough approximation of the ratio in which the male and female eggs are produced at these different tempera- tures. TABLE IX The approximate ratios in which the males and females are produced at different temperatures, as seen in Tables V-VIII. 40 1 | x | Temp. 20°to22°C. Table V 2 1 Temp.24°to25°C. Table VI-VII 4 1 Temp. 26°to 29°C. Table VIII From the foregoing experiments and tables it is evident that temperature has nothing to do directly with determining sex in Hydatina senta but indirectly it determines the number of each sex produced by regulating the number of eggs that each kind of female lays. At a temperature of 20° to 22° C. the male-laying and female-laying females lay about the same number of eggs each, but at a higher temperature of 26° to 29° C. the male-laying females lay about four times as many as the female-laying females. V EARLY PRODUCTION OF MALE-LAYING FEMALES IN A FAMILY OF DAUGHTER-FEMALES None of the previous workers with Hydatina senta have iso- lated the eggs of a female-laying female in the order in which they were produced to determine whether there is any tendency for the earlier laid eggs to produce more male-laying females than the later laid eggs. In my experiments in which all the daughter-females of each individual mother were carefully isolated and the sex character of their immediate offspring was recorded it is clearly shown that the male-laying daughter females appear among the earlier ones in the family rather than among the later ones. In Diagram 1 the plotted line indicates the production of male- laying females among the 472 daughter-females of eleven mother- 14 David Day Whitney individuals, each one of which produced 40 to 44 (average 421% eggs. ‘The eggs were allowed to hatch in the dish with each mother and the young daughter-females isolated soon after hatch- ing. Their different sizes would indicate their relative ages and thus the approximate order in which the eggs were produced. The young daughter-females were isolated in lots from 1 to 8. This manner of isolation is subject to some error but on the whole gives a fairly good approximation of the truth. Pe Pee [ea] CEE PEEEEHE a ae y N HH fs ran Reece poet ducing male-laying females Number and location of eggs pro- NUMBER AND ORDER OF FEMALE EGGS PRODUCED Diagram 1 Record of the egg production of 11 female-laying females from Table I, showing in which part of the egg laying period the male-laying females were produced. Each female laid 40 to 44 (average 42+) eggs. Of the 472 daughter-females 20+ per cent were male-laying females. Nearly all of the male-laying females were produced among the first 28 eggs laid. Only two male-laying females were produced from the twenty-ninth to the forty-second laid eggs. Of the daughter-females 20 + per cent were male-laying females. Diagram 2. ‘This is to show the same point as Diagram 1. oping into male-laying females Number and location of eggs deve!- NUMEER AND ORDER OF FEMALE EGGS PRODUCED Diagram 2 Record of the egg production of 12 female-laying females from Table I, showing in which part of the egg laying period the male-laying females were produced. Each female laid 35 to 39 (average 36+) eggs. Of the 441 daughter-females 16+ per cent were male-laying. Determination of Sex in Hydatina senta 15 Twelve other female-laying females, each of which laid 35 to 39 (average 36+) eggs, produced all their male-laying daughter- females among the first twenty-four eggs laid. This is a clearer case than Diagram 1, because there are no scattering male-laying females among the later produced eggs. Of the daughter-females 16+ per cent were male-laying females. In these two diagrams the mother-individuals were not specially selected but the record of all mothers, in Table I, producing 40 to 44 daughter-females, is shown in one diagram and the record of all mothers, in Table I, producing 35 to 39 daughter-females is shown in the other diagram. The numbers 35 to 39 and 40 to 44 were chosen because they seemed more likely to be the normal than a higher one. These results, together with those obtained at different tem- peratures throw a great deal of light upon Maupas’ results. In -his experiments the highest percentages of males was always obtained from mothers which developed from early laid eggs. In Table I it is seen that an individual mother may produce O to 40+ per cent of daughter male-laying females. This fact must also be taken into account when explaining Maupas’ few experiments. ” VI INFLUENCE OF FOOD Nussbaum supported Maupas’ conclusions that external factors can change the sex ratio in Hydatina but explains this change as being due to poor nutrition of the females and not due directly to the influence of temperature. At the higher temperature the processes of metabolism are taking place so rapidly that the ani- mals cannot eat food and assimilate it fast enough to prevent their tissues from being in a semi-starved condition. Nussbaum’s experiments seemed to show evidence that young females which are starved for several hours as soon as they leave the egg produce a higher percentage of males than those that are fed from the moment they hatch. In many of his experiments he kept many individual females together and did not follow the history of each individual sepa- rately. 16 David Day Whitney Punnett has pointed out that all the starved females of Nuss- baum’s experiments did not produce males, which invalidates his general conclusions. Punnett has isolated female eggsof a “pure female strain,” and after they hatched starved the young females from 2 to 20 hours but no males ever appeared, although the young females were starved for several consecutive generations. I have followed the history of many females which have been starved for several hours immediately after hatching at a tempera- ture ranging from 14° to 29° C. and have found no trace of evidence that a higher percentage of male-laying females is pro- duced. I Temperature 20° to 22°C. Experiment I. Sixty-two eggs were selected at random from the sets of eggs produced by four female-laying females. They TABLE X TABLE XI Record of the sex character of the eggs laid Record of the sex character of the eggs laid by 27 sisters, 15 of which were without food for by 45 sisters, 11 of which were without food for the first 6 to 26 hours after hatching and 12 the first 21 to 26 hours after hatching and 34 were abundantly supplied with food from the were abundantly supplied with food from the moment they hatched. moment they hatched. ~ Temperature 20 to 22° C. Temperature 20 to 22° C. Sister- | Starved from Character of eggs Sister: | Staryedifrom Character of eggs individ- | time of hatch- __ Produced individ- | time of hatch- |___Preduced__ uals | ing a | 9 ‘ials | ing a | ° ee a | hours - % 2 : II 21-26 | 2 ; aS 2 13 fed rol 5 a i 21 fed | 9 I 12 2 a La eS I 16 Q 2 21 g I 23 rol I 2 fof 2 fed rol were placed in “Great Bear” spring water, such as is sold in New York City for drinking purposes, and allowed to hatch. After Determination of Sex in H ydatina senta 17 hatching each daughter-female was kept in this water without food from 6 to 71 hours. Of tog daughter-females from the same mother as the above, 62 were well supplied with food from the moment they hatched. Tables X to XIII give the detailed results and Table XIV gives the summary. The mother-individuals of Tables X and XII TABLE XII TABLE XIII Record of the sex character of the eggs laid enoud Cees Sea of Rea 5 we 3 by 46 sisters, 5 of which were without food for by 53 sisters, 31 of which were without food for Fee Beek: the first 11 to 59 hours after hatching and 22 Gre Te So 0) 7 Ce : ey Aa ng One 2 eereepundantly; supplied wwithfoad) orl the were abundantly supplied with food from the mom hey hatched. moment they hatched. oment they hatche Temperature 20 to 22° C. Temperature 20° to 22° C. | Character of eggs Character of eggs Sister- | Starved from Sister- | Starved from individ Gmeiok barehe | a eeeocuced aaeds Nemreot natch a |e bocetess uals ing ey al 2S uals ing | gt fr) hours Pours I II g 2 50 o . 13 ? I 50 i] 2 | 14 | | ? I 71 2 I 15 | g I 71 ron I ox | ? II fed i xet 5 36 So 30 fed cS) 7 36 g I 38 rol 2 38 ] I 42 g I 47 | @ 4 47 g 2 50 fo) I 54 2 I 59 9 I fed | fot) 21 fed 9 were sisters. The 11 sisters-individuals of Table XI were starved in filtered boiled spring water placed in sterilized test tubes with cotton stoppers. The percentage of male-laying females among all the starved daughter-females is slightly lower than that of those which were fed. 18 David Day Whitney TABLE XIV Summary of Tables X to XTIT, showing the percentage of male-laying females that occurred among the females which were without food for the first 6 to 71 hours after hatching, and also the percentage of male-laying females which occurred among the females that were abundantly supplied with food from the moment they hatched. Temperatnre 20° to 22° C, Starved Character of eggs | Indi- | from produced Per cent viduals! time of | @ hatching fou 4 hours 62 | 6-71 12 50 19+ 1og | fed 27 82 | 24+ 171 | 39 132 | 22+ 2 Temperature 14° to 15° C. Experiment II, October 29. “Two female-laying females were reared at this temperature and their daughter-females isolated. Thirty-five daughter-females were without food from 11 to 64 hours after they left the egg, and 49 were abundantly supplied with food as soon as they hatched. The detailed results are shown in Tables XV and XVI while Table XVII gives the summary. The difference between the percentage of males produced by those starved and those fed is not very great and probably means nothing. 3 Temperature 25° to 26° C. Experiments III, October 31. “Twenty-six female eggs from several individuals were produced at this temperature and as soon as they hatched the young females were starved from I to 13 hours. 19+ per cent of these starved females produced male eggs. Tables XVIII and XIX give the detailed history and Table XX gives the summary. Determination of Sex in Hydatina senta 19 These three experiments, including Tables X to XX, clearly dem- onstrate that food has no influence in determining whether a female shall produce male or female offspring. TABLE XV TABLE XVI Record of the sex character of the eggs laid Record of the sex character of the eggs laid by 49 sisters, 19 of which were without food for by 35 sisters, 16 of which were without food for the first 11 to 41 hours after hatching and 30 the first 20 to 64 hours after hatching and 19 weie abundantly supplied with food from the were abundantly supplied with food from the moment they hatched. moment they hatched. Temperature 14° to 15° C. Temperature 14° to 15° C. Sister- | Starved from Sa CeCNICEES Sister- | Starved from eeoerec cau Ee individ- | time of hatch- __Produced individ- | time of hatch- |__ Produced uals ing 3 ro) uals ing 3 9 | | hours hours | 4 II g 2 20 9 I 20 te) I 21 cr || 2 | 20 fot 2 21 co] 3 21 2 2 23 | @ I 23 9 I 46 fol 5 25 g I 46 } I 25 fol I 48 I 27 9 1 53 | I 41 ge I 53 lees, 4 fed rot I 58 2 26 fed 9 I 61 of v — ae ce I 61 g I 64 fot 5 fed fot 14 fed g VII MALE AND FEMALE STRAINS Punnett says: “My experiments have led me to the conclusion that among the rotifers I used were certainly three different types of thelytokous (female-laying) females, viz: A Females producing a high percentage of arrenotokous (male-laying) females. B Females producing a low percentage of arrenotokous females. C Purely thelytokous females producing no arrenotokous females”’ (p. 226). 20 David Day Whitney TABLE XVII Summary of Tables XV to XVI, showing the percentage of male-laying femal s that occurred among the females which were without food for the first 11 to 64 hours after hatching, and also the percentage of male-laying females which occurred among the females that were abun- dantly supplied with food from the moment they hatched. Temperature 14° to 15° C. Starved Character of eggs | i- | Per aac | from produced vidu- | time of sae | oe = als hatching a | 2 Ge 35 | 11-64 8 27 22+ 49 fed 9 40 18 + 84 H7 |||| 167 20 + TABLE XIX Record of the sex character of the eggs laid by 19 individuals females which were without food for the first 1 to 13 hours after hatching. Temperature 25° to 26° C. | | Character of eggs re Starved from Individ- time of hatch- produc’) = uals : ing rol fo} hours I I | 2 2 2 | Q I 3 | ice 3 3 % I 4 fos I | 5 | of 2 5 2 2 6 | 2 I 8 fof I 3 10 | & 2 13 | 2 TABLE XVIII Record of the sex character of the eggs laid by 15 sisters, 7 of which were without food for the first 7 to 13 hours after hatching and 8 were abundantly supplied with food from the moment they hatched. Temperature 25° to 26° C. | Starved from | Character of eggs suis m- | time of hatch- produced dividuals : ing fot 9 hours | 2 7 | 2 2 10 ee : 10 a | 2 13 | 2 I fed rol | 7 fed ° TABLE XX Summary of Tables XVIII to XIX, show- ing the percentage of male-laying females that occurred among the 26 females that were with- out food for the first 1 to 13 hours after hatch- ing. Temperature 25° to 26° C. Starved | Character of eggs ee | from produced | Per cent ne | time of |————_| gogo ale hatching cof Q | hours | | 2059 )\ieT—13 5 21 | 19 Determination of Sex in H ydatina senta ZT Punnett realized that these conclusions were based on rather scanty data. His data can be shown to be entirely insufhcient. His type 4 is based upon only one experiment which extended through 23 generations and included 109 individuals. 42 + per cent of these 109 individuals were male-laying females. Type C is based upon much more evidence, but it is not sufh- cient to warrant a decisive conclusion. In October, 1906, I started a strain or pedigree culture which extended through 62 generations including 167 mother female- laying females and 3959 daughter-females. ‘This strain was kept at room temperature of 20° to 22° C. Its history is recorded in Tables I and XXI. Table I. Out of 3264 daughter-females from g5 mother-indi- viduals which extended through 12 generations 20+ per cent were male-laying females. Table XXI. In r5 generations, XIII to XXVIII, including 76 daughter-females from 15 mother-individuals only 9 + per cent were male-laying females. In 17 generations, XXIX to XLV, including 208 daughter- females from 17 mother-individuals no male-laying females ap- peared. Ingeneration XLVI the first 327 daughter-females from 18 mother-individuals yielded 48+ per cent of male-laying females. The next 11 generations XLVII to LVII including 58 daughter- females from 11 mother-individuals gave 29 + per cent male- laying females. These results show that a strain producing a higher percentage of male-laying females can develop into a strain yielding a much lower percentage, or even into a strain yielding no male-laying females at all. Furthermore, the apparently pure female-laying female strain can develop into one which will give a very high percentage of male-laying females. Thus the three strains or types of Punnett can be found in one strain and each is capable of giving rise to the other types accord- ing as the data is scanty or extensive. The high percentage of male-laying females in generation XLVI can be readily explained by the results shown in DE seme I and 2 which clearly demonstrate that the male-laying eles are pro- 22 David Day Whitney duced earlier in a set of eggs than are the majority of the female- laying females. In this generation the 18 mothers produced only an average of 18 + eggs each, because the experiment was discontinued at this point. TABLE XXI Continuation of the strain of which the beginning is recorded in the 12 generations of Table I. ay Daugh- 29 | Daugh- Gen. maopkee |. ter 2 ae 2 9 Gen. Mother | _ ter 9 32 99 isolated | isolated XIII I 6 I 5 XXXVUI Tae hy 37, ° 37 XIV I Gye ar 5 XXXIX I | 6 ° 6 RV) ia Parla as x! Mere a6 o | 26 XVI I 6 ° 6 XLI Ta 25 ° 25 XVII I 2 ° 2 XLII Ti] 14 ° 14 XVIII I I ° I XLill rife 78 ° 18 xIX I 5 ° 5 XLIV I 17 ° 17 xx I 6 | ° 6 XLV I 18 ° 18 XxI I 6 ° 6 XLVI 18 327 160 167 XXII I 6 | 3 3 XLVII I I ° I xxmm| 1 Pant ee al XLVI] 1 6 al 2 XXIV I 6 ° 6 XLIX I 6 3 3 XXV I 6 ° 6 L Teal 6 2 4 XXVI I 6 I 5 LI I 6 ° 6 XXVII I I ° I LIL I 6 ° 6 XXVIII I 2 | 1 I LUI I | 6 ° 6 XXTX I Se le 30 5 LIV I 6 5 I XXX I 6 ° 6 LV 1) 5 2 2} XXXI I 4 ° 4 LVI I | 6 5 XXXII I I ° I LVI I 4 ° 4 XXXII I 5 ° 5 LVI 3 6 ° 6 XXXIV 1 Gia sl xe 6 LIX 1 6 ° 6 xXXxXXV I 6 ° 6 LV 2 6 ° 6 XXXVI I 6 ° 6 LXI 6 ° 6 XXXVII I 6 | o 6 LXII 2 4 I 3 How a seemingly pure female-laying female strain is obtained when only a few individuals are isolated from each generation while a parallel strain does not yield the same results is not yet clear. It may be due to some trick of selection in isolating the young females of each generation. Determination of Sex in Hydatina senta 23 If a large number, 45+ per cent, of the daughter-females of one mother produce males it does not necessarily follow that a high or a low percentage of the daughter-females of the next gen- eration will produce males. Nor does it seem to be true that if the daughter-females of one generation produce all female off- spring that the daughter-females of the following generation will do so. ‘Table XXII shows the history of both classes of daughter- females in five generations. TABLE XXII Record of all the female-laying females that were isolated in five consecutive generations, showing that there is no constant relationship between the percentage of male-laying females that are produced by a mother and the percentage of male-laying females that are produced by the daughter-female in the next generation. Off- Moth- Off- Off- Moth Off- Gen. Sisters spring er spring Gen. | Sisters spring | er | spring a2 | 99) 99 | ae |ee|ee I I AV 5 15 15 II 16 ] 36 I 34 ic ° 27 Ut 4 ° 45 | ° 24 2 42 | § 14 I 42 i) | 47 it IV 5 12 20 4 41 ° 36 7 40 22 | 26 VIII THE PRODUCTION OF FERTILIZED EGGS The winter or fertilized egg is supposed to be the male partheno- genetic egg which has been fertilized. This produces a female. The egg has much more yolk material and a thicker shell than the male parthenogenetic egg. A female produces from twelve to twenty fertilized eggs, while a male-laying female produces from forty to fifty parthenogenetic eggs. In order to obtain fertilized eggs males must copulate with very young male-laying females. 24 ; David Day Whitney In a few experiments, comprising several hundred females which had copulated with males when very young, Maupas found that the percentage of females producing fertilized eggs was the same as the percentage of male-laying females from several hundred fe- males that never had copulated with males. He concluded that the fertilized egg is the male parthenogenetic egg which has been fertilized. I have repeated his experiments on a smaller scale and the same results were obtained. Furthermore, the producers of fertilized eggs appeared among the early laid eggs of the mother-individuals. Diagram 3 shows the occurrence of the layers of fertilized eggs among their sister-individuals from five mothers which _pro- duced 125 eggs. Number and location of eggs developing into layers of fertilized eggs NUMBER AND ORDER OF FEMALE EGGS PRODUCED Diagram 3 Record of the egg production of five female-laying females showing in which part of the egg-laying period the layers of fertilized or ‘‘winter eggs” were produced. Each female laid 21 to 27 (average 25) eggs. Of the 125 daughter-females 36 + per cent laid fertilized eggs. Many males, fifteen to twenty, were constantly kept in the dishes with each of the five mothers, so that as each daughter- female emerged from the egg there were many males present. Only one male-laying female appeared among the 125 daughter- females. ‘This experiment was conducted in the fourteenth gen- eration of the strain in Tables I and XXIJ, soon after the isolation of the large numbers in generation XII which gave 21 + per cent of male-laying females. Of the 125 daughter-females 36 + per cent produced fertilized eggs. This percentage is high because the mother-females pro- duced, on an average, only twenty-five eggs each, but if they had produced forty eggs each the percentage would have fallen to Determination of Sex in Hydatina Senta 25 about twenty-three, provided that there had been no more mothers of fertilized eggs to have been produced. “Vhe Diagram 3 shows only one occurring between the eighteenth and twenty-seventh egg. The number and order of occurrence of the mothers of fertilized eggs together with the number and occurrence of the layers of male eggs in parallel sets of daughter-females seem to indicate that the Sayer of male eggs and the layers of fertilized or winter eggs are identical at one stage of their life. In another species of rotifer, Asplancha, Lauterborn has observed winter eggs and male embryos in the same individual. Among the Daphnia, Issakowitsch has found that the same female may produce winter eggs and male eggs. Therefore it is not unreasonable to suppose that the immature male-laying female of Hydatina senta is capable of developing into a layer of fertilized eggs or a layer of male eggs, according to the impregnation or lack of impregnation by the males IX SUMMARY 1 Temperature has no influence in determining the sex of Hydatina senta. 2 About 22 per cent of the females at any temperature from 14° to 29° C. are male-laying. 3 A male-laying female produces eggs faster than a female- laying female and at a temperature of 25° to 29° C. a male-lay- ing female produces more eggs throughout her lifetime than a female-laying female. 4 The male-laying females occur in the early part of a family of daughter-females. 5 Starving the young females for the first few hours after they hatch does not cause them to produce a higher percentage of male eggs. 6 ‘There are no strains that constantly produce a high or a low percentage of male-laying females. 7 The “pure female-laying female strain” can give rise to the normal percentage, 22 +, of male-laying females. 26 David Day Whitney 8 The male-laying female may produce fertilized or winter eggs, provided that she has been impregnated by a male at the proper time. Zoological Laboratory Columbia University May 1, 1907 BIBLIOGRAPHY Hupson, C. T., anpD Goss, P. H., °89—The Rotifera. IssakowrrscH, A.—Geschlechtsbestimmende Ursachen bei den Daphniden. Biol. Centralb., xxv, 1905. LautTersorn.—Ueber die zyklische Fortpflanzung limnetischer Rotatorien. Biol. Centralb., xviii, 1898. LeussenN.—Contnbution a |’étude du developpement et de la maturation des ceufs chez Hydatina senta. 4a cellule, xiv, 1808. Maupas, M.—Sur la multiplication et la fécondation de |’Hydatina senta, Ehr. C. R. AcuSe: ‘Paris; cxi, 1890. Sur la fécondation de |’Hydatina senta, Ehr. C. R. Ac. Sc. Paris, exi, 1890. Sur le déterminisme de la sexualité chez |’Hydatina senta, Ehr. C. R. Ac. Sc. Paris, cxiii, 1891. Nussspaum, M.—Die Entstehung des Geschlechts bei Hydatina senta. Archiv fiir mikroskopische Anatomie, xlix, 1897. Punnett, R. C.—Sex-determination in Hydatina, with some Remarks on Par- thenogenesis. Proceedings of the Royal Society, B. vol. 78, 1906. From the Havemeyer Chemical Laboratory, New York University, New York A NEW EXPLANATION OF THE MECHANICS OF MITOSIS BY ARTHUR B. LAMB Wirth Two Ficures The almost universal recurrence of essentially the same regular arrangement of the chromatin substance in dividing cells indicates emphatically that the same very definitely acting force or complex of forces is operative in them all. Numerous suggestions have been made as to what this omnipresent force may be, but none of them have been able to meet the many requirements of the prob- lem. Moreover, our real knowledge of the whole matter is so scanty that any explanation seems at present a little premature. I am, nevertheless, going to offer still another explanation of this phenomenon; first, because it may prove suggestive to others and may prompt fresh observation, and second, because it calls attention to a phenomenon which deserves the consideration of cytologists, whether it has any application to the present case or not. The marked polarity which mitotic figures exhibit, best de- scribed by saying that they resemble the configuration assumed by iron filings between unlike magnetic poles, together with the move- ments which the chromatin substances execute about one another oblige us to believe that this unknown force is of a polar nature, that is, acts outward from a center and exerts its influence at a distance. ‘The only objection to this conclusion is that a crossing of astral rays has been observed. ‘The lines of force in the field of any polar force cannot, however, cross, and consequently the astral rays which would be assumed to follow these lines of force also cannot, or should not, cross. This crossing, though certainly real, is not, apparently, the prevailing condition, and it can be THe Journar or Experimenta Zo6.ocy, VOL. v, No. I. 28 Arthur B. Lamb explained on the assumption of an intermittent or non-synchronous activity of the centers, as Reinke’ has shown.’ Assuming, then, the existence of some polar force exerting its action at a distance, we are confronted with two possible alterna- tives regarding the sign of this action. “That is, we may imagine either that the centrosomes attract, or that they repel each other. Wilson® has urged that the astral centers represent centers of trac- tion, caused, perhaps, by a volume change at those places. This is in entire agreement with the configuration assumed by the astral rays and the spindle fibers. They simulate the magnetic field between opposite poles, as pointed out above. But this view is quite at variance with the actual movements of the centrosomes. They move apart, even at a stage when astral rays are well devel- oped and hence seem to repel each other and not attract as they ought if they represent opposite poles. Lilliet adopts the other alternative, as did Meves.° He considers the astral centers to repel each other. In this way he explains the movements of the centrosomes satisfactorily enough, but is confronted by the difh- culty of accounting for the configuration of the astral rays. Lillie assumes that electric charges located on the centrosomes are the particular forces which produce the repulsion. He would explain the unexpected configuration of the fibers and the astral rays by the rather dubious assumption of a localized positive inter-astral area which superposes its effect on the purely repellent action of the astral centers. Looking at the matter more closely we see that for every unit of negative electricity on the chromatin sub- stance there should be a corresponding unit of positive electric- 1 Reinke, Fr.: Arch. f. Entwicklungsmech., ix, 1900. *Rhumbler: Ibid., iii, iv and v, 1896, 1897 and 1899, has suggested a non-polar force to explain the astral rays independently progressing rays of crystallization out of a supersaturated solution. While avoiding the difficulty of crossed fibers this explanation encounters the still more formidable one of accounting for the universal occurrence of curved fibers. 8 Tbid., xiii, p. 354-395, 1901. See also his book, The Cell in Development and Inheritance, 3d Ed. The Macmillan Company, New York. It is a pleasure to express my thanks for a most profitable discussion of this whole question with Professor Wilson, who, it seems, had already considered the pos- sibility of a hydrodynamic explanation. 4 Amer. Jour. Physiol., xv, 46-84, 1905. 5 Ergebn. d. Anat. u. Entwick., vii, viii. Merkel u. Bonnet. The Mechanics of Mitosis 29 ity in or on the surrounding aqueous solution. Moreover, since this aqueous solution contains inorganic, ionized salts it must be a conductor of electricity, and the positive charge must be distrib- uted over the whole solution. Any localized positively charged area in the electrolyte, except for the supposed “double layer” around each charged particle seems, consequently, unlikely. Lillie has encountered a similar difficulty in accounting for the configuration of the chromosomes. ‘They ought not only to be repelled from the astral centers but also to migrate toward the boundary of the equatorial plate. This latter thing they do not do, and Lillie is therefore again obliged to make the assumption of a localized positive inter-astral region. There is, however, another force which might well come into play here, which so far as I know has not been mentioned in this connection before, and which involves none of the objections urged against the electrostatic explanation. I refer to the mutual repulsions and attractions, exerted by bodies pulsating or oscil- lating in a fluid medium. We owe our knowledge of this branch of Eydradyaamics chiefly to the two Byerknes, father and son.* They have shown that bodies pulsating or oscillating synchro- nously in.a liquid attract or repel one another cenendinae on whether they pulsate or oscillate in the same or opposite phase. Further- more, these bodies set up lines of flow in the liquid, real hydro- dynamic lines of force, which simulate exactly the lines of force in magnetic or electric fields. The sign of this force is, however, in general, just the reverse of that in electric or magnetic fields. Bodies pulsating synchronously and in opposite phase repel each other, although the form of the jie old the y produce 1s 1dentical with amerocenmnlibe magnetic poles which attract each other. Simi- larly, two spheres oscillating synchronously and in the same phase repel each other, elu they too produce a field like that between opposite magnetic poles. The following experimentally derived figures, taken from Byerknes’ text-book, illustrate this identity of form and reversal of sign in the electric and hydrodyna- mic energies: 6 See Hydrodynamische Fernkrafte, v. Bjerknes; 2 vols. Leipzig, J. A. Barth, 1902. 30 Arthur B. Lamb wt es a AR) XX PATA AWA | 4 ee Fig. 1 Lines of force between unlike magnetic poles. REPuLston. \ i ! pe - ° : t ! \ Sel f ae nee ‘ ; ha / = / y = \ . \ ee Sy \ \ ee yi a Ny 22 Ss a a ea a Se ==, ba aa a = ee! = See —_ SO St ee ad 7 Zi Dae ae Ne ee es N eee sf NR <7) iK , S Games {'\ NX Se, \ 7 ah .\. Se fon tite ‘ 0 : ‘\ \ Lae / \ \ / y \ ‘ i ee C t \ \ ! ean . Z, f ' Fig. 2 Lines of flow between oppositely pulsating bodies. Atrraction. It is this exact reversal of sign of hydrodynamic action at a dis- tance, as compared with electric and magnetic actions, which makes this force peculiarly applicable to the case of mitotic figures, obviating many objections which beset the previous explanations, and particularly the fact that with previously considered polar forces if one made an assumption which would explain the form of the field, the motion of the centrosomes appeared contra dictory The Mechanics of Mitosis 31 and vice versa. If, however, we assume that the centrosomes are pulsating synchronously and in opposite phases, or oscillating synchronously and in the same phase, we obtain the desired repul- sion, and at the same time we get mitotic figures corresponding to the configuration of the lines of magnetic force between opposite poles. That is, we get a configuration of spindle fibers and astral rays precisely like the actual ones. The cases of tri- and multi-polar spindles, so difficult to explain on electrostatic grounds present much less difficulty here. If each centrosome were oscillating along a path radial to the common nuclear center and in the same phase, mutual repulsion, combined with the proper configuration of the astral rays, would be obtained. The movements and configuration of the chromosomes are also better explained on hydrodynamic grounds than by previous assumptions. It is not even necessary to assume that they execute any independent oscillatory or pulsating motions. Byerknes has shown that bodies suspended within the field of force of oscillating or pulsating bodies are attracted or repelled depending on whether they are lighter or heavier than the surrounding medium. ‘This attraction or repulsion is due to oscillations induced in the sus- pended bodies by the permanently oscillating or pulsating bodies. The chromosomes, if heavier than water, or the cell fluid in which they are suspended, would be repelled from each centrosome and would come to occupy a position midway between them in the equatorial plate. Moreover, they would not move outward to the boundary of the equatorial plate. ‘Their induced oscillations, though repelling them from each centrosome, would attract them toward each other, and this action would tend to keep them in the observed axial position. If the chromosomes should become lighter than the cell liquid, the repulsion from the centrosomes would change to an attraction. ‘This immediately suggests that it may be simply a change in specific gravity of the chromosomes which causes them to diverge, after splitting, back toward the cen- trosomes. [t is now of interest to inquire whether this hydrodynamic action at a distance could possibly be strong enough to account for the actual movements of the centrosomes. It is, of course, almost 32 Arthur B. Lamb impossible to decide this by calculation, in the present state of our knowledge, but if we assume that the centrosomes are smooth, hard spheres and that the cell fluid is homogeneous and as mobile as water, it is not difficult to calculate how vigorously they must oscillate or pulsate in order that they shall move apart with the observed velocity.’ Taking the radius as 0.0002 cm., the dis- tance apart as 0.003 cm., the time required for this maximum separation of the centrosomes as fifteen minutes; if the amplitude of oscillation equaled two diameters, the frequency required would be 2000 oscillations per second; if the amplitude were eight diame- ters, the required frequency would be 100 oscillations per second. With similar dimensions, if the centrosome pulsated so that its greatest volume were three times its least volume, a frequency of some 130 pulsations per second would be required.® These frequencies are greater than one would expect. They do not, however, involve any great linear velocities, for the dimen- sions of the particles are very small. ‘Thus the frequency of 2000 oscillations to the second only means a linear velocity of the centro- 7 The formula of Stokes F=6zpur (Brit. Assoc. Report, p. 445, 1887) applying to the motion of spheres though viscous media was used to determine the force needed to give the centrosomes the observed velocity. To find the needful fre- quency of oscillation this was equated to the expression 6 a : 47d! derived by Bjerknes for the attraction or repulsion between oscillating spheres. Similarly, to find the needful frequency of pulsation it was equated to the expression a a) 8/9 — (a°— 1)? p? also based on a formula derived by Bjerknes. In all of these expressions r represents the radius, d the distance between the centrosomes, /t the coefficient of viscosity of the medium (water), p the frequency, S the “action moment,” v the velocity, and a the ratio between the mean and the maximum radius of the pulsating sphere. 8It might also be pointed out here that similar calculations on the hypothesis of electrostatic action show that, if the capacity of the centrosomes is simply that of conducting spheres in an isolating medium, a potential difference of nearly two volts would be required; if the capacity is that of spheres surrounded by a ‘Helmholtz double layer,” a potential difference of only a few thousandths of a volt would be necessary. The Mechanics of Mitosts 33 some of 2 cm. per second. It is, of course, almost impossible to say what effect a viscous, heterogeneous field would have upon the calculation, so we are obliged to leave the quantitative side of the question as quite unsettled. Besides the oscillatory currents produced by pulsating and oscil- lating bodies, Bjerknes has shown the existence of steady currents in the fluid medium toward and away from the centers of motion. Similar currents have been observed in dividing cells, particularly centripetal currents between the astral centers. It does not, how- ever, seem wise to treat this or similarly less pronounced phe- nomena in our present state of ignorance. The assumption of a pulsating centrosome or centrosphere is by no means an impossible one. The assumption of an oscillating centrosome is not even improbable. The assumption of syn- chronous pulsations or oscillations involves no mysterious synchro- nizing mechanism. Random oscillations or pulsations would cer- tainly tend to become synchronous by mutual interaction, while after the closed spindle fibers had formed, whatever their nature may be, any other rate of oscillation would be very improbable. The fact that such oscillation or pulsation have not been de- scribed is not conclusive. Our knowledge of the subject is based almost wholly on dead material, and moreover the oscillations and pulsations may be very rapid and small. In conclusion, I would again like to emphasize that the above is nothing but an ad hoc constructed hypothesis and intrinsically therefore only of hypothetical value. If, however, it calls atten- tion to a little known phenomenon or stimulates fresh observation it will have served its purpose. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Director. No. 193 a THE REACTIONS OF PLANARIANS ITO LIGHT BY HERBERT EUGENE WALTER: Witu Fourteen Ficures Mp te Od CEO ML tejasetlcrassy sieve e/ave os stase aves ste aie soe a1els ate etre areloye avatecera leis evainiereinnsraicaete o eeioreele 37 UG MELISCOLIGAL faetercyatsfetctereroieialeislele eictal-isyaio nreiercte HopduacpaduanducooapoubooD Adasen ousbabop 38 SVT Gee Milter l mere etetey terisiozestortaresetovecoxeiais )caayoictetere cle st cteiete sisiersss/ayesctersrereicieieiarelnere erates sie eteteieee 45 Va Criteriaitormedsuring bel avion ves deratsei= stelaeistars steisiclorsietel cereale rater ae 47 WA © Dsery cl tloriswreretoyesetes staves tetetet rater cie/crsie ese vereiste lvls) s]aisve: «vial crstatactote rents orate cleaver nee cheer 49 Hime E Told) stl Sycooodo o GUD RUE Aa Dao no Reon aUraeacoaed se dopendoceodernadaacassad 49 . LX. AUDEN iO dteh Cela Sapo hbenaeroTgnbenElsiua GoctnceonucdeonocbTd 66500000 50 Rate OfllocOmOtlOnns fatarsiniesctctors oscle) oferctarereyereasieraect=tale ae tereie rarest 50 . ARG bhy355 dap oe on DpH eR pAarAUAehEEnEddutechododacucnootoapeds SI | (ChEpTTSO Ichi sone opodbentandonpaavadpeooduMdcdaasavconDes 51 SUMMA reine lars sisinisia\cisicls oy=taiere oie'aYelaye\ ic /aasysiejaicretratvstcictetetetbersieteicle sr Biss Non=directive light -jsr<\ays/ats ters e)ars(ti 2istsyaistsracei nel te/eicterniclerels\emmiale sereieeie elec 52 Gam AD Paratusara str cecisseleitectciersteleiswiois a cleletsemtanteinet perenne ears 52 De Results Serer rstate;oss,aforesajotelasyalotclaters ayars 2ts1saje eictaieysleisiotsie ste/stanietersteteie aceite 56 IRafelohlocomotioDy «ssh tsisiate/siets vieltersssierars Yeeieceineier eos oeiee eee 56 Alin sth eae ho ceonnotno BouaeRqoEnDooEsmErndanusscoodadsodncuac 58 GChanpeahcourse. poate steletererelals.erais Actors atersthslefeetane eter ere ee eee 59 60 60 61 62 62 63 Beeps ACE srornistassisielaie elctersTasa(eletedate:nialetai Taya os aise oes tiers nrateleraetaectioilerste 64 SUM Maryietatersiancelaterictaists ofeletateetaitearcta cistercierercie etsvete lettereietacsteereterate 71 PRRENOLOCAKIS ARB defector ttcracier ryt cteteve or SS Se Sat ate a ee Tae in eee 72 Avemuconstantidirective lighteacs tists da stetaetditerisicliis ecto seletem ceiseten tetas 72 Orientation seer iceciocctietorsmtere oti eislercareieiereis revels reenter eeicrornieracntiets 72 IRACG OF LOCOMOLION sjeysielcscjerofsiscorsisielore sfercisinicietelcistann eieieteistersinletele ete orale 75 Ghanpeanicharacteroficourse seins ivi-rsicieis sin arsercisierarsiorsieteleeicierersiereve 78 Prcetiracviohorienta tons sre iejeleister-infeci= ce acieie cisistalcieisiciciarsie’s sleistelerevareie 719 IDR ORANG Hioht-adpotde send sodanbarenbasdcnontusaodoedeouua 80 Durationiobiactivityatectuisnviesiccisene einicnis cietsven elatcieciace ice sm ace 81 = Mimeixequired tolleave a mmitcircle. --.icisj-islcidiels cc slalsieis cis ervicecsntys 82 Manner of coming to rest 83 SSN Ea Goh St gonbeko Apopoud oh doen aoanAneUe COUN oneNo ene sea ne 86 Journar or Exrerimenrar ZooLocy, vor. v, No. I. Herbert Eugene Walter B; In.changing: directive laghes <i... 1<t-)-)-1=/o)z)atels/=1=1=/=1al= stele = sl =\oic/etelelotola/eeraiete teeters 87 (Ghanges imlintensitye:jetssce! a= o\aye.c rer tereicssis iotetels sinislessfelonateteteis1<eteyeistere 87 GhanpesimdirectiOninse aoe remiers ce selec s eisia eee n/heioie eters 88 Ritinlol iy sap enna AED Aan DG IuobaupaenandoraaragdaosouAnoDaccda 89 C inycombinationwwith'other responses: <1... .e.0.)nis ins sleleieisinicisie'e.aislsisizisietaisfeieels 89 (Esai Fs shedhoasdaor coded as muMaAadbosbn bocobcunonoDonNAsDOSNUGd go PHI SMOtaxis eecicciai eer eras crohenateletaietapetcte ctataleiste atersiatel= atateraes aceeterete gz Goniotaxissrn..-. «licence Nels e slsesicisier/aee setae see eee eee eile 94 Chemotaxis es vous cctasickes tele eior acetals semaine stratermtelet aces 94 SUMMA YG rors arctasstolecs Grek opatalarevainioleiers tistencte ave otesstoloatet atetete @teteyose 96 gy lRinds/of behavior: joer ace ceiseniee is ae aioe aie. cioisioe sisiseidke See ei stole otete ers sepieeis 97 A (Genencjand:specifie behavior jateressvcreoicretet sry -\alossloles ola lose = ei ele/alevetetaye ateradsfeleiat 98 Percenta re obmegatrveresssistetsiete sta (oy: che utalotelate shetelsieveletateletates tetsteneiets 98 Character of the course in directive light..............0+eeeee0ee 99 Duration of movements (ris sis:aicictetayere l= afa'= ie (ele c's/n)sis/ayeraters =tetslols/oreiare 105 De greeiOl wandering states cic ssotaias<tesetersieisiete aletsiele! sto)<\s ojeleterepateieretetiata 106 Rate of locomonons 4 2.7 << cersierss vives once ¢ vis he vivieue sre mieisieue ieee 107 Tuimexequired;tolleave:aimniticurcles <ste:aiaresets areal= siacecereieleyaclae elem 107 The effects of fatigue....... Rasen TadpeorniadacmianaaicGecnttads 108 Responses to chanpesmintensitycncclaan siete eels sieleholeite ieeereieetel ere 109 Manner ofl coming tonrestisysisjsc/stsrepac se cise ain ctere oratahetetefenstevaystelsteerate 110 Bint ty cor Sen on non EBL on qOscao CeO EEOcabHoInohbeacundoc 110 IB Individwallbehaviorgencyetascissleterie selon <saieeicis sls oslo siet-toisiers reer enter III ‘Rate omisuccessive sd ayss..c dois + sic151s susie, ef- os) arorerenaieto sovatersraiatetetstotniate 112 Relative value of individual behavior.............-+0+seeeeeeeeees 113 Avcave: planariam' <7. cli-tsyatt a/atels niotels c/a stole ete nese ieee eet oars 114 Rilke s Sanco A HOnHOOUS HAC OnronneEnOdD oo Uanomasn dodoncoonss 116 (Pages 117 to 162 are printed in vel. v, no. 2) 4. Basisiofibehaviorss2cescicons-seete ees ane aves aint ee Cine CeaieT ee eee rete 117 A” Morphological’ basisiofibelnawior.-/crcc,ct01</=:e:cjes!/apa)s'a(e =! ntela(o/o\oaielstelsteievala}eetesrataye 117 a), General! formof the bOdy ss sic. s:esseis:thate-<:t1n's1a/e'el<ie'ace siete eareistersteratsitatelenciele 118 be (Photoreceptorss sewiqseectcsecice/icle ois elienryteleieteisya staretstetterctee cre rene 122 SUMUMAY ses aterctercloyaje ceislelaeie ss elie neste eeimetteietelererere sentir 127 B Physiolopicalibasis of behaviors tater. slois/ateieiale vl eyetstetereteyoieteletais vet stersfofetatstere ateiete 128 a Classification of: physiological’states/.c%.. wei vieleloaus aiele ese rlerersieie rete ster 129 b Changes in physiological states induced by light..............+.+s-0005 130 Effect of different intensities i.e: ejere:ctetois: store laietn tots oretetstestet teat etateteeen 130 Effect of'excessivellight: mimesis el slekeielalseiieveloeierileeieie eitater (eters 131 Effect of sudden change in light conditions.............++s+eeeeeee 131 Effect of continued exposure to light............-6 0.0 eee ee seen eee 131 Effect of previous ex posure:to)d ark: jelsteiers\stele ator ster te siatatsteteteperetsr ictal 132 ELCs iqgnappnnodacuacncnuoudan dscancu Joona osaecesteoee 133 G@: Psycholopicalibasisiof behaviors. eve peyre/eseletetatstejesstar ste interac eter etniens oe felotereratofonste 134 a, How,muchican'planarians)see' tise eicje siete! =rasetat=\ sietele)s(aiein e\efeleterers s7els\alele|</s 134 b: “Areiplanarsans/ablejtojlearm ? rarelateetersjereteters tet leterslete ele iets/aratelet=te\e\ele late? 135 The Reactions of Planarians to Light 27 Vili General: Conclusions fare <ls/slats(olayar/afols\elarer/=\elsta\elelatolaIs\ele\e7e dbopondonuecvonooacoRaass ae 138 He PD IFeckOn OLMteL sity jo/ate ys <lnysieteictoi<isl te = 1m eleialarelase]sictelse EES as cbatete ohessi seaponstetsvaae es creieya se 138 Age Historicalusetcmstacpicaste stich ererietec cient viachavsicts eferecitie eerie aie cetarssretes 138 B; Conclusions;with reference to;planariansicc. cic cee sc sence meine weog asincice vee 140 a The distinction between direction and intensity..............--..+2455 140 b “Lhe modifying, influenceiofidirettOn sis. ccicis:= «0 ce wie ee ee rieie cide eee 141 c Instances of behavior due to intensity alone ...............+--+---+00 142 d Theimoditying effect of other factors<.. 5. (-..-..6ce:cree nial @ sieinieisisie anise 144 Wuininih 97 sone vaccndades Some mag tnaUNoh 4 one ssocsDomsesenoon 145 PB aictllriveliGegim@relaa}y hint aohconoshoe codon apNBesResaaamasnopacnpocaDesboodpo 146 Siininkla Honshoshossouppocptuponvedeadtuparnouscacscoconocotc 153 €) YE eit caoo fo cosciponnoddadobenongon dob ocpnunaunocpbos house deeHoGnuCEos 153 Suing gusnp esac ana oo too sbunpEbenDo cH sonmpood CoD doc dao! 155 Wat Tyo) Hoya doh Mabe onaenon bao odparodoesssvenootodnonud BODO SNGOBRAD EAD nOSROCaoUs 155 I InrrRopucTION Light is one of the physical factors which influence the behavior of organisms. ‘The great majority of living things are normally subjected to regular periodic changes in the amount of light to which they are exposed during the alternation of day and night. In addition to these constant periodic changes, there are innu- merable irregular gradations in both the intensity and the character of the light naturally acting upon any organism. An agent of such wide range and almost universal influence as light ought, therefore, when properly analyzed, to prove of material service in interpreting the behavior of animals and plants. The depend- ence upon light of animals provided with organs of sight, is self evident. ‘The direct bearing, too, of light upon chlorophyllaceous plants in the manufacture of their food substance, is plain. But how far light plays a direct part in the life of non-photosynthetic plants and of animals which cannot “see,”’ is less clear. Although possessing eyes, it is very probable that planarians are unable to see in the sense of distinguishing shapes, and it is questionable how far they can distinguish between even large regions of different light intensity. The object of the following paper is to examine the relation of light to animal behavior as applied to certain planarians. 38 Herbert Eugene Walter Il Hisroricar Our knowledge of planarians, as of most other animals, has passed through certain historical phases, during which emphasis has been laid first upon taxonomy and anatomy and latterly upon embryology and zoogeography. The results of these various forms of investigation are highly important since they make the foundation for all future work upon this group of animals. They have, however, only an indirect interest in the present connection and do not, therefore, require review. Perhaps the most modern advance in our knowledge of pla- narians is represented by the school which treats of them as living objects whose individual behavior is to be intimately correlated both with their structure and environment. ‘The most noteworthy contribution from this standpoint has been made by Pearl (03), who has analyzed in considerable detail the reactions of fresh- water planarians (notably Planaria maculata, Planaria doroto- cephala and Dendroccelum lacteum) to various stimuli. He has not, however, discussed the effects of light except incidentally. The earliest reference to the relation of planarians to light is by Dalyell (’14). In his interesting volume on planarians a great number of keen observations upon the general habits and struc- ture of planarians are made, which have since been confirmed, together with certain statements which have not fared as well with the advance of scientific knowledge. He makes the statement (’14, p- 9) that “most planariz court the light indeed;' but P. flexilis rather inclines to shun it, less, we may conjecture, from being warned of its presence by the specks or eyes, than from some disagreeable sensation produced on the body.” Again, referring to P. felina (’14, p. 46), ‘“ This planaria, like the rest of its genus, is powerfully excited to motion by the presence of light. If a number be confined in a glass vessel, the whole assemble in a quiescent state, on the side next the light It is a little surprising that Dalyell should have received the impres- sion that the majority of planarians “court the light,” since he clearly points out the nocturnal habits of these worms. He 1 The italics are mine. The Reactions of Planarians to Light 39 doubts whether the eyes are of service in finding food and says of worms under aquarium conditions (14, p. 107), “If remaining aconsiderable time unchanged, the planariz decrease more rapidly, they become languid, scarcely moving either by the influence of the light or heat, and at last adhere entirely to the side of the contain- ing vessel, where they perish.” Dugés (’28) observed that when light 1s concentrated by means of a lens upon either Dendroccelum (?) or Planaria, movement results which is most pronounced when directed toward the ante- rior end of the worm. He tested the effects of direct sunlight and of diffuse daylight as well as of candle-light, and concluded that the response increases with the intensity of the light. The non- dioptric character of the eyes he has described remarkably well for one working so long before the days of the microtome, and his conclusion, already suggested by Dalyell and later confirmed by Kennel (88), and others, that the eyes play no part in the finding of food, is noteworthy. He also notes that planarians seek the dark. Dalyell (’53, p. 99), in a later volume says, “On April 29 I pro- cured a fine specimen of Planaria cornuta, which spawned soon afterward. The spawn had been breaking up for two or three days preceding May 24, when multitudes of extremely minute yellow specks were seen swimming in the water. Their motion was sufficiently active, without being very quick; it was pursued in all directions and the spawn being contained in a small cylin- drical jar, the specks crowded to the sides next the light whereon numbers remained almost stationary.’’ Again (’53, p. 104), “When withdrawn from the dark the young Planariz rose in great numbers toward the surface of the water, congregating on the sides next the light.”” It is extremely doubtful whether the organ- isms here described were really young planarians. It is more likely that they were the young of some other aquatic animal. Dalyell correctly describes Planaria lactea (Dendroccelum lac- teum?) as being nocturnal. He observed that numbers of this species, beginning activity in the evening, rose on the sides of the jar, although many had descended again by morning. More recently attention has been specifically directed to the 40 Herbert Eugene Walter light relations of planarians in various papers by Loeb, whose important contribution in 18go, ‘Der Heliotropismus der Thiere und seine Uebereinstimmung mit dem Heliotropismus der Pflan- zen,” paved the way in general for all work on this subject. He found (’93b, p. tor) that Planaria torva is not “‘heliotropic”’ in the strict sense, but rather “unterschiedsempfindlich,”’ that is to say, it did not always move away from thesource of the light in the direction of the rays and remain as far removed as possible, but moved about more or less at random, coming torest in some area of lessened light intensity. In a later paper Loeb (’94, p. 255) states that when planarians are suddenly brought into the light they begin to move, an increase in light intensity leading to activity and conversely a decrease, to rest. [he grounds for this conclusion are not made clear. He further confirms the view that planarians are active at night, com- ing to rest in situations of lowered light intensity in daytime. In further experiments P. torva when decapitated was found to react to light precisely as normal worms do with the difference that the reaction required more time. Thysanozo6n brochii, a polyclad, on the other hand, lost its power to respond to light when “the eyes and brain were amputated, from which Loeb draws the conclusion that animals which are closely related morphologically may exhibit wide physiological differences. Hesse (’97), in his classic study of the anatomy of the turbel- larian eye, mentions some experiments and observations on the behavior of planarians and in addition makes valuable contribu- tions to the morphological basis of light reactions. He observed that planarians become active at twilight and he also experimented upon decapitated worms, apparently without being aware of the previous work by Loeb, with whose results his own agreed. He found, as Loeb did, that worms deprived of eyes finally come to rest in areas of lesser intensity much as do normal worms but after a longer time. Hesse found, too, that Dendroccelum lacteum came to rest in the dark 119 times out of a possible 120, whereas with Euplanaria (Planaria) gonocephala the same result was effected in only 88 out of 120 times, notwithstanding the fact that the latter has more highly developed eyes than Dendroccelum. The Reactions of Planartans to Light AI This led him to state (’97, p. 552) that “die Starke der Reaction auf Lichtwirkung nicht der Starke der Lichtwahrnehmung ent- spricht,”” and he ascribes this difference in behavior to a differ- ence in the ‘Gefihlston”’ of the two species. Amongother observa- tions described by Hesse, the two following are of importance in this connection, namely, that a sudden introduction of light caused an almost immediate turning away on the part of the worm, and, that worms with eyes could not be made to remain in the light when escape was possible. In his opinion this apparent perception on the part of the worm is due not to the animal’s ability to distinguish light but rather to unpleasant chemical reac- tions set up within the organism as the result of light stimulation. And, lastly, Hesse showed that the general position of the eyes of a planarian, together with the arrangement of their sensory portion, partly enclosed as it is within pigment cups, affords a device whereby the worm can be oriented to light. By means of this simple apparatus it receives a localized stimulus, which enables it to distinguish the direction from which the light comes. If light, striking the eye of a worm, fell-upon sense cells which were unscreened in any way by pigment, there would result a general stimulation without localization of the stimulus and consequently orientation could not be effected. Parker and Burnett (00) sought, by quantitative methods and with more accuracy than Loeb or Hesse, to establish the part played by the eyes in light responses of planarians. They came to the same general conclusion as these authors since they found that Planaria gonocephala without eyes reacts to light essentially as normal animals do, except that the reaction time is somewhat longer. They also showed that worms when pointed toward the source of light travel at a slower rate than when headed in the opposite direction. With regard to the mechanism of the light , response they say (’00, p. 383): ‘“‘We have seen nothing in our experiments that supports the opinion suggested by Hesse (p. 551) that reactions such as we have described are due to the direct influence of light on the internal parts of the planarians, and we are more inclined to the view that these reactions are initiated by the effect of light on the integument of the animal, 7. e., are 42 Herbert Eugene Walter due to what Graber (’83, p. 229) has called a ‘dermatropic’ function.” Bardeen (’ora, p. 13), speaking of Planaria maculata, states that “susceptibility to light is apt to become lost if worms are kept in captivity,’ and he notes the fact, already brought out by Chichkoff (92), that pigment becomes reduced in sunlight. Hesse had previously emphasized the point that the pigment of the eye of any organism has in itself primarily nothing whatever to do with light perception. Bardeen further found that small pieces of planarians capable of locomotion will respond to light in the same way as uninjured animals, and he notes (’o1a, p. 13), that the worms seem “to move about more by night than by day.” In a later paper (’otb) he speaks of the fact that when a dish containing planarians is brought into light the worms are commonly roused to activity, although how far such activity is due to light and how far to mechanical disturbances he does not make clear. Lillie (’o1), experimenting upon the regeneration of Dendro- coelum lacteum, discovered that posterior headless parts fail to give the typical reaction to light and are incapable of regeneration. He draws the conclusion (’or, p. 132) that “any symmetrical piece of Dendroccelum capable of regeneration tends to come to rest in the shaded part of the dish precisely like a normal individual” and that parts incapable of regenerating ‘‘also become incapable, after a day or two, of performing the usual reactions to light.” These results on Dendroccelum, it will be seen, are similar to those Loeb obtained in experiments upon the polyclad Thysanozoon. Curtis (’02) reports from laboratory observations 42 cases of fission in Planaria maculata, of which number 39 occurred between Io p.m. and6a.m. He adds, however (’02, p. 524), that “this did not seem due to the amount of light to which the animals were subjected during the day, for some of the dishes were so shaded that there was practically no light, day or night, except when they were being examined, and the division was the same in these as in others which were exposed to full daylight.”” A case of division in Bipalium also occurring by night is described by Lehnert (91). In a contribution to the geographical distribution of Planaria The Reactions of Planarians to Light 43 gonocephala, P. cornuta and P. alpina,*? Voigt (’o4) incidentally refers to the manner in which these animals come to rest in the darkest part of a dish. He afhirms that when an aquarium is sud- denly lighted at night only those that are hungry, :. ¢., those with comparatively empty digestive tracts, are found in motion, and he notes that in certain conditions worms may remain quiescent for weeks. The statement made earlier by Duges, that the eyes of planarians play no part in finding food, Voigt confirms. ‘These organs he explains are an aid in distinguishing differences in light intensity as well as the direction from which light comes but are entirely incapable, owing to the simplicity of their structure, of discerning the form of objects. In his opinion worms crawl into hollow stems and similar sheltered places to escape light rather than for warmth, as Wilhelm: ('04) suggests. Neither author, apparently, considers the possible part played by thigmotaxis under such circumstances. Of the delicacy with which worms react to light Voigt says (’04, p. 173): “Die Empfindlichkeit der Planariden gegen plotzliche Belichtung tritt so scharf hervor, das sie fiir den Unterricht eines der anschaulichsten Beispiele zur Demonstration der Lichtflucht bei niederen Tiere darbieten.” Notwithstanding this high degree of sensitiveness to light, he finds that the worms when seeking their food leave the shade and come out even into direct sunlight. And, finally, concerning the bearing which light has on the problem of distribution, he concludes (’04, p- 175): “Auf die Verbreitung im Allgemeinen hat die Belichtung der Bache wenig Einfluss, da sich in der Regel genug dunkele Schlupfwinkel finden, in denen sich die Tiere verbergen konnen.”’ Darwin (’44, p. 242) observed that /and planarians, “especially Planaria tasmania, had an immediate apprehension and _ dislike of light, which they showed by crawling, when the lid of the box was taken off, to the under side of pieces of rotten wood,” and in his enumeration of the places where various species of land pla- narians were found, their avoidance of light is plainly shown. A note by Leidy (’58) refers to finding Rhynchodemus sylvati- cus crawling about on fences frequently at night, but rarely by day. Pp. alpina = P. torva according to Borelli (’93). 44 Herbert Eugene Walter Moseley (’74, p. 111) states that “land planarians are probably all of them nocturnal in habit.” Speaking of the Ceylon land planarians in particular he says: “They are found in dark places, such as under large fallen leaves, and in confnement they coil themselves up away from light.’’ He mentions also the fact that Planaria torva and Dendroccelum lacteum choose the dark side of the vessel in which they are contained. As has already been mentioned, Lehnert (’91) found Bipalium kewense undergoing fission in the dark. Both Bipalium and Geodesmus, hee says, seek continually to hide in shadowy places avoiding even diffuse daylight. Concerning the degree of light perception possessed by planarians, he offers the opinion (’9I, p. 326) that ““Bipalium scheint mit seinem Augen die Umrisse von Gegenstanden in Lichte wahrnehmen zu kénnen.” Hogg (’97) notes that Bipalium is nocturnal in habit, remaining pieae during the day. Only Fiseiel references to the polyclads are found bearing upon the question of light reactions, as for example this sentence, which occurs in Lang’s exhaustive monograph (’84, p. 641), “Die meisten Arten scheuen das directe Sonnenlicht.”’ “The behavior of Thysanozoon with reference to light has already been mentioned (Loeb, ’94). Concerning the light reactions of the rhabdoceeles, especially cer- tain green fone 4 in Shih the green cells are probably symbiotuc, a considerable literature may be found. ‘The principal papers relating to these forms are as follows: On Convoluta schultzii, by Geddes. (79), Barthélémy (84) and Delage (86); on Convo- luta roscoffensis, by Haberlandt | COL), Bohn: Coza, (o3b, Ose); Gamble and Keeble (’03) and Fiihner (’06). Vortex viridis and Mesostomum viridatum (?) are discussed by Schultze (’51), von Graff (’84) and Sekera (’03). A résumé of these papers is, however, out of place here, since the presence of green cells in the organisms involves an entirely different problem from that which is under consideration. The foregoing historical sketch furnishes the basis of the follow- ing general summary of facts which have thus been established with more or less certainty regarding the reactions of planarians to light. The Reactions of Planarians to Light 45 1 Planarians are nocturnal, seeking the dark when exposed to light. 2 The eyes are useless in finding food. 3. The anterior end of the body is the part most responsive to light 4 Decapitated worms act normally except for a slower reac- tion time. 5 Onientation to light depends largely upon the character of the pigment cups of the eyes. 6 The relative energy of the response is dependent upon the intensity of the light. 7 Pigment is reduced in sunlight. 8 Pieces of worms which are large enough to move or regener- ate react to light. g Fission may occur more readily in the dark. 10 Different species respond differently to light. 11 Light reactions diminish during “captivity.” 12 Planariansare “unterschiedsempfindlich” instead of “helio- tropic.” III Marertar The species principally used in the following investigations were Planaria maculata Leidy; Planaria gonocephala Dugés; Phagocata gracilis Leidy; Dendroccelum lacteum Oersted; and Bdelloura candida Guard, all of which are inhabitants of fresh water except Bdelloura, a salt-water species, found living semi- parasitically on the horseshoe crab (Limulus polyphemus). Some observations also were made upon a cave planarian, that as yet has not been identified but which may belong to the genus Phagocata. This interesting worm was kindly placed at my spose oy Dr: A. M. Banta. At any season of the year an ample supply of fresh material was easily obtained except in midwinter, when it was necessary to cut through the ice and dredge up from the bottom water-weeds to which the worms cling. The source of supply for Planaria gonocephala was a small. pond to the west of Fresh Pond in Cambridge, Mass., while Pla- 46 Herbert Eugene Walter naria maculata, Dendroccelum and Phagocata were chiefly obtained from a pond at Falmouth, Mass., where they are especially abun- dant. ‘Twice, through the kindness of Professor Parker, aquaria were generously stocked with Dendroccelum, from a spring on Mount Monadnock, N.H. Bdellourawas obtained from Wood’s Hole, Mass., during the summer from freshly caught horseshoe crabs and, later in the year, from specimens kept in captivity. The setting-up of balanced aquaria in which planarians would thrive did not prove to be a difficult matter. The following method, based largely upon suggestions by Wilhelmi (’04), was used. Jars were filled to the depth of two or three inches with cinders, dirt and dead leaves, over which was spread an equally deep layer of clean sand. Clear water was then poured into the remaining space and the whole allowed to settle, after which afew such plants as Anacharis or Myriophyllum, with whatever micro- scopic life might adhere to them, were added, together with a hand- ful of large pebbles to diversify the bottom. ‘The jars were kept covered from dust in a cool place and occasionally a crushed snail was dropped into each one to supply the worms with food. Planarians require pure water. Whenever for any reason the water in which they are kept becomes foul they will desert their places of concealment and crawl up the sides of the jar, while water that has been standing in lead or iron pipes quickly causes them to disintegrate. Rainwater or water taken directly from some natural source, gives better results than that which has been conveyed through pipes. Naturally the least chemical disturbance takes place when the worms are kept in water dipped up at the time and place of their capture. Planarians will live without being fed for over three months when isolated in jars containing nothing except pure water, but meanwhile they decrease regularly in size. It seems to be impos- sible to “starve” them in the sense in which higher animals may be forced to die from lack of food leaving behind a dead body. These worms instead simply consume their own substance almost to the vanishing point. During a part of the summer of 1905 observations and experi- ments were carried on at the laboratory of the U. S. Fisheries The Reactions of Planarians to Light 47 Bureau at Wood’s Hole, Mass., and I wish here to express my thanks to the director, Dr. F. B. Sumner, as well as to others in authority there, for their uniform courtesies. The bulk of the investigation, however, was made at Harvard University. I am deeply indebted to Professor Mark for the privilege of having a place in his laboratory and particularly to Prof. G. H. Parker, under whose immediate direction the work was done and whose daily counsels and generous suggestions were indispensable. IV Crirerta ror Measurinc BEHAVIOR Both the form and the structure of an animal set a limit to the character and degree of its movements, which no combination of stimuli, external or internal, can force it to overstep. In estimat- ing the influence of light upon planarians, therefore, it is necessary to know not only the normal behavior of the worms but also the possible range of their reactions under any circumstances. For example, the ordinary gliding locomotion of planarians is accom- plished by means of cilia beating in a mucus track and augmented by muscular contraction. It is physically impossible for this sort of locomotion, even under the most favorable conditions, to exceed a certain rate. By the use of excessive stimuli, however, a worm may be forced to abandon this accustomed gliding for a somewhat faster method of progression known as “crawling”’ or “humping,” in which the muscles are used more than the cilia. But when this is done the limit of possible rate of locomotion has been reached, at least for fresh water planarians, which cannot be urged to abandon entirely contact with some support and to swim freely in water, although the marine form, Bdelloura, does have this addition to its repertory of behavior. The following observations may illustrate more specifically what is meant by range of behavior. Planaria maculata, when gliding on the bottom of a dish, was lightly touched on the anterior end with a hair mounted on a glass rod. During one hundred trials of this kind eight different responses resulted, which may be indi- cated as follows: 48 Herbert Eugene Walter Times 1) | Contracted wand turned) asi Garerctetrs:sccxsieteyeraressictesest1oia/ sissies ets ofeiars stat siayarets aistelatet atest steyetateteneterete 32 2 Contracted, lifted up the anterior end, and turned aside........-....0+eeeceeeeeeceeccece 27 3 Contracted, lifted up the anterior end and went straight forward ...........-.......+se005 17 4 Contracted momentarily and then went straight ahead .............020.020seeceeeeeees 5 5» Didmot,contractibutiturmed asides a. cccreerae sclas ace -acigad meine eee CPE ere tEee 2 6 Did not contract but lifted up the anterior end and turned aside..........-.0.00eseeeeeeee 7 7 Did not contract but lifted up the anterior end and went straight forward.................+ 9 8 Did not contract but went straight ahead...... IPR ACO NOD Onan dopeTctn cacdacoacdad I DO tal sicrerayats store state senceloepeteite re tetogen Me oteis lel stoi sveie/o\s0ore co, 312 agsibia, nia sia) el ofer ticle Meiers tear eee meteteore 100 Animals which, like planarians, present a limited range of be- havior are, therefore, more favorable subjects for experimentation than higher forms whose structural complexity increases their possi- ble responses, making in consequence the analysis of cause and effect in their activities more difficult. It is evidently desirable, then, to have as many different ways for measuring behavior as possible, in order not to state these responses loosely from general impressions but in quantitative terms. ‘The principal criteria of planarian reactions to light used in this study, follow: 1 Rate of Locomotion. Since the entire range of possible rates of locomotion depends upon the structure of the worm and is not very great, slight differences become significant. 2. Amountand Character of T urning, that is, whether persistent or irregular, decided or vague, clockwise or contra-clockwise. 3. Change of Course. A change in the character, but not neces- sarily in the direction, of the course is referred to here. “Circus movements,” for example, would not be included under this head- ing because the curving path in such cases, although constantly changing in direction, does not change in character. “Tangents to a circle, however, as well as angular and abrupt deviations from a straight line may properly be regarded as changes of course. 4. Interval of Response. Vhe apparent effect of light is not immediate in all cases, therefore, the time elapsing between the application of the stimulus and the response to it is a valuable measure of reaction. 5 Degree of Wandering. Ina sense the degree of wandering shown by a worm is a measure of its indifference to the stimuli acting upon it. It must be noted, however, that apparent indif- The Reactions of Planarians to Light 49 ference may sometimes be due to a balance of opposing stimuli, in which case wandering or aimlessness is not a true measure of the effect of any single stimulus. 6 Orientation. his is a measure of behavior with reference to the source of the light. It is expressed by the degree of posi- tiveness or negativeness which the worm exhibits. 7 Duration of Movement. The time it takes a worm to tire out when subjected to certain stimuli or, in other words, a measure of fatigue. 8 Effect of Repetition. A measure of response is here referred to which may be expressed quantitatively in units of time or quali- tatively in manner of behavior. g Wigwag Movements. These are waving movements of the anterior end of the planarian, which appear to be a definite attempt on the part of the worm to become adjusted to the stimuli acting upon it. 10 The Time Required to Leave a Unit Circle. This is a rather unsatisfactory criterion because it may indicate in some cases a combination of several conditions as, for instance, latency of response, rate of locomotion and degree of wandering. 11 Manner of Coming to Rest. Included under this heading are such points as the position assumed, the locality selected, and the abruptness of the act. Naturally some of the foregoing measures of behavior will be seen to have more application and value than others in the follow- ing study. V OBSERVATIONS I PHOTOKINESIS The term photokinesis was introduced by Engelmann (’83) to denote the activities which are induced solely by the intensity of light when the directive or orienting factor has been eliminated. In this section will be considered, (A) the behavior of planarians in the absence of light; (B) their behavior in different intensities of non-directive light, and (C) the effect of abrupt changes, both in time and space, in the intensity of non-directive light. 50 Herbert Eugene Walter A Behavior in Dark Darkness may be called the zero point in the scale of light inten- sities. ‘That light is not essential to the activity of planarians is shown by their performances in its absence, as is demonstrated by the following facts. Rate of Locomotion. ‘The average rate of ten individuals of Planaria gonocephala was found to be 0.50 mm. per second in the dark while the same ten worms, subjected to a light from above of 38 c.m.,? with all the other conditions unchanged, averaged 0.82 mm. per second. Again, ten worms of the same species were allowed to travel in the dark ten minutes in one set of experiments and six minutes in another, when their average rates were found to be 0.42 and 0.57 mm. per second, respectively. The method devised for obtaining the above records, previously used in experiments upon fresh water snails (Walter, ’06), although tedious was comparatively accurate. A clean glass plate was submerged in a dish of water and the latter placed in a light-proof receptacle. A single worm was then allowed to travel on this glass for a unit of time, after which the plate was removed and “developed” bk pouring over it powdered carmine shaken up in water. A sufficier.t number of the insoluble carmine particles adhered to the mucus-track left on the glass by the gliding worm to make it possible to wipe dry the reverse side of the plate and to trace thereon in ink the exact course taken by the worm. ‘This permanent ink line was then measured by means of a map meas- urer such as is in common use for measuring sinuous lines. A series of experiments, to be described more in detail later (Table III, p. 57), forms a basis of comparison with the foregoing records in the dark, and further shows that there is an increase in the rate of locomotion in the light. Ten worms, subjected to various intensities of light projected from above and ranging from less than one to several hundred candle meters, showed rates which in all cases were greater than the rate traveled in the dark. $ The abbreviation c.m. is used to denote candle meters. The Reactions of Planarians to Light 51 Turning. That planarians do more turning in the dark than they do in various intensities of non-directive light is apparent from the following table of percentages. TABLE I Per cent of LS of Planaria gonocephala in the dark and in various intensities uf use ——— = Light in candle meters...........-..+ ° .. AI Ir | 39 | i 78 | eel 155 | 217 431 |Av. of all (dark) | | | intensities Percent off turmin pete -ater(eieistesinteeciet= | 87 76 | 66 | 69 | 81 | 67 | 75 | 77 | 65 72 . | Per cent of straight paths............ | 13 24 | 34 | 31 | 19 | 33 | 25 | 23) 35 28 Number of observations............. 71 79 | 67 | 85 | 57 | 62 | 67 | 57 8 Furthermore, out of a total of 40 cases of turnings made by dif- ferent individuals of Planaria gonocephala in the dark 23 were clockwise and 23 contra-clockwise. This perfect balance in be- havior did not recur when the same worms performed turning evolutions in the light. Change of Course. As to what constitutes “definite” and what “indefinite”? changes of path, an S-shaped course is to be regarded as an indefinite efenless wandering, whereas angles in a snare: path or tangents in a curving path are classed as deanite responses because they are what would normally occur if some directive stim- ulus were interposed. It was found that P. gonocephala made indefinite changes in its course more frequently in the dark than in any series of light intensities to which it was subjected for an equal length of time. On the other hand definite changes occurred oftener in the light, although the factor of directive light had been excluded. Table II summarizes 350 recouas on 10 different worms with the results reduced to percentages. It will be seen that the percent of S-shaped (‘‘indefinite”’) paths in the dark decidedly eclipses that which was made in any inten- sity of light, while the per cent of angular and tangential paths (“‘definite’’) laid in the dark is exceeded in every instance by that made in any intensity of light with one exception, viz: II ¢.m., which, however, is not sufficient to change the average result. Summary. Planarians move about in the dark but at a slower rate than in non-directive light whatever the intensity. They 52 Herbert Eugene Walter turn more in the dark than in the light, going clockwise or contra- clockwise with equal readiness. Finally, they make more indef- nite changes in their paths in the dark, but fewer definite changes than in the light. TABLE II Percentage of definite and indefinite changes in the character of the course in dark and in light of different intensities Details of the several intensities employed | Average | | Light in candle meters................-- o | for allin- Jo.94) 11 | 37 | 78 | 126) 155] 217] 431 |(dark)) tensities Definite changes (angular or tangential changes) percents ccoeecasssasanasn| 18 28.5 30°] 15) | 27 |) 32: |i 35;) 21 || 32" |'40 Indefinite changes (S-shaped paths), per) | CONE serretays ee etic este tte stete pein esets 47 | 23 21 | 30 | 23 | 20 | 26 | 34 | 20 | 99 No change in character of course, percent} 35 | 48.5 | 49 | 54 | 50] 48 | 39 | 45 | 48 | 51 Number of observations......../.:..... 34 316 49 | 35 | 48 | 37 | 31 | 48 | 35 | 33 B Non-Drrective Light a Apparatus To test the effect of purely non-directive light, it is of course necessary to eliminate the possible influence of directive light. This may be done by projecting the light upon the moving worms in such a way that they are unable to go either toward or away from the source of the light. Whatever effect is obtained under such circumstances must be ascribed to the non-directive power of light. The elimination of the directive influence of light can be accom- plished by means of various devices. (1) The light may be made to fall vertically from above upon a horizontal field; (2) it may be reflected vertically from below so as to pass through a transparent held at right angles to the plane of the field; (3) methods 1 and 2 may be combined. ‘The apparatus finally used in the majority of experiments with non-directive light, was based upon the method first mentioned. The Reactions of Planarians to Light 53 Fig. 1 shows a diagrammatic vertical section of this apparatus. The light (4), an incandescent electric lamp, was mounted in a black sheet-iron hood (B) to prevent the escape of any lateral light. This hood was suspended from the ceiling of the dark room where the experiments were carried on and was arranged so that it could be easily raised or lowered, thus changing the height and consequently the intensity of the light with reference to any fixed point below. In the hood, beneath the light, was supported Fig.1 4, light; B, walls of hood; C, heat screen; DD, diaphragm; E, roof of hood; F, plate-glass floor of aquarium; G, paraffine wall of aquarium; HH, diaphragm to cut off light reflections from paraffine wall; J, wall of reflector box; ¥, open side of reflector box; K, mirror; L, walls of tunnel; MM, black draperies; N, table. a flat-bottomed, clear-glass dish (C) containing distilled water to a depth of about three centimeters. The heat screenthus obtained effectually filtered out the heat rays, allowing only the light rays to pass through. A few inches under the heat screen was inserted a diaphragm (D), painted black, the purpose of which was to aid in cutting out side reflections besides allowing only a central col- umn of light to escape below. A black sheet-iron roof (£) con- 54 Herbert Eugene Walter fined the upward rays to reflections within the hood itself, at the same time permitting the escape of heated air. Ona table directly under the suspended light lay a horizontal sheet of plate glass (F,) affixed to the upper surface of which was a circular ring (G) made of a mixture of parafine and lampblack. There was thus formed a circular water-tight aquarium twenty centimeters in diameter and two centimeters deep, in which the worms could be observed. On the top of this circular ring rested a black dia- phragm (/7), the aperture of which was sufficiently small to exclude any side reflections which might come from the black paraffine wall. The aquarium, it must be explained, did not rest directly on the table but was mounted as the cover of a box (J), the interior of which had been rendered largely free from reflecting surfaces by the use of black camera-paint. One side of the box was removed and, facing the opening thus made, a mirror (K) was placed at an inclination of 45° with the horizon. The end of a square tun- nel (L), ten feet long and made of black cloth stretched upon a framework of wood, fitted close up to this opening. Suspended from the lower edge of the hood and surrounding the aquarium were adjustable black draperies (/) designed to shut out possible side light and at the same time to allow a hole for the eye of the observer. It will be seen that all light reaching the aquarium comes from the lamp above by passing through the heat screen. After illuminating the field of observation the light passes through the glass floor of the aquarium and is reflected by the mirror into the black tunnel. Most of the light is absorbed in the tunnel, only an insignificant minimum being reflected back to the aquarium floor. Otherwise complications in the character and intensity of the light might arise. By moving the hood (B) up and down and by using lamps of different candle powers a variety of intensities was obtained. The lamps used were tested by means of a Lummer-Brodhun pho- tometer, the loss by reflection from the surface of the water both at the heat screen and at the aquarium being reckoned out in deter- mining the different intensities employed. By simple observation, data for such criteria of behavior as The Reactions of Planarians to Light 55 amount of turning, changes in course, degree of wandering, inter- val of response and manner of coming to rest, could be obtained in this apparatus with approximate correctness. To determine the rate of locomotion, however, required a device which would measure accurately the distance traveled in a unit of time. The Fig.2 ABCF, pantograph; C, fixed point; D, paraffine wall of aquarium; £, plate glass bottom of aquarium; F, place where the arm 4 is grasped by the operator. A style is located at end of arm A, in contact with under side of aquariumfloor. J, style at end of tracing arm B, in contact with smoked paper; , beginning of a course traced on the smoked paper; K, drawing board for attachment of smoked paper; L, sheet of smoked paper fastened to drawing board; M, actual course of the worm. method already mentioned of measuring rate from mucus-tracks developed by means of powdered carmine, proved too tedious and uncertain except for the worm’s maneuvers in the dark, when it seemed the only available way. 56 Herbert Eugene Walter To avoid the inconveniences of this method an attachment was devised for directly duplicating the path of a worm by means of a style traveling over a sheet of smoked paper. The records thus traced were made permanent by immersing the smoked sheets in a weak solution of resin in alcohol and allowing them to dry, after which the paths could be accurately measured and the rates com- puted. The arrangement of this attachment, as seen from above, is shown in Fig. 2. The diaphragm (Fig. 1, H) has been removed for the sake of clearness. At the tip of arm 4F a style directed upward comes in contact with the under surface of the aquarium bottom (Fig. 1, F), while at the tip of arm B a similar style that is pointed downward traces a line on the sheet of smoked paper L at the left. After a little practice it was not difficult to keep the style of arm 4 directly under the posterior end of a gliding worm, thus duplicating its movements with considerable accuracy. The expiration of any time interval can be indicated on the smoked paper record by a crosswise scratch in the path. Arm 4 was rendered as non-reflecting as possible by black cam- era paint as well as by being made triangular in cross section with the apex of the triangle upward. ‘Thus whatever rays struck it from above were mostly either absorbed or reflected in a horizon- tal direction, so that they did not reach the worm under experiment. b- Results Rate of Locomotion. Planaria gonocephala moves somewhat more quickly in non-directive light than it does in dark. Ten apparently normal and representative worms were selected and isolated in individual aquaria. They were kept in the dim light of the dark room in water of the same temperature as that of the experimental aquarium in which they were observed. At the end of thirty-four days of experimentation these worms showed prac- tically the same average rates under the same intensities of light as they did at first. By alternating the individuals these trials were so made that fatigue effects had little part in the results, while the succession of light intensities was varied in such a way The Reactions of Planarians to Light 57 that cumulative effects and the influence of previous exposures were largely avoided. The results obtained in 259 trials are condensed in Table III. TABLE III Rate of locomotion in millimeters per second of Planaria gonocephala in various intensities of non-directive light | ie | a ‘ | =p | Candlejmeterssaacacie sere see | ° | 0.94 | II | 39 | 78 | 126 | 155 217 | 431 Average mm. per sec....... 0.57 | 0.66 | 0.69 | 0.75 | 0.64 | 0.66 | 0.69 | 0.70 | 0.63 Number of records ............ | 30 «|. 28 30 | 29 | 27 || 301) 030 2 28 The mechanical stimulus resulting from the removal of the worms, by means of a camel-hair brush, from their individual aquaria to the observation aquarium was practically the same in all cases as were al] the other external stimuli except light. The difference in the rate of locomotion appearing in these averages is, therefore, clearly due to differences in the light intensity em- ployed. It will be seen also that rate does not increase progressively with intensity. ‘The series of rates and intensities under Table III, if plotted in a frequency curve would give two modes, one at 39 and the other at 217 candle meters, with a slight depression be- tween the two. Still, as has been already pointed out, any inten- sity of light gives a faster rate than no light at all. The slowest average rate was made under the highest intensity of light employed. Certain facts to be brought forward later favor the opinion that this was not an accidental result. Under continuous exposure to one intensity of light the rate of locomotion decreases. ‘The worms seem to “run down” gradually, so that at the end of ten minutes their rate is only about half that during the first minute. Data illustrating this point are given in Table IV. The rate of locomotion depends not so much upon the intensity of light as upon other factors which tend to produce individual behavior upon the part of each particular worm. Stated in another way, there is greater variation between different individuals in the average rate of their locomotion under all intensities than there 58 Herbert Eugene Walter is in the average rate of all individuals collectively under different intensities. [he data for this latter point based upon the average rate of ten worms (259 observations) under different intensities has already been given in Table III (p. 57). The extremes in rate there shown are 0.57 mm. per sec. at zero intensity and 0.75 TABLE IV Average rate of locomotion of Planaria gonocephala in successive minutes of exposure to 39 c.m. of non- directive light Number of minute.......) Ist | 2d | 3d | 4th sth | 6th | 7th 8th | gth | roth | 11th | 12th | | | | | | | | | | | 4 > |b 48 0] Siena aes ee 3 29 | .25 | 29 No. of records averaged. . 17 | 15 | 12 | 7| 5 2 Rate in mm. per second..) .63 | .625) .565) .55 | -53 | -55 | -375] -39 | -39 mm. per sec. at 39 c.m. intensity, which makes a range of 0.18 mm. per sec. When the same data are rearranged to show the average rate for each individual for all intensities, as in Table V, the extremes are 0.49 mm. per sec. and 0.83 mm. per sec. with a range of 0.34 mm. per sec. In fact the individual behavior of these ten worms, despite their apparent similarity, was sufficiently distinct to allow each one to be thereby identified. Turning. Attention has already been called to the fact that there is less turning in light of various intensities than in the dark. A return to Table I will make plain that there fails to be any TABLE V Average rate of locomotion for each of ten worms (Planaria gonocephala) based on trials with non-directive light of various intensities Identification number of worm........ | I 2 3 4 5 6 7 8 9 10 | | Average ‘rate in eight intensities al | | | pressed in mm. persec.............|0.79 |0-57 0.68 (0.64 0.83 |0.70 |o.72 0.58 [0-49 |0.62 ! = ae Seas definite correlation between the degree of intensity of the light and the amount of turning, although the least turning occurs under the highest intensity. This latter point, however, rests upon a very slight difference and may not be significant. It 1s neverthe- less worth mentioning, since it is in line with the effect of the os The Reactions of Planarians to Light 59 highest intensity upon rate, as well as with certain other evidence to be discussed later. The small excess of clockwise over contra-clockwise turnings is not explainable upon the ground of varying intensities of light. A distribution of the cases under the several intensities of light (Table VI) makes it plain that this peculiarity is due rather to individual causes than to light intensities. Indeed it would be difhcult to conceive theoretically how varying intensities of non- directive light could influence a worm in such a way as to affect the direction in which it turns. ‘The natural expectation accord- ing to chance would be an equal number of turnings in either direction. ‘The excess of clockwise turns seems, therefore, un- doubtedly due to internal causes which render certain worms more liable to go one way than another. In fact, when the records were arranged according to individual behavior it was found that of the ten worms seven averaged a majority of clock- wise turns while only three fell in the contra-clockwise column. TABLE VI Character of turning of Planaria gonocephala in non-directive light of various intensities = 7 = ea =n “Sea se aiihea a = | an . Light in candle meters...............| © | 0.94] II 39 | 78 126 | 155 | 217 | 431 |Total Clockwise turns........... deemuaayie 2g bas. Wier mae | 17) x7) |22 | 24 | 22 | 203 Contra-clockwise turns............--| 23 23) ||) 37; 20 | 17 14 | 166 18 17 | 17 occur in the light than in the dark, but fewer “indefinite” changes. This point requires no further exposition as its corollary has already been given. The behavior of the worm in this respect seems to be more closely correlated with the highest intensity (431 c.m.) than with any other. In the highest intensity employed there are indicated (Table II, p. 52) 40 per cent of definite changes, which is con- siderably in excess of the percentage of such changes made in any other intensity. Onthe other hand indefinite, or S-shaped, changes constitute only g per cent of all records taken at the highest intensity, which is less than half the number of indefinite paths made in any other intensity. 60 Herbert Eugene Walter While the extremes of the series of definite changes indicate a general rise in the percentage of their occurrence with an increase of intensity, and while in the same way the extremes of the series of indefinite changes suggest in general a decrease of frequency with the increase of intensity, it can hardly be maintained that the character of the changes in course is definitely correlated in the majority of cases with changes in intensity. Degree of Wandering. Wandering is not closely correlated with the intensities of light. In Table VII, which deals with the percentage of straight paths made by P. gonocephala under dif- ferent intensities of non-directive light, this fact is expressed nega- tively, since it is held that a straight path is a good indication of the absence of aimlessness or wandering and may thus serve as a negative measure of such behavior. TABLE VII Percentage of straight paths made by P. gonocephala in the dark and also in non-directive light of different intensities Light in candle meters... . Hogeoanmnsite o |0.94| 11 | 39 In this respect again the behavior of the worms under the high- est intensity 1s more pronounced than under any other intensity since the greatest number of straight paths were laid at an inten- sity of 431 c.m. Interval of Response. There seems to be some evidence that the interval of time elapsing between the reception of a light stim- ulus on the part of a worm and its consequent response, may be quite considerable. ‘Three facts were established that may sup- port this conclusion. First, when two-minute records were made under various inten- sities, it was found that the worms averaged a faster rate during the second minute of exposure to the light than during the first, in spite of the facts that the mechanical stimulus due to placing the worm in the light machine had a more quickening influence during the first minute and that the fatigue effects were more likely to appear during the second minute. The actual figures The Reactions of Planarians to Light 61 for the above statement, based upon 240 two-minute trials under various intensities, are 0.645 mm. per sec., the average during the first minute, as against 0.713 mm. per sec., the average during the second minute. Secondly, in these 240 trials, the percentage of turning under all intensities 1s greater during the first minute than during the sec- ond, being 87 per cent and 57 per cent, respectively. “This result may possibly be conceived to be due to a greater steadying influ- ence of the light during the second minute than during the first and to a consequent greater turning than during the first minute. But on the other hand a similar decrease of turning, although not so pronounced, took place during the second minute when the worms were in the dark. It must be admitted, therefore, that the fact of less turning during the second minute may have nothing to do with the interval of response. Thirdly, on several occasions a notable piece of behavior was observed, which may have a bearing on the interval of response. The phenomenon in question AGars occurred in connection with a modification of the experimental held within the light machine to be more fully described later. Briefly this modification con- sisted in making a field of two distinct intensities of light, the latter being projected vertically from above in such a way that a sharp line of demarkation formed a boundary between the two areas. Ordinarily when the worms reached this boundary line as they glided from one intensity to another, they responded promptly to the stimulus caused by the change of intensity. Sev- eral times, however, they were observed to travel indifferently exactly along this dividing line for a distance of several centimeters with half the body in one intensity and half in the other. This curious fact lends itself to various interpretations, one of which is that the response to a new intensity may not be, in all cases, immediate. Manner of Coming to Rest. During the experiments made in the non-directive light apparatus previously described, nor- mal worms could never be induced to come to rest in the light. If allowed to remain in the aquarium they would wander about until they reached the shadow under the diaphragm (Fig. 1, H), 62 Herbert Eugene Walter where they finally stopped, usually in the angle formed by the parafhne wall and the bottom. Loeb’s conclusion (’93b, p. 101) that planarians subjected to directive light come to rest in regions of least intensity, seems therefore to be equally true of planarians in non-directive light. Summary. In non-directive light Planaria gonocephala moves faster, turns less and makes more “‘ definite’ but fewer ‘‘indefinite”’ changes than in the dark. Rate of locomotion; amount of turning; changes in the character of the course, as well as the amount of wandering, do not appear to be correlated with varying light inten- sities, unless in the following instance. Under the highest inten- sity employed, namely, 431 c.m., occurred the slowest rate; the least turning; the greatest number of “definite” and the fewest “indefinite”? responses, together with the straightest paths. The excess of clockwise over contra-clockwise turnings throughout the series of intensities is probably not attributable to light. Continuous exposure to light results in a decreasing rate of loco- motion, although in the second minute of movement as compared with the first an increase in the rate of locomotion takes place, while fewer turnings occur. Rate of locomotion is less influenced by differences in light inten- sity than by certain internal factors which go to make up what may be termed the individuality of different worms. Individ- ual worms may sometimes fail to respond for a considerable inter- val of time to light stimuli that ordinarily produce immediate effects. Finally, planarians subjected to non-directive light come to rest in regions of lessened light intensity the same as they do in directive light. “Gi Abrupt Changes in Intensity Abrupt changes in intensity may be of two kinds: either with reference principally to time or to space. First, those changes are abrupt zm time in which light or dark is suddenly thrown upon the worm, and secondly, those changes are abrupt 1m space in which a moving worm passes immediately from an area of one intensity into a sharply defined area of a different intensity. ‘This topic The Reactions of Planarians to Light 63 will be discussed here only in its relation to non-directive light, the effects of sudden changes in directive light coming more properly in a later section. a Abrupt Changes of Light Intensity in Time Whenever worms were left over night in the experimental aqua- rium completely shut off from light, a large proportion of them would be found at rest in the morning when the light in the hood was again turned on. By removing ie diaphragm (Fig. 1, H), under mie edge of which near the penaiine wall the worms were usually enllcceces it was possible without any mechanical disturb- ance to subject resting worms to sudden non-directive light after a prolonged period of complete darkness. This sudden stim- ulus rarely had an instantaneous effect. ‘The interval of response was often several minutes and frequently non-directive light alone proved insufficient to start the worms into activity. No sudden increase of intensity ever proved powerful enough to throw a gliding worm into the more rapid method of crawling. Pearl (’03, p. 551) stated the same fact after subjecting planarians to much stronger intensities of light than were employed in the present experiments. It was found that P. gonocephala showed a decided response— either some change in course or a wigwag motion of the anterior end—more frequently when suddenly subjected to dark than to light. By inserting a key into the electric circuit it was possible to control the light in the hood to a fraction of a second. Worms in complete darkness were by this means subjected to various intervals of sudden light and worms in light to intervals of sudden dark, the results being at once noted. While the worms were in the dark their behavior could not, of course, be directly observed, but by watching them closely just before the light was turned off and also the instant it was turned on again there was no great difficulty in determining whether a response had occurred during the interval. The results obtained from nearly a thousand trials are indicated in Table VIII. It will be seen from this table that there are more responses than failures to respond and that the responses occur more fre- 64 Herbert Eugene Walter quently when the worms are suddenly subjected to dark than to light. It may be further noted that the excess of the responses in the dark over those in the light increases with the interval of exposure, indicating that the worm’s adjustment to a change in the light stimulus affecting it is not in all cases immediate. The effect of previous exposure, whether to several hours of dark or light, is a factor in these results which will be considered more properly later on. TABLE VIII Percentage of the responses of P. gonocephala in various intervals of time when suddenly subjected to dark and to light of 39 c.m. Number of seconds exposed..............-- 5 | 10 15 20 25 30 | Average Percentage of responses in light............ 51 | 59 54 54 48 46 52 Percentage of responses in dark............. 63 66 73 75 71 71 7° Excess of responses in dark....... ROSA OS 12 7 19 21 23 25 18 It should be added that Bdelloura gives a remarkable response when enveloped in sudden darkness. It will frequently forsake its attachment under these circumstances and unattached in the water go through violent contortions. ‘This striking response can be called forth by an exceedingly brief interval of dark, namely, the shortest time required to turn the electric light off and on. Nagel (’94, p. 387) speaks of animals thus affected by sudden shadow as “skioptic.”’ The relation of Bdelloura to light falls into a somewhat different category, however, than that of the fresh-water planarians, since Bdelloura is positive to light, while fresh-water flat-worms are negative. b Abrupt Changes of Light Intensity in Space Several devices were employed to test the behavior of planarians passing abruptly from an area of one intensity of non-directive light into another. ‘The most successful device tried was that in which two lights of different intensities were mounted overhead iy The Reactions of Planarians to Light 65 in the hood of the apparatus already described in Fig. 1, the mingling of their rays being prevented by the insertion of a ver- tical diaphragm (Fig. 3, C), which extended from the region between the lights down to the surface of the aquarium. In order to place the diaphragm in position it was, of course, necessary to remove the heat screen (Fig. 1, C), the presence or absence of which, however, would not have affected the results sought since the water in the aquarium itself was nearly 2 cm. deep and thus Fig. 3. A, stationary light; B, sheet iron walls of hood; C, vertical diaphragm separating the two lights; D, horizontal diaphragm; £, sheet iron roof of hood; F, plate glass aquarium floor; G, paraffine wall of aquarium; HH, diaphragm to shut off reflections from wall of aquarium; J, wall of reflector box; F, open side of box; K, mirror; L, black tunnel; M, black draperies cutting off side light; N, table sup- porting reflector box and end of tunnel; 0, movable light; P, track for movable light; 9, narrow, hori- zontal diaphragm attached at right angles tothe lower side of the diaphragm C, in order to prevent the light rays from the two sources of light, 4 and O, from overlapping. constituted an efhcient heat screen. By keeping the hood sta- tionary and causing one of the lights (Fig. 3, O) to slide up and down at will, it was possible to bring about various contrasts of 66 Herbert Eugene Walter intensity in the field below. The complete plan of the appa- ratus Is given in Fig. 3. The principal variations in the behavior of Dendroccelum and Phagocata upon reaching the critical line separating the areas of two intensities are indicated diagrammatically in Fig. 4. The dotted line represents the boundary separating two areas of different light intensities. The arrows represent the types of paths made by Dendroccelum and Phagocata. For the sake of simplicity the worms are represented as going in one direction; that is, into one of the two contrasting intensities, but the same types of paths resulted as well when the opposite direction was taken. The angles made in crossing the critical line were also more varied than those represented in the diagram. Type 4 represents a passage without re- sponse; B,an angular change of course made at the critical line; C and F, aloop-like return effected after a short excursion into the new intensity, and G,a sharp turning aside, while A indicates a halt at the critical line, as if a barrier had been encountered. Finally D and E represent a temporary pause on the part of the worm accompanied by wigwag move- ments of the anterior end of the body. In the case of D the wigwagging is immediate, but E typifies a case when there occurred in the response an interval of such a nature that the ' significant movements were not made until Fig. 4 the worm had advanced at least its own length into the new area. Of these types all, with the exception of 4, are to be regarded as reactions to differences in intensity encountered. The most questionable are the infrequent types C and F, which may be otherwise explained as arcs in a curving course which might have occurred in a field of uniform intensity. By far the commonest type was D, plainly the least doubtful of the series. The Reactions of Planarians to Light 67 As a result of over 3000 observations on the manner in which the critical line separating the two intensities was passed, three facts become evident. First, responses were considerably more TABLE IX Kind and percentage of responses of Dendrocalum and Phagocata in passing from one intensity of non- directive light to another Turn-backs| No Wigwags and full Loops Angular Total Character of course responses) (Types | stops | (Types courses reece (Type 4) Dand E) | (Types | C and F)| (Type B) G and H) Going into greater intensity, PETCEN Greys eteressfulshessreleat ests 79 II 6 2 2 2I Going into lesser intensity, PEM CODG gararaintatelstetar lererstaroks 5° 36 5 8 I 50 Average responses, per cent.| 64.5 tals 5-5 | 5.0 Aly 35°5 frequent when the worms were passing into the lesser intensity than they were when entering the greater intensity. Secondly, lack of response is more frequent than a visible response of any kind since 64.5 per cent of the crossings made over the critical line were of the type 4. Thirdly, the responses at the critical line were more frequent when the worm was upside down, 1. e.. moving on the surface film, than when it was on the floor of the aquarium. This latter point was illustrated most fully by Phagocata, which, being an active worm, takes quite readily to the surface film, so that it was possible with this species to get a series of observations in which the behavior when crossing the critical line on the bottom of the aquarium could be compared with that when the same line was encountered at the surface film. Table X contains the results of these observations. The doubling of responses when the worm is on the surface film is probably not due to an unequal receptivity of light stimulus by the dorsal and ventral surfaces of the planarian as might at first thought seem possible. As will be shown further on, the worm’s rate of locomotion on the bottom of the aquarium is nearly the same whether the light comes from below or from above, pro- 68 Herbert Eugene Walter vided the amount of light in both cases is equal. Planarians, as Pearl has emphasized, are strongly thigmotactic. Naturally, then, their response to contact is much greater when they are on the glass bottom of the aquarium than when they are suspended on the less resistant surface film. In other words, the less the worm is influenced by the stimulus of contact the freer it is to respond to the stimulus of light. TABLE X Percentage of the responses made by Phagocata at the critical line separating two intensities of non-direc- tive light either on the bottom of the aquarium or on the surface film | Number of No response Response | observations per cent per cent Ounithe surfacedilia!acones chs apeanastosuiss cess 740 45% 544 On: thetbottome. oancrsosen ee eee aneiacg cee ai 1664 76 24 MMotalisiepsannc te oxelateernvctsy se corers/o\sralsinyo: Se co-fiassiecto 2404 60} 39% Finally, a series of experiments was tried in which the contrast between two intensities was varied by raising or lowering one of the lights in the hood. It was found that the responses made by Phagocata under these circumstances increased with the increase in contrast between the two intensities as shown on the bottom line of Table XI, where these contrasting intensities are expressed in a ratio between the constant light taken as unity and the movy- able light. The fact that responses by no means invariably occur when bright light and complete darkness are suddenly substituted for each other (see Table VIII) rendered a further extension of this series unnecessary. The contrasts here used form probably a much greater range of intensity contrasts than the worms ever encounter in nature. Attention to the details presented in Table XI brings to light the fact that, although the number of responses is correlated in a general way with an increase in the contrast between the two illuminated areas, as shown in the bottom line of the table, yet the percentage of the responses is further influenced by the actual degree of the intensities employed. For example, when the two ze fs 7 ot o§ | SE-fF | zh | SLE | S-ob gf sasuodsas yo | . asejuaoiad “ay Lotz rio, Siz 66 ZOz 66 foz 66 goz lof 116 : =| Ss We a oe == | suonearasqo zor | €11 66 ZOz 66 | 101 | zor 66 for | Sor} ztr | €or | zér | 26 | Sar | zex | che | €xx | Ler | oor if jo saquinyy ssdsaz jo'atjog 19 7S ob oS Ly | | eb a4 6S $$$ 9S \ stan 6 5% ISI] apquaoyy +h: g6z of zgz gz biz oz g61 Lg" Lor 69°ztr 61° 9g LS*€9 tL: Lt gi tt If 9 ssdsaa JoaSqg gt | $68 S-1+ | Sz 5A bz S-o1! $*€€| S-cz| S-gz \ aEaionsee WyTty apquaoyyy Lg'9gS | joo of E loz'r1z| Lg: Ltr $L- Lor oS zg SI gg Og°zS |Lg° Lb Lg“ LE if 2 1431) Kapuonvig Ws yuvsuog or: | 1: ap ||) aa ee Wl seb spe | rep || arse sees || sae Ts LOO Gtem | OC lithe |esets Insta |b sag I: ae) WS aquaoyy, 1f-gr 69°21 60°91 | Sb: £1 | Forze Sack) $9°6 | t1'g | LE-9 [es ‘ch-b | O6'£ Sz-€ |€6-z ghz forz g6-1 6-1 zf-1 or -40> Aysuaynt “a . | at} am joonr 1: 61-L1 meL1-Srjr:St—Erin:€r-11| ors 1r-6 |r: 6-L/ rR ES «Il res etter Sect Pasvadut Kyonposd s1 sassuajur asoyr Ma2msag 18P43U0) ayt uayor 14311 aa1oeup-uoU fo sainsuaqut om Surwsvdas aur poosps9 ays yw spor Eobe us pwr0dnyg fo sasuodsas fo aSviuariag IX ATAVL 70 Herbert Eugene Walter areas of light were respectively 33.16 and 68.18 c.m. the ratio was practically the same as when the two intensities were 16.3 and 33-16 c.m., yet the percentage of responses in the two cases is decidedly different, being 10.5 per cent in the former, and 56 per cent in the latter case W hen ve lesser of the two lights was 33.16 c.m. there were invariably fewer responses than aa the lesser light was 16.3 c.m. The latter intensity is undoubtedly nearer the planarian’s optimum intensity, and the apparently inhibitive action of the higher intensities agrees perfectly with certain facts already detailed, as, for instance, that the activities of Planaria gonocephala were less pronounced at 431 c.m. than in lesser inten- sities; and, again, that all planarians show more responses on going into a lesser than when going into a greater intensity. Attempts were made in some other ways to subject planarians to areas of contrasted intensities and, although the results were less satisfactory in general than those obtained by the method of using two buerhead lights of different intensities just described, yet certain facts were ibeatie out that may be worth recording. In the first of these attempts two concentric rings of white paper, each about two centimeters wide and having between them a space of a couple of centimeters, were fastened to the under sur- face of the aquarium floor. The white paper thus arranged reflected the light upward and made areas of gradually increased intensity as compared with the remainder of the aquarium floor through which the light passed without reflection. Worms placed in the center of these circles would consequently be obliged to pass from one intensity of light directly to another, ee er the direction of the radius they might be taking. When worms were actually tested, it was found that they exhibited considerable modification in their movements, particularly when approaching the edge of the paper backgrounds. Owing to the considerable thickness of the plate-glass floor of the aquarium as well as to the fact that white paper is a surface which scatters the light falling upon it, it was found that there was formed, not a sharp line of demarkation between two intensities, but rather a penumbra-like margin of intermediate light. This apparatus was therefore abandoned as unsatisfactory. The Reactions of Planarians to Light 71 The difficulties presented by paper as a reflecting surface largely disappeared by the substitution of a plain mirror in its place, since the surface of a mirror is such that all the light striking it at right angles is reflected at right angles. When, therefore, an unmounted mirror was brought into contact with half of the under surface of the aquarium floor the whole field was thereby divided into two regions sharply separated from each other. Of these one was supplied with light from above only, while the other received the same light plus Aeanl: an equal amount reflected from the mirror below. With the aid of this device an increase of II per cent was gained over the responses obtained when white paper instead of a mirror was used as a reflector. Both Phagocata and Dendro- ccelum were tried by this method. In 76 per cent of the trials made, 7. ¢., in 125 cases out of 165, the worms showed a visible modification in their behavior on reaching the boundary of the two areas of light. It was nevertheless decided that this method was an uncertain test of behavior, since the body of the worm, although fairly translucent, would by no means allow all the light that fell upon it to pass through and be reflected, and consequently the difference of the two intensities to which it was being sub- jected could not be easily estimated. Summary. When sudden light or dark envelops planarians (Dendroccelum, Phagocata and Planaria) the response, if any occurs, is often not immediate. No one of the intensities of light which were employed in these experiments when introduced suddenly was sufficient to make the worms forsake gliding for crawling. Sudden dark calls out more responses than sudden light, while the number of responses increases with an increasing interval of exposure to the stimulus. Bdelloura is decidedly “‘skioptic.”’ Worms encountering the edge of a reflecting area which increases the intensity of the light without introducing any other barrier, show a marked degree of response. ‘The percentage of response is considerably larger when a mirror instead of white paper is used to produce the reflecting surface. If worms are allowed to pass from one intensity to another sharply separated from it, their responses are more frequent upon passing into the lesser intensity 72 Herbeit Eugene Walter than when going into the greater. The average number of fail- ures to respond to these contrasts of intensity reaches about two out of three. Phagocata, at the critical line separating two contrasting inten- sities, responds oftener when on the surface film than when glid- ing over the bottom of the aquarium. The number of responses increases with the increase in the con- trast between the two intensities employ ed, but the percentage of response is greater, regardless of ratio, when one of the lights is of low intensity (13.6 c.m.) than when both are of higher intensity (33 + c.m.) 2 PHOTOTAXIS The term “phototaxis” was introduced by Strasburger (’78) in a study of certain swarm-spores, to indicate movements which were parallel with incident light rays. The term has since been extended by several authors to include similar movements on the part of animals. Any organism is said to be positively phototac- tic when it moves toward the source of light in the direction of the rays and negatively phototactic when it goes in the opposite direc- tion. The purpose of this section is to consider the phototactic move- ments of planarians, as distinct from their photokinetic behavior, (A) when the light remains constant, (B) when the light is changed either (a) in intensity or (b) in direction, and (C) when phototaxis is combined with responses of a different kind. A In Constant Directive Light Orientation. With the exception of Bdelloura all the planarians studied are, under normal conditions, negatively phototactic so far as their first movements in directive light are concerned. To obtain quantitative data for this statement it was necessary to construct an apparatus in which the worms to be tested could be placed quickly and with as little mechanical disturbance as possible in the center of a unit circle with the long axis at right angles to the direction of incident light. The circle was marked off into degrees so that by noting the place at which a worm made its exit a quan- The Reactions of Planarians to Light 73 titative measure of the amount of turning toward or away from the source of the light under the given conditions was obtained. The apparatus finally utilized for this experiment was based upon a device employed by Parker and Burnett (’oo) in testing the relative behavior of normal and eyeless planarians when sub- jected to directive light. Its arrangement is shown in Fig. 5. On the top of a table (4) in the dark room was placed a rectan- gular aquarium (BCDE), the ends of which (BE and CD) were Fig. 5 A, Top of table; BCDE, rectangular aquarium; BE, glass end; FG, round swinging aquarium; H, copper wire attached to ceiling and supporting the swinging aquarium FG; J, movable light; 7, diaphragm; K, surface of water in outer aquarium; L, surface of water in inner aquarium; M, lens. made of glass while the floor and sides were of wood painted with camera-black. Within this aquarium a second cylindrical one (FG), made entirely of thin glass and measuring 20 cm. in diam- eter, was suspended from the ceiling by means of a fine wire (/7) attached to a swivel to allow turning. On the floor of the outer aquarium and directly beneath the inner one was drawn a circle 74 Herbert Eugene Walter 10 cm. in diameter and marked off plainly into arcs measuring 5 degrees each. An incandescent lamp (/), placed on the table at approximately the height of the inner aquarium floor, could be manipulated at any desired distance, while a diaphragm (Ff) prevented much of the light from reaching either the upper surface of the water contained in the two vessels or the floor of the outer aquarium whence it would be reflected. A biconvex lens was then so interposed as to make the light rays practically parallel upon theiremergence fromit. ‘Their course through the inner aqua- rium was kept parallel by means of the medium of water on both its inner and outer sides. A nearly uniform intensity over the entire floor of the swinging aquarium was thus obtained and the objection arising when the inner aquarium is used in air, viz: that it acts as a converging lens, was obviated. Side reflections were eliminated by enclosing the light (/), together with the interven- ing space between it and the diaphragm, with black screens. When a worm introduced into the inner aquarium began to glide, it could with slight mechanical disturbance be quickly cored) by means of moving cen aquarium, into any desired posi- tion with reference to the light, and then swung so as to bring its posterior end exactly over the center of the stationary circle below. Various species of planarians were started in this manner at right angles to the light. Out of 386 cases, 371, or 96 per cent, emerged from the 10 cm. circle at a point farther away from the light than that toward which they were originally directed. ‘This is taken to mean that 96 times out of a hundred the worms were negatively phototactic. If, however, the method of reckoning negativeness employed by Parker and Arkin (’o1) on the earth- worm is used, the foregoing per cent would be somewhat less. These authors assume (’o1, p. 28) that the apparently positive responses of a normally negative animal, such as the earthworm, may be due to causes other than light, in which case an equal number of responses of like nature might be expected to occur on the negative side as well as on the positive. A number equal to the sum of these apparently positive responses should therefore be subtracted from the total of the apparently negative responses The Reactions of Planarians to Light 75 in order to obtain approximately the amount of unquestionable negativeness. By following this method in the case just given, the per cent of negativeness would be g2 instead of 96, but since this method assumes that normally negative worms are never posi- tive, which is contrary to the evidence to be given later, the most accurate estimate of negativeness would probably fall somewhere between these two percentages. Bdelloura, on the other hand, behaves in the same way only three times out of ten, therefore showing itself to be positively phototactic. This difference in orientation becomes more marked if the total number of degrees, that is, the amount of positiveness and nega- tiveness of emergence from the circle is used as the basis of reckon- ing, instead of only the number of times of emergence. Such a quantitative computation is shown in Table XII. TABLE XII Amount and kind of orientation to directive light exhibited by various species of planarians in 396 trials | Number of Total de- | Total de- |Percentage of Percentage of trials grees positive |grees negative) degrees neg. | degrees pos. Negative worms (Dendroce- lum, Planaria, Phagocata). 386 | 566 10157 94.7 ces Positive worms (Bdelloura) .. .| 10 397 50 11.2 88.8 Although the actual number of trials for Bdelloura in this table is small, they are characteristic of what was observed in a large number of unrecorded instances. The amount a planarian may deviate from the direction in which it is pointed, depends upon the direction of the light imping- ing upon it. A negative species deviates from a straight course least when headed away from the source of the light and most when headed toward it, while an intermediate degree of deviation occurs when the direction of the light is at right angles to the long axis of the worm. In the case of Bdelloura the converse is true, as shown in Table XIII. Rate of Locomotion. In obtaining the rate of locomotion of worms subjected to directive light, the double aquarium apparatus 76 Herbert Eugene Walter just described was used. After the worm to be tested had been placed in the inner aquarium and had begun gliding, it was so oriented that the tip of its posterior end came precisely over the center of the subjacent circle 10 cm. in diameter. The exact time of its departure from the center of the circle was then noted and the instant thereafter that the tip of the posterior end passed over the circumference of the circle was again taken and the worm’s course plotted at once on a duplicate circle sheet. Each worm was given four trials in this manner, being started in four different directions, toward the light, away from the light, and with the long axis of the body at right angles to the light, first with one side to the light and then with the other. TABLE XIII Amount of average deviation tn 2400 trials expressed in degrees of a circle, exhibited by negative planarians, (Dendrocelum, Planaria and Phagocata), and a positive one (Bdelloura) when pointed toward, away from, and at right angles to the source of light Direction in which the worm was pointed with regard) tOsthesl ser hiti tess /alejararacstcvatevaveracchey shia. ’eps/s a wierd ontynusith one At right angles Toward Away from Negative planarians, degrees.........-.-+++++++s0-- 48.1 128.7 2759 Positive planarians, degrees..... : eveuarsvele Waxege 49. 39-3 132.1 The time of the worm’s emergence from the circle was not taken with a stop-watch because the observer’s hands were otherwise occupied. Instead a small clock, ticking half-seconds, was placed conveniently near. By counting the number of ticks during the interval of the worm’s transit from the center to the circumference of the circle the time consumed could be determined within less than a half-second. After tracing the worm’s course on a dupli- cate circle sheet and measuring the same by means of a map measurer, a unit of distance was obtained, which together with the known unit of time consumed in covering this distance, fur- nished all the data necessary for computing the rate of locomotion. Ten representatives of Dendroccelum lacteum, Planaria macu- lata, Phagocata gracilis and Planaria gonocephala respectively were given four trials apiece by the method just explained. ‘The results are presented in Table XIV. From the 160 records thus obtained it becomes evident that the average rate of locomotion The Reactions of Planarians to Light fh is greatest when the worms are pointed toward the light, and least when they are pointed in the opposite direction, while an inter- mediate rate occurs when they are started at right angles to the light. This result is at variance with the findings of Parker and Bur- nett (’00, p. 381), who incidentally reported that Planaria gono- cephala when started away from the light traveled faster than when started toward the light. TABLE XIV Average rate of locomotion, expressed in mm. per sec., of various species of planarians when started torward, away from, and at right angles to the source of directive light of 27 c.m. intensity. : Dendroceelum | Planaria |Phagocata Planaria Total Species | lacteum | maculata | gracilis | gonocephala| average Direction in which the worm was pointed with reference to the light At right angles'.....5....2.% 0.855 1.475 1.445 0.980 1.19 LGR Eid logoseoopagnaouneder 0.g10 1.505 1.430 1.205 1.26 wma ye ir OM cra )axerereesey~layate/2)<yei2 0.795 | 1.440 1.310 1.090 1.16 It was further found that, regardless of the direction in which the worms were started, there was a gradual decrease of the rate dur- ing the four successive trials. “The order in which different worms were oriented during the four trials was arranged so as to neutral- ize the possible effect of the sequence in the direction started. In Table XV the data for 200 trials are arranged to express this slowing down of the rate. TABLE XV Average decrease in rate of locomotion for 50 planartans during four successive trials while subjected to directive light of 27 c.m. IN rina exo fi tari all sa sjoyefays)-\sh-00 2 abate ace nayoxste = lava'e) yafelen sie First | Second Third Fourth i} | Averapexatenmimm.) per SeGer cieefeleiers ware laleistaiets 1.140 1.130 1.075 1.070 Various factors influencing the rate of locomotion, such as the intensity of light, the size and species of the worm, the amount of pigment present in the body and the general physiological state of the animal under experimentation, will be more suitably dis- cussed in other connections. 78 Herbert Eugene Walter Change in Character of Course. When several specimens of Phagocata were placed in a square aquarium which received light solely from one side, their first movements were plainly negative, that is, away from the light. After a brief interval, however, it was seen that apparently as many worms were going toward the light as in the opposite direction. In fact an actual count showed that in a certain interval of time 43 worms passed a central point going toward the light while 44 passed the same point in the oppo- site direction. This apparent change in the character of the course was probably due, not to any change in the degree of nega- tiveness of the animal, but rather to the fact that the impulse to keep moving in some direction is stronger than the impulse to neg- ative phototaxis. Consequently when the limit of the aquarium in a negative direction is reached a worm, since it normally travels in straight lines or sweeping curves and does not turn around and around in one spot, continues its locomotion in the direction of least resistance, namely, back toward the light. It will be remembered that Loeb (’93b) has called attention to this fact by saying that planarians are not negatively “heliotropic”’ in a strict sense because they do not remain as far away from the source of light as they can get. Among various observations made with other ends in view, there were numerous incidental cases of a normally negative worm making an unexpected positive response even from the first moment of being subjected to the light stimulus. ‘This occasion] positiveness is clearly apparent from the general fact already noted that four times out of a hundred the average negative pla- narian turns toward the light. Two definite instances of a reversal in the character of response may be cited. The first was the case of a Phagocata in the double aquarium, which became increasingly positive through twelve successive trials. Its average emergence from the circle for the first four trials was 45°, which is a normal negative result, since go° represents com- plete indifference. In the next four trials, however, the average was 100°, that is, slightly positive, and in the last four, 124°, which is decidedly positive, as shown diagrammatically in Fig. 6. The Reactions of Planarians to Light 79 In the other instance an individual worm, Planaria gonocephala, made the erratic average emergence from a circle of 145°, just 35° short of absolute positiveness. ‘This worm was carefully isolated and tested again four days later under identical external conditions when it was found to have returned to a normal nega- tive condition by showing an average record of 56°. Accuracy of Orientation. It was found to be frequently the case that when negative worms were subjected to directive light their first movement instead of being directly away from the source of light formed a path in a diagonal direction. “This tendency to rege GO 90° AS Light 180° O° 90° Fig. 6 The arrow at the left represents the constant direction of the light. In eachof the three sets of trials each worm was headed successively toward 0°, the upper (in the diagram) 90°, 180°, and the lower go°. The point of average emergence for the first set of trials—supposing the records of the lower semicircle to have been transferred to the upper semicircle—was at 45°, of the second set, at 100°, and of the third set, at 124°. travel diagonally away from the light has also been noted in the case of the earthworm by Smith (’02, p. 469). If the negative phototaxis of planarians is to be explained on the theory of tropisms, and if, moreover, the eyes, as Hesse (97) maintains, are the principal organs which, when unequally illumi- nated, cause the directive response, it may be shown that possi- bly the arrangement of the crescentic pigment shields around the sensory cells of the eyes is such that equal stimulation of both eyes is just as certainly received by the worm when it is in a position diagonal to the light as when it is pointed directly away from the light. 80 Herbert Eugene Walter By reference to Fig. 7, in which the relative size of the eyes 1s somewhat exaggerated and made diagrammatic for sake of clear- ness, it will be seen that no more light reaches the sensory cells of either eye from position 4, the diagonal position, than from posi- tion B, and that it is only when the light comes from some source more lateral than 4 that the left eye receives more illumination than the right. This view may furnish a possible explanation of the diagonal paths representing imperfect orientation among planarians, but it can in nowise apply to the case of earthworms since in them direc- tion eyes are absent. Fig. 7 A, diagonal direction of light; B, posterior direction of light; C, location of sensory cells; D, pigment shield. Degree of Wandering. ‘The degree of wandering decreases with an increase of intensity. It may be found approximately through the degree of error in orientation in a unit space under different intensities of light, for perfect orientation signifies the minimum of random wandering and, conversely, the greater the error of orientation the greater the probable wandering. The error of orientation expressed in percentages was computed as follows. With a negative worm emergence from the circle at The Reactions of Planarians to Light SI a point directly opposite the light was reckoned as 0 per cent of error, whereas emergence at a point directly toward the light was reckoned as 100 per cent, or a maximum of error in orientation. The orientation value at these two extremes having been estab- lished, the percentage of error which occurs when the worm emerges at any intermediate position on the circumference of the circle may be easily determined. TABLE XVI Average degree of error in orientation made by various species of planarians during 300 trials in directive light of different intensities | Percentage of error in orientation iene 4 When started Mea etarte! = away from the Average ward the light light 3-3 candle meters......- poopdso ss OnoeandeDane 34 11.5 22.7 FLOIGALI GAG TTNCLENS = ans Vaperalsy/atece(ye/<tatsisraial svete ave4e <ns/e 32 | 12 22.0 BAL Cand lewn etersemrstar sate rsiateisieie/s aleleteteiateiefeinve -| 31 | 10 20.5 | ; MAVCLA RE otatateletaieveyeteteCayetecerctetenesctcus crs| sobs she, ze1 32 II From this table it appears that there is three times as much wandering, or error of orientation, by worms headed toward the light, as by those headed away from it. This doubtless indicates that orientation is a more complicated process in the former case than in the latter. Duration of Activity. Superficial observation is sufficient to establish the fact that different species of planarians when set into activity in directive light show decided differences with regard to the length of time they normally continue in motion before com- ing to rest. Among the forms experimented upon, Bdelloura came to a stand-still in light soonest and Phagocata latest.! Fatigue in itself is by no means the inevitable result of continued activity on the part of anorganism. For instance, Hodge and Aikens (95) observed a Vorticella continuously for 36 hours, during which time its regular ciliary and contractile movements continued unin- terruptedly, while Radl (’or) found that the eye of Daphnia when 82 Herhert Eugene Walter light was flashed upon it vibrated as vigorously after the experiment had been repeated 410 times in close succession as it did at first. An attempt was made with Planaria maculata to see how long activity would continue in a succession of trials in directive light. The worm was started on the middle of an aquarium floor and allowed to glide in any direction. As soon as it stopped and assumed the relaxed contour of the resting worm, the time required for the journey being noted, it was immediately returned to the starting point. Subjected to this treatment, the worm made 39 trips, which in general occupied an ever decreasing length of time, ranging from 18 minutes to 1? minutes, or an average of 5 minutes and 53 seconds each. When returned to the starting point the fortieth time the worm refused to start. Although in this experi- ment, which lasted 43 hours, the worm became gradually less responsive to the mechanical stimulus of the brush by means of which it was transferred to the starting point, its fatigue did not materially affect the negative character of its response to light. Time Required to Leave a Unit Circle. In obtaining the data on this point, the apparatus and method already described (p. 73) were employed. It was found that when worms of different species were subjected to three different intensities in immediate succession the degree of intensity did not prove to be as important a factor as fatigue in determining the average number of seconds necessary for the worm’s exit from a circle 10 cm. in diameter. During the series of experiments upon this point care was exer- cised so to vary the succession of intensities that the effect obtained could not be attributed to any cumulative increase or decrease of intensity. “Thus, on one day the order of intensities was I, 2, 3, on the next 2, 3, 1, and on the third, 3, 1,2. In Table XVII the data obtained are arranged on the left with reference to the actual intensities employed and on the right with reference to the suc- cession of trials made upon the various species which are desig- nated in the middle column. The averages in the table are each made up of four records. It will be noted that Phagocata gracilis and Planaria gono- cephala are, according to these figures, less subject to fatigue than Dendroccelum lacteum or Planaria maculata. The Reactions of Planarians to Light 83 Manner of Coming to Rest. Loeb (’93b) and others have shown that planarians under the influence of directive light generally come to rest in regions of lessened intensity. A few experiments were made bearing on this point. By means of screens and back- grounds, both black and white, a rectangular glass aquarium was arranged so that the area of least intensity was plainly localized and could be varied in different ways. In Fig. 8 are shown (1) the places where worms (P. gonocephala) which had been started together in the middle of the dish finally came to rest; (2) the num- ber of worms in each locality; and (3) the different combinations of backgrounds and screens used in each of the experiments. TABLE XVII Relative effect of fatigue (at right of table) and change in intensity of light (at left of table) as shown b i g g §' y &£ y the average number of seconds required for individuals of various species of planarians to leave a circle Io cm. in diameter INTENSITY | Groups OF TRIALS ————————— SPECIES 3-3¢.m. | 27.0C.m. | 53.0c.m. First Second Third seconds | seconds seconds seconds seconds seconds 63 | 65 64 | Dendroceelum lacteum 54-5 63 ee 40 43 41 |. Planaria maculata | 37 42 45-5 40 | 38 46 Phagocata gracilis | 38 44 42 52.5 | 47 49 Planaria gonocephala 46 52-5 50 = =| E = : 65 | 64 67 Average | 58.5 67 70 Wherever shaded borders are indicated the aquarium was surrounded on five sides by black screens and likewise on the sixth side except for a narrow space admitting the light, the direction of which is indicated by arrows; in a similar fashion, where unshaded borders appear, light-reflecting screens enclosed five sides. It will be seen at a glance that the great majority of the worms placed in directive light come to rest as far from the light as pos- sible. That this is due to the directive power of light is at once apparent by comparing 4, B and C with D, where the light was non-directive. The darkened area was selected whenever the directive force of the light did not prevent, as in 4, C and D, 84 Herbert Eugene Walter The five worms coming to rest on the lighter side of D were carefully examined and found to be mutilated or fragmented individuals, while the same was not true of the others. The reason why the worms in B failed to arrive in the darkened area is probably that, being started near the middle boundary line, their first movements were normal, 7. ¢., away from the light, and carried them into the area of greatest intensity, whence they were unable to escape. In this case the effect of the directive light seems to have more than counterbalanced the locomotive | | Light vertical A B C D Fig. 8 Planaria gonocephala. The arrows represent the direction of the light. The dotted areas were surrounded by black backgrounds, except for a space on the side toward the light, and the clear areas similarly by white backgrounds. The figures represent the number of planarians that came to rest in any particular locality. energy exerted by the worms. Had the species experimented upon been Phagocata gracilis, instead of Planaria gonocephala, the result might have been different, for in the former species, as already shown (p. 78), the phototactic response is secondary to the tendency to a general wandering. It was frequently observed that worms when fatigued after a period of activity apparently lost their phototaxis, with the result that the final movements of a tired worm would sometimes be made toward the light. Such behavior is probably not to be con- sidered as a reversal of phototaxis, but rather as indifference to The Reactions of Planarians to Light 85 photic stimuli, due to the worm’s lowered physiological state and a chance turn toward the light. In fact the final position taken by 49 fatigued worms with reference to the source of light, showed that only five of them, or 10+ per cent, pointed away from the light while 15 (30+ per cent) were headed toward the light and 29 (59+ per cent) stopped indifferently at right angles to tae tars quite probable that among the external fictors that influence a worm to come to a halt, light plays an exceedingly insignificant role, as compared with the stimulus of contact or some stimulus, prob- ably chemical, given out by other worms in close proximity. One curious instance was observed, however, in which light was apparently of more importance than contact or other stimuli in determining the place of coming to rest. A large crystallizing dish half full of water was left over night with a few planarians in Fig. 9 it. Floating on the surface of the water in this dish was a small Petri dish, in which a few more planarians were isolated. In the morning the worms in both vessels were found grouped at the same region on the outside and inside of the smaller dish, as shown in Fig. 9. This curious distribution on both surfaces of the Petri dish could not be due to chemical stimulus exerted by one group of worms on the other, and there seems to be no particular reason why a thigmotactic reaction should have caused them to assemble in such a way. The locality chanced to be one, however, where the intensity of the light was considerably reduced; this seems to offer a reasonable explanation of the observation. Bdelloura in coming to rest shows an entirely different behavior. When left over night free to wander in an aquarium half of which 86 Herbert Eugene Walter had been previously covered with black cloth to exclude most of the light, this species was found in the morning in the light area, a behavior exactly the reverse of that shown by fresh-water pla- narians. Another peculiarity of this species is that individuals in coming to rest arrange themselves in compact rosettes with the anterior end of the body pointed toward the circumference of the rosette, while the sucker-like posterior end remains attached near the center of the group. ‘They are so delicately responsive to me- chanical stimuli that any slight disturbance of one member of such a rosette is sufficient to throw the whole group into activity. The advantage to the individual worm of such a habit of arrangement in coming to rest, is evident. Finally, Bdelloura was repeatedly seen on taking the resting position to point directly toward the light with the anterior end a the body raised and the posterior end Hatencd out into a sucker- like expansion. Summary. Fresh-water planarians (Dendroccelum, Planaria and Phagocata) are negatively phototactic while Bdelloura is posi- tively phototactic. Negative planarians deviate most from the direction in which they are started if pointed toward the light and least if pointed away from the light, an intermediate deviation occurring when they are pointed at right angles to the light. The rate of locomotion is greater when worms are headed toward the light than when they are headed away from it. During successive trials the rate of locomotion decreases. Negative planarians frequently take an apparently positive course because the impulse to move in any direction is greater than the phototactic impulse. The normal negative phototaxis of a worm may change tem- porarily to positive by reason of some physiological state ania is not obviously referable to external stimuli. The greater the intensity of the light the less worms wander in their course. When they are Peaded away from the source of light, there is less error in the precision of their orientation than when they are started toward it. Planarians frequently travel away from the source of light diagon- ally instead of directly. The Reactions of Planarians to Light 87 Bdelloura continues activity in the light for a much shorter time than Phagocata. When subjected to successive trials the period of a planarian’s activity decreases. Change in the intensity of light is less important than the effects of fatigue in determining the time required for a worm to leave a unit circle. When fatigued, worms often become indifferent to light, coming to rest less frequently in an oriented position with reference to the light than in an unoriented one. Fresh-water planarians come to rest as far away from the source of light as possible and, if the directive stimulus does not prevent, in the region of least illumination. Bdelloura candida, on the contrary, comes to rest in regions of greater rather than of less illumination; usually worms of this species arrange themselves in compact rosettes with the anterior ends pointed outward. B In Changing Directive Light The light acting upon planarians in their natural habitat must necessarily be a variable factor of great complexity, since its inten- sity changes constantly throughout the day, while the position of the sun relative to various surfaces which reflect light is also continually shifting. The fact that planarians, to a great extent, keep out of the light, does not diminish the force of this statement, for whatever the part played by light in their behavior, it must always be an exceed- ingly varied and complex one. Changes in the Intensity. When a worm is gliding away from a source of light it shows a more marked response to change of ' intensity when the change is made suddenly than when it is made gradually. In fact, it is possible by exercising patience and care to change the intensity of directive light to a considerable degree so gradually as to produce no corresponding response on the part of the worm, whereas a comparatively slight change, if abruptly effected, immediately results in the animal’s performing some one or more of the acts in its repertory of behavior, such as halting, wigwageging, etc. 88 Herbert Eugene Walter In all the experiments made upon the effects of change of inten- sity in directive light, more responses were found to occur when the intensity was decreased than when it was increased. This is in agreement with the experiments already described relating to the critical region between two intensities, in which it was found that worms show a greater number of responses when going from a higher into a lower intensity than vice versa. Bdelloura is particularly sensitive to changes in intensity. It is necessary to throw a shadow on a moving worm only momen- tarily tg cause it to perform vigorous wigwag movements or to change the direction of its course. Whitman (99), writing of Clepsine, suggests that the extreme agitation of this animal when a shadow is thrown upon it may be the result of natural selection, since any sudden shadow cast upon it in its natural environment may be caused by a turtle swimming overhead, to which the leech, if it is quick enough, may become attached. It may be that Bdelloura, which is also an ecto-parasite, has developed this extreme responsiveness to sudden decrease of intensity in a similar way. Changes i in Direction. The precision with which all the pla- narians in a dish may be made to pass back and forth by shifting a directive light from one side to another is astriking g phenomenon, which is sure to impress anyone who sees it. By careful manip- ulation of the light, it is possible even to make an individual planarian follow a predetermined path in the most undeviating manner. For example, when two lights, placed near the ends of an aquarium, are alternately turned on and off, the worm will zigzag across the field, at right angles to the direction of the lights, while under a moving light it may be made to turn around and around, almost as if its posterior end were a pivot, to trace figure 8’s and curves of various patterns, or to turn abruptly at right angles an imaginary corner. Unlike the changes in intensity previously described the degree of abruptness in any change in the direction of the light made no apparent difference in the quale, of the reaction, since any change in direction, however gradual,-met with an immediate response on the part of the worm. Indeed it was necessary to abandon an The Reactions of Planarians to Light 89 attempt to illuminate one side of the worm alone because the ani- mal invariably turned faster than it was possible to regulate the light. The quickness with which this delicate response to any change in the direction of the light occurred was found to increase upon suc- cessive trials. A square aquarium was arranged so that it could be illuminated instantly at either end, in a room otherwise dark. With one light on, a planarian was allowed to move until it had assumed the characteristic negative direction, whereupon the source of illumination was instantly changed 180° by turning this light off and the one at the other end of the aquarium on. ‘The time required for the worm to become headed about was noted and then a reversal of lights repeated and the interval necessary for re- adjustment again recorded. Ina typical experiment of this kind the number of seconds required by the worm, Planaria maculata, to accomplish re-orientation were for 16 successive orientations as follows: 260, 70, 100, 60, 65, 110, 60, 85, 70, 105, 80, 60, 50, 40, 45, 35. The sum of the first eight is 810 sec., that of the last eight, only 485 sec. Summary. Planarians show a greater response to sudden change of intensity than to gradual change. ‘This response 1s more pronounced when the intensity is lowered than when it is rgised. Bdelloura is particularly affected by sudden changes of in- tensity. Planarians respond with great precision to changes in the direc- tion of the light, and as promptly when the change is gradual as when it is abrupt. The period required for re-orientation to changes in the direc- tion of light, diminishes upon repetition. C [In Combination with Other Responses It is impossible to subject planarians to the influence of light alone. ‘The best that can be done is to render extraneous factors as uniform as possible. For example, so long as a moving worm is kept upon a horizontal surface there can be no directive geotactic stimulation, because the worm is moving in a plane at right angles go Herbert Eugene Walter to the force of gravity. The moment the worm begins to glide up the sides of an aquarium, however, the relation of the axes of its body to the center of the earth changes and directive geotaxis results. No systematic attempt was made to analyze compound stimuli, for such a study would overstep the boundaries set for the present inquiry. Nevertheless certain facts bearing on this point were incidentally noted and these may properly be detailed here. Geotaxis. Ina majority of cases, Planaria gonocephala seems, after several hours of exposure to the dark, to be positively geo- tactic, and after several hours of exposure to light, negatively geo- tactic, as shown in the following series of observations. A cylindrical aquarium jar 20 cm. in diameter and 40 cm. high was placed before a moderately lighted window and stocked with a freshly obtained supply of about 300 worms. No stones, sand, or water-weeds, which would afford places of concealment, were introduced. At intervals during the next 10 days the distribution of the worms was recorded and these records are brought together in Table XVIII. TABLE XVIII The distribution of about 300 planarians (Planaria gonocephala) in an aquarium, as observed forenoons and afternoons during 10 days. The figures express percentages PLACE IN THE AQUARIUM | Top SIDEs | Bottom Time of day a.m. | p-m. a.m. p-m. | a.m. p.m. PSRs aan en ae oncmanenoeoeede arin 5I i | | 38 PA pri o.7erotasecshsfalatesasabelel Seriorarcielee sete aeet 61 13 26 ANpral 28/25. he, grace enatanacet ere a re4ay suesetonssaveostanataiera 74 3 II Is 15 82 IMayitiese nts 72 29 6 7 22 64 IW IEW oa sanntis HPtIg OD RAB OS Criehao OO GOde. oe 63 39 12 | 20 25 41 IMA ypegictstere cia any aitianaxagass ts ahsuive sigiersiaseia\elaters 67 43 16 | 41 May 4.. 50 16 | 34 DMA yi Sara stoseyeteve/=tsferere' foie st eeratsista etotosehs 6 eles e 31 13 56 ——— | ISVET APES yaye ye Soe seiko michele Sie si siete 60.5 30.6 12-3). |j| UI¥-20 || e27 ee 56.2 ‘The forenoon census was taken about 8 o’clock, when the worms were re-arranging themselves after the darknessof the night, while the afternoon records were made about 4 o'clock, when the worms The Reactions of Planarians to Light gi had been all day in the light. The average at the bottom of the table indicates, first, that an approximately equal percentage of worms was found on the sides of the aquarium at both times of day, which may therefore be left out of the reckoning, and, secondly, the occurrence of a significant migration during the interval be- tween 8 a. m. and 4 p. m., demonstrated bythe distribution of the worms at the top and bottom of the jar respectively. Accord- ing to the data obtained, at least 30 per cent of the worms in the top group must have become positively geotactic and gone to the bottom during the day. A later set of experiments in which an aquarium was kept swathed in black cloth during the day showed less migration. The conclusion naturally follows that geotaxis is more likely to occur in the presence of light than in its absence. Whether there is a regular diurnal vertical migration among planarians in nature, as Birge (’97) and Schouteden (’02) found to be true for fresh- water entomostraca, and various authors‘ for different forms in marine plankton, remains unknown. It is probable, however, that planarians ordinarily remain quiescent on the under sides of stones or in other shaded places for considerable intervals of time, coming under the influence of light only when started into activity through some other stimulus. A worm placed in an aquarium with square sides and left free to travel undisturbed on the bottom or the sides occupies the sides more frequently than the bottom. In a trial to test this point, an aquarium was used, the bottom area of which measured approximately five times that of the sides. The course pursued in this aquarium by one worm (P. gonoceph- ala) in directive light and covering 1340 cm., was plotted and the percentage of distance traveled on the sides was found to be prac- tically equal to that traveled on the bottom, notwithstanding the fact that the animal was started in the middle of the bottom, where it had five times as much available territory to travel over as on the sides. Other things being equal, therefore, this worm showed itself five times as ready to travel on the sides of the aquarium as on the bottom. *Groom and Loeb (’g0), Loeb (’93a), and Parker (‘o2). g2 Herbert Eugene Walter The existence of such a decided geotactic tendency should not be forgotten when trying to determine the part light plays in planarian behavior. Again, it was found that there was less accuracy of orientation to directive light while the planarians were on the sides of the aquarium in a position parallel to the light rays than while they were on the bottom. Their behavior in the former case was the resultant of at least two known stimuli, gravity and light, whereas gravity was prac- tically eliminated when they elided on the floor of the aquarium. In the experiment cited under the preceding paragraph 92 per cent of the distance traversed by the worm on the bottom of the aquarium was in a direction in general away from the light, as contrasted with only 79 per cent when it was traveling on the sides of the aquarium. This difference of 13 per cent may represent roughly the necessary correction for geotaxis, in order to ascertain the influence of light alone. Thigmotaxis. Contact with the substratum is an almost con- stant condition of planarian activity. Occasionally worms may be seen dangling free at the end of a mucus-thread, as commonly occurs among many fresh-water snails; sometimes they may fall helplessly from the surface-film to the bottom, but definite con- tact with something firm is the rule during their ordinary loco- motion. A change in the degree of this contact, and consequently a pro- duction of thigmotactic stimulation, may come about in two ways the surface on which the animal glides may present irregularities, such as increased roughness or a different degree of solidity, or the worm itself may vary in the extent of Cae -surface which it brings into contact with the substratum. ‘This latter method of causing thigmotactic stimulation applies especially to Bdelloura, which has the habit of frequently alternating a leech-like looping movement with ordinary gliding, thus ae its contact rela- tions and probably producing a Antec ppling. in conse- quence. As already mentioned, Bdelloura, when subjected to sudden dark, usually detaches itself from its support and wriggles vio- The Reactions of Planarians to Light 93 lently in the water. It is uncertain how far this behavior is attrib- utable to light alone or to some combination of light and thigmo- taxis. This phenomenon of compound stimulation occurs in a less pro- nounced way whenever a change of light intensity results in the “wigwagging” response common to planarians. ‘The same uncer- tainty prevails as to how far the subsequent behavior of the worm may be due to the direct stimulation of light and how far to thig- motactic stimulation primarily and to hal stimulation second- arily. It is evident, then, that under any circumstances there is such a close interrelation of stimuli that an accurate analysis of the consequent behavior 1s difficult. Further evidence of the close relation between different kinds of stimuli is afforded by the fact that planarians are more respon- sive to the mechanical stimulus of a slight jar when the entire ven- tral surface of the body is in contact with the substratum than when the anterior end is lifted up and waving about. Apparently the greater the degree of contact the greater is the effect of a jar- ring mechanical stimulus. This point was demonstrated by means of a small aquarium mounted on a turntable, such as is used in “ringing”? micro- scopic slides, in such a way that it could be rotated with great ease and delicacy. A light from one direction only was projected upon the single pianeian placed in the aquarium. Any attempt to change the. angle of light by rotating the aquarium ever so slightly pesnltecl instantaneously i in a momentary halt on the part of the worm, provided it happened to be gliding with its ventral surface entirely in contact with the floor of the dish. If, however, the rotation was made when the anterior end of the worm was lifted, the halt did not so readily occur. This response was of such deli- cacy that with a little practice it was possible to halt the anterior end of a worm without disturbing the continuous progress of the posterior end! That this halting was due to thigmotaxis rather than to any rheotaxis induced by the movement of the animal against the relatively stationary water particles, is shown by the fact that the reaction was more pronounced when the anterior end of the body was held flat than when it was raised and so brought more under the possible influence of a water current. 94 Herbert Eugene Walter Finally, it may be recalled that in a preceding section data were given (Table X, p. 68) to show that there is more response to light while worms are upside down on the surface-film than when they are in contact with the bottom of the aquarium, a difference probably referable in large measure to the different thigmotactic relations in the two cases. Gomiotaxis. Goniotaxis is a term introduced by Pearl (’03, p. 561) to define a particular kind of thigmotactic response in which the “different parts of the body are brought into such positions that they form unusual angles with each other,” as when a plana- rian occupies the angle formed between a side and the bottom of an aquarium. There is no doubt that the peculiar movements resulting from the goniotactic stimulus directly modify the phototaxis of the worm. Once in the angle of an aquarium a planarian becomes increasingly indifferent to light. In one series of records, show- ing how a considerable number of planarians came to rest, it was found that the majority came to rest in an “‘angle” and that out of this number 78 per cent failed to orient to the light. The stim- ulus of the “angle” was greater apparently than the stimulus of light. Furthermore, it is to be noticed that goniotaxis is always more effective if the worm is in a lowered rather than ina heightened physiological state, for whenever a planarian is freshly intro- duced into an aquarium and is in an aroused condition on account of the mechanical stimulation necessarily given it in transference, it will pass over angles and crevices with total indifference, all the while responding plainly to light. As soon as it has become fatigued, however, if its path chances to cross an angle or crevice it exhibits goniotaxis at once by slowing down and remaining in the new situation, as if caught in a trap, with complete disregard of the continued action of directive light. Chemotaxis. Pearl has made an extensive study of this phase of planarian behavior and suggests that the well-known planarian habit of collecting in groups may be explained on the supposition that a resting planarian is surrounded by a halo of chemical ema- The Reactions of Planartans to Light \O al nations which serve as a direct stimulus to other planarians, attract- ing them and causing them to come to rest in groups. In this connection it is worth mentioning that several times when Dendroccelum lacteum was put im an aquarium with other species of planarians, the individuals of this species would later be found gathered into aseparate group bythemselves. “This manner of isolation was also repeatedly noticed in examining on the under sides of stones taken from the pond at Falmouth, Mass. A similar segregation of species in the case of P. alpina and P. gonocephala, was noted by Collin (’91). He says (’g1, p. 180) “‘liyjima fand diese beiden Arten zusammenlebend, wahrend sie im Harz stets getrennt vorkamen; auch in der Gefangenschaft schien die P. alpina die grossere P. gonocephala in demselben Behalter zu meiden und thr angstlich auszuweichen.”” It would be difficult to explain how these planarians avoid each other so as to fraternize in this fashion, except on the basis of some delicate chemotactic response which caused them to halt when they entered the chem- ical halo of their own kind, but not to do so in the different chem- ical halos of other species. As in the case of goniotaxis, the mani- festations of phototaxis may be entirely superseded by the effect of feeding (Chemotaxis). When once a hungry planarian, driven by directive light into the neighborhood of a crushed snail, becomes subjected to the chemical stimulus arising from the fluids of that object as they are disseminated through the water, it seems to become suddenly indifferent to the light, owing to the greater influence of the chemical stimuli. The same inhibition of the influence of light by a chemotropic response to food has been observed by Parker ('03) on the mourn- ing-cloak butterfly, Vanessa antiopa L. He says (’03, p. 457) “when a butterfly alights on a bough, it orients in the sunlight with the usual precision. Should the sap be running from a near stem, the insect is very soon attracted to the spot, begins feeding, and moves about from that time on with no reference to the direc- tion of the sun’s rays. “Thus, when feeding or near food the but- terflies do not respond phototropically.”’ Furthermore Darwin (81, p. 23) observed that earthworms are less disturbed by light while feeding or during copulation than at other times. go Herbert Eugene Walter The foregoing examples illustrate only a few of the many modi- fications of light responses due to the interference of some other stimulus. Summary. In judging the effect of any stimulus upon an ani- mal it is necessary to have constantly in mind the accelerating or inhibiting effects of other stimuli which may be influencing the organism at the same time. In the case of planarians some ai the responses known to be intimately connected with phototaxis are geotaxis, thigmotaxis, goniotaxis and chemotaxis. Planaria gonocephala shows itself to a certain extent negatively geotactic after several hours of dark and positively geotactic after a similar interval of light. When given horizontal and vertical surfaces of equal extent, worms travel more on the vertical surfaces. Their accuracy in orienting themselves to light while subjected to geotactic stimulus on a vertical surface is less than when they are traveling on a horizontal surface, where the directive geotactic stimulus is eliminated. Thigmotactic stimulus may result either from an environmental change in the substratum, or a change in contact caused by the worm itself whereby its relation with the substratum is varied. There is a close interdependence of the various stimuli which may be acting on an animal at the same time. Behavior may be the direct consequence of light or the indirect result of light combined with the direct effect of a thigmotactic stimulus indirectly brought about by some change in the intensity of the light. The greater the degree of contact with the substratum the more responsive a planarian becomes to the mechanical stimulus of jar- ring, but the less to the stimulus of light, as shown by comparing the behavior of worms on the surface film with their behavior on the aquarium floor. Goniotaxis has an inhibitive effect on phototaxis; this effect becomes more apparent as the worm reaches a condition of fatigue, phototaxis meanwhile becoming less apparent. Dendroccelum lacteum exhibits a remarkably delicate response (Chemotaxis ?) in frequently coming to rest in the neighborhood of its own kind. The Reactions of Planarians to Light 97 Hungry planarians in the presence of food have their photo- taxis entirely obscured. 3 KINDS OF BEHAVIOR In the two preceding sections, treating of Photokinesis and Pho- totaxis, respectively, animal behavior, as illustrated by the effect of light upon planarians, has been taken up from the point of view of the stimulus. In the two following sections, on the other hand, the reactions of planarians will be dealt with from the stand- point of the animal rather than from that of the stimulus. To this end a classification of the behavior of planarians in light is here presented based upon (A) generic and specific differences, and (B) individual differences. That there are morphological differences which fall naturally within the lines of this classification has long been recognized, indeed, the criteria used in classification by systematists are based almost exclusively upon such differences, while relatively little importance has been attached to differences in the behavior of animals. As already mentioned in the historical review, Loeb (94), in dealing with the differences of behavior which characterize the two genera, Planaria and Thysanozoon, pointed out that decided physiological variation may appear in forms closely related mor- phologically. The same fact had been previously emphasized for the case of the pulmonates by Willem (’g1). Obviously such physiological variations do not furnish reliable criteria for the systematist, since they are so largely dependent upon environ- mental causes, and furthermore the work of the systematist is usually done upon dead animals. Nevertheless some interesting relations between behavior and systematic position await the stu- dent who approaches the study of animal behavior from this direc- tion. Strictly speaking, all behavior is individual behavior. In this sense it is manifestly incorrect to speak of the behavior of a genus or of a species per se. The behavior of individuals may, nevertheless, be classified into responses which are characteristic of all the members of a genus, 98 Herbert Eugene Walter or again into responses which are characteristic of only one species of a genus and not necessarily of other species of the same genus, and, finally, into those peculiar to the individual as such, which may not in all particulars be shared by other representatives of the species to which the individual in question belongs. It is in this sense of the terms generic, specific and individual, that behav- ior will be taken up in the present section. A Generic and Specific Behavior In the present inquiry a basis for generic comparisons is afforded by a study of the behavior of edad: of four different genera, namely, Planaria, Dendroccelum, Phagocata and Bdelloura, while some idea of specific differences is ee possible by compar- ing the behavior of individuals of the two species Planaria macu- Tet and Planaria gonocephala. In the cases of Dendroccelum, Phagocata and Bdelloura it is obvious that the conclusions drawn are based in each instance upon the behavior of representatives of a single species under each genus. ‘The question may be properly raised as to how far such conclusions indicate generic behavior and how far specific behavior. Conceding that from the data obtained exact deductions may not be drawn, the fact still remains that the three species, Dendroccelum lacteum, Phagocata gracilis, and Bdelloura candida, are separated from each other by generic gaps, such that the differences exhibited by these species may be regarded as generic in degree. The point unestablished, then, is whether other species of the genera in question if examined might not show that the behavior, which in these single representative species seems generic in nature, is not characteristic of other species of the same genus as well. It will be convenient to present the data of both generic and specific behavior at the same time. Percentage of Negativeness. The manner of obtaining this _ criterion of behavior has been explained in the section on Phototaxis (p. 72). It will be remembered, too, that in Table XII a comparison was made between positive and negative worms, showing the degree of their orientation to directive light. The The Reactions of Planarians to Light 99 data there used are rearranged for the present purpose in Table XIX. TABLE XIX Percentage of generic and specific negativeness in worms started at right angles to incident light, as deter- mined at the circumference of a circle 10 cm. in diam. by the average amount of their deviation from the directions in which they were started SPECIFIC GENERIC DIFFERENCES DIFFERENCES F Planaria Mende eee Boe hpi aried |iEdellowealy seca gono- coelum cata maculata | cephala Number of observations........... 78 80 158 10 78 | 80 Total number of degrees positive... 155 238 165 397 5 | 160 Total number of degrees negative...) 2112 1964 4070 50 | 2102 | 1968 Percentage of negativeness........ 93-1 89.6 96.1 II 99-9 84.6 Comparing the figures given in this table, a greater range of difference is seen to obtain between the two species of Planaria (P. maculata and P. gonocephala) than between the genus Planaria and either of the other negative genera, namely, Dendroccelum and Phagocata. Although not indicated in this table, similar results appear when the number of times the worms went in a negative direction is used as a basis of comparison, instead of the total num- ber of degrees of negative deviation. Character of the Course in Directive Light. When worms were placed on the middle of a rectangular aquarium floor and sub- jected to a directive light their movements showed both generic and specific differences. By experimenting with one worm at a time it was possible to plot on a sheet of paper with sufficient accuracy for general comparison the entire course of the worm during a considerable period. This was done many times and typical records of such observations are given in Figs. 10-14. — In such instances the worm was exposed to a light of approximately 147 c.m., placed so as to correspond to the right side of the figures. The central rectangular area bounded by the broken lines indicates the limits of the floor of the aquarium, while the smaller exterior adjacent areas represent its vertical sides so rotated as to 100 Herbert Eugene Walter bring them into the plane of the floor. The course taken by a planarian is indicated by the tortuous line. The full line shows the course taken on the solid surface of the aquarium; the dot- Fig. 10 Dendroccelum lacteum. The shortness of the path shows comparatively little persistence in locomotion, and the direction, considerable indifference to the source of light. and-dash lines, the course of the worm on the surface film. The dotted line indicates a hiatus in the path, made necessary by the attempt to represent on a flat surface a continuous line which tra- verses vertical as well as horizontal surfaces. A succession of The Reactions of Planarians to Light IOI abrupt kinks in the line signifies that at that point the worm exe- cuted decided wigwag movements with its anterior end. The figures are reduced in size from the original records. In Fig. 10 is given a specimen record of a Dendroccelum’ which 4 Fig. 11 Phagocata gracilis. This path shows great activity on the part of the worm, and, although it is mostly laid down away from the source of the light, it shows that the worm experienced no great difficulty in moving toward the light. came to a standstill after 18 minutes of locomotion. ‘The first movement of this worm was diagonally away from the light, but It soon came back toward the light traversing almost the entire 102 Herbert Eugene Walter width of the aquarium and in doing so showed considerable indif- ference to the directive influence of the light. Its susceptibility to goniotactic stimulus is plainly shown by its behavior upon reach- Fig. 12 Bdelloura candida. This path was traversed with much ‘‘wigwagging;” there was indif- ference to the source of light and locomotion was not of long duration. ing the angle formed at the junction of the sides and floor of the aquarium, as well as by its manner of finally coming to rest. A typical Phagocata (Fig. 11), on the other hand, exhibited The Reactions of Planartans to Light . 103 almost no goniotaxis, although the worm repeatedly crossed the line of the angle. The response to the directive influence of the light, too, was in this case even less than that of the Fig. 13 Planaria maculata. Considerable activity was shownover this course and a decided inability to approach the source of the light beyond about the middle of the aquarium. Dendroccelum just described, as is evident from the general wan- dering character of the course. Although the Phagocata in ques- tion frequented both sides of the aquarium—that which was toward 104 Herbert Eugene Walter the light, as well as the opposite side—its wanderings were in the main on the side away from the light. An hour’s activity is chron- icled in the record, at the close of which the worm was apparently as energetic as ever. Fig. 14 Planaria gonocephala. The path is of the same generic type as with Planaria maculata (Fig. 13), and is easily distinguishable from those of Dendrocelum, Phagocata and Bdelloura. Fig. 12 gives a characteristic record of the way in which Bdel- loura behaves. The first movement of this specimen was more The Reactions of Planarians to Light 105 toward than away from the source of the light, but very soon wig- wagging motions set in, and after every exercise of these move- ments, which were apparently in the nature of explorations, a change in the direction of the course was effected. As might be expected, such abrupt changes in direction were more difficult of execution when the worm was on the surface-film. Characteristic movements of individuals of the genus Planaria are shown in Figs. 13 and 14. From these two typical records it would be difficult to select any diagnostic points which would dis- tinguish the behavior of P. gonocephala from that of P. maculata. There is no doubt, however, that taken together the behavior of representatives of these two species presents a distinct (generic) dif- ference from that of the representatives of the other genera studied. The most striking feature of the Planaria records ( (Figs. 13 and 14) is the high degree of response exhibited by members of this genus to the decane action of light. Although many attempts were made by the individual worms to penetrate the half of the aqua- rium nearer the light, yet they seemed as unable to keep to that direction as they would have been had a solid barrier been inter- posed between them and the light. This characteristic respon- siveness to directive light helps to explain why (as shown in Fig. 8, B, p. 84). P. gonocephala was unable to come! to rest In the area of lessened illumination as it would naturally have been expected to do. From the cases cited in this section, at least, it may be afirmed that the generic differences are so pronounced that one could take a miscellaneous, unidentified assortment of such records and correctly assign the great majority of them to the proper genera. Duration of Movement. When worms of different genera are subjected to the same light intensity there is considerable variation in the time required to bring them to a standstill. Bdelloura is usually the first to stop, followed in order by Dendroccelum, Pla- naria and Phagocata. Of the two species of Planaria, P. gono- cephala, although averaging somewhat smaller in size, usually keeps in motion for a longer time than P. maculata. The individ- ual records of the duration of movement given in Figs. 10-14 may be taken as a typical set of records. “They were as follows: 106 Herbert Eugene Walter Fig. 10. Dendroccelum, 18 min. Fig. 11. Phagocata, 60 min. (still moving). Fig. 12. Bdellourz, 15 min. Fig. 13. Planaria maculata, 47 min. Fig. 14. Planaria gonocephala, 60 min. (still moving). Woodworth (’97) in contrasting the activity of Planaria macu- lata, P. gonocephala and P. dorotocephala states that individuals of the latter species remain in motion longer than individuals of the other two—an observation confirmed by Pearl (’03). Degree of Wandering. fa worms started at the center of a circle parallel to the direction of the light and pointing away from its source, then the more devious its course the more it may be said to wander. Both generic and specific differences were obtained bear- ing upon this phase of behavior. Selected instances of such dif- ferences are given in Table XX, expressed in average degrees of deviation upon emergence from a circle 10 cm. in diameter. TABLE XxX The average generic and specific differences between individuals of four genera and two species of Janarians expressed in degrees of deviation upon leaving a circle 10 cm. in diam. In ever y instance the worm was started away from the source of the light SPECIFIC GENERIC DIFFERENCES DIFFERENCES Ded Pl PI _ | Planaria os 248°" | Planaria Bdelloura ae gono- celum cata maculata cephala Average degree of deviation....... 9-4 30.0 24.6 132.0 21.6 27-7 Number of observations........... 56 46 gz 10 46 46 The remarkably large deviation shown by Bdelloura is due to the fact that it is a positive worm. When pointed toward the light its deviation was only 39.3°, a number which would perhaps be more justly comparable with the other records in this table. But even so, it will be seen that Bdelloura, of all the forms observed, is the least oriented by directive light. Specific differences in the degree of wandering are in general less marked than the generic differences, according to the records in Vable XX. The Reactions of Planarians to Light 107 Rate of Locomotion. As regards rate of locomotion the records of specific differences exhibit a wide range, although not as great as that of the generic differences existing between Dendroccelum and Phagocata. TABLE XXI The average rate of locomotion expressed in millimeters per second SPECIFIC GENERIC DIFFERENCES DIFFERENCES Dendro- Planaria | Planaria Phagocata | Planaria celum maculata |gonocephala Rateinvmm. per seG...feincslsteieesis|-)2)- 1] (O50 1.395 1.272 1.470 Number of observations........-..... 40 40 80 40 40 It is interesting to note in this connection that Parker and Bur- nett (’00, p. 385) give the average rate for Planaria gonocephala as 1.08 mm. per sec., while Pearl (’03, p. 546) records for P. macu- Jataler: 48 mm. per sec. in the case of a worm Ir mm. in length and 1.23 mm. per sec. for one 6 mm. in length. An average of these two records, that is, 1.355 mm. per sec., might per- haps be comparable with the average (1.272) given in T able XXI, since an equal number of large eed small worms from each genus formed the basis on which these averages were calcu- lated. Time Required to Leave a Unit Circle. If planarians invari- ably took a straight radial path in going from the center to the circumference of a circle, the time required to leave a unit circle might be used in computing the rate of locomotion. Such a path, however, is not taken. Nevertheless, records of this kind, although untrustworthy for purposes of accurate calculation, furnish a relt- able criterion for the comparison of generic and specific behavior. The differences in behavior in the representatives of three genera and two species are given in Table XXII. Bdelloura failed so frequently to emerge from the circle that it is excluded from the list. Each genus and species was tried an equal number of times in light of three different intensities. 108 Herbert Eugene Walter As might be expected, Table XXII presents a close parallelto Table XXI. The only difference in the relative values of behav- ior, expressed by the averages of rate and time in these two tables, appears in the case of P. maculata. ‘This species, though first in the scale as regards actual rate of locomotion, is second as regards the time required to leave a unit circle, a condition indicating relatively more wandering on its part than was shown by any of the other worms. TABLE XXII The average time in seconds taken in passing from the center to the circumference of a circle 10 cm in, diameter in directive light GENERIC DIFFERENCES | SPECIFIC DIFFERENCES Dendro- | | Planaria | Planaria Phagocata Planaria celum : maculata | gonocephala Average number of seconds. . . 62 | 40 54 | 47 60 Number of observations...... 120 120 240 120 120 The Effects of Fatigue. ‘Yo obtain an idea of generic and spe- cific differences in the effects of fatigue, two sets of averages have been combined. First the average rate of ten worms of each kind, when subjected to four successive trials, was first ascertained and the difference between the first and the fourth rate was then ex- pressed as a percentage of increase or decrease in rate, as the case might be. Secondly, the time required to leave a unit circle in twelve successive trials was next recorded and the average percentage of increase or decrease in time of the last four trials, as compared with tne first four trials, was computed. By com- bining these two kinds of percentages the relative differences in the effects of fatigue upon the individuals of the various genera and species, are clearly brought out. If the results of this computation be compared with the con- clusions reached in another way under the preceding paragraphs on ‘‘duration of movement,” it will be seen that there is a com- plete agreement in the relative behavior of the different genera and species. ‘That is, the worms most subject to fatigue are the The Reactions of Planartans to Light 10g first to come to rest and those least affected by fatigue continue longest in motion TABLE XXIII The average generic and specific difference in fatigue GENERIC DIFFERENCES SPECIFIC DIFFERENCES Dendro- ’ Planaria Planaria Phagocata Planaria ceelum maculata gonocephala Percentage of change in rate of the fourth trial as compared Withrthesfirstasj/sjcrcisieies ie) — on +9.5 i) =13 i Percentage of change in time required to leave a unit circle of the last four as compared with the first four of twelve consecutive trials.......... 2) —10 —15.8 =23 —8.7 Average percentage of fatigue =22 —0.25 —12.4 —18 = 6.5 Total number of comparisons 30 20 40 20 20 Responses to Changes in Intensity. When worms in non-direc- tive light pass from a given intensity to one 2} times as great, decided differences appear in their behavior, the generic differences being plainly of wider range than the specific. TABLE XXIV Average differences in response at the critical line separating two areas of non-directive light of which one (82.50 c.m.) is approximately 2% times as great as the other (33.16 c.m.) GENERIC DIFFERENCES SPECIFIC DIFFERENCES | Dendro- : | Planaria | Planaria Phagocata _ Planaria } celum | maculata |gonocephala | | Number of observations. ..... 45 | 202 | 206 50 156 Percentage of responses....... 17 37 52.5 55 50 Percentage of failures to re- SPO Glee respec tartar steyacaidiaicye 8 63 47-5 45 50 In this table Dendroccelum is shown to respond in only 17 per cent of its passages across the critical line separating the two dif- ferent intensities of light, while Phagocata responds in 37 per cent IIo Herbert Eugene Walter and Planaria in 52.5 per cent of the cases. ‘These are differences in degree of response that are great enough to be of unquestion- able significance. When, however, Planaria maculata and Pla- naria gonocephala are compared in the same way, only a slight difference in the degree of response, namely, that between 55 per cent and 50 per cent, is to be observed. Manner of Coming to Rest. Although little attention was paid to this point during the series of observations taken up for the present study of planarian behavior, still a few indications of gen- eric difference in the manner of coming to rest appear from the foregoing data. Bdelloura, it will be remembered, has a distinc- tive manner of coming to rest in close rosettes within an area of increased illumination, while Dendroccelum shows a considerable tendency to collect in exclusive companies during periods of inac- tivity. With regard to the two species of Planaria studied, nothing at all definite was observed in this connection which could be called a true specific difference in behavior. Between Dendro- coelum and P. gonocephala, however, a decided generic difference seems to exist, as several series of records on orientation in direc- tive light show. Dendroccelum according to these records came torest in an unoriented position in 70 per cent of the cases observed, while P. gonocephala failed to take up an oriented resting position in only 59 per cent of the observations. In other words, P. gono- cephala is more liable to come to rest in a position oriented with reference to the light than Dendroccelum. Summary. The essential points brought out in the foregoing section are condensed for the sake of brevity and clearness in Table XXV. In certain instances, namely, in changes in the character of the course (2), the influence of fatigue (7), and the percentage of responses to change in light intensity (8), specific behavior shows a more intimate correlation than generic behavior, otherwise the range between the behavior of P. gonocephala and P. maculata is greater than the generic differences separating Planaria from the other genera under observation. It might be expected a prior: that generic differences would exhibit a greater range than specific differences and that similarly, The Reactions of Planarians to Light III specific behavior would include more phases of action than indi- vidual behavior. In the present series of records hardly enough representatives of different genera and different species were under consideration to establish any convincing generalization on this point. TABLE XXV Comparisons in behavior, generic and specific SPECIFIC GENERIC DIFFERENCES DIFFERENCES CRITERION OF BEHAVIOR ? Planaria Dendro- ; Planaria Phagocata| Planaria Bdelloura | gono- celum maculata | cephala (1) Percentage of negativeness 93.1 89.6 92.2 II 99-9 | 84.6 (2) Character of course in di- rective light without — with great with with Turns toward the light much indiffer- much ease No | contrast difficulty ence difficulty | Shows goniotaxis...... plainly slightly slightly none slightly slightly Wigwag movements ... few few few many few few (3) Average duration of move- MENGE jee cise acl oe cee. 18min 60+min. ? 15 min. 47min. 60+min. (4) Amount of wandering... . 9-4 30. 24.6 39-3 21.6 27.7 (Av. deviation in degrees) ! (5) Rate of locomotion ....... 0.85 1.395 1.270 ? 1.470 | 1.075 (In mm. per sec.) | (6) Seconds required to leave | axzocm. circle.......... 62a 40 54- ? 47 60 (7) Comparative influence of fatigue, percent....... 22 0.25 12.4 ; 18 6.8 (8) Percentage of response to change in light intensity.) 17 37 §2.5 2 55 50 (9) Manner of coming to rest.) in dark in dark in dark in light in dark in dark in exclu- in rosettes | sive groups | B Individual Behavior The analysis of specific behavior leads to the study of individ- uals, since it is the average activity of different individuals that makes up the behavior typical of the species. In biological litera- T12) Herbert Eugene Walter ture animal behavior, particularly among the lower forms, is ordinarily referred to in its specific or even generic aspect. “The distinctive actions of individuals, as such, it seems, have usually been outside of the purpose of the observer.° Individuals, however, even among such comparatively simple forms as planarians, do not always act with machine-like uniform- ity. Until it is possible to predict with exactness what behavior will result under any given set of conditions, an accurate knowledge of the behavior of any kind of organism must be based upon repeated observations of individuals as such rather than as representatives of species and genera. Individual variations in behavior constantly appeared through- out the course of the present investigation. It will be sufficient, however, for the purpose of making clear their importance to cite only a few instances of such variations. It should be noticed that whenever “exceptions to the rule” of behavior occur, as in the case of negative planarians coming to rest in the light or Dau Poritvely phototactic for a time (see the two cases cited on p. 78), they are ordinarily simply abnor- mal cases of individual elavior standing out against the back- ground of average specific or generic behavior. Exceptional cases of this kind, however, are not so typical of what really constitutes individual behavior as the less aberrant actions making up the majority of the movements which the animal performs. The main point to be recognized, then, is that the individual presents unknown factors, which, even in the simplest forms of life, where the range of variation is least, have never yet been reduced entirely to hence: physical terms, a fact which i impairs somewhat the conclusions of those writers who would draw a complete parallel between an organism and a machine. Rate on Successive Days. When the rate of locomotion of cer- tain isolated individuals is averaged from four trials, for example, and the same experiment is repeated on the following day with the same individuals, thereby eliminating the effects of fatigue, under as nearly identical conditions as possible, the two sets of figures °Frandsen (’o1), who was impressed by individual differences in the phototaxis of Limax, and Smith (’02), who worked with the earthworm, are exceptions to this generalization. The Reactions of Planarians to Light 113 thus obtained show more variation than would be expected if the organisms experimented upon responded in a machine-like way. If not all, at least a part of this variation may, then, be due to differences in individual behavior. TABLE XXVI The differences among tsolated individuals of different species in the average rate of locomotion, based on 4 trials each on each of two different days, expressed in mm. per sec. Dendrocelum Phagocata Planaria Planaria lacteum gracilis maculata gonocephala INTs Ohi: gaan do oodanouocenonoun 1.52 1.22 1.76 0.67 niscentlie rl Aoheacanrn qonAaads0 dpe 0.70 0.96 ay 8 0.73 The Relative Value of Individual Behavior. In the three fol- lowing tables individual behavior will be compared with light intensity with respect to (1) rate of locomotion, (2) range of rate and (3) manner of turning. First, the individual behavior of 10 worms belonging to the species Planaria gonocephala under all intensities of non-direc- tive light showed greater range in the rate of locomotion than the average behavior of the same ten worms showed under any single intensity of non-directive light. TABLE XXVII The relative effect on rate of locomotion of individual behavior and light intensity. The averages are expressed in mm. per Sec. A Variation or INpIvipUAL BEHAVIOR Identification number of worm ..............-- I 2 3 4 5 6 7\ 8 9 10 Average rate in all the intensities given in B..... 0.79|0.57.0.70 0.64 0.83 0.700.72 0.60'0.49 0.62 e Range = 0.34 [0.83 (No. 5) —0.49 (No. 9) ] B Variation IN Dirrerent LicHr INTENSITIES Light in candle meters ......... °o | 0.94 ial 39 78 126 155 | 217 | 43% Average rate of the 10 worms given misao deine oo coc adonepsonaeey 0.57 | 0.66 | 0.69 | 0.75 | 0.64 | 0.65 | 0.69 | 0.70 0.63 Range = 0.18 [0.75 ‘39 cm.) — 0.570 (0 c.m.) | IIl4 Herbert Eugene Walter Secondly, the range between the maximum and minimum rates of ten individuals in all intensities of non-directive light was greater than the average range of rate of the same individuals under different intensities of non-directive light. TABLE XXVIII The relative effect of individual behavior and light intensity on the range of rate of locomotion, expressed in mm. per sec. A VariaATIoN or INDIvIDUAL BEHAVIOR Identification number of worm............... I 2 3 4 5 (SOS; 8 9 | 10 Maximum rate in all intensities given in B..... 2.58 1.67/2.00 1.67/2.17 2.08 2.20 1.78 1.82 1.58 Minimum rate in all intensities given in B..... 0.92 0.67/0.92 0.83/1.03 0.42/0.67 0.28,0.28 0.75 AN Pe? Obst Aterstaysrarsiseare slsistaratersrotle,o}sjexstors 1.66 1.00)1.08 0.841.14 1.66 1.53 1.50 1.54 0.83 Range 0.83 = [1.66 (No. 1 or 6) — 0.83 «No. 10)] B_ Variation IN DirrereNT Licut INTENSITIES Light in candle meters ......... ° 0.94 | II 39 78 126). |) USS. A217) (0 43T Maximum rate for all worms given ee cB ee HBOS ee OMe 1.§8 | 2493} 1292 | pTL77 |) W92s| -79)) 1.87.) 200%) res Minimum rate for all worms given Trna A terer fats letelaie arate eidavertsewsstacints 0.71 | 1.07 | 0:74 | 1.04 | 0.77 | 0.62 | 1.15 | 1.03 | 0-79 Rangsiafirate’sjesaic.+:s;sto,<f=%6 0.87 | 0.86 | 1.18 | 0.73 | 1.15 | 1.17 | 0.72 | 0.97 | 0.76 Range 0.46 = [1.18 (11 c.m.) — 0.72 (155 c.m.)] Thirdly, with respect to clockwise or contra-clockwise turnings, individual factors were found to be of more importance than differences of intensity of non-directive light in determining the direction of turning. It should be added that the ten worms concerned in the three preceding tables were as similar in size and external appearance as it was possible to select. A Cave Planarian. This specimen came from an Indiana cave, where it probably had always lived in darkness up to the time of its capture. When first made the subject of experiment, it could be briefly described as a white worm, about 6 mm. in The Reactions of Planarians to Light 115 length, devoid of any dark pigment except in the two eye spots. Although most nearly resembling Dendroceelum lacteum_ in color, it showed some differences from this species in the contour of its body and particularly in its behavior. It was thought prob- able, therefore, that this was a representative of some species peculiar to a dark habitat. The absence of sexual organs made its exact identification impossible. In the present connection it will be referred to simply as “the cave worm.” As a unique sub- ject for the study of individual behavior, it proved to be very TABLE XXIX The relative effect of individual behavior and light intensity on the direction of turning, expressed in a ratio of contra-clockwise to clockwise movements A Variation or InpDIvIDUAL BEHAVIOR , 2 : I I I I z I I I I Ratio of contra-clockwise to clockwise turn- f | 2 P ° ane ; : 4| to | to to to | to to | to to to | to ings in all the intensities given in B ..... | 1.52|1.400.42|4.00|2.08 4.21/2.23/4.02/0.85 0.93 Range = 1 to 3.60[1 to 4.02 (No. 8) — 1 to 0.42 (No. 3)] B_ Variation IN DirreRENT Licut INTENSITIES Light in candle meters ......... ° 0.94 II 39 78 126 | 155 | 217 | 431 Average ratio of contra-clock- ( I I I I I z I I I wise to clockwise turnings of 4| to to to to to to to to to the ro worms given in A... | I 1.10 | 1.24 | 1.65 I 0.93 | 1.32 | 1-40 | 1.58 Range = 1 to 0.72 [1 to 1.65 (39 c.m.) — 1 to 0.93 (126 c.m.)] interesting. A comparison of its activities with those of other planarians is given in Table XXX, where it will be seen that this cave worm was considerably more active than any other kind of worm under observation, both with respect to locomotion and to the average time required for it to leave a unit circle. Re- garding the degree of negativeness which it presented, no new feature appeared, though its average in this point was rather higher than that of all the other worms studied. However, its degree of wandering quite exceeded anything shown by planarians which had been reared in the light. 116 Herbert Eugene Walter If the relationship of an animal could be determined by behay- ior alone, there need be no hesitancy in saying that this unidenti- fed planarian should not be classified under the species Dendro- coelum lacteum, since in all the criteria mentioned in the foregoing table it stands at an opposite extreme to Dendroccelum. In point of fact its behavior more closely resembled that of Phago- cata gracilis, a species which, according to Dr. A. M. Banta, who kindly furnished the cave planarian for this study, is common in the streams in the vicinity of the cave where the latter was found. TABLE XXX The behavior of a cave planartan compared with that of planartans accustomed to light. THE CAVE OTHER PLANARIANS PLANARIAN CRITERIA OF BEHAVIOR ear LE Te Number Average Average) Maxi- Mini- Se Range of trials record record mum mum Average amount of negativeness, expressed in the percentage of deviation upon leav- ing a circle 10 cm. in diameter when started at right angles to incident light 7o 99-6 91.8 99-9 84.6 15-3 (B delloura omitted) Average rate in mm. per sec............ 60 2.00 1.203 1.473 | 0.853 0.62 Average seconds required to leave a circle, LOw CUEING LAN so ctyeeelo,s cose Saat Tees a go 27.8 | 52.2 62.2 39-7 22.5 Average deviation in degrees when pointed away from the light. (Amount of wan- ering ee sacberuaees eee cece 46 47 25°.6 | 39°.3 9°54 29°.9 Summary. Average individual behavior constitutes typical specific behavior. Variations in individual behavior make accu- rate predictions of responses to stimuli under given conditions, impossible. The rate of locomotion of the same individuals varies from day to day even under apparently identical conditions. Individual variations in the rate of locomotion, in the range between maximum and minimum rates, and in the percentage of clockwise turnings, are more variable than the average behavior in these particulars under different light intensities. An unidentified cave planarian showed greater activity and more inclination to wander than any of the other planarians under observation. (To be continued) CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Director. No. 193. THE REACTIONS OF PLANARIANS TO LIGHT BY HERBERT EUGENE WALTER With Fourteen Ficures (Concluded) 4 BASIS OF BEHAVIOR In the sections on Photokinesis and Phototaxis certain con- ditions of illumination were shown to be variable factors in influ- encing the movements of planarians. Following this treatment of the subject an attempt was made under “Kinds of Behavior”’ to classify the effects of light according to the way in which the responses of planarians become manifested in a generic, a specific or an individual sense. It now remains to consider the nature of the factors which cause different individuals to present charac- teristic differences in behavior. ‘There are at least three ways of approaching the matter. “These may be roughly indicated as the point of view of the morphologist, the physiologist, and the psychologist. A Morphological Basis of Behavior The structure and shape of a planarian, its muscular and cil- lary equipment, together with the kind and distribution of its light- receiving apparatus, are some of the morphological factors defin- itely restricting the kind and range of its reactions to light. “These morphological factors may be grouped into, first, those which determine the general form of the body and consequently influ- ence the animal’s activities in a broad sense, and, secondly, those directly concerned with the reception of the light stimulus, the photoreceptors. Tue JourNAL oF ExPEriMENTAL ZOOLOGY, VOL. V, No. 2. 118 Herbert Eugene Walter a General Form of the Body A normal, full-grown planarian may be expected to give typ- ical reactions to any stimulus. Fragments of a planarian, on the other hand, whether occurring from natural or artificial causes, would not be expected to behave as perfectly developed worms do, and observation shows that they do not. As previously mentioned, Loeb (’94), and later other investi- gators, established the fact that planarians with eyes and brain removed are still able to give characteristic reactions to light, while Lillie (or) found that any fragment capable of regenera- tion would respond to light. In all cases of mutilated worms, however, the response to light is slower and less precise than that exhibited by normal individ- uals, and therefore different in degree if not in character from that of the latter. It has been repeatedly observed that worms mutilated unilaterally perform circus movements regardless of the light. This seems to be a plain case of morphological limi- tations on the part of the crippled animal, whereby the cilia and musculature of one side, on account of injury, are less efficient than those on the other side. Since it is practically impossible in nature to select at random a dozen planarians of which at least one specimen does not show some sort of mutilation, the modi- fied behavior of morphologically imperfect animals becomes a factor of considerable importance in any general analysis of planarian activities. Again, with regard to the general form of the body, it seems reasonable to suppose that a mature planarian loaded down with sexual products, or one gorged with food, must encounter mechan- ical difficulties in responding to light, so far at least as locomotion is concerned, which the same animal when unencumbered would not experience. A few experiments were performed to test this supposition, in which a comparison of the behavior of large and small worms was attempted. Pearl (’03, p. 546), it will be recalled, has shown that in the case of Planaria maculata, a worm II mm. long travels at a faster rate than one 6 mm. long. This experi- ment was repeated with a confirmatory result but, as will be seen The Reactions of Planarians to Light 119 upon examining Table XXXI, the same result did not occur when Dendroccelum lacteum was used. TABLE XXXI The average rate of locomotion in mm. per sec. of 5 small and 5 large individuals of each of four species, subjected to identical light conditions Larce SMALL SPECIES , A Size Average Size | Average mm. rate mm. Tate Dendroccelum lacteum (first trial).......... II 0.695 4 1.01 Dendroccelum lacteum (second trial ........ II 0.74 4 0.77 pha po Cataigraciliss sees sjereieieie.s\cisiele <icieievels 9 1.58 4 Te2T Planariaimaculatas |. jectccle ccs ce swarm eic}e/-\vin/\s 13 1.57 8 1.37 Planaria gonocephala....................- 10 1.17 5 | 0.98 The worms selected for the experiments detailed in Table XXXI were carefully chosen as to length and did not vary more than a millimeter in any case from the size recorded in the table. The result obtained with Dendroccelum was so unexpected that the same ten individuals were put aside and tried a week later under as nearly identical conditions as possible. As will be seen by the table, the result of the second experiment was in general the same, though not so pronounced, as that obtained in the first set of trials. In the cases of Phagocata gracilis, Planaria gonocephala and Planaria maculata, the larger worms traveled faster than the smaller ones. Why the factor of size should give a different result in the case of Dendroccelum lacteum from that common to the dark-pigmented planarians is by no means clear. It is conceivable that a planarian with undeveloped sexual organs or one whose size was reduced through starvation might have a better ciliary equipment 71 proportion to its mass than a normally adult animal and that in consequence it could travel faster. “This sup- ; position explains the behavior of Dendroccelum lacteum, but it does not throw light on that of the other species, of which the smaller individuals, instead of traveling faster than their larger associates, moved at a slower rate. It is possible that in the case of the dark-colored worms reduction in size is accompanied by a 120 Herbert Eugene Walter corresponding reduction in the photoreceptive elements, which, according to the experiments of Loeb (’94) and of Parker and Burnett (’00) seem to be in some degree at least distributed over the entire body. If this is true, there would result less stimulation from the light and consequently a slower rate. That Dendro- ccelum lacteum when reduced in size does not suffer a similar reduction of its photoreceptive apparatus is probable. ‘The work of Lillie (’o1), wherein he showed the inability of headless individ- uals of Dendroccelum lacteum either to regenerate or to respond to light, suggests that the photoreceptive apparatus of this species is not scattered over the entire body, but is rather concentrated anteriorly, in all probability consisting of the eyes only. If this is true, a reduction in the size of the body would not necessarily cause a proportionate reduction in the photoreceptors, and, indeed, the proportion of the light-receiving elements as com- pared with the mass of the body might increase as the worm became smaller. In this connection it is interesting to note that Gissler (’82) pointed out that in the case of Bdelloura candida increasing size of the body is accompanied by a decrease in the size of the eyes, and so far may this inverse ratio be carried that the eyes sometimes disappear entirely inlarge individuals. If there actually exists some such inverse ratio between the size of the photoreceptors and the mass of the body in the case of Dendro- ceelum lacteum, it is easy to see why the smaller worms travel faster than the larger ones. By another series of experiments it was found that the smaller worms of all four species, with the possible exception of Dendro- ° ceelum lacteum, orient with less accuracy than the larger worms under the same external conditions. In these experiments, as in the previous ones already described dealing with orientation, each worm was placed at the center of a circle 10 cm. in diameter and headed successively toward, away from, and at right angles in both directions, to the incident light. ‘The average amount of deviation at the circumference of the circle from the direction in which the worms were started, reckoned in degrees, gives a crite- rion of their accuracy in orientation. ‘The averages of behavior obtained are indicated in Table XXXII. The Reactions of Planarians to Light I21I The lessened accuracy in orientation among the smaller worms, as compared with the larger ones, helps to support the hypothesis that reduction in size entails proportionate reduction in the photoreceptive apparatus. The fact that Dendroccelum lacteum forms an apparent exception to this general rule may also be taken as evidence that in this case the photoreceptive apparatus is more localized than in the other worms studied and_ conse- TABLE XXXII The average deviation (expressed in degrees), at the circumference of a circle 10 cm. in diameter, of large and small worms, each lot consisting of 5 individuals. Each worm was headed successively toward, away from and at right angles in both directions to incident light. The actual sizes of the worms were the same as in Table XX XI DENDROC@LUM LACTEUM PLANARIA | Phagocata| Total S1zE OF WORMS s q | x Sion gono- | gracilis average First trial tale | Average | maculata eeerate | Large, degrees... 67 { 7° | 68.5 57 64 63 } 63 | Small, degrees... 57 } 85.5 68.5 61 69 72 67.4 quently does not suffer a proportionate decrease when the size of the body becomes less. It is furthermore quite possible that a sexually mature planarian may on that account behave differently in light than an immature one. For instance, Yerkes (’03) states that in the case of the hydromedusa Gonionemus murbachii, the sexually mature individuals are the ones most sensitive to light, and Schouteden (’02) found the young of Daphnia positive, while - the adults were negative to light. Finally, Harper (’05) has shown that in the case of the earth- worm the degree of sensitivity to light depends upon the degree of contraction or expansion of the body, since the photoreceptor cells—which in the earthworm lie interstitially at the bases of the epithelial cells—are more exposed to stimulation when the worm is expanded and conversely more shielded when it is contracted. It is more than likely that planarians offer a parallel instance and that their comparative indifference to light stimulation when in the relaxed resting position is due to the fact that then they pre- sent a more rounded contour and consequently their photoreceptors 122 Herbert Eugene Walter are more deeply buried from the light than when they are in the expanded position assumed while gliding. b_ Photoreceptors What is the photoreceptive apparatus of the planarian? Is it made up of the eyes only, or partly of nerve-endings or of some special morphological elements homologous perhaps to the pho- toreceptor cells in the integument of the earthworm as described by Hesse (’96). Or does the central nervous system, the ciliary apparatus, or the musculature receive the stimulus directly with- out the mediation of special sense organs ? Although these questions were not made the subject of parti- cular investigation in the present study of planarian behavior, certain facts incidentally appear from the observations made for other purposes which bear directly upon these inquiries and may serve as a basis for a brief discussion of the nature and location of the photoreceptive apparatus of planarians. ‘The presence of eyes in the anterior part of the body, together with the wigwag movements which often take place in the same region when a variation occurs in the light conditions, point directly to the con- clusion that the anterior end of the worm is more responsive to light than the posterior end. ‘Uhe fact that many planarians con- tinue to react to light with considerable definiteness after the whole anterior end of the body is removed, indicates that this region does not necessarily contain the entire photoreceptive apparatus. Decapitated individuals of the species Dendro- ceelum lacteum, it should be noted, seem to be exceptional in this respect since, according to Lillie, they fail to react to light. In further support of the supposition that the anterior end of the planarian is the portion most sensitive to light it was found that the skioptic response of Bdelloura candida is confined not only to the anterior end but particularly to the region directly including the eyes. Observations repeatedly showed that if Bdelloura was allowed to come to rest in a field illuminated from above only, a sharp narrow shadow thrown across its body pro- duced no visible response unless the shadow included the eyes. The Reactions of Planarians to Light 123 The moment, however, that the eyes were in shadow the worm would elongate and frequently begin strikingly active movements. It has already been shown (Table XIV, p. 77) that all the different species of planarians upon which experiments were made, traveled at a faster rate when they were started with the ante- rior end pointed toward directive light than when away from it. A reason may be offered for this characteristic increase in rate on the ground that the anterior end was plainly subjected to stronger stimulation when directed toward the light than when pointed away from the source of the stimulus. In the latter instance it was not only turned away from the source of the stimulus but was shielded also from the light to a considerable extent by the shadow formed by the posterior part of its own body. Again, when a small beam of sunlight passing through a_ pin- hole in an opaque screen was directed locally to different parts of a gliding Planaria maculata, it was found that tropic response would occur in case one side of the anterior end was illuminated, and that it was not necessary for the eye itself to be included in the illuminated area to obtain such responses. However, when the middle of the body or the posterior end was similarly stimu- lated the worm could not be made to turn. From the foregoing observations it seems probable that the photoreceptive apparatus of planarians is mainly but not exclu- sively located in the anterior end of the body and that considerable specific or generic difference may exist with respect to the extent of the distribution of additional light-receiving organs over other parts of the body. It is interesting to note in passing that Gamble and Keeble (’03) found that in the case of the green rhabdoccele Convoluta roscoffensis the sensitiveness to light was at the ante- rior end of the body only. Concerning the relative sensitiveness to light of the dorsal and ventral surfaces of planarians, a set of experiments was performed on Planaria gonocephala in which the results show an absence of any marked differentiation in this regard. It is well known that in the matter of response to a thigmotactic stimulus the dor- sal and ventral surfaces of a planarian show a very striking differ- ence. Indeed, the dorsal surface is negatively thigmotactic to 124 Herbert Eugene Walter such a degree that it is practically impossible to make a worm remain with its dorsal surface in contact with any surface, while its ventral surface is just as strongly positive in its thigmotaxis. In contrasting the receptivity of these two surfaces to light stimulation a field of two adjacent intensities, similar to that used in the experiments on abrupt spacial changes in light intensity (Fig. 3, p. 65), was arranged in such a way that, in the first instance the source of the two lights was below, and in the second above, the field in which the worms were placed. ‘The intensities of the light in each case were approximately 66 and 33 c.m. By this means the responses of the worms could be tabulated as they glided from one intensity of light to another and those given when the light impinged on the dorsal surface compared with similar responses made when the light struck directly on the ventral sur- face. It will be seen in Table XX XIII that the results do not indi- cate any particular difference for the dorsal and ventral surfaces with respect to the distribution of the photoreceptors. ‘This con- dition of affairs, however, may be largely due to the translucency of the planarian’s body, which would render light-receiving organs accessible from whatever direction the light primarily comes. TABLE XXXIII A comparison of responses made by Planaria gonocephala to a change in light intensity, tabulated with reference to the source of the light and its relative degree of stimulation upon the dorsal and ventral surfaces of the worm respectively Number of | Percentage of Perce eeok ition of light wigha Position of lig’ observations | total responses | Bee movements | IOI | 5° Gre 156 | 53 36 An exact determination of photoreceptors other than the eyes was not made. Both Iijima (’84, p. 438) and Carriére (’82, p. 167) in their histological researches upon planarians found “‘Neben- augen’’ frequent and these occasional accessory eyes have also been described by Janinchen (’96, p. 259). Such structures may The Reactions of Planarians to Light 125 possibly be interpreted as the connecting link between undifferen- tiated light-receiving organs and the normal eyes of the planarians. The part that pigment plays in the reception of light is not as yet clearly defined. It is not probable that pigment in itself con- stitutes a photoreceptor, though it is usually found associated with sensory cells which are directly concerned with light reception. That it is not an essential factor of a photoreceptor is evident, inasmuch as it is absent from the eyes of albino animals. The secondary role of pigment in the reception of light by organisms is admirably pointed out and discussed by Beer (’01). The presence of pigment in a planarian may, however, modify the animal’s response to light stimulation by shielding the sen- sory cells from light, and since its distribution in general is near the exterior, it may afford some clue to the relative receptivity of internally and externally situated photoreceptors. In other words, if pigmented and non-pigmented worms, for example, ex- hibited the same behavior in light, it might reasonably be assumed that the photoreceptors were not located internally, since they would be partially shielded from light in the case of the pigmented forms and consequently would give rise to a different response. It is of interest, therefore, to contrast the behavior of dark- pigmented worms with those in which the dark pigment is absent except in the eyes. ‘This is done in Table XXXIV, but it by no means follows that the contrasts there given between the behavior of dark and light worms are due to the presence of dark pigment in the one case and its absence in the other. Other factors than pigment may very probably have been influential in bringing about variations in the light reactions tabulated. Furthermore, it is inaccurate to refer to a white worm as being non-pigmented, since in that case it would be entirely transparent. ‘The question, then, so far as planarians are concerned, is confined not to differ- ences between pigmented and non-pigmented but to differences between dark-pigmented and light-pigmented forms. It will be seen from Table XXXIV that when subjected to light stimulation dark-pigmented worms in general show more activity than light-pigmented forms. A single exception to this rule occurred in the case of the cave planarian experimented upon. 126 Herbert Eugene Walter This marked difference in behavior might possibly be explained on the hypothesis that the direct effect of light on the deeper lying nervous system is inhibitive; that is, so excessive as to produce a sort of light rigor. ‘Thus the more the central nervous system is shielded from light by pigment the less the inhibitive effect becomes apparent. Certain it is that Bdelloura candida, which has dark TABLE XXXIV The behavior of dark-pigmented worms contrasted with that of worms not possessing dark pigment dis- tributed over the body. The number of observations in each case ts not given since the details of this table have already appeared elsewhere Dark PIGMENTED LiGuT PIGMENTED s eae} &, a — | s 3 s = Cole at re aa eae Ur me ctsyentedill ieee fra sell cx ey po iy Or | a9 oOo Our os oo heey arr oG x Fa iors een s [-rsy e 6 cm be oa | aU > b s s Le Leo eH [pet eee =| os vo eed ae s by 5 Sgilze|sa > a S S| & le as|A =< los Duration of movement in a typ- ical set of experiments, minutes) 47 60+ | 60+ 56+ 18 15 | 6s Percentage of orientation to light upon coming torest.......... 41 41 30 30 Wigwag responses at the critical line separating two intensities of non-directive light, per cent 39 39 8.5 | 8.5 Average number of seconds re- quired to escape from a circle | 10 cm. in diameter ........... 48.8 | 59.6 | 39-6 | 49.3 | 62.2 27.6 | 44.9 Precision of response Deviation in degrees upon emerg- ing from a circle 10 cm. in dia- meter when headed away from | the ightsrcyarcievaa iterate s(cte(=teieiets= ay Hae D ey 29.1 26.3 10.1 11.4 | 10.7 Rate of locomotion in mm. per SECanaciteccsee el tteretersiace 1.47 | 1.075| 1.395| 1.28 | 0.85 0.85 pigment in its eyes only, may be brought to a standstill very read- ily by means of light stimulation. With the exception of the eyes it may be possible that the photoreceptive apparatus is not differ- entiated to such an extent that it could fairly be said that any part of the translucent planarian body is entirely free from the direct stimulation of light. The relation of pigment to light reactions The Reactions of Planarians to Light 127 is, however, by no means settled in the foregoing observations. This matter should be finally tested by comparisons in the behay- ior of different individuals of the same species showing variation in pigmentation or of identical individuals at different times when their phases of pigmentation are unlike, rather than upon indi- viduals of different species. It has proven impossible to include such a consideration in the present paper, but the preliminary steps toward attempting an analysis of the function of pigment with reference to light reactions have already been made and it is expected that a discussion of this phase of planarian behavior will be presented later. It may be stated here that when Planaria maculata is fed with a drop of human blood, a decided increase in pigmentation makes its appearance within a few days, due probably to the oxidation of the hemoglobin in the blood corpuscles with which the planarians have Become gorged. ‘This single observation suggests an experi- mental means oe controlling che amount of pigment in a single individual and it may reasonably be supposed that tests of eae ior before and after excessive pigmentation will contribute direct evidence upon the part played by pigment in reactions to light. Summary. Mutilated planarians in general respond to light with less accuracy than normal individuals. Small worms move more slowly than large ones in the case of those species whose photoreceptive apparatus 1s not solely confined to the anterior end of the body. In the case of Dendroccelum lacteum, whose photoreceptive apparatus is relatively greater in small individuals than in large, the rate of incomouonet faster among the smaller than among the larger. Small worms orient with less accuracy than large ones. Pla- narians in the relaxed, resting position are less responsive to light than when they are stretched out in the act of gliding, a result probably of the greater exposure of the photoreceptors to light in the latter instance. The anterior end of the body 1s the chief photoreceptive region and in certain worms, such as Dendroccelum lacteum and Bdel- loura candida, the anterior end is apparently the exclusive seat of this function. 128 Herbert Eugene Walter No marked difference in response to light is shown between worms stimulated on the ventral surface and those equally stim- ulated on the dorsal surface. Aside from the eyes, which form at least a part of the photo- receptive apparatus, no definite light-receiving organs were recog- nized. Planarians possessing dark-colored pigment distributed over the body show in general greater activity when subjected to light than forms in which there is no dark pigment except in the eyes. The central nervous system, as well as the more exterior parts of the planarian, may possibly be stimulated directly by such light as passes through the translucent body. B The Phystological Basis of Behavior The continually changing adjustment in any organism between the incoming and the outgoing energy gives rise to varying phases of metabolic balance, which may be designated as different “ physi- ological states.” Such physiological states form a noticeable fac- tor in the behavior of any animal, a fact towhich Jennings (’o4b, p- 109) in particular has called attention. That the difference between such states is great may be readily demonstrated. A planarian’s response to directive light when it is in a relaxed, quiescent condition is plainly different from that exhibited after it has been vigorously disturbed by a brush. In fact, it is extremely difficult to get two animals that are in precisely the same physiological condition, or the same animal in precisely the same state at two different times, since the exact adjustment of physiological states is too. delicate a matter to be controlled by the present gross experimental methods. The attempt is ordinarily made to eliminate from experiments, so far as possible, the disturbing element of changing physiological conditions, that is, to keep constant all the factors except the one which is being subjected to test, and those results are counted as most successful in which such disturbance is reduced to a minimum. It is the purpose of this section first, to give a possible classifica- tion of the different physiological states in which a planarian may The Reactions of Planarians to Light 129 be, and, secondly, to pass briefly in review some of the many ways in which light may change the physiological state of such a worm. a Classification of Physiological States It is by no means easy to define even a simple physiological state, since the subtle changes form a continuous series of conditions which pass imperceptibly into each other. An arbitrary classification for convenience may, however, be made as follows: 1 Relaxation, or rest. 2 Slight activity, without locomotion. 3. Normal activity. 4 Violent activity. 5 Rigor. 6 Exhaustion. In the first of these states there is a minimum expenditure of energy caused by the ebb of the katabolic processes. The second and fourth states indicate what are often referred to as conditions of low and high “tonus,” but as this term has a technical significance with reference to muscle reactions, it will not be used in this classification. Thethird state, thatof normal activ- ity, is the average condition; it is the most desirable one to main- tain in testing the animal’s responses to different stimuli. By rigor is understood a state wherein there may be an excessive outgo of energy, but unaccompanied by movement, while under exhaus- tion is cneleded the condition when energy is not being released because there is none to release. That excessive or continuous light stimulation may go beyond the point producing rigor or exhaustion and may actually cause death, has been repeatedly proven in the case of bacteria by a long line of observers. The inhibitive effect of excessive light upon other organisms than bacteria has been pointed out by Berger (00) with reference to Cubomeduse; by Pearl and Cole ® Tyndall (’78), Downes and Blunt (’77, ’78), Jamieson (82), Duclaux (’85a,’85b, ’g0), Arloing (’87a,’87b), Roux (’87), Dandrieu (’88), Raum (’89), Pansini (’89), Janowski (’90), Buchner (’92) , and Ward (94). 130 Herbert Eugene Walter (02) in the case of various infusoria as well as Hydra, Hyallela, Clepsine, Stichostema and Physa; by Yerkes (’03) for Goni- onemus and by Carpenter (’05) for Drosophila. b Changes in Physiological States Induced by Light A variety of stimuli besides light may cause an animal to pass from one physiological state to another. For: example, the sense of phototaxis was reversed through mechanical stimulation by Towle (’00) in Cypridopsis and by Holmes (’or, ’o5b) in Orches- tia and Ranatra. The following typical illustration of the manner in which changes from one physiological state to another succeed each other is offered as a basisof comparisonwith the responses tolight itself, which are about to be described. In the absence of mechanical stimuli a planarian may be in a state of relaxation. Very gentle mechanical stimulation causes the worm to lift its anterior end and move it cautiously about, bringing the animal into a state of slight activity without locomotion. If, now, the mechanical stim- ulus is prolonged or increased in intensity, enough energy is released to put the animal into gliding locomotion, when it may be fairly said to have passed into the state of normal activity. Pro- vided the stimulation is made still more pronounced, the worm can next be forced to forsake gliding for crawling or humping, so passing into the state of violent activity. Further, it is possible by vigorous shaking to throw the worm, temporarily at least, into a condition of inactivity through excessive stimulation, during which the animal would remain quiet, not because it is failing to release any energy, but because it is unable for the time to set free its energy in the form of locomotion. In other words, it is in the state of rigor. Last of all, if mechanical stimulation is repeatedly applied a condition of exhaustion will appear when the worm has no more available energy and so is unable to move at all. Effect of Different Intensities. As already pointed out, no inten- sity either of directive or non-directive light was found sufficient to change the condition of normal gliding into crawling. The Reactions of Planartans to Light 131 Moreover, light of any intensity or direction frequently proved ineffective in arousing a quiescent worm into any state of apparent activity, particularly if the worm had but recently passed into the state of rest after a prolonged period of exercise. Effect of Excessive Light. In the experiments with non-direc- tive light it appeared that Planaria gonocephala, when subjected to an intensity of 431 c.m., showed somewhat less activity than at lower intensities, both with respect to rate of locomotion (Table III, p. 57) and to the number of turnings made (Table VI, p. 59); yet, so high a degree of intensity of the light stimulus was appar- ently not sufficient to cause a change into the physiological state of light rigor. It was comparatively easy, on the other hand, to transform Bdelloura candida by means of excessive light from the state of normal activity into that of light rigor. Effect of Sudden Change in Light Conditions. A sudden change in light intensity either by increase or decrease is more effec- tive in producing a new physiological state than an equal grad- ual change. ‘The sudden withdrawal of the lamp to a consider- able distance, for example, is usually sufficient to throw a worm from a normal state into violent activity, that is, from a gliding movement into a disturbed state in which the anterior end is waved actively about. But if the light is gradually withdrawn the same distance the worm will usually not pass into a different physiological condition. The sudden introduction of complete darkness was never found sufficient to reduce an active worm more than temporarily to the resting position. Sudden dark might temporarily halt a moving worm, but it would not cause it to come to rest and assume the relaxed contour. In Bdelloura candida sudden dark, instead of checking the animal’s movements, threw it into violent activity. Effect of Continued Exposure to Light. Continuous exposure to light results in fatigue, which finally causes planarians to change from the state of normal activity to that of relaxation. ‘The tend- ency toward such a change is shown in Table XXXV, where the responses of a number of worms newly subjected to light stimu- lation are contrasted with the responses made by the same worms after they had been moving about for several hours in the light. 132 Herbert Eugene Walter The fresh worms show more activity than the fatigued worms do. Otherwise expressed, the worms have a tendency to change into a lowered physiological state upon continued exposure to light. TABLE XXXV Fatigue effects due to continuous exposure to non-directive lights forming adjacent fields of different intensities, as shown in the behavior of Phagocata gracilis Ratio oF THE TWo -96: 13-4501 Average INTENSITIES iene ae 3-45 3 Percentage of the re- Going Going Going | Going Going | Going sponses at the crit-| into into FF into into 4 into into a ical line separating greater lesser & | greater | lesser Pe greater lesser is = iS ' 0 f 2 & the two intensities intensity intensity 2 intensity intensity 2% intensity intensity oa Fresh worms........ | 10.5 | 21 16 45-5 47-5 | 46.5 | 28 34-4 | 31+ Fatigued worms.... 2.5 9-5 32-5 33-5 | 33 17.05 21.5 | 19.5 It may be incidentally noted in Table XXXYV that, as has already been pointed out in another connection, the percentage of responses is greater when the contrast between the light intensities is greater, and that both fresh and fatigued worms respond oftener upon going into the lesser intensity than when going into the greater intensity. The time required for a worm placed in directive light to come to rest; that is, to run the gamut from the state of normal activity to that of rest, becomes gradually shorter with continuous expo- sure. As fatigue increases the worm shifts down the scale of physi- ological states in less time than when freshly subjected to directive liohe A specific case of this kind has already been described in the paragraph on “duration of movement”’ (p. 105); where in 39 consecutive trials the change from normal activity to relaxation was first made in 18 minutes, but the thirty-ninth time in Ir} minutes, while the fortieth time even mechanical stimulus failed to arouse the exhausted worm from the resting position. Effect of Previous Exposure to Dark. Worms kept several hours in complete darkness make a larger percentage of re- sponses to changes in their light environment than those which previous to experimentation have been several hours in light. Individuals removed from the stimulus of light for any consider- The Reactions of Planarians to Light 133 able time are more responsive when subjected to it, for the reason that they are in a physiological state farther removed from fatigue than those worms which have remained a long period in the light. This point is brought out in Table XXXVI. TABLE XXXVI Percentage of reactions of two worms, Planaria gonocephala,to a sudden change in light intensity both when previously kept several hours in the dark and also when previously exposed for several hours to light Percentage of responses Number of observations After several hours in the light........... 54 100 After 48 hours in the dark............... 66 100 Summary. Physiological states grade imperceptibly into each other, but may be tentatively divided into: 1, relaxation; 2, slight activity; 3, normal activity; 4, violent activity; 5, rigor; 6, exhaus- tion. Various stimuli besides light may induce a change from one physiological state to another. No light intensity lower than 431 c.m. is sufficient to throw a worm into a higher state than that expressing normal activity, nor is the absence of light sufficient to bring a planarian to rest. Excessive light intensity shows a tendency to carry Planaria gono- cephala from a state of normal activity to one of rigor. Bdelloura candida is easily changed into a condition of rigor by light. A sudden change of light intensity acts more immediately than a gradual change in causing planarians to pass from one physio- logical state to another. Continuous exposure to light induces fatigue, finally resulting in the passage of the worm into a state of continuous relaxation, in which condition it becomes practically indifferent to light. Repeated trials of the time required in constant light to come to rest show that a progressively shorter interval occurs between the state of normal activity and that of relaxation until a point of complete inactivity is reached, the worm finally remaining in the latter state for a prolonged period. 134 Herbert Eugene Walter Planarians kept for some time in darkness pass into a state in which they are more’responsive to light than individuals exposed for a similar length of time to light. G Psychological Basis of Behavior Among the first questions that naturally arise concerning the behavior of planarians in light are those which approach the mat- ter from a psychological point of view. How much can planarians actually see, and can they, by repeated experience, “learn” to adapt themselves to changes in the light surrounding them ¢ To this kind of inquiry it is most difficult to give a satisfactory answer, for the reason that it is impossible to go beyond conjecture and inference in judging what any animal, aside from man, can see or know or experience. It is only possible to state, in more or less definite terms, the responses which animals make to light, since it is beyond man’s power ever to experience how animals “feel” under any circumstances. a. How Much Can Planarians See? Broadly speaking it may be said that planarians can distinguish light from darkness. The experiments described on pp. 84, et seq., relating to planarians placed ijn aquaria so surrounded by backgrounds as to produce regions of different light intensity, point to this conclusion, since when subjected to such differential environments the worms come to rest in the darkened areas. Again, the numerous responses made at the critical line separa- ting two light intensities may be regarded as evidence of some power of discrimination on the part of the worm between dif- erent intensities of light. It is probable, furthermore, that planarians can distinguish a moving object when that object is of sufficient size and contrasts with its surroundings in its degree of illumination, for the reason that a moving object from which light is reflected, means the same to a worm coming into the vic:nity of the object as any other change in the direction of light, such as might be caused by moving a The Reactions of Planartans to Light 135 lamp from one position to another. ‘To changes in directive light planarians are known to respond very definitely, and consequently they may be said to distinguish the motions of objects. With regard to true seeing, however, in the sense of distinguish- ing the forms of objects, it is safe to assume that planarians have almost no power whatever, since their eyes are optically unable to form images even if the central nervous system were highly enough developed to interpret images when formed. In the case, there- fore, of Planaria alpina, which, according to Collin (91, p. 180), “shuns” Planaria gonocephala when the latter has been put into the same aquarium with it, seeking “‘strenuously to escape” from its larger relative, the conclusion does not necessarily follow that P. alpina sees an enemy and experiences the sensation of fear. As previously pointed out (p.95), the whole matter is probably explainable on the basis of negative chemotaxis alone. ‘To attri- bute fear, therefore, or any other similar complex sensation, to an organism whose responses are so plainly of a simple reflex nature, is to go quite beyond the evidence. In the performance of the two great life processes of nutrition and reproduction, light is apparently in no way a direct aid to pla- narians, since they thrive in situations from which light is entirely excluded, as in caves, and since they habitually frequent places where this factor is reduced to a minimum. Light cannot, then, be regarded as a directly essential factor in the life of planarians. That light is not essential to the activity of protoplasm has more than once been demonstrated. Engelmann (’79), for example, showed that the streaming protoplasm of plant cells occurs nor- mally in darkness, while Maupas (’87) found ciliates multiplying as rapidly in the dark as in the light. b~ Are Planarians Able to “Learn” ? With regard to the ability of these worms to acquire upon repe- tition an abbreviated form of response; that is, to “learn,” a few suggestions may be drawn from experiments already described in other connections. It will be remembered (p. 93) that when a small aquarium 136 Herbert Eugene Walter was delicately mounted upon a turntable, such as is used in “‘ring- ing’’ microscopic slides, a very slight rotation was sufficient to bring to a halt momentarily a gliding worm in this aquarium. It was possible to control this momentary response to such a point of nicety that the anterior end of the worm could be made to halt for an instant without interfering with the onward loco- motion of the posterior end. If this slight rotation was repeated at intervals of a second it was found that the worm under obser- vation halted with less and less certainty, until after a dozen or more trials it continued to glide on without halting at all. In ordinary phraseology the worm had learned by experience not to be alarmed by a sudden mechanical shock. ‘The lesson, how- ever, was always very soon forgotten, for after an interval of less than a minute, during which the aquarium remained stationary, the worm responded exactly as it did at first, whenever a slight rotation was made. In a similar way the skioptic response of Bdelloura candida became less pronounced upon repetition, until it was possible to throw a shadow upon the animal without obtaining any response at all. Again, when worms were placed in a field of non-directive light, parts of which were of two different intensities, the number of wigwag responses made at the critical line separating the two intensities grew less after the animals had repeatedly crossed the line. At first the new condition of sharply contrasted light intensities in the worm’s field of locomotion called out a large percentage of wigwag responses. Later, however, by repeated experiences the worm became familiar with this feature of its environment and made fewer wigwag motions. A definite instance of such a decrease in response is given in Table XX XVII. TABLE XXXVII Responses of Planaria gonocephala on crossing the line separating two intensities of non-directive light Wigwag movements No response Percentage of response First 25 crossings....... 21 4 84 Second 25 crossings..... 19 6 76 Third 25 crossings...... 12 13 48 Fourth 25 crossings ..... | 8 17 32 The Reactions of Planarians to Light 137 It will be seen that when Planaria gonocephala was first intro- duced into a field of contrasted intensities, it made the wigwag response at the critical line marking a change of light intensity, in 84 per cent of the first 25 crossings, while during the second, third and fourth sets of 25 crossings, the per cents uniformly decreased until at the fourth 25 crossings the number of wigwag responses fell to 32 per cent. It may be objected that the instances thus far cited in this section find a more reasonable explanation upon the hypothesis of fatigue, but the same surely cannot be said of the fol- lowing case. It was found that Planaria maculata oriented itself to directive light at successively shorter intervals when the position of the light was suddenly changed. To produce such a series of re- sponses there was placed in the dark room a shallow aquarium with an electric lamp at either end, under the control of the right and left hand, respectively, of the experimenter. A planarian was placed in the middle of the aquarium and the right-hand light turned on. As soon as the worm was fairly oriented to this light and gliding away from it, the right-hand light was turned off and at the same instant the left-hand light turned on. The time in seconds required for the worm to orient to the new light; that is, to turn 180° and begin to glide away, was recorded. On p. 89 a typical series of records of such responses is given, in which the number of seconds required for re-orientation when the source of light was reversed, varied from 260 seconds, at first, irregularly down to 35 upon the sixteenth trial. It will be seen from this series that the worm acquired by experience some degree of facility in adapting itself to certain variations in its environment which it would never be liable to encounter in nature, and that this adapta- tion cannot be explained as due to fatigue. Davenport and Cannon (97, p- 32) found similarly that “‘ Daphnias respond more quickly and accurately to light after having made several trips to it.” It is quite certain, however, that any educative attainment which a planarian may experience, or which a planarian may acquire, is exceedingly evanescent and also that there is no evi- dence that the worm emerges from reflex behavior into responses connected with consciousness. 138 Herbert Eugene Walter Summary. The existence of feeling or consciousness among planarians is a matter of pure conjecture. From their responses it may be inferred that they are able to distinguish dark from light, as well as objects in motion, but it is not clear that they can distinguish the forms of objects. The knowledge which planarians have of objects in their imme- diate environment, such as food, enemies, etc., depends largely upon chemical and tactile means. ‘They are, therefore, as well able to go through the entire range of their activities in the dark as in the light. Upon repetition planarians may in some instances become accus- tomed to, or acquire greater facility in, responding to stimuli, but this result of experience is almost instantly lost, so that it is doubt- ful whether these animals possess more than the merest rudi- ments of the primary criterion of consciousness, namely, the ability to learn. VI GENERAL CONCLUSIONS Probably the questions which have occupied the greatest share of attention throughout the literature dealing with the reactions of organisms to light, are the following: 1 Is the direction or the intensity of light of more importance in orientation ? 2 Which theory best explains orientation and phototaxis, the theory of trial and error or that of the tropisms ? 3. How far is behavior with respect to light, adaptive ? I DIRECTION OR INTENSITY Before the part played in the behavior of planarians by either the direction or the intensity of light can properly be discussed, it will be necessary to present a brief historical résumé of certain general conclusions reached by investigators along this line. A Historical Cohn (’53), Strasburger (’78) and Loeb (’g0, ’93a) attributed the directive effect of light to the action of the rays. In a later The Reactions of Planarians to Light 139 paper Cohn (64) abandoned his first position and came to regard intensity as the important element in light, a position also main- tained by Famintzin (67), Engelmann (’83), Oltmanns (’92), Verworn (’or) and even by Loeb (’93b) in the case of Planaria torva, which he found came to rest in accordance with the intensity, and regardless of the direction, of the light. Davenport and Can- non (’97) modified this point of view by attempting to show that direction and intensity may each operate independently, producing, respectively, “phototaxis”’ and “photopathy.”’ Holt and Lee (’or) followed with an excellent summary of the whole controversy, emphatically maintaining, in opposition to Davenport and Can- non, that intensity alone is the only possible operative factor in light stimulation and that direction of the rays has no effect what- soever except in determining a greater intensity of light with refer- ence to one part of an organism as compared with other parts. Among more recent investigations Holmes (’03), experimenting with the same organism that fled Oltmanns to ascribe the greater importance to intensity, namely, Volvox, declares himself in favor of direction, while Zeleny (05), on the other hand, gives an instance of Serpulid larve going both toward the source of the light and away from it; that is, moving regardless of direction, in order to arrive in regions of increased intensity. Carpenter (’05) found that the pomace fly, Ampelophila droso- phila, will orient to the direction of light after it has first been sufhciently aroused by the intensity of the light, while both Yerkes (99) and Towle (’oo) maintain that direction and intensity are by no means mutually exclusive, and that each may play a part simul- taneously in determining the behavior of an organism. Lastly, it has been made clear by Parker (’03) that, besides direction and intensity of light, the size of the source of illumina- tion may determine the orientation. ‘This theory explains why butterflies alight upon a patch of reflected sunlight which produces a large but faint retinal image instead of flying toward the sun itself, which forms only a small but intense retinal image. In the case of planarians, however, this phase of light stimulation is not operative, since the eyes of these animals are incapable of forming retinal images. 140 Herbert Eugene Walter B Conclusions with Reference to Planarians The behavior of planarians may in general be more satisfac- torily explained by regarding, with Loeb, the intensity rather than the direction of the light as the principal operative factor in light reactions. At the same time there is much evidence that the inten- sity utilized by the organism, is intimately associated with, and powerfully modified by the direction of the light. As a basis for these conclusions the following points will be considered. First, the distinction between direction and intensity; secondly, the way in which directive light modifies the intensity with reference to planarians; thirdly, the action of intensity without the modifying effect of direction, and finally, modifying effects of factors other than light. a. The Distinction Between Direction and Intensity Theoretically it is plain that light per se with respect to any fixed point, may be regarded in two distinct aspects, namely, that of intensity and that of direction. ‘The intensity of light under ordi- nary circumstances varies inversely as the square of the distance and is independent of the position of the source of light. That is to say, at any points equidistant from its source, light has the same intensity, but the more remote the less is the intensity at any given point. ‘The direction of light, on the contrary, is depend- ent solely upon the position of the source of the light and in no way upon the distance. When intensity and direction are con- sidered with reference not to a fixed point but to an organism presenting three dimensions and made up of differentiated pro- toplasm, the basis of light relations becomes more complex. Light cannot here be treated as a phenomenon per se but must be considered in relation to a differentiating organism. It is true that intensity in the case of the organism, as in the case of a fixed point, varies with the distance from the source of the light. A decided difference, however, appears in the case of the organism inasmuch as, owing to its structure, the intensity received by it varies also in accordance with the position of the light. ‘This The Reactions of Planarians to Light I4I second form of variation in intensity is directly due to the fact that the organism has a solid form and is not homogeneously photo- receptive. The direction of light with reference to the organism, presenting as the latter does a structurally diversified form, is influential only as regards the position of the source of light, just as in the case of a fixed point. Any change in the position of the source results, then, in a redistribution of the intensities falling upon the organism, so that again the intensity received varies in accordance with the position of the light. It is this factor of posztion in light that has been termed the direc- tive influence of light and it is seen to be due to variations in the intensity of light with reference to the organism, and not to any peculiar property of light iself. By “‘non-directive light,” on the other hand, is understood those conditions which secure for the organism equalized or symmetrical intensity with respect to the parts stimulated. If this interpretation is correct there can be no response, strictly speaking, to the direction of light exclu- sive of intensity although the factor of intensity may be continually modified by that of direction in the light relations of organisms. b The Modifying Influence of Direction It is undeniable that the planarians experimented upon exhib- ited without exception a definite characteristic phototaxis, that is to say, they habitually go either toward or away from the source of light according as they are respectively positive or negative. In analyzing this phototaxis it seemed desirable to eliminate so far as possible the factor of intensity, but the attempt to do this was only partially successful owing to physical limitations. A step was made, however, toward subjecting worms to directive light without at the same time exposing them to a variation in intensity by inserting a biconvex lens between the source of the illumination and the aquarium, thus making the diverging rays of light parallel throughout their course in the aquarium. By this “means was formed a field equal in its amount of illumination at 142 Herbert Eugene Walter the two ends of the aquarium, the one opposite and the one next to the source of light, with the exception that there was a slight difference at the two ends due to the fact that light in its passage through water is partially absorbed. But modification of light in any degree results in producing less intensity at the farther end of the aquarium, though this difference is less pronounced when a lens is employed. ‘Therefore, although worms placed in this apparatus went with considerable precision in the direction of the propagation of the light, there is no certainty that their behavior was not due simply to differences in intensity. Worms which are thus apparently traveling directly in accordance with the direc- tion of the light, are meantime being subjected to different inten- sities at the anterior and posterior ends of the body, for the reason that the anterior end is more or less shadowed by the rest of the body, since the latter cuts out a certain portion of the light received at the posterior end. That direction of light is a factor by no means to be disregarded, even if it cannot be proven to be the immediate cause of phototaxis, is apparent when it is recalled that slight changes in direction call out corresponding changes in the course of the gliding pla- narian, whereas considerable changes in intensity when the direc- tion remains constant and particularly when such changes are gradually made, may fail entirely to produce corresponding changes in the worm’s behavior. ‘This is due to the fact that slight changes in direction may cause considerable changes in the asym- metry of illumination. When a worm, for example, is receiving horizontal light from behind, its head is more or less in shadow, the sides of its body being at the same time equally illuminated. The moment the light is shifted in even a small degree to one side, one entire side of the animal may receive an increase of illu- mination and the opposite side be thrown into shadow. Thus a slight change in position initiates a fundamental change in the distribution of intensity over the planarian’s body. c Instances of Behavior Due to Intensity Alone The effect of intensity as a separate factor from the directive influence of light is clearly demonstrable in certain phases of The Reactions of Planarians to Light 143 light reactions. Yo isolate intensity by excluding the possibility of directive light; that is, to secure equalized intensity with refer- ence to the organism, is not difficult and the manner in which this was done, with non-directive light falling upon a horizontal field from above, has been sufficiently detailed in the body of the paper. It may be briefly recalled that planarians experimented upon by this method showed a certain unmistakable degree of response which could be referable only to differences in equalized intensity. For example, the rate of locomotion was found to be faster in any non-directive intensity up to 431 c.m. than in darkness, although light in itself was not always sufficient to start a worm into ac- tivity, nor was its absence sufficient to check an animal already in motion. Again, though no close correlation between behavior and the degree of intensity was found to exist, there appeared cer- tain general results which were plainly referable to intensity differences only. Instances of such results are the behavior of Planaria gonocephala (which was modified in several particulars at 431 c.m. as compared with its behavior at. lower intensities) ; the coming to rest in regions of diminished intensity of individuals of all species except Bdelloura; and the increase of wigwag re- sponses corresponding to an increase of intensity differences when a field of contrasted intensities was used. It is interesting to observe that increase in the intensity of non- directive light, and continued exposure to non-directive light of constant intensity, both tend to produce the same behavior that would result in directive light. Under any of the three conditions just mentioned there resulted by actual experiment fewer turnings, fewer ‘indefinite changes” and more nearly straight paths on the part of planarians than occurred when the worms were (1) placed in non-directive light of lower intensity, (2) subjected a short time to non-directive light of constant intensity, or (3) left in darkness. Now, fewer turnings, fewer “indefinite changes,” and more nearly straight paths are ordinarily characteristic results of directive light, so that here is a case of reactions, which if resulting from the employment of directive light would be termed phototaxis, occur- ring in non-directive light as the result of intensity alone. Mast (03) experimenting upon the reactions of planarians to thermal 144 Herbert Eugene Walter stimuli obtained a similar result. He observed that apparently “negative” as well as “positive” responses resulted when the animals were subjected to non-directive thermal stimult. Another noticeable phenomenon with reference to responses to intensity is, that more wigwag responses occurred at the critical line separating two different intensities when the lesser of the two intensities was 16 c.m. than when it was 33 c.m. (Table XI, p. 69). Similarly responses were more frequent when planarians were subjected suddenly to dark than when they were flooded suddenly by light, and, throughout a large number of series, responses were invariably more frequent ben the worms were passing into a region of diminished intensity than when they were entering an area of increased intensity. It is to be inferred that all these phases of behavior are due to the probable fact that the lower inten- sities compared are nearer the worm’s optimum as regards light than the higher ones, since the latter apparently have a tendency to inhibit activity. Lastly, the relative part played by intensity of light varies decidedly in different species of planarians. ‘The relative inten- sity in different parts of an aquarium, when no lens is used to les- sen the contrast, has comparatively little influence upon Phagocata gracilis, as its extensive wanderings (typically reproduced in Fig. 11) toward and away from the source of light, indicate. Planaria maculata and Planaria gonocephala, on the contrary (Figs. 13 and 14), notwithstanding their ability to come toward the light in the direction of the “rays” throughout the farther half of the dish, seemed invariably to encounter an impassable barrier as soon as they approached within a certain intensity, thereby showing a more delicate responsiveness to intensity differences. d The Modifying Effect of Other Factors In attempting to analyze the relative bearing of the intensity and of the direction of light upon the behavior of planarians there must be constantly kept in mind two general sources of error which are always present when these factors of light are in operation. ‘These The Reactions of Planarians to Light 145 are (1) the physiological state of the organism at the time of obser- vation, and( 2) the simultaneous effect of other stimuli. A physiological state may be directly traceable to known causes, such as previous exposure to other stimuli or the condition of meta- bolic balance in which the animal chances to be at the time of observation, or, again, it may be the result of factors at present unknown, which consequently, although in active operation, are not susceptible of analysis. In any case it is certain that the uncontrolled factors comprehended under the term “ physio- logical state” prove individual planarians to be not identical Sigeha nists, but organisms possessing a more or less definite individuality. Moreovens it has been shown that differences in physiological state play a greater part in the determination of behavior than do intensity differences in the light stimulus. When a planarian is approaching a state of fatigue, for example, it becomes indifferent to differences of intensity. With regard to the simultaneous effect of other stimuli acting in conjunction with light, it has already been pointed out that behav- ior is the resultant of all the factors, external as well as internal, which may be acting upon an organism at a given time, and that consequently the effect of any one of the operating factors, such as that of light, for example, cannot be determined unless the value of the other factors involved is also taken into account. In support of this view, which is so self-evident, it will be recalled that some of the ways in which the responses of planarians to light may be modified by geotaxis, thigmotaxis, goniotaxis and chem- otaxis, were touched upon. Summary. Direction and intensity are separable qualities of light. Direction is dependent upon the relative positions of the light and the organism, whereas intensity depends upon the dis- tance between the light and the organism as well as the initial intensity of the light. When applied to living organisms intensity may act independ- ently of direction, or in conjunction with it. Direction cannot act independently of intensity upon organisms, since the latter possess definite form and consequently cannot receive the light at a single point. 146 Herbert Eugene Walter With reference to an organism, directive light is resolvable into unequalized intensity and non-directive light into equalized intensity. Asymmetrical intensity in directive light is largely due to the partial shadowing of that part of the body farthest away from the source of the light. Slight changes in the position or direction of the light may cause considerable changes in the symmetry and the degree of the shadow effects and consequently in the relative intensity of the light on different regions of the body of an organ- ism. To different degrees of equalized or symmetrical intensity pla- narians show considerable response, but the correlation between their behavior and the degree of intensity is not so close as it is in the case of asymmetrical intensity. Increase in intensity of non-directive light, continued exposure to non-directive light of constant intensity, and change from dark- ness to non-directive light, all tend to bring about apparent photo- taxis similar to that occurring in directive light. Responses are more frequent on the part of planarians in inten- sities approaching the optimum than in higher intensities, where there is a tendency to inhibition. . Relative differences in responses to various intensities are due to specific differences between planarians. The physiological state of an organism together with the influ- ence of known stimuli other than light are constant sources of error in estimating reactions to light. ‘These factors taken to- gether play a more important part in planarian behavior than light stimulus. Finally, the action of light upon planarians is a function of its intensity, which, under certain conditions, is emphasized by the direction of the light. 2 TRIAL AND ERROR OR TROPISM? It is apparent from the preceding section that light may have two effects upon organisms. Of these, one is a kinetic effect, arising from the intensity of the stimulus and resulting in a gen- The Reactions of Planartans to Light 147 eral activity termed photokinesis, while the other, connected indi- rectly at least with the direction from which light impinges upon an organism, is called phototaxis. In the case of planarians these two phases of light stimulation have been shown to be intimately associated and both operative. Carpenter (’05) pointed out in the case of the pomace fly that phototaxis occurs only when pre- ceded by photokinesis or some other reaction, and such an inter- relation of the two is undoubtedly of wide occurrence. The object of this section is to inquire into the causes underlying pho- totaxis. Loeb (’93b) has shown that phototaxis is the result of orientation. It does not necessarily follow, however, that orien- tation invariably results in phototaxis. In fact Dearborn (’oo) found that crayfishes would orient to an electric light introduced into the water near them without making any considerable loco- motor movements in consequence.’ To the question of how orientation of organisms to light is caused, three possible explanations may be presented: 1, Chance result of photokinesis; 2, reflex response to directive stimuli; 3, voluntary action. Since the first hypothesis seems entirely inadequate to account for the uniformity of orientation in pla- narians, and the third alternative is out of the question with refer- ence to these animals, a consideration of the reflex responses to directive stimuli may be taken up at once. There are two general theories which attempt to explain the way in which orientation occurs through reflex responses to stimull. ‘These theories are first, the trial and error theory of Jennings and Holmes, and secondly, the tropism theory of Verworn and Loeb. By the trial and error theory orientation, with its consequent pho- totaxis, 1s interpreted as the result of repeated attempts on the part of an organism to become adjusted to any given stimulus. Those attempts which fail to result in adjustment to the stimulus are “errors,” and as such are followed by other attempts until finally some one secures the necessary adjustment. ‘Trials of this kind may be made in different ways according to the organism 7 Throughout the following discussion orientation will be understood as a position assumed with refer- ence to the light while phototaxis will be made to include motion toward or away from the source of the light. 148 Herbert Eugene Walter in question. Among the infusoria and rotifera, as Jennings has shown in a masterly series of papers,® such attempts at orientation are made by means of a “ motor reflex,” consisting in (1) a sudden withdrawal from the stimulus, (2) a rotation toward a structurally defined side of the asymmetrical organism, and (3), lastly, an advance in a new direction. In the case of organisms which do not possess marked asym- metry the trial and error method, as pointed out by Holmes (’o5a), resolves itself into a series of “‘random movements;”’ that is, a number of apparently experimental movements are made, which finally result in the best adjustment to the stimulus. In both of these methods the organism acts as a unit and not in response to localized stimulation received asymmetrically. The tropism theory, on the contrary, 1s based upon asymmetrical action as the result of asymmetrical stimulation. If an organism receives a stronger stimulus on one side of its body than on the other, the result, whether direct or indirect, is that it moves in such a way that this asymmetrical stimulation becomes symmetrical. In other words, orientation occurs. It is unfortunate that the tropism theory was made to apply to the behavior of the infusoria, since it has been shown beyond doubt by Jennings that exact observation of the behavior of these organisms and an analysis of its details does not admit of the tropic interpretation, but is, on the other hand, explained by the trial and error theory of motor reflexes. It is also to be regretted that the unquestionable rout of the tropism theory, as applied to cer- tain protozoa and other asymmetrical forms, should have led to an attempt to exclude it from the remainder of the animal king- dom. In a paper on the tropism theory Jennings (’o4a) names as an essential criterion of tropism the direct unilateral stimulation of the motor organs. After showing how inadequate such an assump- tion is to explain the orientation of animals, particularly that of Infusoria, he continues (’o4a, p. 104), “We should perhaps con- ® See bibliography in Contributions to the Study of the Behavior of Lower Organisms. Carnegie Inst. of Washington. Publication No. 16. 256 pp. 1904. The Reactions of Planarians to Light 149 sider here a modification of the original form of the tropism theory that has been proposed by some authors. ‘This is in regard to the assumption that the stimulating agent acts directly on the motor organs upon which it impinges. For this it is sometimes proposed to substitute the view that the action of the stimulating agent is directly on the sense organs of the side on which the stim- ulus impinges and only indirectly on the motor organs through their nervous connection with the sense organs. When thus modi- fied the theory of course loses its simplicity and its direct explain- ing power, which made it so attractive. In order to retain any of its value for explaining the movements of organisms, it would have to hold at least that the connections between the sense organs and the motor organs are of a perfectly definite character so that when a certain sense organ is stimulated a certain motor organ moves in a certain way. When we find, as we do in the flatworm (see the following paper), that to the same stimulus on the same part of the body, under the same external conditions the animal reacts sometimes in one way, sometimes in another, the tropism theory, of course, fails to supply a determining factor for the behavior.” It seems to me that the mechanism by means of which the asymmetrical response is brought about is immaterial, so long as that response can be shown to be the result of asymmetrical stimu- lation. Asymmetrical response might occur either from direct stimulation of the motor organs as was implied in the earlier papers on the infusoria, or by means of a more complex method, consisting of stimulation of the sense organ, transmission to the central nervous system and thence to the motor organs. The outcome in either case would fulfill the demands of the trop- ism theory, if asymmetrical response to asymmetrical stimulation be taken as its criterion. In the quotation just cited, the objection that such transmission compels stereotyped behavior is hardly valid, since stereotyped reaction is by no means the only alterna- tive of asymmetrical stimulation. ‘That flatworms do not respond uniformly to directive stimuli cannot be disputed, but that fact does not exclude the possibility of all tropic reaction on their part. The imperfection of response may be simply the result of imper- 150 Herbert Eugene Walter fections in the worm’s nervous circuit, assuming that planarian reactions are due to indirect rather than to direct stimulation of the motor organs. In fact, repeated evidence of the failure of a constant and perfectly invariable orientation on the part of pla- narians has been given in the preceding pages. Such failure, moreover, is quite as likely to occur in the application of the trop- ism theory to behavior as it is in the case of the trial and error theory, since stereotyped reactions and forced movements, as Holmes (05a, p. 112) has emphasized, are no more characteristic of tropisms, which depend upon a differentiated stimulation and response, than they are of trial and error movements, resulting from a single motor reflex given in response to all kinds of stimu- lation. Furthermore, it has been urged that tropism indicates a simpler form of reaction than trial and error for the reason that it in- volves only a local part of an organism while the motor reflex of trial and error requires that the organism act asa whole. Conse- quently, since motor reflex has been indisputably demonstrated as the method of infusorian phototaxis, Jennings (’04a, p. 95) asks, “Should we conclude that the reactions in the higher metazoa are simpler and less unified than in the protozoa f” That the motor reflex, which occurs with machine-like uni- formity, regardless of the point where the stimulation is received, is more complex in character than the stimulation of an asym- metrical part of an organism which may depend for its response upon sense-organ, nervous transmission and motor apparatus is an assumption difficult to sustain. It seems more reasonable to agree with Harper (05) in placing tropism higher in the evolu- tionary scale than trial and error. The fallacy that “tropism leads nowhere; it is a fixed final thing like a crystal’ (Jennings, ’o4c, p. 251), while trial and error alone offers possibilities of the higher evolution of phototaxis, has already been answered by Holmes, who points out that trial and error, at least that phase of trial and error depending upon motor reflex, is even more fixed and stereotyped than the reactions occurring in accordance with the tropism schema. To quote: “The end result of both methods is the same, 7. ¢., to get the organism away The Reactions of Planarians to Light 151 from the stimulus. In the one case it is accomplished by direct reflex without more ado; in the other, only after a considerable waste of energy in inconsequential vermiculations” (Holmes, "05a, p- 110). It is at least conceivable that under the tropism schema, as the nervous differentiation of an animal becomes more complete, the ability of the organism to interfere with and modify its machine- like responses to external stimuli might also increase, resulting in a flexibility of behavior which would present quite as much variation for natural selection to act upon as that evolved by the trial and error method. ‘This point of view by no means denies that trial and error is the usual “method of intelligence” (C. L. Morgan ’o0, p. 139). It is simply an attempt to recognize in the method of tropism also one of the possibilities of evolutionary progress in behavior and as such holding a higher position in the scale of evolutionary methods than trial and error by motor reflexes, It has been shown (p. 143) that planarian responses of an appar- ently asymmetrical character may occur as a result of symmetri- cal stimulation. Similar instances in the case of planarians have also been demonstrated by Mast (’03) with reference to thermal stimuli. This, however, is no exception to the validity of the tro- pism theory, in which asymmetrical responses result from asym- metrical stimulation. Because a planarian may make an appar- ently phototropic response when subjected to symmetrical stim- ulation, is not evidence against the supposition that the usual phototropic response is due to asymmetrical stimulation. The “wigwag’’ movements of planarians, to which repeated reference has been made in the preceding pages, resemble super- ficially the “random movements” of the earthworm as described by Holmes. They do not, however, ordinarily appear to be the basis of trial and error selection resulting in orientation, since in a majority of cases, after a worm Hales and makes wigwag movements it continues on its way without a change of direction. The movements of Bdelloura candida, as shown in Fig. 12 form an exception to ordinary planarian behavior in this respect As a rule wigwag movements are probably occasioned by a general Eeambence arising from some stimulation which thowrs 152 Herbert Eugene Walter the worm into a different physiological state. Exploring move- ments, such as these seem to be, may bring about asymmetrical stimulation, in which case the worm makes a tropic response. It was particularly noticed that when planarians received light from below, the anterior end of the body was frequently tilted back and forth as if to make it possible for the light when coming from such an unusual direction to enter the pigment cups of the eyes. The phenomenon suggested the craning of necks and bob- bing of heads among a crowd of people who are all trying to see the same object at once. Wigwag movements seem to be oftener connected with changes in the intensity of light than with changes in its direction. When the latter occur, tropic response is immediately the result. In the course of the experiments previously described wherein the worms glided from an area of one intensity of non-directive light into another it was noticed that in a majority of cases when the critical line was not crossed at right angles, no change in course occurred, even when the worm halted and made wigwag move- ments. Of course at a certain instant of any diagonal crossing of the critical line one eye must receive more stimulus than the other, in which case according to an inflexible tropism theory asymmetrical response ought to occur. But such a response does not frequently appear aa the reason for this becomes clear when it is remembered that a considerable number of responses were shown to occur which were called “latent wigwags”’ (Fig. 4, £), be- cause they failed to make their appearance until in some instances the worm had passed more than the length of its body beyond the critical line. Since, therefore, latency of response to intensity is by no means uncommon, it is evident that the brief interval of asymmetrical stimulation occurring when a worm glides diagonally into an area of different intensity is not sufficient to result in an asymmetrical response. Two conclusions, then, seem reasonable, namely, that phototaxis as related to planarians is primarily due to asymmetrical response resulting from asymmetrical stimulation, and that wigwag move- ments, together with similar apparent trial and error forms of behavior, contribute chiefly to this end, 7. ¢., to phototaxis. The Reactions of Planarians to Light 153 Summary. Orientation may occur without phototaxis. Two theories have been advanced to explain orientation and phototaxis in lower organisms, namely, the trial and error theory and that of the tropisms. “The former may be based upon “‘ motor reflexes” or upon “random movements” according to the sym- metry of the animal. The tropism theory rests upon asymmetrical response to asym- metrical stimulation. It does not necessarily depend upon the direct stimulation of the motor organs, nor is it essentially stereo- typed in its character any more than are trial and error responses by motor reflex or random movements. The tropic form of response may, and probably does, require a more complex mechanism than that which causes the motor reflex, consequently it is the form of response to be logically expected among planarians, since the motor reflex has been proven to be the form utilized by the protozoa. Tropisms, as well as trial and error movements, provide, through the modifying control of an evolving central nervous system, sufh- cient latitude of variation for natural selection to work upon in the evolution of higher forms of behavior. Asymmetrical response may, in certain cases, result from sym- metrical stimulation, but ordinarily its cause is asymmetrical stimulation. Wigwag movements are occasioned most frequently by changes in intensity, and they may result in orientation and phototaxis by assisting an organism to secure asymmetrical stimulation. Latency of reaction accounts for some of the failures in orien- tation which often occur even when asymmetrical stimulation is acting upon an organism. Finally, the orientation and phototaxis of planarians is more consistently explained by the theory of tropisms than by the theory of trial and error. 3. ADAPTATION. It remains, finally, to inquire how far the reactions of planarians to light are adaptive; that is, how far the response to light is “of 154 Herbert Eugene Walter such a kind that it better insures the existence of the individual, or of the race” (T. H. Morgan ’o03, p. 1). It is evident that the generally negative character of the reac- tions of planarians to light indicates a tendency on the part of these worms to reduce as much as possible the amount of light stimulation received or to avoid it altogether. The rigor effects of excessive stimulation furnish evidence also that light is a factor in a planarian’s environment which it finds unavoidable and unwelcome and to which it is adapted only in a negative fashion. In fact the vague distinction separating “lower” from “higher” animals consists largely in the ability of higher animals to assume an active aggressive rather than a passive defensive relation toward the factors making up their environment. For example, the evolution in animals of the visual organs, which in the planarians is only inceptive, enlarges the possible range of photic responses until light becomes an essential factor in an animal’s environment, contributing largely to its welfare by enabling it to see its food, to avoid its enemies and to select its mates. It is plain that light plays no such important part in the activities of planarians, for, as has already been pointed out, light per se is not essential to pla- narians, since they are known to ihe successfully in dark caves. Moreover, so far as known, light does not influence the regenera- tive or reproductive processes of planarians in any way whatso- ever. The formation of pigment may perhaps be regarded as an adaptation to light conditions, inasmuch as anna possessing pigment are thereby shielded to a certain degree from excessive stimulation. With reference to activities connected with nutrition and repro- duction, planarians are not dependent upon light stimulation. They are otherwise equipped, since they doubtless find their food by chemotactic means and avoid whatever enemies they may have, not aggressiv ely nor activ ely by retreating from visible foes but malen in a passive way by remaining Concealed from enemies that might see them. They have no organs of defense but survive by escap- ing attention. In this sense their negative phototaxis may be regarded as of protective value and consequently adaptive. Furthermore, the geographical distribution of fresh water pla- The Reactions of Planarians to Light 155 narians has been shown by Borelli (’93) and Wilhelmi (’04) to be chiefly dependent upon temperature and almost not at all upon the amount of illumination to which they are subjected. Voigt (’04) noticed that worms when hungry may be seen wandering about even in patches of bright sunlight with apparent disregard of light. This seems to be a case of the light reactions becoming over- balanced by other responses. Summary. Light is not an essential factor’ in planarian activities, since the behavior necessary to the welfare of the individual and the race is mainly referable to other factors. A planarian’s response to light is of a passive character, which may have an adaptive significance only in so far as its phototaxis tends to conceal the worm from its enemies. The presence of pigment may also be regarded as an adaptive condition induced by the animal’s relation to light. 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REGENERATION IN COMPOUND EYES OF CRUSTACEA BY MARY ISABELLE STEELE, M.A. Witu Sixteen Pirates AND Two Ficures IN THE TExT SRM TEC OCILICESO MI cice svete ec iozarai ores eras teysle ate royens Tenctesel sve ie sie sc\siers, oe stay 0ia,oye0u oh, arare/are\s,\ur0 ofove\aseisherslens 164 MibmmIVeth ods andtmaterial ses sctra cen cicrea ns etoietelsievers Totelefer cece sete 2's. ohclelovesc nrcis, yeloioieteretelsies eloierele 165 iMit 'iheagaenlcuhi Ge Pes sas aoecpacpede aso conan ad soccer e EEE EC erOE Seo cuSadaneaneacEs 168 IV The preliminary regenerative processes...... 26200. 2. 0 cee sence ese ete ese c teers eee ees 172 A Healing of the wound and formation of new cuticle.............-.--.0.00-02.0-055 173 Bae Removaljotthenny ured tissue cjasiajis rete aie\a/aicisvace <ratesares ses aiata atagstolecovafafcvatcrmiagaate/ starerayere 179 CmiMethodloficellidiysistomt terete retcts ates sectors sever sieves sheieteleye ater relia aiciatelsiexstateve/seievatescta (ao 183 V_ Regeneration occurring after destruction of upper part of eye ........- 0-000. eee eee eee 185 Ac Repenerationlofifinctional eyez ioi.)..c/- o)o/~\+ 101 55+ (0/2 e1e\-1210.vipie nhs e}sinseiels ee [esereieiciessisisiotsiaie/ aie 185 meme epeneratinpieye trom Suita ce PLOW ate tere alm ayate lore <1 2.51= beta ials\cletet-i-.s erepasiieioresetete 186 2 Details of the development of regenerating ommatidia.....................-. 190 a) regeneration ofiretinulee = <2. <eteeine1s ec sie ences = Seed cron Sats tee ee 190 6) WRegeneration‘oficrystalline cOneS s<.s\e:ci ersie «ccs misug vicieie /aicieiels a. tieyainte/aiete 192 c Regeneration of rhabdoms 3 Comparison of ommatidia in regeneration and ontogeny...........-.--.-..-.- 4 Differences in the regeneration of eyes among Palemonetes Crangon and Grn Gard Sxpppba0 550 odgnd0 odup so bodosuocdennsccptudasonsudoa 199 5 Comparison of normal and regenerating eye...... 6.6 +0 eee eee erence ees 202 B Cases of aberrant regeneration of ommatidia .......5.2+.c0serececscecsercercecses 203 C_ Eye stumps that show an abnormal development or no regeneration................- 210 1 /Abnormalidevelopment of pigment. oi.0% 5.6.0.5 1c o.2csefsinie)ciere t= sjensjals oe nie oie ele 210 2 Eye stumps that show no regeneration.......-.----+--++.s+5+ APopabteean 2s 207) VI Regeneration after removal of the yreater part or all of the optic ganglion................ 219 PACMEL ELEC crabs tery steerer terieteraioicielotateieie ares. stevessisye.evslelsreysTeceuegsislelsyetatetetaictcterfaqepere fate ere 219 1 Regeneration of heteromorphic appendages ........- +--+ -00+02+e ees seen 219 2 Cases that show no special regeneration...........+-6+2+-ce erect sees ences 221 1D (Gea Forinsgaosene oo Cedbad oA aero Rb Ses cen oUbUrE eae cro parties coger onaar honmaan 223 1 Regeneration of heteromorphic appendages. .........-. 060004 e eee eee ee eens 223 2 Cases that show no special regeneration......... 0.0.8 eneerer eects tee eee es 223 @ Trib moncicsysdosoondapasaandn onouncotepseaan cde cdsnes SoeEEAoeenpeacs onan 225 D Histology of the heteromorphic appendages...0....-.. +++ 2020520 sees eee eee eens 227 E General consideration of the regeneration following removal of entire eye........-..-- 230 VIE Regeneration after splitting the eye longitudinally......... 26.60.0600 eee e eee eee eee eee 237 Wilt SummetyApadechcncenecusdvoncbubobordsdsbadco6d kann sno Rssaanedeor ooeaEr Sr eSooa: 238 TDG EET B00 6d denned dup pokdesueuUDABCOeesnaaocur cabsemapgnoeEb concen Sanmodeon 241 xylan tomomplatesntdtelci« sietstercisiere ojecorersis ciate > ete eyeieljs.o sjoim Aiaaisieie) s/s cfm ie/njelsinis[n elelaielais 243 Tue Journat or ExreriMENTAL ZOOLOGY, VOL. V, NO. 2. 164 Mary Isabelle Steele I INTRODUCTION The regeneration of the Crustacean compound eye has been made a subject of observation by a number of investigators. But for the most part end results alone have been described. Very few details of the processes involved in regeneration have been given. ‘To give an accurate description of the histogenesis of the regenerated structures is the chief aim of this paper in the belief that it will contribute something toward a more accurate under- standing of the more general problem of regeneration. The problem has separated itself into three main divisions: first, the regeneration of a functional eye; second, a search for the causes of no regeneration and abnormal regeneration, and third, observations upon the heteromorphic regeneration which may follow the removal of the entire eye. Herbst (’96, 00) and Morgan (’98) have made the principal observations upon the regeneration of the Crustacean compound eye. But so far as the particular phases dealt with in this paper are concerned Herbst’s descriptions are not sufficiently detailed to be of especial assistance in this work. Herbst’s observations upon the regenerated heteromorphic structures are somewhat more extensive and in some respects furnish an excellent basis for comparison with the results to be discussed in this paper. For the most part, however, where results have been compared with the work of others the comparison is made between the regenera- tive and embryonic development and between normal and regen- erated structures. The terminology employed is in great part that used by Parker (91) in his work upon the compound eyes of Crustacea, it being the terminology in most general use. A few minor deviations have been made but no wholly new terms have been introduced. The following plan has been followed in this paper. First, a brief description of the normal adult eye is given to furnish a basis for comparison. ‘This is followed by a description of the prepara- tory regenerative processes which in reality constitutes one of the most important phases of the subject. Then the regenerative processes proper are described under the three main divisions already suggested. Regeneration in Compound Eyes of Crustacea 165 The experimental part of the work was begun at the University of Pennsylvania. Observations upon living material were also carried on during two summers at Woods Hole, Mass., and one summer at Cold Spring Harbor, L. I. The greater part of the detail work of examining the preserved material has been done at the University of Missouri during the present year. In closing I wish to express my thanks to Dr. E. F. Phillips and Dr. D. B. Casteel for the care of experiments; to Dr. E. G. Conklin, University of Pennsylvania; Dr. C. B. Davenport, Carnegie Institute at Cold Spring Harbor, and Dr. George Lefevre and Dr. W. G. Curtis, of the University of Missouri, for their interest and valuable suggestions during the course of the work. II Marerrat anp Metuops The small hermit crab, Eupagurus longicarpus, the common shrimp, Palzmonetes vulgaris, and the sand shrimp, Crangon vulgaris, afford the greater part of the material used in the series of experiments to be described in this paper. For comparison two species of crayfish, Cambarus virilis and C. gracilis, a species of fresh water Ascellus, the common (wood-louse), Oniscus, and the fresh water Gammarus were used. Other emacs also were experimented upon but since no decisive results were obtained they need not be considered here. The work has been confined chiefly to the eyes of the forms used although experiments upon the appendages, particularly the . antennz, were conducted at thesame time. “These were, however, largely for the purpose of comparing relative rates of regeneration of the different parts, especially the rate of regeneration of the appendages as compared with that of the eyes. The experiments upon the eyes consisted in either the removal of a part of the eye or of the whole eye. ‘The part removed varied greatly in the different series of experiments and more or less in individuals of the same series. A limited number of experiments upon Palzemonetes included the removal of both eyes or the removal of one eye with a part of the brain; these operations in most cases resulted fatally. The effect of splitting the eye was 166 Mary Isabelle Steele also tried upon two series of Palamonetes, each series was com- posed of a considerable number of individuals. Results, however, were not particularly different from those obtained after removing a part of the eye. A total of 600 Palamonetes and hermit crabs had either one or both eyes operated upon. A much smaller number of Crangon and crayfish were used. No accurate account of the Ascellus, Oniscus and Gammarus was kept. More than 50 per cent of the Palamonetes and hermit crabs died immediately, or within a short time, after the operation, many of them dying within a few minutes after the eye was injured. Of the survivors about 58 per cent lived through one or more moults. Forty-two Crangon had the eye operated upon and of these one died of the operation. The crayfish used were for the most part C. gracilis, measuring from 12 to 15 mm. in length, that had moulted but once after hatching. Palamonetes and Crangon moult once about every ten days to three weeks. The hermit crabs moult much less frequently, often but once in two or three months. The hermit crabs regenerate, however, as rapidly as Palemonetes or Crangon. Considerable difficulty was experienced in keeping the animals alive and in keeping individual records. Finally the plan of keeping each animal in a separate finger bowl was adopted. This method was fairly satisfactory except in very warm weather. Then the water became warm and unless it was changed often the animals soon died. Chopped-up bits of clam were fed to them two or three times a week. Great care had to be used in warm weather for the water became foul, if the food was left in more than three or four hours, and caused the death of the animals. In spite of all precautions various accidents occurred which resulted in the death of promising material. Twice attempts were made to transfer the experiments from Philadelphia to Woods Hole or vice versa with disastrous results in each case. The failure was due in part no doubt to the extreme warm weather. For although every known precaution was taken most of the animals died within twenty-four hours. A number of the ordinary fixing fluids were used to preserve Regeneration in Compound Eyes of Crustacea 167 material. Among those most frequently used were Fleming’s osmic fixative, Perenyi’s fluid, Kleinenberg’s picro-sulphuric, picro-aceto-sulphuric, Petrunkewitsch’s fluid and alcohol acetic. Other fluids also were used and boiling water was tried. ‘The best results were obtained from picro-aceto-sulphuric and Perenyi’s fluid. In any case it is difficult to obtain a fixative that does not shrink the inner tissues from the chitin, the regenerated tissues being much more easily affected in this respect than normal tissues. The embedding was done altogether in hard parafiine 54° to 58° C. melting point. It was necessary to embed the material for a long time in order to cut it without tearing the chitin from the softer tissues. [he most satisfactory infiltration was obtained by placing the objects first in equal parts of oil and paraffine, leaving them on top of the water bath over night and then replacing the oil and paraffine with pure parafiine, leaving them on top of the water bath from eight to ten hours longer. Finally they were put in the parafiine bath from one to two hours. Even after the most thorough infiltration it was well nigh impossible to obtain complete series of good sections, because of the difficulty of cutting through the different textures of the material. In dehydrating preparatory to embedding, cedar or bergamot oil was usedin preference to xylol as these oils made the tissues less brittle. The chief stains employed were Fleming’s triple stain and Heidenhain’s iron hematoxylin. Various counter stains were used with the iron hematoxylin but the most generally satisfactory were acid fuchsin and orange G. In most instances no attempt was made either to soften the chitin or to remove the pigment before the eyes were sectioned. As a rule, however, the material was fixed shortly after a moult so that the chitin was as soft as could be obtained. Any sort of a reagent used to soften the chitin seemed to be more or less inju- rious to the softer parts, particularly the regenerating tissue. When the chitin was removed, however, 2 per cent nitric acid in 70 per cent alcohol was found to be most satisfactory. It was generally disadvantageous to remove the pigment because this destroyed many of the landmarks both in respect to the regener- 168 Mary Isabelle Steele ating tissue and the condition of the remaining old parts. Sections were sometimes depigmented on the slide. For this Mayer’s chlorine method was used. Table I gives in brief a record of the individuals operated upon, the character of the operation and the end results. III Tur Normat Aputt EYE Before taking up the discussion of the regeneration of the eye it will be perhaps well to give a brief description of the structure of Text Fig. 1 The lines, ab, a-c, a-d, a-e, a-f, a-~g and a-h represent approximately the different levels at which a part or whole of the eye has been removed. When the cut was at level as low as a-e a part of the optic ganglion was usually involved. In Palemonetes the eye never regenerated when the cut came as low as the level a-e. Hermit crabs may regenerate an eye from the level a-f and a hetero- morphic appendage from the level a-h. the normal eye. The nature and extent of the operations and the subsequent changes in the eyes will then be more easily under- stood. The eye of Palamonetes has been concisely described by Parker (’97) and the following description is adapted from his. It is to be noted that the eye structure of the three forms experi- mented upon is practically the same. Regeneration in Compound Eyes of Crustacea 169 TABLE I. PALAEMONETES | No. of | Died Lived | Showed Series | individ- Operation | Date from and _regenera- | uals | operation| moulted | tion I 16 part of cornea removed May 10 12 4 ° TS || £2. part of cornea removed july II 8 bs I Ti 38 part of cornea removed July 20 29 3 | 8 IV 40 part of cornea removed July 28 28 II 8 Vv 15 part of cornea removed July 30 3 12 ° VI 16 part of cornea removed August I 12 z: I VII 15__| partof cornea removed August a 10 2 3 Vill 12 part of cornea removed November 5 5 5 ° Ix ez 7am part of cornea removed | November 9 8 9 ° ibe ||) 4G entire cornea removed | March 10 4 7 ° |) 38 entire cornea removed July 20 8 9 I Ty, || 30 entire cornea removed July 29 28 2) ° v | 30 entire cornea removed July 30 20 9 ° VI 13 entire cornea removed August 4 8 5 ° VII 12 entire cornea removed December 30 II I ° entire eye removed | | I 12 both eyes | January I 4 6 ° II 12 entire eye | March 5 9 Hie | ° 100% 1) ae both eyes March 5 12 On eo DV; 18 both eyes March 10 10 5 ° Vv 45 entire eye April 19 12 20 ° I 20 eye split March 5 5 12 3 I 28 eye split May 24 nO) Ee | ° | HERMIT CRABS ee | ara, | cornea removed March 25 2 8 | 3 I 12 | cornearemoved May 26 2 7 | 2 TIL 25 | cornearemoved July 9 2) eer O nel em nis I 15 entire eye removed | May 26 I | 72.) | I I 25 entire eye removed July 9 | Aan era 9 Ut 8 entire eye removed October 16 oe || fo) IV 12 entire eye removed November 27 | 2 6 | ) CRANGON Ty) 20 part of cornea removed August 4 | ° 19 10 rE 22 eye removed August 4 | I 19 I Note.—Removing the entire corneal portion of any of the forms was always accompanied by the removal of part of the optic ganglion, In many cases as much as half of it. It frequently resulted that all of the corneal portion subsequently degenerated after a part of it had been removed, and it also frequently happened that the removal of the entire corneal portion resulted in the loss of the whole eye stalk, consequently the above table can be used only as a very general indication of the character of the operation. 170 Mary Isabelle Steele The compound eye may be regarded as that part of the optic apparatus contained in the eye stalk. It consists of a large num- ber of ommatidia occupying the distal end of the stalk and a series of four ganglia which extend through the axial portion of the stalk. This series of ganglia for present purposes may be regarded as a compound ganglion composed of four rather distinct sections or ganglionic masses united to each other by nerve fibers. The ommatidia are connected with the distal section of the optic ganglion by the retinular nerve fibers. ‘The optic nerve passes inward from the proximal section of the ganglion to unite the eye with the brain. “The basement membrane forms a sort of partition between the ommatidia and the optic ganglion. ‘The transparent chitinous covering over the ommatidial region is known as the cornea. Each ommatidium is composed of the following cells: two cor- neal hypodermal cells, four cone cells, two distal retinular cells, eight proximal retinular cells one of which is rudimentary, and a variable but small number of accessory pigment cells. Black pigment granules are contained in both proximal and distal retinule and are found only in these cells. “Uhe yellowish pigment is confined exclusively to the accessory pigment cells. “The differ- ent cells enumerated above give rise to the structures that consti- tute a complete ommatidium. ‘The two corneal hypodermal cells secrete the square corneal facet which covers the outer surface of the ommatidium. Immediately beneath the corneal hypodermal cells is the crystalline cone formed by the four cone cells. The nuclei of these cells are located in their distal ends. ‘The main body of the cone appears as a dense hyaline secretion. Proximally the cone is less dense in structure and tapers to a slender stalk lying between the coneandrhabdom. ‘The rhabdom, according to my observations, is a swollen spindle-shaped structure proximal to the inner ends of the cone cells. ‘The distal retinular cells lie near the inner end of the cone; the proximal retinule surround the distal end of the rhabdom. ‘The proximal processes of the retin- ulz extend over the rhabdom and pass through the basement membrane as the retinular nerve fibers to enter the optic ganglion below. The accessory pigment cells lie both above and below the basement membrane. Regeneration in Compound Eyes of Crustacea 7 Before closing the description of the normal eye mention should be made of another point. In young individuals in each of the species examined in this series of experiments there is present a growth zone in the ommatidial region. From this zone the num- ber of ommatidia is increased as the animal grows older and increases in size. In longitudinal sections cut in a horizontal plane this zone is apparent on the inner edge of the eye as a narrow band of elongated cells situated above the basement membrane and between it and the completely developed ommatidia. In some instances partially differentiated ommatidia can be recog- nized in this growth zone. This zone has been mentioned by Parker and others. It is briefly described in this connection now because it is probable that in some instances ommatidia that have apparently regenerated have in reality developed from the growth zone. The terminology used in the above description and in the dis- cussion of the regenerating eye is that used by Parker. ‘The series of optic ganglia described by him as occupying the eye stalk, however, have been referred to in this paper as different divisions of a single ganglion, it being thought that the matter could be treated with less confusion in this way. Also, the distal portion of the eye stalk, called the retina by Parker, is referred to here as the ommatidial portion. It is composed of a large number of individual ommatidia and the more general use of the term, retina, does not imply all the structures composing the ommatidia. The structure of the rhabdom as described in the present paper is not in full accord with Parker’s description. While the above description applies especially to Palazmonetes it is sufficiently accurate for the other forms described in this work to serve all purposes. The most marked differences in the struc- tures of the eye of Palamonetes and of the other forms used are as follows: In Crangon and in crayfish the eye stalk is much shorter and proportionately greater in diameter than in Palamonetes. Also, in Crangon some small glands are found located just below the basement membrane. Hermit crabs have long slender eye stalks similar to those of Palamonetes except that at the base and occupying the dorsal inner edge there is a small pointed squame 172 Mary Tsabelle Steele bearing a number of sensory hairs. (Fig. 37, 0-5q.) And finally, there are no accessory pigment cells in the hermit crab’s eye. These cells are very conspicuous in the eyes of Palamonetes. IV THe PRELIMINARY REGENERATIVE PROCESS The preparatory stages leading to the regeneration of an eye stump consist chiefly of the following processes; the healing of the wound, the removal of the injured tissues and the active prolifera- tion of new cells of a comparatively undifferentiated character. In removing any part of the eye the injury to the remaining soft, inner tissues is considerable. Especially is this true when the cut passes through the ommatidial region. Much of the tissue sur- rounding the wound is crushed and torn out of place. On this account the process of healing over the cut surface is much more difficult to follow than the healing of the wound after an antenna or leg has been removed. Before taking up the description of the preliminary regenerative process it will be perhaps of interest to give in brief the immediate effects of the operation. The death of the animal which so fre- quently follows close upon the operation seems often to be due chiefly to nervous shock. It cannot be caused by loss of blood alone for usually there is no profuse bleeding. When the eye of Palzmonetes is operated upon the animal often turns over and over ten to forty times as soon as it is released and returned to the water. Many of the animals die before they succeed in nghting themselves. Others lie upon their sides several hours after they have ceased revolving and die without showing any normal activi- ties or regaining their equilibrium. It very seldom happens that animals which whirl over and over many times after the operation ever recover from its immediate effects. “These apparently help- less motions indicate that the operation has caused the loss of equilibrium. Crayfish sometimes exhibit these uncontrolled whirling move- ments. Similar movements are also noted in fresh-water Gam- marus and in Ascellus. In Gammarus and Ascellus these move- ments are executed after the removal of the antennz or some of \ Regeneration in Compound Eyes of Crustacea 173 the mouth parts. This shows that the effects are not specific- ally connected with operations upon the eye. Whatever its ini- tial cause the effect is transmitted to the whole nervous system. In many cases the operation seemed to affect the animal more seriously when only the upper part of the eye was removed than when the entire eye was cut off. Frequently the relative number of survivors was greater in the latter than in the former case. In other instances the animal did not seem greatly affected by the operation regardless of whether the whole or a part of the eye was removed. (See Table I.) The immediate visible effects upon the eye may be briefly described as follows. As soon as any part of the corneal covering is removed or even as soon as a rent is made in it a considerable amount of the soft, viscous, inner tissue flows out through the opening. It is perhaps carried out by the escaping blood. Much of the pigmented, retinular tissue seems to escape, perhaps because it is softer and more viscous than the other tissues. After an hour or so the remaining, inner tissues are seen bulging out and above the general level of the surface. This is probably on account of the destruction of the normal tension of the tissues due to the changed pressure conditions at the wounded surface. A similar appearance is obtained when the surface injury consists of a rent torn in the cornea with a needle. A HEALING OF THE WOUND AND FORMATION OF NEW CUTICLE In a few hours after the operation most of the pigmented tissues have disappeared from the surface of the wound and the swollen surface takes on a whitish appearance. ‘This white swollen surface is apparent for several days. Not until the fourth or fifth day 1s there any sign of the characteristic red-brown crust which generally forms over wounds in Crustacea. Sections of an eye fixed six and a half hours after the operation show no definite indications of the healing of the wound. A great deal of the broken and mangled tissue lies outside the wound and hanging to the cuticle about its edges. Inside, the tissues are twisted and misshapen. At the edges of the wound there are 174 Mary Isabelle Steele slight indications that it is preparing to close over. But the quantity of material lying inside and out makes it impossible to determine what tissues are taking part in closing the wound. During the next twelve or fifteen hours the changes are still not clearly defined. ‘The interior still presents a rather badly confused mass of injured tissue. Near the edges of the wound, however, there are evidences that the hypodermis has begun to push outward to cover the cut surface. For the most part ‘the wound shows a smooth even surface which indicates that the passage outward of the injured tissues has ceased and that a sort of equilibrium had been established. The mass of tissue closing the cut seems to be made up of a few hypodermal cells and cytoplasmic strands, a considerable accumulation of blood cells and the nuclei of the breaking down tissues of the eye. Around the edges of the cut occasional strands of hypodermis with a very few nuclei can be distinguished. Sections of an eye fixed about forty hours after the operation show the beginning of crust formation. Almost the whole surface has been covered. Judging by the reaction to stains, the part which may be considered the matrix of the crust is formed by an attenuated, chitinous secretion of the hypodermis. In this matrix are embedded numerous nuclei of the injured tissues together with a great many blood cells and a few hypodermal cells. In some parts the crust is sharply marked off from the underlying tissues by a space filled with coagulated plasma. Over one part of the wound the crust is not yet fully formed. At this point hypodermal strands containing elongated flattened nuclei are seen stretched across the space still uncovered. The strands appear in two or three layers with very few nuclei in each layer. No definite centers of cell proliferation can be recognized at this time. After the crust has covered the cut surface it continues to increase in thickness for two or three days, then hardens, turns a bright reddish brown color and remains over the stump until a moult has occurred. ‘The crust takes no further active part in the healing and regenerative processes. Fig. 46 represents in a semidia- grammatic manner the crust formed over the wound in a crayfish eye about sixty hours after the operation. ‘The crust is continu- i Regeneration in Compound Eyes of Crustacea 175 ous with the inner surface of the cuticle covering the rest of the eye. There are still masses of the injured inner tissues that have been excluded by the formation of the crust clinging to its outer surface. ‘The old tissues in the interior of the eye stump have shrunk back from the crust leaving a considerable space occupied by coagulated plasma. ‘The old cuticle and the matrix of the crust both stain deeply either with orange G or acid fuchsin. No distinct cuticle can be recognized for several days, from six to eight, after the operation. In the eyes of Palamonetes that have moulted seven or eight days after the infliction of the injury a cuticle which corresponds approximately in thickness with the cuticle covering the remainder of the eye has formed over the wound. ‘This new.cuticle is much looser in texture than the old cuticle. Regeneration does not take place so rapidly in crayfish as in the marine forms examined so that a new cuticle is somewhat longer in forming. Frequently a considerable space intervenes between the overlying crust and the cuticle which has formed beneath it. This is probably due to the recovery of the tissues of the stump from their early swollen condition during which time they were gorged with blood and occupied more than their normal amount of space. It is not unusual to find considerable spaces between different layers of the new cuticle as if a shrinking of the tissues had taken place during the process of forming the new layers of cuticle. The shrinking of the interior tissues without doubt also accounts in part for the folds and wrinkles which often appear in the cuticle over the wound. The secretion of the new cuticle which grows over the wounded surface begins some little distance back from the cut edges of the old cuticle and is continuous with its inner layers. Fig. 47 1s a semidiagrammatic representation of the relation of the old and the new cuticle and the exclusion of the broken down tissue by the development of the new cuticle beneath it. Only eight nuclei appeared beneath the new cuticle in the section as shown in Fig. 47. This figure is taken from an eye that had been injured by tearing through the cuticle with a needle. Reference to the figure shows that very little of the old cuticle had been removed. Fig. 50 shows part of a section near the edge of the wound, Fig. 51 part of a 176 Mary Isabelle Steele section near the center of the wound. Both figures show the distribution of the hypodermal nuclei beneath the new cuticle that had formed over the cut surface of the eye of a Palamonetes ten days after the operation. Examination of Figs. 47, 51 will show that a new cuticle may be secreted before a complete hypodermis can be recognized. Recently hatched Cambarus gracilis, 12 to 15 mm. in length had eyes injured by tearing through the cornea with a sterilized needle. The eyes operated upon in this manner were fixed at different times, varying from eleven to thirty-five days. All of the eyes, however, were fixed before a moult had taken place. In this way it was possible to determine the precise position of the original injury. Sections of such eyes show one point conclusively, at least for Cambarus gracilis. ‘That is that the proliferation of new cells begins from the hypodermis immediately surrounding the rent. From the edges of this proliferating center new cells push out to replace the cells that were removed or that have broken down. Previous observations made upon the regeneration of the eye in crayfish (Steele ’04) indicate that crayfish probably do not regenerate a functional eye. It appears, however, that the prelim- inary regenerative processes are essentially the same in crayfish as in the other forms examined. It is frequently the case that a much greater proportion of the soft inner tissues are destroyed than of the outer cornea or the hypodermal layer beneath it. When the cut is made the retinule and the lower ends of the cone cells press out of the wound and leave the outer ends of the cones and the hypodermis practically undisturbed. Such a condition is particularly noticeable in eyes that were operated upon by tearing the cornea with a needle. In such cases the hypodermal cells 1m situ secrete the new cuticle. This cuticle is, however, without corneal facets, a fact which shows that while the operation neither removed nor caused the disintegration of the hypodermal cells it still affected their activity to such an extent that they no longer function in their usual specialized manner. ‘They now function as the ordinary hypo- dermal cells over the general surface of the body. Sections frequently show a morphological transformation of the Regeneration in Compound Eyes of Crustacea 177 Ps TEV corneal hypodermal cells in situ that are engaged in the secretion of the new cuticle. In the normal adult condition the pair of corneal hypodermal cells that belong to each ommatidium appears as much flattened cells crowded between the distal ends of the cones and the corneal facets. Their nuclei stain faintly and appear to be slender oval bodies lying flat against the cuticle. As the distal ends of the cones in an injured eye break down the nuclei of the corneal hypodermal cells enlarge, become rounded, stain deeply and in every way show signs of increased activity. Fig. 52 includes a series of figures showing the transformation of the corneal hypodermal nuclei into the larger, more deeply staining type seen in the regenerating eye. a and / inthis figure represent the corneal hypodermal cells as they appear in the normal omma- tidia. The other figures of this series, c,c’,d and e, show corneal hypodermal cells, belonging to ommatidia that have degenerated either wholly or paral oie c, c’, e the distal ends of “he cones still remain almost intact and in c the nuclei of the corneal hypo- dermal cells appear but little larger than those associated with nor- mal ommatidia. In c’ and e, however, the nuclei of the corneal hypodermal cells are much enlarged, stain deeply and the cyto- plasm surrounding them appears granular and loosely reticular. One nucleus to the left of the figure in d appears to be preparing to divide amitotically. That the nuclei shown in d are the trans- formed nuclei of the original corneal hypodermal cells is deter- mined by the fact that on either side of these nuclei are others still associated with partially disintegrated cones. Their original character is also suggested by the fact that they are grouped in pairs. A regenerated, rather than a transformed, hypodermis over the ommatidial region never shows the nuclei arranged in pairs in the early regenerative stages. “That the new cuticle has been secreted by ieee Paneionued hypodermal cells is shown by the relations of the two structures. The cytoplasmic strands of the hypodermis are continuous with the inner layers of the cuticle (Fig. 52d). Of course it is not absolutely proved that the transformed hypodermal cells take part in the later regenerative processes. This could not be done without examining a very great number of 178 Mary Isabelle Steele stages. But even the examination of a series of sections from the same eye will show that the indications are strongly in favor of the view that these hypodermal cells remain active and constitute the hypodermis during the subsequent regenerative processes. Fig. 53 1s taken from the same eye as d in the series shown in Fig. 52. In the later figure it is evident that regeneration is in progress. The hypodermal cells, however, show a tendency toward a paired arrangement indicating that they are the original hypodermal cells. After a part of the corneal covering has been removed it is evident that an entirely new hypodermis must be regenerated over the wounded surface. In crayfish it was seen that active cell proliferation began near the edges of injured hypodermis and that new cells pushed outward from these centers. ‘The early stages have been examined in a number of eyes of Palemonetes. The centers of cell proliferation in this form are not so apparent. The nuclei which in the early stages appear beneath the cuticle that covers the wound are very few and lie far apart. “Their number increases not chiefly by the repeated multiplication of nuclei at the edges of the wound but by the repeated division of the nuclei that are pushed out onto the wounded surface. During these early stages while the nuclei are actively dividing the cytoplasm is very loose and reticular and the cell boundaries are indistinguishable. In many cases the new cuticle which becomes apparent after the first moult is somewhat definitely separated into a dense outer portion and a semifibrous inner division. The inner division often appears as an interlacing network of fine fibers, many of which can be traced into the hypodermis beneath (Fig. 53). The loose incompact character of the hypodermis over the wounded surface probably indicates a high degree of activity of the hypo- dermis in this region. It is apparent from the foregoing description of the formation of the hypodermis covering the wound that the regenerated hypo- dermal cells may arise in two ways. They may arise by a trans- formation of the old corneal hypodermal cells im situ in which they assume a less specialized réle or they may arise by the migra- tion of a limited number of hypodermal cells from the edges of the Regeneration in Compound E yes of Crustacea 179 cut, which later multiply until a complete hypodermis is formed. In either case the first new nuclei must be contributed by the remaining hypodermal cells. Whenever the cut has not removed the entire ommatidial portion the remaining corneal hypodermal cells must assume a somewhat less specialized role in order to form the first new nuclei of the regenerated hypodermis. B REMOVAL OF THE INJURED TISSUE The fact that the inner tissues of the eye are so much softer than the chitinous outer covering renders it impossible to operate upon the eye in any way or to remove any part of it without serious injury to the remaining softer tissues. It is evident that, before any considerable regeneration can take place, the injured tissues must be either repaired or removed. All the observations made upon regenerating eyes tends to show that none of the injured tissues except the hypodermis ever repair themselves. Sections of an eye fixed six and one-half hours after the operation show that considerable changes have already taken place in the injured tissues. The effect is particularly noticeable in the retinula. Many of the retinular nuclei have become separated from the pigmented retinular processes and appear as rounded bodies surrounded by a dense mass of cytoplasm. ‘These are irregularly scattered among the other tissues. Some parts of the interior have become fairly clear of the broken down structures and are occupied chiefly by coagulated plasma. In other parts of the eye the injured tissues lie in confused masses. The changes in the next twelve or fifteen hours do not show much advance over the earlier stages. ‘The interior still presents a badly confused mass of broken down tissue. In some parts, however, the cone nuclei appear larger, the bodies of the cones have begun to dissolve and the number of rounded retinular cells appear somewhat more numerous than in the earlier stages, their nuclei showing irregularities in shape (Fig. 75a and b). During the earliest stages, six to sixty hours after the operation, the ommatidia that are still intact always appear bent and twisted out of their normal positions. Later stages show that these 180 Mary Isabelle Steele : uninjured ommatidia have regained their original shape and position. ‘This temporary contortion of the ommatidia seems to be due to the immediate effects of the operation, reduction of pressure, destruction of normal tension relations, etc. That in the later stages they appear normal again indicates that they have adjusted themselves to the new conditions imposed upon them by the operation. Besides those ommatidia that are actually injured by the operation a large portion of those remaining frequently degenerate. The destruction of the tissues of the eye is, consequently, much more extensive than the original injury. ‘This fact is strikingly illustrated by eyes in which the original injury consisted in thrust- ing a needle into the ommatidial portion. In several such in- stances the entire, or almost the entire, eye has degenerated. Instances of this kind have been observed in the eyes of sev- eral Palamonetes and also in the eyes of young Cambarus gracilis. Similar phenomena have been observed in eyes of fresh water Gammarus. To be sure the eyes of Gammarus are quite small which may account in a measure for the fact that in six or eight eyes examined in serial sections only one showed any of the old ommatidia intact. In Cambarus gracilis there were two instances out of six in which none of the ommatidial portion remained. In one of the cases the entire eye had degenerated; not even the vestige of the stalk remained. All of this degeneration took place in about thirty days and without a moult. In the other case all of the ommatidial portion and more than half of the optic ganglion had degenerated during the same period. In other eyes of the same series very little degeneration followed the operation. In these extreme cases it seems probable that some infection played a part. In the degeneration and removal of the injured tissues the retinula) degenerate most rapidly. In many cases they break down within the first few hours after the operation. ‘Their long pigmented processes become separated from the cell body and collapse into shapeless masses of pigment, which become scattered through the other tissues. The greater part of this pigment finally gathers in clumps near the level of the basement mem- "i Regeneration in Compound Eyes of Crustacea 181 brane. Although the retinulze are the first to collapse their remains are the last to be gotten rid of. Evidently the pigment is absorbed and removed only with difficulty. Often in regenerating eyes that have completely regenerated new ommatidia much of the old pigment remains. The cell bodies of the retinulz after losing their pigmented processes appear as large nuclei surrounded by a narrow zone of condensed cytoplasm. Within a short time these cells become scattered widely through the eye. After a few days their nuclei appear irregularly shaped and soon afterward become conspicu- ously polymorphic (Fig. 75a and b). In the usual course of events these retinular cells disintegrate and disappear. But under some conditions they apparently remain and later multiply and give rise to an abnormal development of tissue that secretes pig- ment. The rhabdoms and the inner ends of the cones also degenerate within a short time after the retinula. The cones continue to dis- solve from the proximal ends distally. The last parts to disappear are the outer ends in which are embedded the cone nuclei. Before the dissolution of the cones is complete the cone nuclei appear greatly enlarged and stain deeply. Their enlarged appearance is probably largely due to the disintegration of the cone substance from about them. As the disintegration of the tissues proceeds the interior of the eye becomes filled with a granular mass containing scattered nuclei and masses of pigment. This granular mass which is usually very much vacuolated is made up of the remains of the old tissues together with more or less of coagulated plasma and blood cells. Sometimes the remains of the old cones appear as long tapering bands of granular material extending from the periphery inward. Fig. 51 represents a small area of the disintegrated ommatidial structures as it appeared in the eye of Palamonetes ten days after the operation. Only two or three nuclei lie close beneath the cuticle and a few others lie scattered deeper in. Most if not all of these more deeply located nuclei are the remains of the old omma- tidial structures. In the lower part of the figure are some old 182 Mary Isabelle Steele pigment remains. ‘The granular mass occupying the greater part of the figure is made up chiefly of partially dissolved cones. The slender strands which can be traced through the granular mass are in part at least made up of new cytoplasm. At certain points the cytoplasmic strands are seen to be continuous with the inner layers of the cuticle. Fig. 48 is taken from a section of an eye of Palamonetes seven days after the removal of part of the ommatidia. A surface view from which this figure is taken is shown in Fig. 1. Practically all the material represented in Fig. 48 except the cuticle is made up of disintegrated ommatidia. The long band extending inward shows approximately the position of a former cone. ‘The large vacuolate spaces in the upper part of the figure are old nuclear remains. In the process of disintegration the nuclei at first enlarge and stain deeply. Later the nuclear contents disappear although the nuclear membrane persists for some time longer, often becoming shrunken and folded into a variety of shapes. It has been seen that the disintegration of the injured tissues begins immediately after the operation and that the greater part is accomplished in from one to three days. ‘The distal ends of the cones alone remain intact for a much longer time, in some cases from two to three weeks and occasionally even longer. ‘The removal of these disintegrated tissues is much slower than their dissolution. Regeneration proper may and usually does begin within a few days after the old structures have broken down and progresses simultaneously with their removal (Figs. 49,50). One part of the eye may show ommatidia differentiating while another region is still occupied by disintegrated old structures. The individual differences in the rate of regeneration of such frequent occurrence is probably largely dependent upon the variations in the length of time required for the removal of the old structures. This prob- ably also accounts for the fact that regeneration does not take place uniformly throughout the same eye. ‘The part of the eye that gets rid of the injured tissue soonest regenerates first. The above observations apply in general to all the forms used. Crangon, however, offers a significant exception in that the injured Regeneration in Compound Eyes of Crustacea 183 tissues disintegrate much more slowly than in Palzmonetes, her- mit crabs or crayfish. C METHOD OF CELL DIVISION In the earlier preliminary stages of the regenerative processes it is impossible to distinguish cell outlines. We should there- fore speak of nuclear divisions, perhaps, instead of cell divisions. In later stages the cytoplasm becomes differentiated about the individual nuclei. In all cases of the regeneration of the eye the nuclei are increased by amitotic division. Before a definite hypodermis is established the nuclei can be seen in various stages of constriction, separating off new nuclei for the development of the future underlying structures. ‘There seems to be no perfectly definite manner in which the constriction and separation of a nucleus into two parts takes place. One or two characteristic forms, however, appear so frequently as to be readily distinguished. The two most usual types are seen in the three nuclei occupying the extreme right of Fig. 53. When a nucleus divides in a plane parallel to its long axis it usually assumes the form of the outer one of the three referred to. The formation of the notch on one side gives the nucleus a peculiar heart-shaped appearance which seems characteristic and is easily recognized. Figs. 49 and 53 show nuclei of this same type further advanced in division. ‘The other type referred to is represented by the other two nuclei of the three at the extreme right of Fig. 53. Apparently these two nuclei were recently formed by the longitudinal division of one of the heart- shaped nuclei, like the one lying beside them. Each of these two is now dividing unequally by a transverse constriction. A dividing nucleolus can sometimes be seen but more frequently a definite nucleolus cannot be distinguished. When nucleoli are seen in dividing nuclei they usually appear with a darkly staining strand of material connecting them. In sections of young Cam- barus gracilis eye, nine and sixteen days after the operation, two nucleoli can be recognized in many of the nuclei. In those nuclei that are dividing one nucleolus lies in each part. In all except in the later stages of regeneration of the eye nuclei dividing amitot- 184 Mary Isabelle Steele ically can be found in great abundance. But at no time are mitotic divisions seen. In a careful examination of a large num- ber of sections only one cell has been found that suggested the possibility of its being in mitotic division. ‘The appearance of this one suggests a late anaphase or an early telephase. Conse- quently it is not certain that this is mitotic division. “This nucleus appears near the left edge of Fig. 53 at K. In any case it must be admitted that amitosis is the regular method of cell division in regenerating eyes of the forms studied, since in every specimen examined during the stages of cell divisions amitosis has been observed and mitosis has not been seen. That such is the case is somewhat unexpected since Miss Reed (o4) found mitotic division abundant in the regenerating leg of a crayfish. Miss Reed, however, observed that there were no mitotic figures during the early stages of regeneration, although new cells were being eapily formed. Perhaps we may infer from this that amitosis took place in the regenerating leg of the crayfish during the preparatory stages at ee But one would hardly expect such differences in cell division in forms so closely related for example as crayfish and Palemonetes. No observations have been made upon histogenesis in the regenerating appendages of hermit crabs, Palemonetes or Crangon. Hence it cannot be said whether or not the eye furnishes an unique exception to the regeneration of other parts in these forms. Recently, however, many have come to regard amitosis as a phenomenon of more frequent occurrence than has been generally supposed. Meves (’g1 and ’94), McGregor (’99), Child (’04 and ’07), all describe amitosis as a normal phenomenon and con- sequently no longer accept Vom Rath’s view that a cell is nearing its final dissolution when it begins to divide amitotically. On the other hand many of the more conservative investigators are unwilling to admit that amitosis occurs as a normal phenom- enon and believe that the apparent cases of amitosis can be explained on some other grounds. But in all the instances hitherto described amitosis has been found occurring along with mitosis. In the present case, however, all of the cell divisions are amitotic and they all take place in cells derived from the hypodermis; in Regeneration in Compound Eyes of Crustacea 185 these respects the regenerating eyes investigated in this series of experiments offer a unique instance in their method of cell division. V REGENERATION OcCURRING AFTER DESTRUCTION OF DistaL Part oF EYE Under this heading will be discussed the results obtained from eyes injured in varying degrees but not exceeding the destruction of more than the distal two sections of the optic ganglion. ‘The injury originally inflicted varied from tearing the surface of the cornea with a needle to cutting off the whole top of the eye so that at least the first part of the second section of the optic ganglion had been removed. Frequently, however, as has been explained in Section IV the part of the eye ultimately lost was much greater than that originally removed. For in cutting the eye with scissors or tearing it with a needle much of the tissue surrounding and under- lying the wound was so injured that it afterward degenerated. In the series of experiments to be described under this division nearly two hundred Palamonetes, twenty Crangon and fifty hermit crabs were used. Fifty per cent of the Palamonetes, 5 per cent of the Crangon and Io per cent of the hermit crabs died of the operation. Of those that survived the loss of blood and the nervous shock of the operation a considerable number died from other causes without having undergone a moult. Forty-five Palamonetes, seventeen Crangon and twenty-nine hermit crabs, however, lived through at least one moult. Among these a num- ber showed no regeneration either from surface examinations or from sections. The greater number of those that gave surface indications of regeneration and for comparison a number that showed no regeneration have been sectioned and examined. A REGENERATION OF THE FUNCTIONAL EYE Experience has shown that the number of days an experiment covered serves to indicate only in the most general way the stage of regeneration. While the time element naturally constitutes a most important factor the rate of regeneration is also dependent upon the season of the year, the age and the physiological activity 186 Mary Isabelle Steele of the individual and perhaps other factors not so apparent. Besides individual differences displayed between members of the same species there also appeared to be differences in the ability to regenerate and the rate of regeneration. among the three species chiefly used, these differences being much more marked in the regeneration of an eye than in the regeneration of an appendage. The hermit crabs seem to completely regenerate an eye in shorter time than either Palamonetes or Crangon. But the final results were similar in all three forms. The few significant differences will be pointed out and discussed later. rt Entire Preparations of Regenerating Eyes A careful examination of the regenerating eye frequently reveals a number of important features and is absolutely essential to the later interpretation of the sections. Therefore outline surface drawings have been made of all eyes later sectioned. In most cases the normal eye was drawn in connection with the regener- ating eye for comparison as to size, shape, etc. Frequently the two eyes were sectioned together. These surface views which had better be regarded as optical sections were drawn with a camera after the eyes had been brought into oil preparatory to embedding. Figs. 1 to 25 represent various stages of regenerating eyes. Fig. 1 shows dorsal and ventral views of a Palamonetes’ eye seven days after an operation which removed the ventral corneal surface and immediately after a moult. A comparison of the dorsal and ventral views shows that the injury is confined chiefly to the underside. In shape the eye is practically normal. It is not even flattened on the underside as the thinning out of the pigment near the center would seem to indicate. As is usually the case the injured eye is much smaller than the uninjured one. Not only the region operated upon but the whole eye decreases in size after the operation. ‘This indicates that however localized the operation may be the effect is much more extended. In this particular case the part of the eye proximal to the injury measures only three-fourths of the length of the same region in the unin- jured eye. Regeneration In Compound Eyes of Crustacea 187 Fig. 3 represents the ventral view of a Palamonetes eye ten days after being injured. In this case also the injury extends across the ventral side of the ommatidial portion. The pigment of the broken down ommatidia can be seen scattered in flakes and patches through the upper part of the eye. From the dorsal side the eye appeared nearly normal but sections show that almost the entire eye is in process of degeneration. The specimen represented in Fig. 4 shows a regenerating eye nineteen days after the removal of almost all of the ommatidial portion. It is readily seen that very little remains except the eye stalk. The pigment patches are remains of the original eye. Across the end of the stump the cuticle is wrinkled and folded, indicating that comparatively little new tissue has been formed and that the cuticle follows more or less closely the rough uneven out- lines of the wounded surface. When the entire ommatidial portion has been removed or has degenerated regeneration seems to be considerably slower than when a large part of it remains uninjured. ‘The two specimens shown in Figs. 9 and 1o afford a striking illustration of this fact. Both of these eyes were operated upon at the same time. Each animal moulted twice, the first time on the same day and the second, a day apart. Both were fixed in picro-acetic at the same time, thirty-two days after the operation. In Fig. g the injury involved only the posterior ventral side, less than one-half of the ommatidia. While in Fig. 10 the injury included all of the structures lying distal to the basement membrane. Examination of the sections shows new ommatidia completely differentiated in Fig. 9 while in no part of Fig. 10 are they yet defined. The next specimen, Fig. 20, presents a rather striking appearance and suggests immediately that the regeneration taking place is not altogether of the normal type. ‘This is a thirty-eight day specimen and belongs to the same series of experiments as the preceding two specimens. All of the part distal to the dark pigmented band is regenerated tissue. [he pigment consists chiefly of the remains of the old retinulz. It is evident even from a surface view that no ommatidia have developed. Sections show, however, that on one side the differentiation of cones is beginning (Fig. 69). Here it 188 Mary Isabelle Steele may be well to mention a fact that has been observed a number of times. The regeneration of the new ommatidia never presents a uniform stage of differentiation in any case whether or not all of the old ommatidia have disappeared. In fact it may be possible to select several stages from the same eye and sometimes two or three stages from the same section. An eye thirty days after the injury, Fig. 6, shows some interesting features in comparison with the one just described. From a ventral view this eye shows no signs of remaining ommatidia and from the dorsal side only a very few cones and facets are evident. The upper part of the eye is transparent. Below this transparent area are scattered patches of pigment representing the remains of the old eye. Sections show that no new ommatidia have been entirely differentiated, that the retinular cells have differentiated and are establishing connections with the optic ganglion, that new cone nuclei are being separated from the hypodermal nuclei, that on one side a few of the old ommatidia remain and that in the tissues lying nearest the old ommatidia new cones are being developed. The most striking feature presented by this specimen is the clearness with which the connections between the retinulze and the ganglion cells can be made out. ‘These connections will be discussed at length in the consideration of the detailed structures of the regenerating eye. Fig. 17 shows the ventral view of a regenerating Palamonetes eye from a thirty-five day specimen. Examination of the dorsal side shows that a large number of the ommatidia on that side appear uninjured, the greater part of the injury being confined to the ventral side as indicated by a surface examination. Sections show, however, a gradation:of regeneration from a stage in which there is no signs of cone differentiation to the complete formation of a new ommatidium. It was said above that hermit crabs regenerated an eye more rapidly than either Crangon or Palamonetes even in instances where a considerable portion of the optic ganglia had been removed. Figs. 5, 12 to 15 show regenerated eyes of hermit crabs after one or more sections of the optic ganglion have been de- stroyed. Fig. 12 had at least the distal division of the ganglion Regeneration in Compound Eyes of Crustacea 189 removed. ‘The regenerated eye shown in the figure was developed within hive hrees days and after only one moult. This moult occurred on the thirty-second day and the eye was fixed in Perenyi the thirty-third day. Sections show that ommatidial structures have been fully differentiated although incompletely developed. One point is particularly noticeable in these sections; the omma- tidia are very much shorter thaninthe normaleye. ‘This condition was probably caused by the mechanical pressure of the covering cuticle which forced the developing ommatidia into less space than they would otherwise have occupied. Fig. 13 shows the eye of a hermit crab regenerated from a stump in which not more than half of the optic ganglion remains. ‘The regeneration in this eye took place in thirty-eight days. One moult occurred twelve days after the operation. Although extremely small the eye 1s practically perfect except that the corneal facets have not yet developed. Figs. 14 and 15 show two other regenerated eyes of hermit crabs forty-one and sixty-seven days respectively after the operation. Sections of the forty-one day eye do not show ommatidia as fully developed as the thirty-eight day specimen previously described. The sixty-seven day specimen shows the eye complete in all its details even to the corneal facets. Whether a younger regenerated eye might not show the corneal facets has not been determined since no specimens were available between the forty-one day and the sixty-seven day specimens. A noticeable feature in all the regenerated eyes of the hermit crabs is their small size in comparison with the normal eyes. It is probable that the regenerated eyes would have increased in size if the experiment had covered a longer period of time. Sections of the eye shown in Fig. 12 indicate that the definitive size has not been reached. For, lying outside the fully formed ommatidia are others in the process of development. In the case of the eye shown in Fig. 13 sections do not show any indications of partially developed ommatidia and it may be that this eye would never have reached the size of the normaleye. Perhaps this is what we should expect since the amount of nervous tissue present is considerable less than is normal. It is more difficult to interpret the actual condition of a regener- Igo Mary Tsabelle Steele ating eye in Crangon than in either Palamonetes or hermit crabs. The whole of the eye stalk in Crangon is thickly covered with branching pigment cells. So that even after the eyes are brought into oil very little can be seen in detail by examining them in toto. Figs. 7 and 8 thirty-one and thirty-two days, respectively, are fairly characteristic of the regenerating eye of Crangon. From all that can be determined from the outside, regeneration seems practically complete in each of these cases. Sections show, however, that comparatively little regeneration has taken place. A fuller discussion of the regenerating eye of Crangon will be taken up elsewhere. 2 Details of Development of the Regenerating Ommatidia The complete regeneration of the ommatidial portion of the eye involves three stages. ‘These can be separated from each other rather sharply although they overlap more or less. The first stage consists in getting rid of the broken-down tissues and the healing of the wound; second, the active proliferation of new cells; and third, the differentiation of the new ommatidia. The first and second stages have been discussed in Section IV. a Regeneration of Retinulze All the observations support the conclusion that the regenerated ommatidia are derived entirely from the hypodermis. Before the hypodermis covering the end of the stump has been clearly differen- tiated, however, the proliferation of cells for the new structures lying below has begun. So that at the same time hypodermal cells are dividing in two planes, one at right angles to the periphery to increase the number of hypodermal cells, and the other parallel to the surface. The inner nuclei of the latter division migrate inward and become the first retinular cells. As they migrate they become elongated with their long axes radially arranged. Fig. 54 shows the early stages of the separation of retinular nuclei, some separating from the nuclei at the periphery and others migrating in. At a comparatively early date these retinular nuclei have migrated a considerable distance below the surface Regeneration in Compound Eyes of Crustacea IgI and may be seen in a relatively well defined row (Fig.68). Soon after the retinular nuclei have been separated from the nuclei at the surface they themselves begin to divide (Fig 68) in a plane at right angles to their plane of original division. ‘These longitudinal divisions may begin before the nuclei have reached their definitive position. ‘This division continues until a band composed of many nuclei has been formed. Figs. 60 and 62 show portions of such bands. Here and there single nuclei are found lying much nearer the basement membrane than the retinular band (Figs. 60, 62). These are occasionally seen constricted. It has been impossible to determine with certainty the fate of these scattered nuclei but there are evidences which suggest that they become the nuclei of the accessory pigment cells. The retinular nuclei even in the early stages of their migration stain much more deeply than the nuclei at the periphery. But at this stage no definite cytoplasmic outlines can be distinguished. Very faintly staining delicate strands of cytoplasm, however, can be found extending between the retinular nuclei and the periphery. These strands form an intermingled network and with the nuclei lie in a granular substance. Soon after the nuclei have reached their definitive position the cell bodies of the retinule can be recog- nized. Each nucleus appears surrounded by more or less definite strands of cytoplasm which are radially arranged and extend outward toward the periphery and inward to the basement mem- brane (Figs. 60, 65). These can now be definitely recognized as retinular cells. Usually it is easier to see the proximal than the distal strands for at an early stage these lower processes begin to secrete pigment and are consequently more conspicuous (Fig. 65). At the stage represented by Fig. 62 there is only the merest begin- ning of pigment deposition. ‘The fibers are but little differentiated from their background which still seems to be composed largely of a homogeneous granular material, probably to a great extent coagulated plasma. : At a stage such as 1s represented by Figs. 60, 62, in which there is only the merest beginning of pigment secretion delicate cyto- plasmic processes can be traced from the retinular nuclei inward 192 ; Mary Isabelle Steele through the basement membrane beneath which they can be found branching over the ganglion cells. Fig. 62 shows several isolated retinular cells and their proximal processes which are seen extend- ing through the basement membrane and reaching to the ganglion cells beneath. There is no evidence that these retinular fibers are directly connected with the ganglion cells; they seem merely to twine around them. When the fibers have reached the basement membrane they may pass directly through it as the two shown in the left side of Fig. 62, or they may extend along the upper face of the basement membrane before entering the ganglionic mass below. ‘The retinular processes frequently branch shortly before entering the basement membrane or just as they emerge below it. No special nerve methods were employed yet numbers of these fibers are seen branching among the ganglion cells and many are readily traced from the retinular nuclei to the basement membrane (Fig. 65). It is only in very favorable specimens that the fibers can be traced, however, through their whole length. b Regeneration of the Crystalline Cones Up to and including the stages shown in Fig. 65 and described above there is no evidence of any differentiation of the crystalline cones. A definite hypodermis, however, has been formed with an increased number of nuclei, from which other nuclei are separating, These are the cone nuclei (Fig. 63). The formation of the cone nuclei does not appear to take place in a uniform manner. The usual method, however, is by the division of the hypodermal nuclei in a plane parallel to the periph- ery, the inner nuclei thus formed being the cone nuclei. But since in every ommatidium there are four cone nuclei and only two hypodermal nuclei, it is evident that either the hypodermal cells must divide twice or the first cone nuclei must themselves divide in order to make the cone nuclei just twice the number of the hypo- dermal nuclei. Observations indicate that in some cases the second pair of cone nuclei arise by the division of the first pair. In other cases, however, it is uncertain whether they arise in this manner or whether they arise from the hypodermal nuclei. It is Regeneration in Compound Eyes of Crustacea 193 probable, however, that they