‘n le OMe Solel) Sonl4S) llMKalledvatien 7a G1 77 ee BR nx. nx. 11 | 30) 11 | 56 | 46 | Killed at 56 INT aia ory. cise Se ./88.0/382.0} 29) 13) 28 | 138) 26 | 48 | 44 | Killed at 48 Aver. max. { 1916-17... .|18.3/17.2] 260] 218] 43 | 21] 31 or total i 1916-18.... 91 | 119) 75 | 58 | 531 02 N. B.—The 1918 larvae were not measured at the 18 to 20-mm. stage. The 5/5 group of thyroidless larvae had defective jaws. Abbreviations: Aver., average; Con., control larvae; Op., thyroidless larvae; Nz., not measured; Mazx., maximum; Met., metamorphosis or metamorphosed. 1 Only one of all the control larvae studied became over 50 mm. in length. 10 E. R. HOSKINS AND M. M. HOSKINS 1917. The frogs which formed from the larger larvae were larger than those formed from smaller larvae. In Rana sylvatica there is thus a great variation in the size of the newly formed frogs. As shown in table 2, the body length of these normal frogs of the same stage of development varies more than 25 per cent and their volume nearly 100 per cent. The mortality in 1917 was very great, but was about the same in both the thyroidless and control animals. However, 42 of the former and 20 of the latter reached the period of normal metamorphosis. Of these (table 1) the controls all metamor- phosed normally. Of the operated animals, 12 metamorphosed and 30 failed to do so. By autopsy and sectioning it was later ascertained that every one of the 30 was devoid of thyroid, and in every one of the 12 the gland had regenerated after the oper- ation. In the 1918 work, out of 47 thyroidless animals which reached the period of normal metamorphosis, 44 failed to meta- morphose and in only three experimental larvae did the thyroid regenerate permitting metamorphosis. In the three seasons a total of 91 experimental larvae were reared to or beyond the time of normal metamorphosis, and of these 75 remained in the larval condition. In addition to these, many which were killed in the early stages for purpose of study would doubtless have survived long enough to be added to the 75. From these experi- ments and those of Allen (’18) we are justified in the conclusion that removal of the thyroid from young frog larvae will delay if not entirely prevent metamorphosis under ordinary laboratory conditions. Most of the 75 thyroidless larvae just referred to were killed after it became evident that they would not develop into frogs, but a few were kept alive. Of these, six survived the winter and the second season of normal metamorphosis with- out becoming frogs. It is more or less common knowledge that if Amblystoma larvae kept in aquaria are not properly fed, they may fail to undergo metamorphosis their first season, but do metamorphose the second season. The same thing is true also in nature. Dr. T. G. Lee, of the University of Minnesota, has told us that he has collected in the early spring Amblystoma larvae which should have metamorphosed the previous season. GROWTH OF AMPHIBIA AFTER THYROIDECTOMY 11 Normal Rana sylvatica larvae kept in aquaria and well fed metamorphose normally, but with our thyroidless frog larvae the second season was passed without their metamorphosing. Concerning the growth of the thyroidless larvae, it may be noted from tables 1 and 2 and figures 10 to 19 that they ulti- mately became much larger than the control larvae. As stated above, the thyroidless animals grow more rapidly than the con- trols, but the difference is not very great until near the time of metamorphosis. When the controls at this period have nearly reached their maximum length, their growth seems to slacken,,. and their length does not change for a short period of time before it starts to decrease during metamorphosis. It is during this time, as was noted also by Allen (18), that the experimental animals gain very much in size over the controls. In some of our 1918 series measurements of volume of the animals were made at this time and in some cases (table 2) the thyroidless larvae were more than twice as large in volume as the control animals of the same age. The volume was determined by the amount of liquid displaced by an animal. After the metamor- phosis of the controls (1917) the growth rate of the experimental larvae decreased greatly and growth nearly ceased during the winter, but the larvae grew more rapidly during the second spring and summer. For example, the animal shown in figure 19 became 55 mm. in length by the time the controls had meta- morphosed in early August, 1917. It was 62 mm. in length on September 2 and 66 mm. on September 27 when next measured. On October 19 the length had not increased. The animal began growing again in May, 1918, but it increased its length by only 5 mm. by June 25, and on July 5 had reached its maximum length of 72 mm. with a volume of 2.38 cc., or nearly three times that of the normal larva at the time of metamorphosis. In this growth after the first summer the body length (nose-anus) in- creased 2 mm. and the tail 8 mm. the animal thereby becoming relatively long-tailed. Its hind legs increased after the first season about one-half of a millimeter in length, reaching 5 mm. In these miniature legs the normal segments and digits are slightly differentiated. 12 E. R. HOSKINS AND M. M. HOSKINS One 1917 operated larva deserves special mention. Its thyroid regenerated and then hypertrophied. The animal metamor- phosed about a month before the controls and after metamorpho- sis it was less than one-third the normal size (fig. 20). The length of the controls at this time was 40 mm. (fig. 21). An attempt was made to duplicate this experiment in 1918 by cutting to pieces the thyroid anlage and leaving the pieces in the animal. In other specimens transplantation of the thyroid was performed, but in neither group did these experimental larvae metamorphose earlier than the controls, although a few were smaller than nor- mal. In three 1918 specimens from which the thyroid had been _ removed, the gland regenerated and the animals metamorphosed at a size about one-half that of the average control larva, but not at an earlier date. Figures 10 to 19 show the changes in body form exhibited by the control and thyroidless animals. Figures 12 and 13 show a normal tadpole in the process of metamorphosis. The hind legs are well developed and the skeleton of the body has begun to acquire the adult shape. In figure 12 it may be noted that the head is flattening and narrowing. The larva shown in fig- ures 14 and 15 is a thyroidless specimen of the same age as that of figures 12 and 13. Its total length is greater than that of the control larva, the hind legs are very small and the body (fig. 14) is more nearly cylindrical than that of the control (fig. 12). The eyes are more laterally placed in the control larva and, although its head is narrower than that of the other (fig. 15), the eyes are further apart. The head of the control speci- men is slightly more pointed than that of the thyroidless larva and is more like that of the frog (fig. 17). The experimental animal retains this larval form for some time, but it slowly under- goes changes in shape which tend toward those of the controls. Normally, during the period of metamorphosis, the form of the animal changes as follows: The body decreases very slightly in length; the anal canal shortens 2 to 4 mm. (compare figs. 12 and 16); the transverse and horizontal diameters decrease nearly one-half; the tail atrophies; the dorsal and ventral sides flatten; the eyes come actually closer together, but are relatively farther GROWTH OF AMPHIBIA AFTER THYROIDECTOMY 13 apart; the rima oris increases in size and changes in shape; the nostrils become relatively more lateral in position and larger, and the legs increase from one-half to two-thirds (table 2). The thyroidless larvae after the season of normal metamor- phosis still resemble young larvae in general shape, but they TABLE 2 Length and volume of larvae and frogs. Showing the variations in the length and volume of living and fixed larvae. The animals were fixed in picro-formo-acetic and preserved in alchohol. Note the small size of the frogs, which are only one- third to one-half the volume of the larvae from which they are transformed. Vari- ations in shrinkage occur during fixation. THYROIDLESS LARVAE CONTROL LARVAE IN FROGS IN ALCOHOL PME TEE: Alive In alcohol Length Length Length Length Nolte |e ee VO Lure |) cae Volume |e ee IVGlume N-A. Tot. N-A | Tot. N-A. | Tot. N-A. | Tot. mm. mm, ccs mm. | mm. ce, mm. | mm, CGe mm. | mm. ce. 20.0 45.0 | 1.10 | 25 | 72.0) 2.88 | 24.5) 68.5} 1.92 | 16.0] 20.0) 0.42 19.0 46.5 | 0.81 25 | 65.5) 2.40 | 23.0} 60.0) 1.80 | 16.0] 18.0) 0.40 19.0 41.5 | 0.76 | 25 | 65.0) 2.45 | 20.0) 54.0} 2.19 | 15.0) 19.0} 0.35 18.5 46.0 | 0.95 25 | 65.0) 2.41 | 22.5) 58.0) 1.95 | 15.0} 17.0} 0.39 18.5 46.5 | 0.86 | 25 | 63.0) 2.37 | 23.0} 60.0) 1.80 | 14.5) 15.0] 0.31 18.5 46.5 | 0.79 | 24 | 64.0) 1.98 | 22.0) 57.0) 1.78 | 14.0) 15.0] 0.28 18.0 46.0 | 0.99 | 22 | 63.0} 1.88 | 21.0} 56.0) 1.58 | 14.0} 15.0) 0.23 18.0 42.0 | 0.78 | 20 | 54.0) 1.19 | 17.0} 48.5] 0.98 | 13.5] 16.0] 0.30 18.0 41.0 | 0.68 18 | 46.0) 0.85 | 17.0) 42.5) 0.75 | 18.0] 19.0} 0.26 17.0 r 42.5 | 0.74 16 | 39.5) 0.60 | 15.0} 36.5) 0.50 | 13.0) 13.5] 0.22 17.0 39.0 | 0.55 14 | 38.0) 0.51 | 18.7; 34.5} 0.41 | 12.5] 14.0} 0.22 16.5 40.0 | 0.73 13 | 32.5) 0.38 | 12.0) 30.0) 0.32 16.0 39.5 | 0.52 Averages 18 | 43.2 | 0.79 | 21 | 55.6] 1.57 | 17.6] 50.5} 1.33 | 14.2] 16.5] 3.07 N-A., Nose-anus length; Tot., total; Vol., volume. have undergone a few changes (figs. 18 and 19). The tail has become relatively long, the eyes are wider apart, the dorsum of the head is slightly flattened, and the legs have increased very slightly in length. During the winter and the second spring and summer the thyroidless larvae (figs. 10 and 11) make a still nearer approach in form to that of the frog. The legs 14 E. R. HOSKINS AND M. M. HOSKINS increase very slightly in length, the head and back become very flat on account of skeletal changes, and the eyes are relatively more lateral in position than in normal larvae. Other changes noticeable are a relative increase in the length of the tail and a blunting of the anterior end of the head. Allen (718) states that the head is relatively long, but this is not the case in our specimens. In his comparison Allen (’18, fig. 3) failed to super- impose the centers of the heads of the larvae. At the end of the period of metamorphosis the thyroidless larvae are from three to five times as large in volume as the frogs of the same age and some become ultimately more than six times as large as the average frog after metamorphosis and nearly three times as large as the average control larva before metamorphosis (table 2). Table 2 shows the variation in the volume (and hence weight) of amphibians as compared on a basis of length both in the living and fixed condition. These animals were fixed in Bouin’s fluid and preserved in 70 per cent alcohol. The data here were se- lected from a very large number of such measurements and published in order to demonstrate a source of serious error in attempts to compare relative volumes of organs on a basis either of length or volume of the animal, as well as to show the relative sizes of larvae and frogs. The volume of fixed specimens varies greatly on account of variations in the amount of shrinkage during fixation, and similar variations in the size of fixed organs must also occur. The volume (or weight) of a living larva of a given length is only fairly constant and varies not only with the amount of foreign substances within the intestine, but also on account of variations in shape produced by changes in the tail-body re- lationship. The intestinal contents cannot be removed without destroying the gut, and this would prevent the measurement of the volume (or weight) of the entire animal. In addition to loss of water, a considerable portion of the natural decrease in size suffered during transformation from the tadpole to the frog is due to discharge of intestinal contents, and the exact amount of this is difficult to determine. This loss of fecal matter in- GROWTH OF AMPHIBIA AFTER THYROIDECTOMY £5 creases the difference between the relative volume of larvae and frogs. The shortening of the tail and anal canal during meta- morphosis prevents comparison of the larvae and frogs on a basis of the body length (figs. 12 and 16). Given a frog and larva of the same body length (i.e., nose-anus) the latter is two to three times the volume or weight of the former (table 2). If the body length be considered as the distance between the tip of the head and the posterior limit of the body cavity, a larva is more than three times the size of a frog of the same body length. In table 2 the measurements of living larvae are given only in the case of the thyroidless animals, as the variation in the controls is similar. The increase in the volume of the control larvae and frogs on account of growth of the legs is offset by a slight increase in the relative length of the tail in the thyroidless animals. Regeneration. On April 13, 1918, 12 mm. of the tail and the entire hind legs were removed from a large thyroidless larva from the 1917 series. After fourteen days the tail had increased in length 11 mm. and the body 2 mm. One hind leg had not grown at all, but the other had grown 2 mm. and regenerated a foot that was about half the size of the one removed. The part of the tail that regenerated contained less muscle than normal but was otherwise like the part removed. On April 27 an additional 13 mm. of the tail and the regenerated leg and foot were removed. The tail increased again 14 mm. in length in twenty-eight days. The leg did not again regenerate. The body again increased 2 mm. in length. On June 24 an additional 12 mm. of the tail was removed and it regenerated 7.5 mm. in twenty-three days. During this period a twist developed in the tail near the body. On July 17 an additional 7.5 mm. of the tail was removed. In five days, 2 mm. of the tail had regen- erated when the animal died. During the three months of this experiment none of the other thyroidless larvae had increased more than 6 mm. in total length, 2 mm. of which growth was in body length, while the experimental animal had regenerated 34.5 mm. of tail and its body grew 4mm. Had it not died, the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 1 16 E. R. HOSKINS AND M. M. HOSKINS larva might have grown still more. Only one hind leg showed any sign of regeneration, and it grew to only half the size of the one removed. When this regenerated leg was removed the animal was unable to regenerate it again. The steadily increasing amount of time required to regenerate the removed caudal tissues and the failure in regeneration of removed legs indicates that, although thyroidless larvae are able to grow and regenerate tissues more than a year after the ex- tirpation of the thyroid, the larva will not continue to regener- ate removed tissues indefinitely or at the same rate as the first regeneration it produces. Allen (718) has noted that young thyroidless larvae are able to regenerate removed parts of the body. Zeleny (’09) found that in normal Salamander larvae repeated amputation of the tail causes an increase in the rate of regeneration. This is influenced, however, by additional in- juries to the body, such as removal of one or both hind legs. If the additional injuries are severe, a decrease in the rate of regeneration is to be observed. Forced metamorphosis. An attempt was made to force a thy- roidless larvae into metamorphosis. The total length of the animal was 59 mm.; the nose-anus length, 24 mm.; the length from the nose to the end of the body cavity, 20 mm.; that of the hind legs, 4.5 mm., and the volume was 1.90 cc. This larva was placed in a moist chamber on a bed of wet spirogyra, where it lived for two days. It is probable that with care animals might be kept alive in such conditions much longer. This speci- men breathed at irregular intervals. In the forty-eight hours during which it lived in the moist chamber, its tail shrank 24 per cent, its anal canal about 50 per cent in volume (due mostly to discharge of contents), and its volume decreased 18 per cent. The gut shortened little, if any, but was strongly contracted. The lungs were filled with air. The shrinkage was not due to inanition, for if a larva similar to the one described is kept in water without food it will maintain its volume for a much longer period than that given to the experiment. Moreover, the shrink- age which finally results from inanition is uniform, while in this experiment the tail shrank first of all, and very rapidly, as it does GROWTH OF AMPHIBIA AFTER THYROIDECTOMY vs in normal metamorphosis. 40. 65 Thymus of thyroidless larva larger, than that of figure 64, but same age. 66 Small lymphoeytes and large thymie cells from thymus shown in figure 64, xX 670. 67 From thymus shown in figure 65. 670. 68 Epithelioid body from same larva as figure 64. Cap., capsule. X 670. 69 Epithelioid body from larva of figure 65. 670. 70 Small regenerated thyroid, showing unusual structure. XX 160, 71 Normal thyroid of young frog. The structure and size are the same as before metamorphosis. 160. 2 From figure 70. > 670. 3 From figure 71. X 670. 74 Thyroid of half-grown normal larva. > 160. 5 Thyroid of precocious frog (fig. 20). This thyroid regenerated and hy- pertrophied. Compare with figure 74 from a normal larva of the same age. 60} 76 Spleen of small normal larva (23 mm.) showing the two types of splenic cells XG O70, 77 Spleen of control larva at maximum length before metamorphosis. X 670. 78 Spleen of thyroidless larva of same age as that of figure 77. X 670. 62 PLATE 6 GROWTH OF AMPHIBIA AFTER THY ROIDECTOMY E, R, HOSKINS AND M. M, HOSKINS Osim es : hes (Mths rs ae A 63 PLATE 7 EXPLANATION OF FIGURES R. sylvatica. Representative specimens from sixty-nine complete and forty partial autopsies. All removed organs were drawn with a camera lucida. Abbreviations: Ov., ovary; T., testis; S., spleen; K., kidney; F. B., fat body. Other abbreviations and dates as in plate 2. ] Ovaries, kidneys, and spleen. X 8 79 Control larvae. 4/14 to 6/2/18. Alive: Tot., 45 mm., B., 18.5 mm.; fixed: Tot., 42 mm., B., 17.5 mm. 80 Thyroidless larva. 5/2 to 6/8/18. Alive: Tot., 45 mm.; fixed: Tot., 2 Tee dee ley), Oe 81 Control frog. 4/14 to 6/5/18. Alive: Tot., 15 mm., B., 14 mm.; fixed: Tot., 14 mm., B., 138 mm. 82 Thyroidless larva. Alive: Tot., 53 mm., B., 22 mm.; fixed: Tot., 53 mm. B., 21 mm. 83 Thyroidless larva. 5/3 to 10/17/17, Alive: Tot., 65 mm., B., 22 mm.; fixed: Tot., 60 mm., B., 21 mm. 84 Thyroidless larva. 4/28 to 12/21/17. Alive: Tot., 54 mm., B., 22 mm.; fixed: Tot., 50 mm., B., 21 mm. 85 Thyroidless larva. 4/28/17 to 7/10/18. Alive: Tot., 68 mm., B., 22 mm.; fixed: Tot., 56 mm., B., 22 mm. 86 Control frog. 4/7 to 8/12/17. Alive: Tot., 14.5 mm., B., 14 mm,; fixed: Tot., 14 mm., B., 13.5 mm. 87 Thyroidless larva. 4/14 to 7/13/18. Alive: Tot., 65 mm., B., 24 mm.; fixed: Tot., 60 mm, B., 23 mm, d Testes. < 8 88 Thyroidless larva. 5/2 to 6/8/18. Alive: Tot., 45 mm., B., 18 mm.; fixed: Tot., 42 mm., B., 17.5 mm. 89 Control larva. 4/18 to 5/28/18. Alive: Tot., 45 mm., B., 18.5 mm.; fixed: Tot., 42.5 mm,, B., 18 mm. 9) Control frog. 4/14 to6/5/18. Alive, 15 mm., fixed, 14 mm, 91 Thyroidless larva. 4/14 to 7/15/18, Alive: Tot., 54 mm., B., 20 mm.; fixed: Tot., 48.5 mm., B., 17.5 mm. : 92 Thyroidless larva. 5/8 to 10/17/17. Alive: Tot., 68 mm., B., 23 mm.; fixed: Tot., 63 mm., B., 21 mm. 93 Thyroidless larva. 4/28/17 to 7/6/18. Alive: Tot., 72 mm., B., 25 mm.; fixed: Tot., 68.5 mm., B., 24.5 mm. 64 GROWTH OF AMPHIBIA AFTER THY ROIDECTOMY PLATE 7 £. R. HOSKINS AND M. M. HOSKINS AN [Da \\ IG \)\ 65 PLATE 8 EXPLANATION OF FIGURES R. sylvatica. Ovaries, X 36 94 Half-grown normal larva (fig 21). Ooeytes, 50 pu. 95 Precocious frog (fig. 20). Oocytes, 75 pu. 96 Young normal frog (1917). Oocytes, 120 zu. 97 Young thyroidless larva (1918). Large as control larva at metamor- phosis. Oocytes, 50 » (smaller oocytes than those of 1917 larvae of same size). 98 Thyroidless larva (1918) (5 weeks old). Much younger than that of figure 97, but of much larger size, oocytes (60 uw) are relatively of the same size. 99 Thyroidless larva one-half the size of larva of figure 98, but more than twice as old (eleven weeks) Oocytes (130 u) are relatively about four times as large in diameter as those in figure 98. 100 Thyroidless larva (1918). Very little older than larva of figure 99 (twelve weeks), but nearly four times as large. Oocytes (180 u) are relatively smaller than those of figure 99. 101. Thyroidless larva (6 months old), killed in October, 1917. Oocytes, 200 pu. 102. Thyroidless larva killed December 21, 1917 (eight months old). Oocytes, 250 p. 103. Thyroidless larva fifteen months after thyroidectomy. Oocytes, 300 u. Measurements represent the diameter of the largest oocytes. 66 PLATE 8 GROWTH OF AMPHIBIA AFTER THYROIDECTOMY E. R. HOSKINS AND M. M. HOSKINS SEER, PLATE 9 EXPLANATION OF FIGURES R. sylvatica. Testes 104 A, T. S. Control larva at beginning of metamorphosis (July 29, 1917). < 34. B, same, showing beginning of tubule formation, but no synapsis. > 240. 105 A, T..S.° Young control frog. X34. 'B, same. x 240. 106 A, T. S. Thyroidless larva larger but of same age as larva of figure 104. 34. B, same, showing advance in tubule formation, but no synapsis. x 240. 107 A,T.S. Large thyroidless larva, killed four months after thyroidectomy (August 20,1917). 34. B, same, spermatogenesis begun. X 240. 108 A, T.S. Large thyroidless larva, six months after thyroidectomy. x 34. B, same, showing spermatozoa in tubules. > 240. 109 Large thyroidless larva one year older than that shown in figure 106. Shows fully matured testis. V. #., efferent tubule leading into kidney and con- taining spermatozoa. X 34. 68 GROWTH OF AMPHIBIA AFTER THY ROIDECTOMY PLATE 9 E. R. HOSKINS AND M. M. HOSKINS 69 Resumen por la autora, Helen Dean King. Estudios sobre ‘‘inbreeding.”’ IV. Nuevos estudios sobre los efectos de ‘“‘inbreeding”’ sobre el crecimiento y variabilidad de peso de la rata albina. Los datos publicados en el presente trabajo demuestran el crecimiento y la variabilidad de peso de mas de 600 ratas albinas pertenecientes a las generaciones comprendidas entre la 16 y 25 generacion de un tronco “inbred”? hermano con hermana, per- tenecientes ambos a la misma cria. Los principales puntos de interés son los siguientes: 1. El ‘inbreeding’ continuo no ha producido efecto perjudicial alguno en el tronco albino original en lo referente a la marcha y extensién del aumento de peso del cuerpo, ni tampoco ha alterado la forma de la grafica de creci- miento de los dos sexos. 2. Las relaciones normales de peso de los sexos no se han alterado después de 25 generaciones de “inbreeding.” 38. La variabilidad de los pesos de estos animales es relativamente alta en todas las edades y no decrece cuando el “inbreeding” avanza. 4. Una comparacién de la variabilidad de los pesos de diferentes series de albinos del mismo tronco con los de las ratas ‘“‘inbred”’ indica que el aumento de variabilidad en las tltimas se debe a la accién del medio ambiente y a la accion de la nutrici6n, no al “inbreeding.”’ Translation by José F. Nonidez Carnegie Institution of Washington AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JUNE 30 STUDIES ON INBREEDING Iv. A FURTHER STUDY OF THE EFFECTS OF INBREEDING ON THE GROWTH AND VARIABILITY IN THE BODY WEIGHT OF THE ALBINO RAT HELEN DEAN KING The Wistar Institute of Anatomy and Biology EIGHT CHARTS In order to complete the series of records for the first twenty- five generations of inbred albino rats, data showing the growth and variability in the body weights of individuals belonging in the sixteenth to the twenty-fifth generations are given in the present paper. Five litters from each generation of the two inbred series (A and B), comprising a total of 296 males and 310 females, were used for this study. The rats in these litters were selected in the same manner, and they were weighed at the same age periods, as were the individuals of the seventh to the fifteenth generations for which body-weight records were taken (King, 718). The data for the animals in the different generations of the inbred strain are therefore strictly comparable. During the past three years, when most of the weighings were taken, it was not possible to rear the animals under environ- mental and nutritive conditions that were as favorable to growth and to fertility as those existing previously. Owing to economic conditions incident to the war, it became necessary to make a radical change in the character of the food that the rats received. The ‘scrap’ food (carefully sorted table refuse), on which the animals of the earlier generations seemed to thrive exceedingly well, had to be replaced by a ration that consisted, for the most part, of oats and corn, with the occasional addition of various kinds of vegetables and a little meat. Some of the available 71 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 1 (P HELEN DEAN KING substitutes that from time to time were added to the diet in order to vary it, such as alfalfa, linseed and cottonseed meal, proved very injurious to the rats and very materially affected their growth and fertility. For some time, therefore, the food given the animals has been largely in the nature of an experiment, and it has not even yet been possible to work out a ration that produces as rapid and vigorous growth and that is as favorable to reproduction as was the ‘scrap’ focd given previously. Extremes of temperature, either of heat or of cold, have a very marked effect on the body growth of the rat, as they have on that of mice (Sumner, ’09), and many of the animals in the later generations of the inbred strain suffered considerably from this cause. During the excessive cold of the winter of 1917-1918 it was impossible to keep the colony house above the freezing point for days at a time, and in consequence the rats ceased growing at a normal rate and many of them developed pneu- monia. The periods of intense heat experienced during the summer of 1918 also had a very deleterious effect on the vitality and on the body growth of the rats. Asa result of the combined action of these various factors, all inimical to growth as well as to reproduction, the rats of the eighteenth to the twenty-fifth generations were severely handicapped, and they did not increase in body weight as rapidly, nor did they attain as great a maxi- mum body weight, as did the individuals of the earlier gener- ations. That this decrease in the size of the inbred animals was caused by unfavorable conditions of environment and of nutrition, and not by continued inbreeding, is shown conclusively by the fact that the body weights of hundreds of rats in the outbred-stock colony were just as seriously affected by these adverse conditions as were those of the inbred rats, as will be shown later. Data showing the average body weights at different ages of 179 males and of 130 females belonging in the sixteenth to the twenty-fifth generations of the A series of inbred rats are given in table 1 and in table 2: similar data for 117 males and for 180 females belonging in the same generations of the B series of inbreds are given in table 3 and in table 4. TABLE 1 Showing, by generations, the average body weights at different ages of 179 males belonging in the sixteenth to the twenty-fifth generations of the A series of inbred rats GENERATIONS AGE 16 17 18 19 20 21 22 23 24 25 days ey estes 13 TOR OU Sh LOM Peas malian 20) LOM imaliaalt ete eats 30 44 44 49 41 42 45 46 44 39 43 60 al PAL sss OF se lel edule 96 83 | 104 90 188 | 186 | 192 | 142 | 186 | 164 | 165 | 126 | 121 | 163 120 282) 228) 220 Oe |) Zoe i aide | 203: |) LEON 159" 1F 200 151 255 | 253 | 259 | 2382 | 259 | 244 | 226 | 207 | 188 | 233 182 274 | 268 | 286 | 262 | 280 | 271 | 250 | 231 | 216 | 252 yA 296 | 277 | 309 | 286 | 289 | 295 | 276 | 243 | 231 | 277 243 302 | 288 | 328 | 298 | 297 | 311 | 291 | 254 | 246 | 279 273 321 | 310 | 352 | 298 | 308 | 310 | 308 | 264 | 265 | 292 304 317 | 321 | 356 | 301 | 309 | 305 | 305 | 279 | 272 | 303 334 327 | 322 | 365 | 305 | 313 | 311 | 313 | 290 | 276 | 316 365 333 | 319 | 378 | 308 | 324 | 322 | 320 | 288 | 284 | 325 395 336 | 332 | 394 | 304 | 334 | 318 | 306 | 298 | 287 | 327 425 331 | 339 | 376 | 295 | 340 | 319 | 293 | 297 | 290 | 332 455 320 | 332 | 361 | 295 | 357 | 316 | 296 | 289 | 293 | 322 Number rats weighed Lifan|aeelefan ea Nel eel Ge 2a eis ess eT TABLE 2 Showing, by generations, the average body weights at different ages of 130 females belonging in the sixteenth to the twenty-fifth generations of the A series of inbred rats GENERATIONS AGE 16 17 18 19 20 21 22 23 24 25 days 13 DSS Yh abe Lee atric aia! eas) yh ate a aes tly Le 7s 30 41 44! 48} 39; 40| 44] 438] 42] 388] 41 60 99 |} 105 | 110 | 94] 97] 95} 99} 81} 79 | 94 90 141 | 168 | 148 | 184 | 153 | 135 | 142 | 107 | 109 | 131 120 170 | 178 | 179 | 163 | 177 | 166 | 170 | 135 | 187 | 153 151 187 | 197 | 195 | 182 | 195 | 192 | 179 | 163 | 160 | 177 182 206 | 211 | 208 | 197 | 205 | 203 | 183 | 184 | 178 | 180 212 210 | 214 | 219 | 205 | 215 | 209 | 202 | 190 | 185 | 191 243 214 | 225 | 222 | 212 | 221 | 218 | 207 | 194 | 187 | 194 273 230 | 221 | 224 | 214 | 222 | 218 | 208 | 197 | 190 | 204 304 225 | 233 | 224 | 221 | 216 | 230 | 206 | 206 | 199 | 212 334 229 | 232 | 220 | 225 | 215 | 236 | 215 | 209 | 189 | 215 365 240 | 231 | 221 | 223 | 214 | 242 | 217 | 211 | 194 | 223 395 242 | 227 | 224 | 219 | 210 | 239 | 217 | 209 | 195 | 234 425 235 | 231 | 224 | 214 | 216 | 231 | 216 | 208 | 190 | 240 455 243 | 236 | 223 | 216 | 212 | 229 | 215 | 204 | 186 | 230 Number rats weighed 13 11 12 13 13 14 14 13 13 14 74. HELEN DEAN KING Tables 1 to 4 are inserted mainly for reference, but a compari- son of the data for the males and females in the various genera- tions brings out clearly the relation between the two sexes as regards their relative body weights at different age periods. In some few instances the average body weights of the males and of the females in a given generation were the same when the animals were thirteen or thirty days old, but after this age the males were the heavier at each period for which records were taken. A similar relation between the body weights of the sexes was also noted for the inbred animals of the seventh to the fifteenth generations (King, ’18; tables 1 to 4). Investigations in which large series of stock Albinos were weighed at stated periods (Donaldson, ’06; Jackson, 713; King, ’15; Hoskins, ’16) have shown likewise that, with few exceptions, the average body weight of the males exceeds that of the females at each weighing period. Since the data for all generations of the inbred strain is in full accord with that for various series of stock Albinos, it is evident that inbreeding through twenty-five generations of brother and sister matings has not changed the normal relative body weights of the sexes at any age period for which records have been taken. For the purpose of analysis and to facilitate a comparison between the growth in body weight of the individuals in the later generations of the inbred series with those in the earlier generations, the body-weight data for the animals belonging in the sixteenth to the twenty-fourth generations of each inbred series were combined in groups of three generations each: the data thus combined are shown in tables 5 to 7. In each of these tables the data for the individuals of the twenty-fifth generation are given separately in order to show the status of the animals at the end of this period of inbreeding. Data indicating the growth in body weight of males and of females belonging in the various generation groups of the A series of inbreds are shown in table 5. As a graphic representation of series of data greatly facili- tates their comparison, the body-weight data for various groups of albino rats, given in tables 5 to 11, have formed the basis TABLE 3 . Showing, by generations, the average body weights at different ages of 117 males belonging in the sixteenth to the twenty-fifth generations of the B series of inbred rats GENERATIONS AGE 16 17 18 19 20 21 22 23 24 25 days as 13 UGH Ze PAO aL eT Te) Wey). aI aR Te 30 52 44 51 48 41 43 48 48 42 42 60 TOF Pe LT Oe 23. LOS) bit 96 | 114 95 | 114 90 178 | 165 | 181 | 175 | 161 | 168 | 159 | 154 | 143 | 147 120 213 | 222 | 225 | 197 | 203 | 220 | 188 | 180 | 182 | 180 151 23a 2A azole P22 |p2oonl oto 22Oh 212 2O4 a0 FT! 182 266 | 266 | 284 | 251 | 263 | 267 | 263 | 235 | 228 | 231 212 284 | 279 | 297 | 277 | 272 | 277 | 279 | 252 | 240 | 252 243 289 | 289 | 310 | 304 | 279 | 305 | 284 | 258 | 259 | 279 273 296 | 297 | 319 | 323 | 285 | 312 | 293 | 265 | 272 | 2890 304 304 | 303 | 323 | 324 | 289 | 317 | 300 | 276 | 271 | 299 334 BLOM Palm neoOn Poor U2Ioulco2o I oOln Zon 2920 S12 365 SPAN OU er7 || oR |) Pa) eer || BUley | 20) |) SOs a eee 395 337 | 326 | 311 | 352 | 296 | 322 | 309 | 293 | 303 | 339 425 3834 | 354 | 305 | 364 | 290 | 320 | 305 | 300 | 298 | 352 455 340 | 299 | 299 | 350 | 279 | 322 | 299 | 297 | 287 | 349 Number rats weighed WPA aa fy at TL |p IY | aed ah TABLE 4 Showing, by generations, the average body weight at different ages of 180 females belonging in the sixteenth to the twenty-fifth generations of the B series of inbred rats GENERATIONS AGE 16 Lio 18 19 20 21 22 23 24 25 days 13 TOR TOR | haniZeels 2OM mle else OS eesti 7ante tes 30 47 |) Ca wets ee TO) ea EE le Zot ly oeS 60 LOZ Pe OSM LOOM OZ TAO Sh 9e | sare 93 90 I Zia ella leon lesen 4 Om lelsern leet hOm ne iD 120 166 | 165 | 179 | 167 | 162 | 192 | 151 | 149 | 148 | 152 151 189 | 180 | 199 |} 183 | 179 | 188 | 177 | 166 | 167 | 166 182 199 | 194 | 212 | 194 | 198 | 199 | 194 | 177 | 177 | 178 212 211 | 205 | 214 | 205 | 201 | 200 | 199 | 187 | 185 | 187 243 215 | 214 | 216 | 212 | 216 | 210 | 199 | 196 | 191 | 191 273 221 | 226 | 218 | 216 | 220 | 210 | 203 | 196 | 194 | 194 304 DAW 2hie\e2l | 218) 20 2005 198%) 196" |.202 334 225 | 224 | 215 | 225 | 217 | 209 | 207 | 208 | 203 | 204 365 : DBS) || BR PR) PRP OA ABA AON Pes) Pall || ize 395 239 | 216 | 216 | 232 | 212 | 210 | 207 | 212 | 201 | 213 425 243 "| 238 | 218°} 233 | 207 | 203 | 203 | 210 | 199 | 220'- 455 243 ..| 253 | 217 | 228 | 201 | 202 | 202 | 211 | 200 |} 218 Number rats weighed 16 14 18) 19"); 19 1S ol Shie elS 19 | 20 76 HELEN DEAN KING for the construction of the graphs shown in figures 1 to 8. The graphs in figure 1 show the growth in body weight of four gener- ation groups of male rats belonging in the A series of inbreds (data in table 5). In this, as in some of the other figures, the graphs should properly run very close together or overlap in various places. If, however, the graphs had been drawn in this manner, it would be difficult to follow their course, and therefore TABLE 5 Showing the average body weights at different ages of inbred rats of the A series, separated into groups according to the generation to which the individuals belonged MALES FEMALES aT Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- tions 16-18|tions 19-21)tions 22-24) tion 25 |tions 16-18)tions 19-21|tions 22-24] tion 25 days 13 19 18 17 18 18 17 16 lye 30 46 43 42 43 44 41 41 41 60 129 110 96 103 105 95 86 94 90 189 163 137 163 150 139 120 131 120 228 214 WeZ 200 175 169 144 153 151 255 244 206 233 192 190 168 172 182 275 271 231 252 208 202 185 180 212 291 290 249 PAT 214 209 193 191 243 301 302 264 279 220 215 196 194 273 322 306 280 292 225 218 198 204 304 326 305 286 303 227 2211 204 212 334 333 310 294 316 227 222 204 215 365 337 317 298 325 232 224 208 223 395 340 316 299 327 251 220 208 234 425 345 313 294 331 229 218 205 240 455 338 316 293 322 231 218 202 230 the space between them has been arbitrarily widened in some places in order to keep the lines distinct. While the general course of all of the graphs in figure 1 is much the same, their relative position clearly shows the progressive decrease in body weight that has resulted from the action of unfavorable conditions of environment and of nutrition. The rats in the sixteenth to the eighteenth generations were fed, for the most part, on ‘scrap’ food, and, as graph A in figure 1 shows, EFFECTS OF INBREEDING ON BODY WEIGHT it the males of the A series that belonged to these generations were heavier at all ages than were the males in the later generation groups, excepting at the 243-day period. Rats in the nineteenth to the twenty-first generations were not greatly affected by the change in diet, as for some months it was possible to give them ‘scrap’ food part of the time. The males of this generation in bedy weight. Albino Rat. Series A Males Fic.1 Graphs showing the increase in the weight of the body with age for males belonging to various generation groups of the A series of inbred rats. A, graph for males of the sixteenth to the eighteenth generations, inclusive; B, graph for males of the nineteenth to the twenty-first generations, inclusive; C, graph for males of the twenty-second to the twenty-fourth generations, inclusive; D, graph for males of the twenty-fifth generation (data in table 5). group, as the position of graph B indicates, were nearly as Jarge as were those of the earlier generation group during the adoles- cent period, but in the adult state their body weights fell off rapidly. Individals in the twenty-second to the twenty-fifth generations of the inbred strain suffered most severely from the altered food conditions as well as from extremes of temperature, and the males of the A series were very inferior in body weight 78 HELEN DEAN KING to those of the preceding generations, as graph C and graph D in figure 1 show. Since the number of weighed individuals in a single generation was comparatively small, it is not surprising that the course of graph D should be rather erratic. At its beginning this graph runs very slightly higher than graph C, but at the 90-day period it begins to rise rapidly, and at 334 days it crosses graph B and subsequently runs above it until the final weighing. In the A series of inbreds the males of the BECHER [tt] Growth in body weight Albino Rat CTH SeresA F co Body weight in grams _ Fig. 2 Graphs showing the increase in the weight of the body with age for females belonging to various generation groups of the A series of inbred rats (data in table 5; lettering as in fig. 1). twenty-fifth generation were, as a group, superior in body weight to the males of the generation preceding. The superiority of these individuals can be attributed in part to an improvement in the nutritive conditions and in part to the fact that the ma- jority of animals in this generation were born at the time of year that experience has shown is most favorable for body growth in the rat, i.e., the winter months. Graphs showing the growth in body weight of females belong- ing to various generation groups of the A series of inbreds are . EFFECTS OF INBREEDING ON BODY WEIGHT 79 shown in figure 2. The data from which these graphs were constructed are given in table 5. In general the relative position of the graphs in figure 2 is much the same as that of the graphs in figure 1. Graph A, represent- ing the body weight increase with age for females of the sixteenth to the eighteenth generations, runs higher than any of the other graphs for the greater part of its course, while the position of the other graphs indicates that there was a gradual decrease in TABLE 6 Showing the average body weights at different ages of inbred rats of the B series, separated into groups according to the generation to which the individuals belonged MALES FEMALES AGE a a a ae Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- tions 16-18)tions 19-21|tions 22-24] tion 25 |tions 16-18/tions 19-21|tions 22-24| tion 25 13 19 19 19 18 17 18 18 18 30 49 44 46 43 46 42 43 41 60 116 115 102 114 102 98 89 93 90 175 168 152 147 146 141 128 127 120 220 207 184 180 170 173 148 152 151 249 235 216 211 190 183 170 166 182 271 260 243 231 202 197 183 178 212 286 275 258 202 211 202 190 187 243 295 297 268 279 215 213 195 191 273 303 308 277 289 221 215 198 194 304 309 311 283 299 220 216 198 202 334 319 319 296 312 222 217 206 203 365 320 319 304 322 227 219 207 210 395 326 319 301 339 230 217 207 213 425 328 323 301 302 234 213 204 220 455 321 318 296 349 236 211 204 218 the body growth of the animals as inbreeding advanced. ‘The females of the twenty-fifth generation (graph D) were, on the whole, slightly heavier than were the females of the preceding generation group (graph C). Table 6 gives data showing the average body weights at differ- ent age periods of males and of females belonging to various generation groups of the B series of inbreds. 80 HELEN DEAN KING The data given in table 6 served as the basis of construction for the graphs shown in figure 3 and in figure 4. A comparison of the graphs in figures 3 and 4 with the corre- sponding graphs in figures 1 and 2 shows that there was very little difference between the two inbred series (A and B) as regards the pody-weight increase with age in the animals of the various generation groups. In the B series, as in the A series, males and 360 Growth in body weight Albino Rat 4 SeriesB Males 5 pepaesiey y, soscees Oo 20. 40 60 80 10 Fig. 3 Graphs showing the increase in the weight of the body with age for males belonging to various generation groups of the B series of inbred rats (data in table 6; lettering as in fig. 1). females in the sixteenth to the eighteenth generation groups (graph A) were heavier animals at any given age than were those of subsequent generations; while the rats of the twenty-second to the twenty-fourth generation groups showed a much less vigorous growth than did the animals in the earlier groups. The rats in the twenty-fifth generation of the B series increased in body weight very slowly during the adolescent period, as the position of graph D in figures 3 and 4 indicates; but in the adult EFFECTS OF INBREEDING ON BODY WEIGHT 81 state their growth was much more vigorous, and their body weights, especially those of the males, compare favorably with the weights of the animals in the group comprising the rats of the sixteenth to the eighteenth generations (graph A). An examination of figures 1 to 4 brings out one fact of con- siderable interest: all of the graphs have the same general form, although they vary somewhat in height. As the form of these graphs is practically the same as that of the growth graphs for COCCeeeeet ae r|Body weight in grams a2 Buseseeenssecesaseessnecscessesseszseseecenecsenseseescccsesccceeseeeeneces 0 20 40 60 80 100 120 40 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Fig. 4 Graphs showing the increase in the weight of the body with age for females belonging to various generation groups of the B series of inbred rats (data in table 6; lettering as in fig. 1). stock Albinos as determined by Donaldson (’06) and others, it follows that close inbreeding, continued through many genera- tions, does not alter the character of the growth graph for the albino rat. Theoretically, it might be expected, perhaps, that long-continued inbreeding would cause a slowing up of the growth processes, since the animals totally lack the stimulus to growth that a condition of heterozygosis seems to give in many cases (East and Hayes, 712; Jones, 718). The body weights of the animals in the sixteenth to the twenty-fifth generations of the inbred strain tended to lag somewhat during early postnatal 82 HELEN DEAN KING life (figures 7 and 8, graph B), but this was undoubtedly due to the action of environmental and nutritive conditions, not to inbreeding. Any agency influencing growth, whether it be bene- ficial or detrimental, naturally produces its greatest effect during the period when growth is normally most rapid and vigorous. Since unfavorable conditions of environment and of nutrition unquestionably limited the extent of body growth in the animals of the later generations of the inbred strain, it is very probable that these factors also lessened the rate of growth during the early life of the individuals. If body growth in the inbred rats of future generations is retarded during the adolescent period, although the environmental and nutritive conditions under which the animals live are such that they produce rapid and vigorous growth in outbred stock Albinos, the change in the rate of growth can be ascribed to the effects of inbreeding. As far as the ex- periment has gone at present, the evidence does not warrant the conclusion that inbreeding per se has altered the form of the growth graph to any appreciable extent. The body-weight data for the animals in various generation groups of the two inbred series, as given in table 5 and in table 6, were combined in order to show the weight increase with age in the individuals of the inbred strain as a whole. The combined data are shown in table 7. The data in table 7 are not presented graphically, since there was such a close agreement between the corresponding records for the various generation groups of the two series that graphs constructed from the combined data would not differ materially from those given for the separate series (figs. 1 to 4). Table 8 gives data showing the increase in the weight of the body with age for all of the individuals in the sixteenth to the twenty-fifth generations of the A series of inbreds for which growth records were taken; table 9 shows similar data for in- dividuals of the B series. A comparison of the data in table 8 with corresponding data in table 9 shows that the rats in the two inbred series were much alike as regards the rate and extent of their growth in body weight. To show this similarity more clearly, weight data for the males of the two series are presented graphically in figure 5. TABLE 7 Showing the average body weights at different ages of inbred rats of the two series (A, B) separated into groups according to the generation to which the individuals belonged MALES FEMALES AGE nn nn Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- | Genera- tions 16-18|tions 19-21|tions 22-24 tion 25 |tions 16-18/tions 19-21|tions 22-24] tion 25 13 19 19 18 18 18 18 17 17 30 47 43 44 43 45 41 42 41 60 124 112 98 107 103 97 88 94 90 183 165 142 157 148 140 125 129 120 225 211 179 193 173 171 146 152 151 253 240 210 225 191 186 169 168 182 274 266 235 244 205 199 184 178 212 289 283 252 268 212 205 192 188 243 298 299 265 279 217 214 196 192 273 313 307 279 291 223 217 198 198 304 318 308 285 301 222 218 200 206 334 326 314 294. 314 224 219 205 208 365 328 318 300 324 229 221 208 215 395 312 318 300 302 231 218 207 222 425 336 317 297 339 232 215 205 229 455 329 317 294 332 234 214 203 223 TABLE 8 Showing the increase in the weight of the body with age for 179 males and for 130 females belonging in the sixteenth to the twenty-fifth generations of the A series of inbred rats MALES FEMALES AGE Body weight Number Body weight Number Of n= s, || seems Ae ee eles IA EI Of amn= Average | Highest) | Boweat. dividuals dividuals Average | Highest | Lowest days grams grams grams grams grams grams 13 18 24 14 179 17 24 14 130 30 43 54 36 179 42 57 33 130 60 109 205 58 179 95 158 62 130 90 161 268 92 179 135 187 80 121 120 203 294 128 179 162 218 108 125 151 232 321 163 178 182 235 133 123 182 256 361 196 178 196 238 162 122 212 274 382 192 170 203 246 159 116 243 285 404 199 162 208 268 157 110 273 298 432 215 148 212 268 178 100 304 302 413 213 141 215 279 169 95 334 308 410 2138 127 216 273 174 91 365 314 418 223 116 220 283 168 86 395 308 421 231 103 220 305 164 80 425 313 485 227 85 218 298 169 68 455 311 447 223 75 214 269 162 58 84 HELEN DEAN KING The relative position of the graphs in figure 5 shows that during the early growth stages males of the B series of inbreds were slightly heavier at any given age than were the males of the A series; in the period from 100 to 300 days the advantage in body weight was with the males of the A series; beyond this age males of the B series were again the heavier. In the adult state the space between the graphs represents a difference of only about TABLE 9 Showing the increase in the weight of the body with age for 117 males and for 180 females belonging in the sixteenth to the twenty- jifth generations of the B series of inbred rats MALES FEMALES AGE BODY WEIGHT Number BODY WEIGHT Number a a Re of in- of in- Average | Highest Lowest dividuals Average | Highest | Lowest dividuals days grams grams grams grams grams grams 13 19 24 15 117 18 22 14 180 30 46 62 36 117 44 60 34 180 60 111 147 64 A? 96 137 63 180 90 163 230 110 117 136 188 98 161 120 201 281 153 Rly 162 218 22) 169 151 230 326 165 117 179 236 136 164 182 255 358 189 116 186 247 143 176 212 270 367 195 115 199 250 yf 163 243 285 392 219 113 205 261 169 163 273 294 415 227 106 208 277 168 148 304 300 410 236 104 209 290 172 148 334 311 459 258 96 PALS 287 181 136 365 315 460 259 85 216 280 180 126 395 SIS 449 239 78 216 293 177 114 425 319 455 246 64 215 293 171 99 455 315 450 238 56 213 279 168 78 2 per cent in the average body weights of the. two groups of animals. Graphs showing the increase in the weight of the body with age for females of the two inbred series are shown in figure 6. These graphs are based on data given in table 8 and in table 9. In figure 6, as in figure 5, the graphs lie very close together throughout their entire course. Females in the B series of in- breds were slightly heavier animals than those in the A series Growth in body weight. Albino Rat. [5 Body weight in grams Fig. 5 Graphs showing the increase in the weight of the body with age for males belonging in the sixteenth to the twenty-fifth generations of the two series (A and B) of inbred rats (data in table 8 and in table 9). Fig. 6 Graphs showing the increase in the weight of the body with age for females belonging in the sixteenth to the twenty-fifth generations of the two series (A and B) of inbred rats (data in table 8 and in table 9). 85 86 HELEN DEAN KING during early life, but in the adult state this relation was reversed and the females in the A series were about 2 per cent heavier, as the graphs in figure 6 indicate. In the seventh to the fifteenth generations of the inbred strain, also, the animals of the two series had about the same average body weight at corresponding age periods, although, as a group, the individuals of the B series were slightly heavier (King, 718; tables 11 and 12). Throughout the period of over nine years that this experiment has been in progress, therefore, body growth in the individuals of the one inbred series has closely paralleled that of the individuals in the other series. If the varying con- ditions of environment and of nutrition to which the animals of the inbred strain have been subjected have had any influence on the heritable factors on which growth depends, it is evident that they have acted on the animals of both series in a similar way. I am strongly inclined to the opinion that environmental and nutritive conditions do not influence genetic growth factors directly, but that they act by either stimulating or retarding the growth processes. Body-weight data for a total of 606 individuals, 296 males and 310 females, belonging in the sixteenth to the twenty-fifth genera- tions of the inbred strain are given in table 10. Reference to this table, which is a combination of the data in table 8 and in table 9, will be made later. In connection with another problem I have recently taken a series of body-weight records for a second group of outbred stock Albinos. Supposedly these rats represented the best stock in our colony at the time that the investigation was begun (1916), as care was taken to select for breeding the largest and apparently the most vigorous individuals from the large number available for this purpose. These stock Albinos were reared simultane- ously with, and under the same environmental and nutritive conditions, as the inbred rats of the twenty-first to the twenty- fifth generations. The body-weight data for these animals are given in table 11. A comparison of the body-weight data for the stock Albinos (table 11) with that for the inbred group (table 10) shows that EFFECTS OF INBREEDING ON BODY WEIGHT 87 the inbred rats, both males and females, were much heavier than the stock rats at every age for which records were taken. Not only were the animals in this stock series very inferior in size to those in the first stock series reared in 1913 to 1915 as controls for the inbred strain (King, 715; table 3), but their average body weights during adult life were no greater than those of the rats in the first six generations of the inbred strain which suffered severely from malnutrition (King, ’18; table 3). TABLE 10 Showing the increase in the weight of the body with age for 296 males and for 310 females belonging in the sixteenth to the twenty-fifth generations of the inbred rats (Series A and B combined) MALES FEMALES AGE BODY WEIGHT Number BODY WEIGHT Number eRe EN, 0 | Vo fin of in- Average | Highest Lowest dividuals Average | Highest | Lowest dividuals days grams grams grams grams grams grams 13 18 24 14 296 18 24 14 310 30 A 62 36 296 43 60 33 310 60 110 205 58 296 95 158 62 310 90 161 268 92 296 136 188 - 80 282 120 202 294 128 296 162 218 108 294 151 232 326 163 295 180 236 133 287 182 255 361 189 294 187 247 143 298 212 PAG? 382 192 285 201 250 157 279 243 285 404 199 275 206 268 157 273 273 296 432 215 254 210 277 168 248 304 301 413 213 245 PALI 290 169 240 334 310 459 213 223 214 287 174 227 365 314 460 223 201 218 283 168 212 395 312 449 231 181 218 305 164 194 425 315 485 227 149 216 298 169 167 455 312 450 223 131 213 293 162 136 To facilitate a comparison between the body growth of inbred rats belonging in various generation groups and that of outbred stock Albinos, graphs showing the weight increase with age in two groups of inbred rats and in two groups of stock rats are given in figure 7 and in figure 8. Growth graphs for various series of male rats are shown in figure 7. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 1 ' 88 HELEN DEAN KING In figure 7, graph A runs considerably above all of the other graphs, except at the thirteen-day period, thus showing that the growth of the males in the seventh to the fifteenth generations of the inbred strain was exceptionally vigorous. Males in the sixteenth to the twenty-fifth generations were relatively small: in the adult state their average body weights were about 9 per TABLE 11 Showing the increase in the weight of the body with age and the coefficients of vari- ability for 165 males and for 139 females belonging to a series of stock albino rats that were reared under the same environmental and nutritive conditions as the inbred rats belonging in the twenty-first to the twenty-fifth generations MALES FEMALES ve Average Coefficients of Number | Average Coefficients of Number Be A variability suidiwiaanla Bae variability eeteacats days grams grams 13 15 15.8+0.92 165 17 16.0+0.84 139 30 40 18.41.01 165 39 17.61.04 139 60 94 21.3+0.83 150 83 20.2+0.83 131 90 126 20.0+0.97 149 116 17.4+0.75 122 120 173 19.6+0.76 149 137 14.9+0.66 118 151 195 18.1+0.71 149 152 12.0+0.52 120 182 213 15.9+0.63 147 164 11.3+0.51 ial 212 226 18.0+0.71 143 il7(il 13.70.65 102 243 232 17.6+0.61 137 174 13.1+0.61 105 273 239 18.3+0.76 129 185 13.0+0.74 101 304 243 19.6+0.87 116 186 14.4+0.71 94 334 247 17.9+0.89 108 189 14.3+0.72 87 365 254 15.8+0.77 94 188 14.4+0.74 86 395 258 15.6+0.76 75 195 15.4+0.86 aL. 425 263 19.1+1.24 54 192 14.8+0.93 57 455 269 18.01.35 39 195 15.2+1.02 49 18.0+0.85 14.8+0.76 cent less than those of the males in the earlier generations, as the position of graph B indicates. A comparison of graph B with graph C in figure 7 shows that the body-weight increase with age in the males of the later gener- ations of the inbred strain was, on the whole, very similar to that in the males of the series of stock Albinos reared in 1913 to 1915 as controls for the inbred strain: stock males grew somewhat EFFECTS OF INBREEDING ON BODY WEIGHT 89 more vigorously during the adolescent period, but they were not as heavy as the inbred males in adult life. Since the inbred males were fully as large as the males in the stock series that had been reared under much more favorable conditions of environ- ment and of nutrition, it is evident that continued inbreeding Fig. 7 Graphs showing the increase in the weight of the body with age for males belonging to four series. A, graph for males of the seventh to the fifteenth generations of the inbred strain (series A, B); B, graph for males of the sixteenth to the twenty-fifth generations of the inbred strain (series A, B); C, graph for males of the selected series of stock Albinos reared in 1913 to 1915 as controls for the inbred strain; D, graph for males of the stock series reared simultaneously with the individuals of the twenty-first to the twenty-fifth generations of the inbred strain (data in table 10 and in table 11 of the present paper and in table 13 of ‘Studies on inbreeding I;’ King, ’18). has not produced a deterioration in the original stock as regards the normal weight increase with age. The males in the seventh to the fifteenth generations of the inbred strain were much su- perior in body weight to outbred stock males reared under simi- lar environmental and nutritive conditions (compare graph A with graph C in figure 7). Likewise, inbred males of the six- 90 HELEN DEAN KING teenth to the twenty-fifth generations, living for the mest part under the handicap of inadequate nutrition, were considerably heavier at all ages than the males in a stock series that were reared simultaneously with them, as a comparison of graph B with graph D in figure 7 shows. The space between these graphs, at the 200-day period, indicates a difference of about 17 per cent in favor of the males of the inbred group. Growth graphs for various groups of female rats are shown in figure 8. EEECEEEEEEE EEE Gwin weight. eno Rat! ae58) Females ,>—,,-> SSSeSeee08Rn' SESee een eeeeeeeees SRGeeeeneeese 0 100 120 140 220 24 O 0 Fig. 8 Graphs showing the increase in the weight of the body with age for females belonging to four series (data and lettering as in table 7). The growth graphs for various groups of females, shown in figure 8, have the same relative positions as have the graphs for the corresponding groups of males (fig. 7), but they lie some- what closer together. Inbred females of the seventh to the fifteenth generations, as graph A shows, were heavier at all ages (except thirteen days) than the females of the other groups; in the adult state their average body weights were about 2 per cent greater than those of the inbred females belonging in subse- quent generations (graph B). Body weight increase with age EFFECTS OF INBREEDING ON BODY WEIGHT 91 in females of the sixteenth to the twenty-fifth generations of the inbred strain closely followed that of the females in the first series of stock controls (compare graph B with graph C in figure 8). The animals in both of these latter groups were about 14 per cent heavier in adult life than the females in the stock series reared during the past two years (graph D). In explanation of the remarkably vigorous growth of the animals in the seventh to the ninth generations of the inbred strain it was suggested in the first paper of this series (King, ’18) that: “favorable nutritive conditions following a period of semi- starvation greatly increased metabolic activity and so stimulated the growth impulse that the animals attained an unusually large size. After the maximum effect of the stimulus had passed there was a gradual decline to more normal conditions of metabolism and a corresponding decrease in the average size of the individ- uals.” Rats seem to be particularly sensitive to changes in food conditions, more so than is generally supposed, and only by feeding them constantly on a proper diet can their normal weight and fertility be maintained. In light of the valuable researches of McCollum (18) and his associates, it is evident that the ‘scrap’ food that the rats received during the period when they exhibited their maximum growth and fertility not only furnished a well-balanced ration as regards the basic food stuffs, but that it also gave a sufficient quantity of the essential accessory foods, ‘fat-soluble A’ and ‘water-soluble B,’ to greatly stimulate the growth processes. The experimental diets recently used in our colony have very evidently been deficient in ‘fat- soluble A.’ As a result the rats have shown marked evidence of malnutrition, although they have received an abundance of food. By rectifying the mistakes of the past and feeding the animals on a properly balanced ration, it is hoped that body growth will again respond to the stimulus of adequate nutrition and that it will be possible to obtain inbred animals that are as large as those in the seventh generation. As after twenty-five generations of brother and sister matings the animals in the in- bred strain were fully as large as were the best stock animals obtainable, it is evident that close inbreeding does not inevitably 92 HELEN DEAN KING cause a decrease in body size, as Darwin (’75, ’78), Crampe (83), Ritzema-Bos (’93, ’94), and others have asserted. Inadequate nutrition, seemingly, is far more detrimental to body growth than is close inbreeding, even when continued over many generations. VARIABILITY IN THE BODY WEIGHTS OF INBRED RATS At the end of fifteen generations of brother and sister matings the rats in the inbred strain were over 96 per cent homozygous, according to the calculations of Fish (14). Animals of the later generations, which had attained a degree of homozygosity prob- ably greater than that ever before reached by any group of labora- tory mammals, might be expected, perhaps, to show a very great uniformity in body weight at different age periods, if the body weight increase with age in the rat is entirely dependent on the action of genetic growth factors. But just as the rate and extent of body growth in this animal seems to be largely a matter of environment and of nutrition, so also the variations in body weights at different age periods are apparently greatly influenced by these conditions. As it is impossible, at present, to distinguish the variability due to environmental and nutritive action from that resulting from a difference in the genetic factors for body growth, one can only calculate the total amount of variability in given groups of animals and then, by comparison, determine the relative variability of the groups. No very defi- nite conclusions can be drawn regarding the effects of close inbreeding on the variability in the body weight of the rat until the animals can be kept under environmental and nutritive conditions that are so uniform that their effect is practically constant and therefore negligible. In order to obtain some idea regarding the relative extent of variability in the body weights of the animals in various genera- tions of the inbred strain, coefficients of variability, with their probable error, were determined for the body weights of the individuals in the sixteenth to the twenty-fifth generations of each of the two inbred series and for the weights of the animals in the two series combined (A, B). These coefficients, with EFFECTS OF INBREEDING ON BODY WEIGHT 93 their probable error, were calculated from the data summarized in tables 8, 9, and 10 according to the formulae given by Daven- port (714); they are shown in table 12. During early postnatal life, as the coefficients in table 12 show, the females in both inbred series were slightly more vari- able in body weight than were the males, but after thirty days of age the males, as a rule, were the more variable. TABLE 12 Variability Showing the coefficients of variation, with their probable error, for the body weights at different ages of the two series of inbred rats (sixteenth to the twenty- fifth generations, inclusive) SERIES A SERIES B COMBINED SERIES (A, B) AGE Males Females Males Females Males Females days 13 12.6+0.45)138.5+0.56/12.2+0.54)12.2+0.44)12.4+0.36)12.9+0.33 30 12.1+0.43}11.9+0.50)14.3+0.63)14.4+0.52/138.5+0.37/13.3+0.36 60 22.9+0.82|/18.0+0.75/16.3+0.71/16.4+0.60/20.6+0.57|)17.1+0.46 90 20.1+0.72|15.8+0.69}14.8+0.65/14.0+0.53)18.4+0.51)15.5+0.44 120 19.1+0.68]13.7+0.58|14.3+0.63)11.9+0.44)16.1+0.45)12.7+0.35 151 14.5+0.52)10.2+0.45]13.7+0.60/10.7+0.39)14.2+0.39/10.5+0.30 182 13.3+0.48] 9.1+0.39]12.6+0.59}12.3+0.44/13.1+0.36)10.4+0.29 212 13.3+0.49] 9.6+0.43/12.0+0.53} 8.7+0.33)12.8+0.43) 9.2+0.39 243 13.3+0.50) 9.8+0.45/11.8+0.51| 8.5+£0.32/12.4+0.36}) 9.30.27 273 12.6+0.49) 9.9+0.47)10.9+0.50) 9.1+0.35/11.8+0.35}) 9.4+0.29 304 11.3+0.45)10.8+0.53)10.2+0.48) 8.6+£0.34/11.2+0.34) 9.7+0.30 334 12.3+0.52)10.1+0.50/11.2+0.55) 7.9+0.32)11.7+0.37| 8.9+=0.28' 365 12.3+0.54/10.3+0.53)12.0+0.62) 9.0+0.38)12.2+0.45) 9.6+0.31 395 12.9+0.61/11.3+0.62/12.4+0.67| 9.8+0.44)12.7+0.45)10.5+0.36 425 14.2+0.73)10.2+0.59}/12.9+0.77/10.5+0.50)13.8+0.54/10.7+0.39 455 14.3+0.79)11.7+0.73)13.6+0.69/11.5+0.62/14.1+0.59/11.6+0.47 Average........ 14.4+0.58/11.6+0.55)12.8+0.60/10.9+0.43/13.8+0.43)11.8+0.35 was at its maximum for both sexes at the sixty-day period, and then tended to decrease with advancing age for some time. In table 12 the average coefficient for the male group in each of the two inbred series, taking all ages together, exceeds that for the corresponding group of females by over two points. Since this difference is over three times the probable error, it is sufficiently large to indicate that the males had a greater range of vari- 94 HELEN DEAN KING ability in body weight than had the females. Coefficients of variability for the body weights of the individuals in the earlier generations of the inbred strain (King, 718; table 15), and also those for various series of stock Albinos (Jackson, 713; King, 715), all show that the males are more variable than the females. Such a relation between the sexes as regards the variability in their _ body weights would seem to be a characteristic of the albino strain of rats in general, and from the results obtained in the present study it is evident that this relation has not been changed by twenty-five generations of close inbreeding. Males in the sixteenth to the twenty-fifth generations of the A series of inbreds had a somewhat greater range of variability in body weight than had the males of the B series, judging from the relative size of the coefficients for the two series as given in table 12. Between the average coefficients for the two series there is a difference of 1.6 points in favor of the males of the A series; a similar relation between the two series existed also at an earlier period (King, 718; table 15). Throughout all genera- tions of the inbred strain, therefore, the range of variability in body weights was greater in the males of the A series than in those of the B series. This difference persisted even during the periods when body growth and variability were greatly influ- enced by environmental and nutritive conditions. A comparison between corresponding coefficients for the fe- males of the two inbred series (table 12) shows that, as a rule, the females of the A series were slightly more variable in body weight at different age periods than were the females of the B series, but, taken as a whole, the one group of females was about as variable as the other, since the difference between the average coefficients for the two groups is only 0.7 point. As the study of variability in the females of the earlier generations of the inbred strain led to the conclusion that ‘‘the range of variability in body weights was practically the same for the females of the two inbred series,” it is evident that long-continued inbreeding has not altered the relative variability of the females in the two inbred series any more than it has that of the males. EFFECTS OF INBREEDING ON BODY WEIGHT 95 Table 12 shows that in each inbred series the coefficients of variability for both sexes decrease in size with advancing age until the animals attained an age of about 300 days, and then tend to become somewhat larger; a similar change in the size of the coefficients at various age periods was also noted for the animals in the earlier generations of the inbred strain as well as for those in the two stock series reared as controls. After reach- ing the height of their reproductive activity at the age of from seven to ten months, certain individuals, especially males, tend to accumulate an excess of adipose tissue; while other individuals, even members of the same litter, will show little change in body weight for a period of several months, or they may even decline steadily in body weight although they are apparently in good physical condition. The increased variability in the body weights of older rats is, therefore, due in great part to the ac- cumulation of a greater or less amount of adipose tissue; it is not a growth phenomenon comparable to that shown during early postnatal life. In order to make a closer analysis of the relative variability in the body weights of animals in successive generations of the inbred strain, coefficients of variability were calculated from the body-weight data for the animals in three generations combined as summarized in table 7. This series of coefficients is shown in table 13. In table 13 the average coefficients for the male groups com- prising the individuals of the sixteenth to the twenty-fourth generations vary by less than one point, so it is evident that in the later generations of the inbred strain the variability in the body weights of the males did not decrease with the advance of inbreeding, as was the case in the earlier generations (King, ’18; table 16). The series of coefficients for the males of the twenty- fifth generation are, as a rule, smaller than the corresponding coefficients for the males of the preceding generation group. But the difference between the average coefficients for the,two groups is less than three times the probable error, so it cannot be considered as significant, especially as the number of body- weight records used in calculating the coefficients for the animals HELEN DEAN KING 9L°0*S°8 | SS'OFF OL | IS'0+Z2'6 | 29°0+8'0I | 98°06 OT | 9 OFF ZI | 9L'0F8 SI | 6L OF E'S | WSBIOAY 66.0=9'8 | 29°02 OT | 02:0FP76 | SO TF6 IT | C8 TO ST | 82° 0F2Z TL | Cl TT PT | SE Te eT GST cO'I+6'6 | 99°0+9'6 | 89°0+E'S | 08 OF8'6 | IC T¥CCT | $9 OF9 OT | co T+8 ST | SO TFS TT SGP OO'T=1T'IT | 2¢ 088 | 99° 0F6'8 | 92°07 OT | 46°08 01 | 89°0+F OT | $8 OFF GI | LE 16 GT S6E 99°0=Z'4 | 8h 0F9'8 | 088 | 99026 | 86°09 OT | TO OFE TIT | 18 OFO CI | $8 OFF IT G9E 8¢°0=8°9 | FY OFI'S | HO0FOL | 6S°0FE6 | 66:06 01 | 99°09 OT | TZ: 08 TIT | 69 0FF OL PEE 1Z 082 | Ch OFT 8 | SS OFS | 09 096 | 00 T+O TT | co OE OT | 62 OFT OL | 19066 POS co O=6'¢ | 88OFT LZ | 88°0F6'9 | 69°0F8'6 | S8°0F66 | 99 0FP TT | 19 OFE TIL | £9 OFE OL E16 29 OF1T'L | OF 00'S | 8E°0+0'L | 6F OFFS | 28066 | 69 OFC CI | 09:06 OL | 69: OFF OF €FG 99°0=9'2, | SF OF9 8 | SE0F8 9 | 27 0FF'S | 8 OFT'OL | 79: 0FG EI | 890-2 01 | 19 OFT TE GIG 99°02" 98 | &F0- 9 84|\ ee O=79—) IS OFP-6 OS 0F9 6 je8o OI cl | 69.01 TT 9 0+7 IT cs LL:0+8'8 | 9F'0*0'6 | 6€°0*6'L | 19° 0F2 OL | 8°07 OT | 249 00'S | £9 O06 TT | $9 OFF GI TST L9°0=Z'8 | 69°09 IT | 2o'OFE OL | 69° OFT IT | 48°09 OL | 89 OFE FI | €4°0*6'ST | £9 OF8 IT OZI 6 0-1 TT | 82:0=2°S. | 19 OF ECL | PO OFT TT | OOL=TI CL | P8 OFS ZT | c8:0F9 ST | G2 0F2 TI 06 02 T+9'FI | 68 OFE ST | 249°OFLZ ET | 08 OF8'ST | 92°O0F8'8T | ZL: OFT ST | 06°00 'ZT | 16:06 LT 09 29'0+9'2 | 99°09'ST | L908 IT | 6L'0FL FL | Z9°0*6'9 | 19 OFF ST | 69 OFT EL | OL OFE ET O& 8o°0OFT'L | 29°08 ZI | 249°0F8'ET | CL OFG'ET | PO OFLL | 19 OF8 SI | CL OFL EL | 99 OFS GI &1 shop GS $2-CZ 12-61 8I-9T iG FC-GS 12-61 81-91 WO1yB.19Ue4) SUOT} BIOMOL) SUO1}B.10U9) suor}e1euay WOr}B1dUdL) SuUOI}BI9UO4) SUOT}BIBUaL) SUOI}BIBUa) adbV 96 Sa IVAN SH1IVW pabuojaq syonprarpur ay7 yoryn 07 woYnsauab ay) 07 Burpsooon sdnowb oyur paynuvdas (g ‘W) saiuas ony ay) fo sou pauqua fo sabp quasaffyp yo sqybvam fipog ay? sof ‘4o1sa apqnqoud wray? yun ‘uoynreva fo szuarayfaos ay; Burnoyy &l HTAViL £ EFFECTS OF INBREEDING ON BODY WEIGHT 97 of a single generation was only about one-third of that used for a group of three generations. The average coefficients for the three groups of females com- prising the animals in the sixteenth to the twenty-fourth gener- ations of the inbred strain are all lower than those for the corre- sponding groups of males (table 13), and they also fail to show a significant decrease in size as inbreeding advanced. The average coefficient for the body weights of the females in the twenty-fifth generation is considerably smaller than that for any of the three generation groups, but here also no definite conclusion seems warranted, since the small number of records on which the coefficients are based may be responsible in great. measure for the result. The animals in the seventh to the fifteenth generations of the inbred strain lived under environmental and nutritive conditions that were fairly uniform and seemingly very favorable to growth and to fertility. The body weights of these individuals showed a slow decrease in variability with the advance of inbreeding, as the relative size of their coefficients of variability indicates (King, 18; table 16). During early life the rats in the sixteenth and seventeenth generations lived under the same environmental and nutritive conditions as the animals of the preceding generations, and at this time they were all seemingly somewhat less variable in body weight than were the individuals in the fifteenth gener- ation. Before the weight records for these rats were completed, a change in diet became necessary, as ‘scrap’ food of the required quality and quantity could no longer be obtained. The effects of the change in food became very apparent in the course of a few weeks, and, as individual rats responded differently to the altered conditions of nutrition, there was a marked increase in the variability of the body weights in the animals of all ages. When the coefficients of variability were calculated from the series of body-weight data obtained for the animals in the six- teenth to the eighteenth generations, they were found to be somewhat larger than those for the animals in the fifteenth generation, as was expected from the observed appearance of the animals. The animals in the later generations of the inbred 98 HELEN DEAN KING strain have shown a variability in body weights considerably greater than that found in any group of inbred animals since the tenth generation. By comparing the corresponding coefficents for the two series of outbred stock Albinos that were reared in the colony on differ- ent diets, one can determine whether the variability in the body weights of these animals was influenced by the nutritive con- ditions under which they lived. By a further comparison of these coefficients with those for the animals in the later generations of the inbred strain, it will be possible to determine whether the increase in the variability of the inbred animals was due to al- tered conditions of nutrition or to the effects of long-continued inbreeding. All of the stock Albinos reared in 1913 to 1915 as controls for the inbred series were fed on ‘scrap’ food. As has already been recorded (King, ’15; table 4), the coefficients of variability for the body weights of the fifty males in this series range from 10.2 to 17.0, with an average of 13.6 for the entire group, taking all ages together; coefficients for the fifty females vary from 8.9 to 15.7, with an average of 11.5 for the entire group. The second series of stock controls was reared in 1916 to 1918 simultaneously with the inbred rats of the twenty-first to the twenty-fifth generations, and they, as the inbred rats, were fed on various experimental diets. These stock Albinos came from the same general stock colony that furnished animals for the first series of controls, so the coefficients for the two series are strictly comparable. An examination of the coefficients for the body weights of the rats in this control series, as given in table 11 of the present paper, shows that all of them are much larger than the corresponding coefficients for the animals of the first stock series, while the difference between the average coefficients for the two series is over four times the probable error. It is evident, therefore, that the rats in the second series of stock controls were much more variable in their body weights at all age periods than were the animals in the first stock series. Since both of these stock series were outbred, the increased variability in the animals of the second series cannot be attributed to the EFFECTS OF INBREEDING ON BODY WEIGHT 99 effects of inbreeding; nor can it be ascribed to a difference in the genetic constitution of the two series of animals, since no new ‘blood’ was introduced into the general stock colony from 1913 to 1917. From the evidence given, one seems warranted in assuming that the marked difference in the variability of the two series of stock animals was due, in great part, to the effects of changed conditions of nutrition which so greatly influenced the body growth of the individuals in the second series. It is probable also that the extremes of temperature to which many of these rats were subjected also affected their variability in body weight to some extent, although the effects of temperature changes were very much less than those of nutrition. Since the variability in the body weights of outbred stock Albinos was seemingly greatly affected by nutritive and environ- mental factors, one would naturally conclude that these factors would likewise influence the variability in the body weights of inbred animals reared simultaneously with and under the same conditions as the stock Albinos. The increased variability in the inbred animals of the sixteenth to the twenty-fifth gener- ations is, on this assumption, the result of environmental and nutritive action, and it cannot be cited in support of Walton’s (15) contention that continued inbreeding tends to increase variability. ‘It is interesting to note in this connection that a comparison between the average coefficients for various groups of inbred rats and those for stock Albinos indicates that changed conditions of nutrition produced a much greater effect on the variability in the body weights of stock Albinos than it did on that of the animals in the later generations of the inbred strain. In this experiment, owing to the action of environment and of nutrition, it is impossible to determine the changes, if any, that inbreeding per se produced on the variability in the body weights of the animals in the later generations of the inbred strain. This study of variability is of value, therefore, mainly because it shows that in the later generations of inbreds there existed between the two series (A and B), and between the two sexes, the same relative variability in body weights as that found in the earlier generations. Twenty-five generations of brother 100 HELEN DEAN KING and sister matings have not, seemingly, altered the relative variability in the strain, whether the total amount of variability has been influenced by inbreeding cannot be determined until it is possible to rear a number of generations of these animals under uniform conditions of environment and of nutrition. GENERAL CONCLUSIONS As a whole, this experiment has shown that the closest form of inbreeding possible in mammals, the mating of brother and sister from the same litter, is not necessarily inimical either to body growth, to fertility, or to constitutional vigor, provided that only the best animals from a relatively large number are used for breeding purposes. Selection, seemingly, is able to hold in check any tendency that inbreeding may have to bring out the undesirable, latent traits inherent in the strain. In the course of this investigation it has been shown that adverse conditions of environment and of nutrition produce far more detrimental effects on growth and fertility in the albino rat than does inbreeding. These factors, apparently, do not alter the genetic constitution of the individual, since the animals soon resume their normal growth and fertility when environ- mental and nutritive conditions are again favorable. The sex ratio in the rat is seemingly a character that is amen- able to selection, since through this process the inbred strain has been separated into two lines: one line (A) showing a high sex ratio, the other line (B) showing a low sex ratio. The effects of selection on the sex ratio seem to be limited, however, since there has been no cumulative effects of the selection, although the two lines have been kept distinct for eighteen successive generations. Whether it will be possible to change the sex ratio in the two lines by reversing the selection is the chief prob- lem in view in the continuation of this work. Throughout the entire course of this investigation there has been a great similarity between the two inbred series as regards the variability in the body weights of the animals at different age periods. In the earlier generations the variability in body EFFECTS OF INBREEDING ON BODY WEIGHT 101 weights seemed to decrease with the advance of inbreeding, but in the later generations the variability was greatly influenced by environmental and nutritive conditions. Until these latter factors can be controlled, it will not be possible to draw any definite conclusions regarding the effects of inbreeding per se on the variability in body weights. SUMMARY 1. The data given in the present paper show the growth and variability in the body weights of 296 males and of 310 females belonging in the sixteenth to the twenty-fifth generations of two series (A and B) of albino rats that were inbred, brother and sister from the same litter. 2. Owing to economic conditions, many of these rats were not reared under very favorable conditions of environment and of nutrition, and in consequence they did not grow as rapidly nor did they attain as great a maximum body weight as did the individuals in the earlier generations of this inbred strain. 3. In every generation from the sixteenth to the twenty-fifth the males were heavier than the females at all age periods after thirty days (tables 1 to 4). This result agrees with the finding for the inbred rats of the earlier generations, and also with that for various series of stock Albinos. Apparently, therefore, long- continued inbreeding has not changed the normal body-weight relations of the sexes at any age period for which records have been taken. 4. In the A series of inbreds the rate and extent of growth in body weight were much the same as those in the B series of inbreds: in the adult animals there was a difference of only about 2 per cent in the average body weights of corresponding groups of males and females in the two series (tables 8 and 9; fig. 5 and 6). 5. Close inbreeding for twenty-five generations has not altered the form of the growth graph for the albino rat to any extent. 6. Rats belonging to the later generations of the inbred strain were not as heavy at any age period as were the animals in the earlier generations, but they were much superior in body weight to stock Albinos reared under similar conditions of environment and of nutrition (figs. 7 and 8). 102 HELEN DEAN KING 7. Individuals in the sixteenth to the twenty-fifth generations of the inbred strain had about the same average body weight at different age periods as had the individuals of the stock controls reared in 1913 to 1915 under favorable conditions of environ- ment and of nutrition (figs. 7 and 8; compare graph B with graph C). Seemingly, therefore, inbreeding has as yet pro- duced no deterioration in the original Albino stock as regards the rate and extent of growth in body weight. 8. Variability in the body weights of the animals in the later generations of the inbred strain followed the same general trend as that in the animals of the earlier generations and in those of the two stock series studied: in both sexes it increased from birth to sixty days, and then decreased steadily until the animals were about 300 days of age, tending to rise again in older rats (table 12). 9. In the later generations of the inbred strain the males were more variable in body weight than the females. This result agrees with the finding for the animals of the earlier generations and for various series of stock Albinos. 10. In the inbred animals of the sixteenth to the twenty-fifth generations variability in body weights was relatively high, and it did not tend to decrease with the advance of inbreeding as in the earlier generation (table 13). 11. Outbred stock Albinos, reared simultaneously with and under the same environmental and nutritive conditions as the inbred rats of the twenty-first to the twenty-fifth generations, showed a variability in their body weights at all ages much greater than that in the animals of the earlier stock series reared under more favorable conditions of nutrition. It appears, there- fore, that the increased variability in the body weights of the animals in the later generations of the inbred strain was due to the action of environment and of nutrition, not to the effect of continued inbreeding. 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Resumen por el autor, Harley Nathan Gould. Universidad de Pittsburgh. Estudios sobre el sexo en el molusco hermafrodita Crepidula plana. III. Transmisién del estimulo productor de machos por el agua de mar. El molusco gaster6podo Crepidula plana pasa durante su vida por una fase de macho, una fase de transicién y una fase de hembra. La fase de macho es inestable y se presenta solamente como resultado de un estimulo susministrado por un individuo de la misma especie mds grande que el individuo estimulado. El aislamiento completo de los individuos pequefos no desarrol- lados sexualmente, durante largos periodos, demuestra que bajo tales condiciones no tiene lugar mas desarrollo de los caracteres machos que la formacién de unas pocas espermatogonias. En su debido tiempo aparecen los caracteres de la hembra. Los individuos pequenos y no desarrollados sexualmente confinados a distancias fijas de 4 a 7 mm. de hembras grandes, impidiéndose de este modo todo contacto, desarrollan en la mayor parte de los casos caracteres del macho en varios estados de madurez sexual. Bajo tales condiciones se producen menos machos y peor desar- rollados que cuando los animales pequefios estan mas cerca del origen del estimulo. Los individuos grandes de Crepidula forni- cata, una especie préxima a Crepidula plana, no inducen desar- rollo alguno sobre los individuos pequenos de esta ultima especie, excepto en unos cuantos casos dudosos. El estimulo que provoca el desarrollo de machos acttia de tal modo que indica que es una substancia que sale de los cuerpos de los individuos grandes de Crepidula plana, la cual substancia es difusible en el agua de mar, pero es muy inestable. Translation by José F. Nonidez Carnegie Institution of Washington AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AUGUST 4 STUDIES ON SEX IN’ THE HERMAPHRODITE MOLLUSC CREPIDULA PLANA Ill. TRANSFERENCE OF THE MALE-PRODUCING STIMULUS THROUGH SEA-WATER HARLEY N. GOULD Marine Biological Laboratory, Woods Hole, and School of Medicine, University of Pittsburgh ONE TEXT FIGURE The second paper of this series! described a number of experi- ments showing the instability of the male phase in the marine gastropod Crepidula plana. In common with other members of the family Calyptraeidae, C. plana passes through a sperm-pro- ducing phase during the early part of its life while it is small (up to about 15 mm. in length) followed by a transitional phase (15 to 20 mm.) and later by an egg-producing phase (20 to 40 mm.). Growth goes on with varying degrees of rapidity during life. The functional females are the largest and oldest. The species has a peculiarity in that the development and mainte- nance of the male phase requires a stimulus from the outside, which is furnished by the presence of a larger individual, usually transitional or female, in the immediate vicinity of the potential male. The animals are most commonly found in colonies adhering to the inner surface of shells inhabited by hermit crabs. The younger, smaller Crepidulas have various degrees of male devel- opment, those directly attached upon the shells of the large fe- males as a substratum, or close beside them, being nearly all fully developed males, while those at a distance of 5 mm. or over are more likely to have only partially developed male or- gans; the degree of development being less in the specimens far- ther from the source of the stimulus, i.e., the large individuals of the colony. 1 Gould, 1917, II. 113 « a 114 HARLEY N. GOULD In a group in which there are no females and all the members are less than 10 or 12 mm. in length, there are seldom any adult males; the majority being, instead, sexually undeveloped (neu- ter); but often the smaller members of such a group have a rudi- mentary male development, evidenced by the presence of many spermatogonia in the sex gland, even some spermatogenesis and a rudimentary penis. In fact, wherever two members of the - species are attached close together, however insignificant the difference in size between them, the smaller tends to begin male development. ISOLATION OF NEUTERS The adult male stage is never developed in isolated animals, nor can it be maintained after removal of a male from the colony, Wishing, however, to determine whether any partial develop- ment of male characters would take place in completely isolated specimens, the writer allowed young neuter animals to attach themselves to the inner surface of glass vials, one to each vial. These were all kept in salt-water aquaria. Selection of the speci- mens for the experiment was made with care from hermit shells contaiming only-atew small C. plana. Each was examined with a lens, and only those quite devoid of rudimentary male charac- ters were used. After isolation a few specimens were taken from time to time, examined, then fixed and sectioned for study of the gonad. At the beginning all were from 5 to 12 mm. in length, and were thus at the size when male development can easily be induced. They grew during the period cf isolation, and the last lot, taken at fifty days, were much larger. Slides were made from twenty-four specimens; two at twenty-two days’ isolation, four at twenty-four days, three at twenty-six days, five at thirty- three days, five at forty-three days, and five at fifty days. The results may be summarized as follows: External male characters: In three animals only, two twenty-four days and one at thirty-three days, there was a very small stump at the spot where the penis forms. No other exter- nal signs of the male condition appeared. STUDIES ON SEX IN CREPIDULA 1s) Gonad: In three cases there were a few spermatogonia in the - sex gland; one at twenty-two days, one at twenty-four days, and one at forty-three days. None of these corresponded with any one of the three having a rudimentary penis. In sixteen cases the gonad was inactive (containing only primordial male and female cells). The remaining five were the animals sectioned after fifty days’ isolation. They had passed from the neuter to the incipient female condition, having various stages in early growth periods of oocytes, and had grown considerably in size, being now from 14 to 23 mm. in length. A similar record was made of males removed from colonies and kept isolated in vials. As was shown in a former paper, all males lose their male characters after removal from the colonies. Four samples were taken from the vials after thirty-six days, four after forty-six days, four after fifty-three days, and four after sixty days. There was no resumption of spermatogenesis or redevelopment of external male organs after the degeneration in any case. The only hint of any such activity was the presence of a few dividing spermatogonia in the gonad of one isolated forty-six days. It should be recalled that previous experiments demonstrated the ability of degenerate males to reassume the functional male state under stimulus from larger individuals. It is thus indicated that the gonads of isolated small specimens may produce a few spermatogonia, but proceed no further toward spermatogenesis; and the spermatogonia so formed later degener- ate, as sections show. The isolation experiment is meant to clear the way for others, i.e., to show, in cases where partial male de- velopment is induced under weak stimulus, how much of this is due to internal causes. The writer concludes that rapid sperma- togonial multiplication, formation of spermatocytes, or any later stage of spermatogenesis is an indication of an external stimulus. In previous experiments where they developed male charac- ters under observation, the neuters were placed as closely as possible to the larger animals. Only in this way could the stimu- lus be clearly shown. The writer failed to find positive evidence 116 HARLEY N. GOULD of a stimulating secretion thrown into the sea-water. The ques- tion arose whether physical contact is necessary for the trans- ference of the stimulus. STIMULUS WITHOUT CONTACT A simple apparatus (fig. 1) was devised to hold a large female Crepidula at a definite distance from a small neuter without al- lowing them to touch or to move farther apart. The female was removed from the inner surface of a hermit crab’s shell and al- lowed to attach herself to the concave surface of a watch crystal. The small neuter was placed on the floor of the flat-bottomed de- pression in a hollow-ground slide. Mosquito netting was fas- tened over the depression to prevent the neuter from escaping. a b c d Fig. 1 Diagram showing method of preventing contact between specimens. a, large female; b, small neuter; c, depression slide; d, watch crystal. The depression slide was inverted and fastened over the watch crystal containing the large female; leaving the neuter animal, imprisoned in its cell, at a distance of from 4 to 7 mm. from the top of the female’s shell. There was no possibility of contact, yet there was little hindrance to diffusion currents in the sea- water between the two. The variation in the distance between female and neuter was due to irregularities in the curvature of the watch crystal and in depth of depression of the slide. The average distance was 6 mm. After various periods, samples of the originally neuter C. plana were fixed and sectioned. The results are tabulated below (table 1). Those specimens the gonads of which showed any male de- velopment beyond the mere presence of spermatogonia are marked ‘male.’ ‘Inactives’ are specimens with primordial germ cells only, or with these plus spermatogonia. ‘Females’ are animals where some development of oocytes could be detected. STUDIES ON SEX IN CREPIDULA 117 TABLE 1 Bete |e Rear eage2x i), MEME Oa ly RR days 14 if 4 3 0 15 20 11 3 6 ibd 21 14 3 4 Pa 19 14 5 0 ———— So | MRO tales easy 67 43 14 ; 10 Thus, forty-three out of sixty-seven, or about 64 per cent, showed spermatogenetic activity of some sort more than isolated neuters show. Classifying these forty-three with regard to de- gree of male development, we have: fully developed testis, twenty- five; testis containing sperm, but with some missing stages of spermatogenesis, four; testis developed as far as spermatids, eight; spermatogonia and spermatocytes, two; spermatogonia in multiplication period, four. The occurrence of incipient female development in some of the specimens will be understood if we assort them all in the order of their size, indicated by the length of the shell in millimeters. This is done in table 2. All those having early stages of developing oocytes (‘female’) are seen to be among the larger animals used for the experiment. TABLE 2 LENGTH NUMBER OF MALES | NUMBER OF INACTIVES | NUMBER OF FEMALES mm. 8 6 0 0 9 10 2 0 10 12 2 0 11 6 1 0) 2 5 3 1 13 3 1 2 14 1 3 1 15 0 2 3 17 0 0 1 18 0 0 1 2 0 0 1 118 HARLEY N. GOULD Female development is much slower than male, and it is likely that the most of these animals were already in the course of fe- male differentiation when selected as neuters. The percentage of ‘inactives’ is also greater among the larger specimens. It has been evident to the writer from many observations that the tend- ency to male development under stimulus gradually wanes as the period approaches when female development may set in. It is, however, sometimes possible to superimpose male on early female development, as shown in the former paper. In the watch-crystal experiment forty-three animals out of a possible sixty-seven showed some degree of male activity in the sex gland, twenty-five of them being fully developed males. Compare this with the result obtained when neuters were placed on and closely around females. In the latter case (from records in previous paper) fifty-one out of a possible fifty-three showed some degree of male development, and thirty-four of them were adult males. It is clear that more males develop when the neuters are close to the source of the stimulus than when sep- arated by several millimeters; and furthermore, the difference in the results of these two experiments cannot be adequately set forth in tabular form. Examination of the gonad under the mi- croscope shows it more strikingly. Many marked ‘adult testis’ in specimens from the watch-crystal experiment are only a fraction of the size of the gonads developed in those placed close to or on the large females. There are often signs of arrested development in the former, shown by the paucity or absence of some stages of spermatogenesis. An examination of the small individuals in a large number of normal colonies shows about 62 per cent adult males (determined from external characters). By placing neuters on and close to females, about the same percentage of adult males was obtained, and this could have been raised considerably by rejecting all those specimens which had moved several millimeters from the females during the course of the experiment. In the watch- crystal experiment only about 38 per cent became adult males. The development of the male phase by neuters imprisoned in depression slides thus shows that the male-producing stimulus is STUDIES ON SEX IN CREPIDULA 119 able to act in the absence of physical contact and through several millimeters distance in sea-water. A comparison with other ex- periments indicates that fewer and less fully developed males are produced under such conditions than when the stimulus acts more directly. The writer has tried several times to determine whether a large female of Crepidula fornicata, another species of the same genus, could furnish the stimulus for male development in a small neuter Crepidula plana. The experiment has been difficult to carry out, as the little C. plana neuters were generally crushed by the twisting and turning movements of the great C. fornicata before sufficient time elapsed to make the experiment valuable. The writer has, however, slides made from thirty-two C. plana selected as neuters and kept near the C. fornicata for various periods. Of these, twenty-one remained entirely neuter and six became incipient females. The remaining five show traces of male development. Two of these must be counted out because the microscopic appearance of the gonad shows that the few prod- ucts of spermatogenesis there must have been formed and fur- ther activity must have ceased before the experiment began. This leaves only three which seem to have developed any male characters during association with C. fornicata, and they are as follows: a. Penis partly developed and small testis as far as spermatids, not very active. ‘Time, seventeen days. b. No penis. A few spermatogonia and spermatocytes. Time, eleven days. c. Nopenis. Spermatogonia and afew spermatocytes. Time, eight days. Thus, there are no adult males developed out of twenty-six neuter specimens (leaving out of consideration those which had begun female differentiation), but there are three with partial male development during the experiment. This result is rather perplexing: One would naturally expect either an appreciable proportion of males, if the C. fornicata exerted any influence, or none at all, if they did not. However, we may draw the conclu- sion that the male-producing stimulus is not due to any general THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 1 120 HARLEY N. GOULD change in the medium (sea-water) caused by C. plana which would be similarly caused by other species. The C. fornicata females used for the experiment were larger than the largest C. plana females, and would be expected to throw into the sea- water at least as much of the general katabolic products, for in- stance, as the latter, yet they had almost no effect in stimulating development of the testis. It should be emphasized that the power of large animals of the species C. plana to stimulate spermatogenesis in the smaller is not limited to females. A number of unusually large males were removed from a colony and imprisoned in a watch erystal with nine small neuters. In eighteen days five of the nine showed some degree of male development, mostly immature. The large males were in the meantime losing their male characters. They were kept forty days after this losing all signs of maleness and growing larger. A second lot of nine small neuters was placed with them. In sixteen days eight of the nine had some degree of male development, averaging nearer the mature male phase than the first nine. The numbers are too small to speak for the relative effectiveness of large males and large transitionals, but show the ability of both to produce the stimulus. SUMMARY The stimulus passing from larger to smaller Crepidula plana, causing the latter to assume and retain the male phase, can be transmitted for several millimeters through sea-water, though its effectiveness is reduced at this distance. Indication that the stimulus may be given faintly by Crepidula fornicata, a related species, was given in only three out of twenty-six cases. The stimulus acts in such a manner as to suggest that it is a specific substance given off from the bodies of the animals, dif- fusible in sea-water, but very unstable. BIBLIOGRAPHY Goutp, H. N. 1917 Studies on sex in the hermaphrodite molluse Crepidula plana. I. History of the sexual cycle. Jour. Exp. Zodl., v. 23, no. 1. II. Influence of environment on sex. Jour. Exp. Zodl., v. 23, no. 2. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AuGusT I1 UROLEPTUS MOBILIS ENGELM. II. RENEWAL OF VITALITY THROUGH CONJUGATION GARY N. CALKINS Columbia University, New York City. ONE CHART AND ONE FIGURE In a previous paper I have described the morphology and the cytology of division and conjugation stages of the rare hypo- trichous ciliate which forms the subject of the present paper.! Lending itself admirably to the cultural method which has been employed, Uroleptus mobilis is the most satisfactory organism for experimental work I have yet encountered. Paedogamous conjugation, in epidemic form, occurs readily, under the proper conditions, in the culture medium. Ex-conjugants, upon iso- lation, live and thrive in this culture medium in practically 100 per cent of cases—a rare phenomenon among the hypotrichs. Asexual reorganization, or parthenogenesis, called by Woodruff and Erdmann ‘endomixis’, occurs at fairly definite periods, un- der the protection of a cyst membrane. Such reorganizations, therefore, are advertised by the form assumed by the organisms and do not interfere with the study of comparative vitality throughout the life cycle. Starting with a single individual which was isolated imme- diately after conjugation, on November 16, 1917, I have followed the life history of thirteen different series, all beginning as ex- conjugants of pairs of normally conjugating individuals, and all were progeny of the original ex-conjugant which was isolated November 16, 1917. Three additional series, derived from encysted individuals, have also been studied in similar isolation cultures. The different series were started at various periods 1 Calkins, 1919. Uroleptus mobilis, Engelm. I. History of the nuclei during Division and Conjugation. Jour. Exp. Zoél., vol. 27, no. 3, p. 293. 121 122 GARY N. CALKINS of the life history of parental series and at different stages of vitality, so that I have abundant data for the study of com- parative vitality of parent and offspring. It is with pleasure, based upon admiration for the genius of that gifted pioneer in this field of research, Edouard Maupas, that I can say these data convincingly prove the truth of his conclusions that conjugation in ciliates restores vitality and prevents the phenomena accom- panying ‘old age’ which we include under the terms senescence and natural death. METHODS AND RECORDS On October 3, 1917, a rich culture in an old hay infusion was found to contain a large number of individuals of Uroleptus mobilis. The normal structure of this rare ciliate is described and its systematic position given in my earlier paper. It is illustrated again in figure 1 of the present paper. Attempts were immediately made to cultivate the organism on the usual hay- infusion culture medium. A better medium was obtained by mixing boiled flour water, hay infusion, and spring water, and this was used until the middle of January, 1918. It was in this medium that the first pairs of conjugating individuals were found and isolated, giving the ex-conjugant destined to form the first, or A series. AZ 5. Reguoeet. DESO e oo Oo AT sa UGS 6. Migrations; Aero cation IME PTOUPSs 4 sss . gs Qe Brcedin pina rise RPA MESES) Sry 44.0 (LEN Geil peel oka 179 Se IONOMIUCECOMKE | AtlOMShery © tee eae = revo eens oko ot ee SI IJt. Movements and reactions... els? zs Local movements. : 5 1 . Movements of Tie: uahitits Re iL OIG agen oe ens ie ae 185 Sst ON ae Ons | eens aaa 187 ive, Mechanicaliexcitutlony sss se cere monet we LOZ If Ma ctilerstimuUla trons. Ae Ae ass Ave ks ee bs hae See LOE A. Dorsal surface. . >, JR B. Ventral parts. . ne Ne U94 C. Distribution of Beart iia . 198 D. ieee Peco toes hk MN OP Ton aoe oll 3 galt Vlve 200 2ke Nalbrartony-s timailiiny 5 eho ed. Rae Ne ohahen ls Cee haere. Were ara an) 2 oF Bhiomlotaxist (ais sak MPO ie Maire a2) 1tOed Aer. OPES ae Va 2 OF Ae theotropisiles: Aiton joie Sar cite Roa aay SOG oitine naiee see 200 HeAGCOUrOpISMU As. cee A AES. TERT). ed. CR a Sk aR) 6 210 SEO UMA T Vee MO tARles eden Ode AER MIRE eee EN ARES Tee) a, AI ghee 1 Contributions from the Bermuda Biological Station for Research, no. 110, and from the Anatomical Laboratory of the Northwestern University Medical ‘School, no. 66. 157 158 LESLIE B. AREY AND W. J. CROZIER V. Thermal excitation......... Se Tha ee che eee eee es 1. Behavior at digeeont Paperanires. itt apaeen Watsine a see RLS 2. Tepea applica ian taulien cardi colds are ee nee 221 Be SUMMA ic fal Fe Repo haslat orale roi ice Te ee ANS Sens IEE eS Vii. Photrevexcita tome cine. tea tece ace 5.5003 cul SO tort cet oh ta ee 1. Effects of hone REE ee 224 a. Behavior in an Plunmaned Geld PASI AAS Se OO es ee b. Results of partial illumination of ie OC ec ree 2 (as tae 2s NOMLCRCOGIAIUSENSULIV TY. yee cys ae, c These matters are touched upon at this point for a reason which will become apparent further on in this paper. They are discussed with some completeness in a subsequent paper, by one of the present writers, dealing with the ethology of chiton. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 2 166 LESLIE B. AREY AND W. J. CROZIER kinds and number of these epizoites depend upon the size of the Chiton and the character of its habitat. The most conspicuous of them are barnacles (Tetraclita, fig. 1), Spirorbis, and Serpula. To this group must be added algae, comprising not merely the thin coating upon the valves, but also the Enteromorphas, which (in appropriate habitats) grow plentifully upon and between the HL. 128.3 oe oS) 6.2 cms. 129.44 8.8 cms. Nf py Baas 4.Coms ee C2 (x/4 KH. 123,/ XZ.33.38 & J 77 cms. 1/1 cms. 3 Fig. 3 Outlines of the fourth valves of five chitons of increasing ages (sizes) ; dorsal aspects, anterior up; see text. X 3. Fig. 4 Illustrating protective growth of the valve substance resulting from the presence of epizoic barnacles; the outline is that of the valve. X 1. scales of the girdle. They are very important for the production of homochromie effects, because the periostracum of the scales is but little eroded, even in large chitons. The valves are rarely much overgrown with these algae, except among the largest chitons. In the felted matting of algae various young mollusks, nematodes, archiannelida, and protozoa abound. ‘The serpulas THE SENSORY RESPONSES OF CHITON 167 affect only the very largest chitons. Barnacles remain attached to a valve in some cases until they have formed three growth lines (‘year lines’). One effect of the barnacles is important in connection with our preceding remarks regarding the forward erowth of the tegmentum as correlated with the erosion of the superimposed umbo. Instances such as that illustrated in figure 4 show how it is possible for the shell to grow in a protecting manner. In studying shell variation in the chitons it must be remembered that the attached barnacles may be removed, before or after their death, and leave no obvious trace, although they may have been responsible for irregular growth of a valve. At sexual maturity the female Chiton tuberculatus is colored in a different way from the male: its tissues are impregnated with a salmon-pink substance concerned in the metabolism of the ovary. If the shell plates are separated, this differential color- ation of the sexes may be detected in dorsal view. Normally, it is quite invisible. This is the first instance of its kind which seems to have been described among mollusks. Its importance has been discussed in another place (Crozier, 719). In the gill channels and under the girdle of chitons obtained on sunlit shores where Enteromorpha and associated plants are growing in a felted covering over the rock, there are nearly always to be found considerable numbers of a commensal isopod. It appears to be the Eusphaeroma (Sphaeroma) crenulatum of Richardson (’02, p. 292; ’05), described by her from specimens collected at Bermuda many years before by Goode, but concern- ing which no information as to habitat or local manner of occur- rence has previously been recorded. The association of this isopod with C. tuberculatus is general throughout the Bermuda area, but the commensalism is of a more or less facultative kind, since the isopod is found sometimes among the algae at some distance from a chiton. Even where the supply of algae is scanty (as in crevices within the walls of caves), the isopods are also sometimes found, but usually not in such abundance, under the girdle of Chiton. As many as twenty or more are to be found under a chiton 8 em. long. The association is quite independent of the sex and sexual coloration of the chiton. The isopods are 168 LESLIE B. AREY AND W. J. CROZIER small (2 mm. long), and when taken from a chiton at low tide their coloration is quite pale, of a yellowish cast, with minute black markings. The coloration becomes darker in the light, and then reproduces on a small scale something of the greenish-to- black color pattern of the chiton girdle. The sphaeromas fre- quently remain in place under the chiton at high tide, and in a glass aquarium they will reassume a position within the gill channel or under the girdle of medium-sized or large chitons. There they take up stations chiefly along the lateral margins of the girdle, which are slightly raised during the respiration of the chiton. The isopods remain with heads pointed outward, into the incoming respiratory current. Sudden shading causes them to dart back into the ctenidial channel. When in their resting position a small portion of the anterior end may project beyond the edge of the girdle of their host, and under these cir- cumstances their coloration, resembling that of the dorsal tu- bercles upon the girdle, renders them very inconspicuous. Occa- sionally one of these isopods creeps in between the gill filaments, usually at the posterior end of a chiton, and under these condi- tions is forcibly shot out at the posterior extremity of its host by means of the water current; the isopods appear, moreover, to react negatively to currents of this strength, and continue vio- lently to swim away, in a spiral path, from the region of the anal current even after it has ceased to act upon them in a gross mechanical way. At high tide, and when under water in aquaria, the isopods creep freely over the dorsal surface of the chitons; under these circumstances their coloration is to a very high degree homochromic and concealing. This relation between Sphaeroma and Chiton will be made the subject of further study. At present it can be said that there does not appear to be any precise ‘attraction’ (chemical, for example) exerted by the chitons upon the isopods. This commensal isopod is involved in the very complex envir- onmental correlations, which may be clearly analyzed, in the life-history of Chitons. Therefore we mention it here, although detailed work on its behavior and relations must be deferred for the present. THE SENSORY RESPONSES OF CHITON 169 2. Growth and duration of life As with animals in general, the rate of growth of Chiton de- creases with advancing age. For the area considered in this report the growth of Chiton tuberculatus appears to be ade- quately represented in figure 5. A detailed analysis of the material upon which this curve is founded will be given elsewhere. A ge(estimated ). ~< Ny ce Oe ie Ole = eure 1S ems. 3 4 5 Length, Fig. 5 Showing the relation between size (total length, in centimeters) and estimated age (years) in Chiton tuberculatus. These est mates are not in- tended to be of final significance. The normal average growth curve differs in shape from that shown. The two lines shown include between them most of the variations found in the Chiton population of Great Sound (April to May, 1918). The maximal duration of life seems normally to be from eight to nine years (fig. 6). A length of existence so great as this appears not to have been suspected previously for the chitons. This species probably comes to reproductive maturity in the second (or third?) year of life (Crozier, 719). The general rate of growth 170 LESLIE B. AREY AND W. J. CROZIER corresponds with that of some other chitons (Heath, 799, ’05 b 05 c), at least for the early years. It is also true that in certain other chitons the second year marks the incidence of sexual maturity (Heath, ’05¢). The large Cryptochiton stelleri grows more rapidly than Chiton tuberculatus does, but also matures in the second year (Heath, ’05 c). The curve in figure 5 is introduced here for the purpose of correlating some statements to be made subsequently regarding behavior at different ages (ef. Crozier, ’18b). in@y iA 56 70.60, Gnas: Age, estimated. Fig. 6 The frequency distribution of estimated ages in the chiton popula- tion on the north shore of Long Island, Great Sound (April, 1918). It is neces- sary to consider each local population separately. The one here plotted is fairly representative. 3. Destructive agents The power with which chitons may adhere to the rock surface is well known. When disturbed the girdle is firmly applied to the surface and the shell plates closely approximated. Except by means of a very powerful lateral push, it is impossible to dis- lodge them, once they are ‘set’ (which may happen very quickly) ; there is no projecting part of the smooth depressed animal which could offer a ‘hold.’ The dead plates found in the field have almost without exception been those of animals eight to nine years old. THE SENSORY RESPONSES OF CHITON HZ Chitons are eaten as the main constituent of ‘suck-rock soup’ by some of the poorer people of Bermuda. One of us has observed rats quickly seizing chitons and devouring them. From the attacks of many other carnivorous animals of the shore zone, C. tuberculatus is relatively immune. About 7 in 1000 were found with oyster-drill holes in one or more valves. The animals whose shells were so attacked were always stillalive. The holes pierced merely the tegmentum, the dense, hard articulamentum being impervious to the oyster-drill’s efforts. Although as many as 125 barnacles have been noted upon one chiton of medium size, it does not appear that they produce a deleterious effect. After the death of the chiton, the barnacles drop off, frequently without leaving any trace; they are never very firmly attached, except in the case of very old chitons with thoroughly eroded valves, and it does not seem as though they can even pierce the periostra- cum. The same applies to Spirorbis. Serpulids grow only on very old chitons; they become incorporated in the substance of the shell, and appear to be in some instances responsible for a local increase in its thickness. Injuries suffered by the girdle can be slowly repaired. Several animals were examined four weeks after they had been marked upon the girdle by having a deep notch cut init. The notch had been partly filled in by new mantle tissue, the new dorsal surface bearing small, irregularly distributed plates. The new plates were at first widely separated and irregular in shape. After six weeks they were still irregular, but had become more closely set together. The power of regenerating the plates (‘scales’) may be related to the fact that the periostracum of these plates appears lustrous and uneroded long after the shell plates have been intensely weathered. Chitons are sometimes found in the field with small groups of the girdle scales removed, exposing the bare mantle, as well as with notches or ‘bites’ removed from the girdle. The general impression derived from the consideration of destructive agents in relation to Chiton is that these mollusks are very efficiently protected. The length of life which they seem to attain, the variety of habitats which they frequent, and the ie LESLIE B. AREY AND W. J. CROZIER character of their sensory responses, which determine certain features of their life in these habitats, afford important evidence to this effect. 4. Feeding All the chitons, probably, are vegetable feeders. They rasp the thin coating of algae from the rocks by means of the radula (H. Jordan, ’13), which is long, armed with powerful black teeth, and operated by a complex arrangement of muscles (Plate, ’97). The body musculature is also involved in feeding. The whole body ‘lurches’ back and forth synchronously with the use of the radula, the forward swing coinciding with the retraction of the radula; the foot remains stationary. In rock crevices C. tuber- culatus occurs frequently in groups, piled one animal upon another. Investigation has shown that under these circum- stances they may feed on one another’s backs upon the algae growing there. The radula removes not merely the algae, but some of the rock surface as well. The chitons may be of some slight geological importance in this way, and they may also be in small part responsible for the destruction of the periostraca of their associates and thus for the weathering of their shell plates. Most of their feeding seems to be done at high tide. It is when covered with water that they move about most freely, although in damp places they also move to some extent at low tide. The great majority of the individuals are found well confined within tidal limits. While exposed to the air as the water falls they defecate copiously. The feces are discharged in the form of tiny cream-colored, cigar-shaped masses, varying in length with the length of the animal (fig. 1); in an animal 8 em. long the fecal masses are 3.1 mm. long and 1 mm. in greatest diameter. The masses consist for the most part of minute granular bits of sand, but contain also undigested plant remains and fatty globules. When treated with acid, bubbles of CO: appear; all but a slight meshwork of algae fragments is dissolved. The mass of plant fibers holds the fecal matter together in a pellet, which persists for as much as twelve hours under water in nature. When it is considered that, along the north shore of Long Island, for THE SENSORY RESPONSES OF CHITON 173 example, more than 700 chitons, averaging 7 cm. length, were found within a strip three-eighths of a mile long, their eroding importance will be admitted to deserve examination. (A study of this matter is being made.) By the time the tide has risen one-quarter, every chiton in an intertidal group is found to have deposited a considerable mass of fecal matter within the anal region of the mantle cavity. At high tide they do not appear to defecate to any great extent. There would seem, therefore, to be some rhythmic sequence of feeding operations roughly coérdinated with tidal events. This might assist in the determination of a metabolic rhythm, which might in turn receive expression in (tidal) rhythms of behavior. 5. Respiration The respiration of most individuals of C. tuberculatus is also subjected to the influences of tidal events. Under water, Chiton obtains oxygen by means of a water current, passing inward laterally along the girdle, through the gills, and escaping at the anal end (fig. 7). Out of water, the gills are more or less con- tracted against the dorsal wall of the ctenidial channel. Some oxygenation may, however, occur out of water, since the gills remain damp, and in nature the girdle is usually lifted from the substrate to some slight extent, unless the creature be disturbed. The girdle is important for respiration, as the region in which it is lifted from the substrate localizes the intake for the water current. When completely submerged, this is commonly at the anterior end. The incoming water then impinges upon the dorsal surface of the proboscis (‘palp’). Water is also taken in at the sides of the body. The latter is exclusively the case when the chiton is but partly submerged (i.e., with the anterior end out of water). The girdle may be locally lifted in the form of channels (fig. 7) or may be completely lifted. The water passes up between the gills, and escapes under an elevation of the girdle at the posterior end. This elevation is of somewhat variable form, although always located between the posterior ends of the right and left gill series. It is formed as a direct result of the water current impinging on the inner ventral margin of the girdle. 174 LESLIE B. AREY AND W. J. CROZIER Figure 8 illustrates this point. When a chiton, partially out of water, on the wall of an aquarium, swings from a vertical posi- tion (fig. 7) to one such as that shown in figure 8, the posterior, elevated part of the girdle travels to one side as a smooth wave. The water current also enables a chiton to sample the surround- ing water. It is of importance for reproduction, since the stim- ulus to egg laying is provided by the diffusion of sperm from near-by males; these sperms are carried past the openings of the oviducts, past the ‘osphradia’ (p. 253), and eggs are liberated Fig. 7 Illustrating the course of the water current n Chiton. Diagram- matic. Fig. 8 The course of the water current in Chiton when the animal is par- tially submerged. Diagrammatic. in their company (Metcalf, 92; Heath, ’99, ’05 cc). The ne- phridia also discharge their excretions into the respiratory cur- rent. These excretions, together with the water that has been ‘used,’ are usually shot to a considerable distance, because, the anal opening being smaller than the incurrent openings, the velocity of the outgoing current is high; here also, as in Ascidia (Hecht, 718), the ‘used’ water is discharged in such a way that it is not readily employed again for breathing purposes. The ventral surface of the girdle is transversely ribbed, pro- viding minute channels through which water is taken in, even THE SENSORY RESPONSES OF CHITON 175 when the girdle is not detectably lifted; this can be demonstrated with suspended carmine. The girdle can, however, be very tightly applied to a smooth surface. A chiton, if attached to the wall of the dish, will live for two or three days completely submerged in an aquarium containing other dead and decaying chitons. During this time no water is taken into the gill chan- nels. Hence, although chitons appear to frequent regions where, by wave action, the water is well aerated, it does not appear that they are particularly sensitive to want of oxygen.® 6. Migrations; association in groups The larger chitons rarely engage in creeping movements unless they are at least partly under water. Occasionally they are seen to creep about when the wet under surfaces of rocks on which they may be situated are turned over and exposed to the light. They also creep, slightly, on wet rocks covered with algae. In dark pockets within the walls of caves, where compact groups of chitons may be found, they may be seen, if watched carefully, to move slightly upon one another; such places are, however, decidedly damp. When the tide comes in and covers a chiton, it may become active immediately. Conversely, when left by the receding of the tide, a chiton usually stops creep- ing and remains where it happens to be. If, however, water be splashed over it, it will continue creeping for a longer time; if the splashing be stopped, the animal stops creeping immediately. Even when left in the sun to dry, upon the tide’s falling, Chiton is not entirely immovable. In the case of the larger animals, if they be partially covered by a shadow, they will, even in this condition, move forward, or backward, or turn slightly, so as to become more evenly adjusted with reference to the ight. The possibility of such movements suggested that an 6 According to Heath (’05c¢, p. 392), the gills of Trachydermon raymondi, which employs the gill cavities as breeding chambers, may become occluded during the breeding season by the 200 or more young trachydermons therein sheltered (Plate, 99, Taf. 6, fig. 218); under these circumstances the lateral proboscis lappets become (like the whole proboscis) much distended with blood, and may then be concerned in respiration. 176 LESLIE B. AREY AND W. J. CROZIER ‘anticipatory’ creeping toward the rising water of the incoming tide, based upon some form of hydrotropism, might be dis- covered in Chiton, and was accordingly looked for. None was found. Chiton is in this regard analogous to the actinians (Parker, ’17 b); there is no ‘memory’ of recurring tidal events. Having in mind the possible metabolic basis of tidal rhythms in behavior, discussed in connection with feeding and respiration, the behavior of Chiton has been studied for the occurrence of other tidal rhythms—inactivity as to creeping, movement out of water, and the like. Nothing of this kind seems to occur in C. tuberculatus. Chiton, unlike a limpet, does not settle down into a de- pression closely conforming in outline to the impression of its shell. Neither does it, like a limpet, leave evidence upon the rock surface of wanderings and returnings to a ‘home station’ (Orton, 14). Inasmuch as a number of chitons seemed always to be present in certain depressions, or ‘pockets,’ which were ex- amined at low tides, and since observation of the behavior of other chitons showed that they usually began to move about as soon as the rising tide had wetted them, data were sought to answer the question as to whether chiton exhibits in one form or another ‘homing habits’ of the type which have been described for Patella and its allies (Kafka, 714). . An experiment of this sort is here recorded: June 15, 1914. Observations were restricted to a definite area of smooth rocks below the boat house on Agar’s Island. The chitons were marked for subsequent recognition by means of a deep notch cut in the girdle on either side of the body. (As noted elsewhere, about four weeks were required for such notches to be even par- tially obliterated through regeneration.) In the area of shore con- cerned in this record there were several deep crevices and niches into which chitons crept. The observations begun at this date were con- tinued until July 14 (see table 1). This table shows plainly that for a period of twenty-six days no material additions were made to the chiton population of this particular section of the shore, although it did appear that there were occasional new arrivals. In all, twenty-four chitons were marked, and at the end of the experiment, twenty-four days after THE SENSORY RESPONSES OF CHITON eT the last one had been marked, eight of these still inhabited the restricted region which was examined. The occasional arrival of a new chiton in this area is consistent with the gradual and fluctuating disappearance of the marked individuals. Perhaps the handling and stimulation due to cutting for marking pur- poses caused an initially increased wandering of the marked TABLE 1 Concerning the migration and ‘homing habits’ of Chiton g g TOTAL | PREVI- f DATE | HOUR weak uae MARK: REMARKS SEEN ED 6/15 | 11.30} 6 0 4 (1 lost; 1 injured and rejected) 10 found in 6/15 6.00) 16 3 10 deep crack; these overlooked before? 6/16 | 10.00} 12 11 1 6/16 | 5.30} 18 12 6 | A new niche found, overlooked before; both marked and unmarked animals were in it. 6/17 | 11.00] 10 9 1 6/17 6.00} 18 12 0 | One individual in a crevice, could not be seen well. A new one? 6/18 | 11.30) 14 13 1 6/18 6.15) 7 7 0 | Tide not completely down; hard to see clearly. 6/19 9.30} 9 9 0 6/19 6.30} 10 9 1 6/20 | 10.00) 7 i 0 6/21 | 11.00) 8 a 0 | One hidden; impossible to distinguish whether marked or not. 6/29 | 11.00) 12 9 0 | Three others seen but inaccessible; marked? Cif Al 6.00) 10 8 0 Two others seen but inaccessible; marked? 7/14 6.00} 9 8 0 specimens. The general result is clear, however: Chiton is not stationary, it does move about to some extent, but adult ani- mals, such as those used in this experiment, do not move fre- quently from place to place. Further observations showed that there is probably some correlation with age in the matter of migration. The youngest 180 LESLIE B. AREY AND W. J. CROZIER animals tend to occur in isolated groups, and further from high- water level than the younger individuals, there results a type of segregation which is favorable to the occurrence of some degree of homogamy (assortive fertilization). This would result in the economical utilization of sperms, and might possibly have ad- ditional effects of an ‘adaptive’ kind. Some further correlation between habitat and breeding habits in other chitons have been noted by Heath (05 e, 07). PH. Poet Shorey = { Fig. 9 A group of Chitons in a shallow depression. X 3. 8. Bionomic correlations Chiton tuberculatus is strictly intertidal in habitat. It, there- fore, becomes possible to examine the details of its natural history rather extensively. The complexity of the catenary systems of relations revealed by such examination renders orderly description difficult. Some of these relations we have referred to in the preceding sections. Numerous others remain THE SENSORY RESPONSES OF CHITON 181 to be considered. They concern phenomena of coloration, re- production, determination of ‘choice’ of habitat, and similar features, comprising some of the things which involve explanation in terms of the animal’s sensory physiology. In the matter of coloration, for example, the chitons in‘ general exhibit homo- chromic (‘concealing’) characteristics which are commonly of some precision (e.g., in Cryptochiton, Heath, ’05 b, p. 213, and in other genera which we have observed; cf. also Plate, 1901 a, p. 376). In C. tuberculatus this homochromic correla- tion is decidedly evident—in most cases it is unmistakable. It involves several pigments, their mode of distribution, the over- growth of the shell by algae, barnacles, ete., a shifting of the chiton during growth to stations further below high-water level, the erosion of the valves, and a further shifting to more exposed habitats, with corresponding changes in the appearnace of the creature. There is little reason to doubt that in the later growth of the chitons (three to four years old) conditions of food supply directly determine through the course of metabolism the char- acter of the. pigmentation displayed in the periostracum. The most fundamental factor concerned in the changing habits and appearance of chiton with advancing years, however, is its move- ment into more illuminated areas. The whole problem of its bionomic correlations becomes, from this standpoint, somewhat more directly open to attack. In general, it is not: How are the bodily processes kept going by the aid of movements? and, How does it happen that the movements are of such a character as to keep the processes going? (Jennings, ’07, p. 57), but rather: What is the relation between the sensory capacities which deter- mine and direct the bodily movements, on the one hand, and on the other hand the way in which the bodily processes are found actually to be going? Naturalists have long been content to assign a given ‘reaction’ to some one or another of the categories of adaptation, and to rest satisfied that progress had thus been made in explaining it. No progress can be made in this way. Neither are we greatly helped by placing the responsibility for the adaptation in a general way upon the environment. The situations requiring THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 2 182 LESLIE B. AREY AND W. J. CROZIER analysis are too specific. The real problem is to trace to their sources of origin some of the harmonious correlations—involving habits, coloration, and the like—which specific organisms display. For studies of this kind Chiton tuberculatus affords eminently advantageous material. The analysis of its sensory character- istics, forming the body of this report, is taken therefore as the starting point for a series of quantitative investigations in ethology. III. MOVEMENTS AND REACTIONS For the analysis of the sensory capacities of Chiton we depend upon its motor reactions under various forms of activation. It is therefore necessary to outline the different modes of response exhibited by these animals. The movements of chitons present, in fact, a certain degree of diversity, somewhat at variance with the traditional epithet ‘sluggish,’ so frequently applied to them. Slow, as a rule, the movements undoubtedly are, and for that reason particularly favorable for examination, as the responses can be studied with precision. The motor reactions of Chiton comprise movements of local parts of the body, bendings and twistings of the animal as a whole, and pedal locomotion. This classification of movements is largely artificial, but convenient. Each of these classes may be dealt with in further detail. 1. Local movements Local responses may be obtained from almost every part of Chiton. Since the muscular organs concerned in these move- ments are described in Plate’s monograph (’97, ’99, ’01 a), they will not be considered here. The girdle (fig. 10) reacts locally by puckerings and by bending movements. The individual shell plates may be pushed apart from one another, elevated, depressed, and closely approximated. These local responses are involved also in the general movements of the whole animal. The local movements of the ventral parts are less directly in- volved in responses of the animal as a whole. At the anterior end (fig. 11), the mouth, a transverse slit, is situated upon a THE SENSORY RESPONSES OF CHITON 183 proboscis (fig. 14), clearly marked off from the foot. The pe- riphery of the proboscis is thin and very mobile, and reacts by local contractions and bending movements when irritated. During feeding the mouth opens in rhythmic fashion to permit the extrusion of the subradular organ and lingual ribbon, as Heath (03) observed in Cryptochiton. The whole proboscis may be retracted and temporarily covered by the forward ex- tension of the anterior part of the foot; Heath (’99, p. 579; sep., p. 4) noted that the proboscis of Ischnochiton magdallensis was completely exposed in animals up to 4 mm. in length, but that with further growth it became normally covered by the pig- mented anterior part of the foot. The surface of the foot itself is locally reactive, as shown by puckerings upon its surface and along its margins due to contraction. The substance of the foot may be swung, either as a whole or in any local part, to one side or the other, and may also be considerably extended as well as tightly contracted. The gills respond singly to local stimulation by contracting in such a way as to be pulled dorsally toward the wall of the gill channel. They may also, under certain circumstances, exhibit synchronous movements. The anal papilla is capable of movements of extension, retraction, and sidewise bending. The neuromuscular mechanism of these movements is to a large extent locally contained. If the head segment or the tail segment be cut off, the tissues in the piece removed (head, sole of foot) are reactive to touch; stimulation of the foot or of the mantle causes the foot to be drawn toward the source of irri- tation. The part of the chiton remaining after the amputation also gives the customary responses, although the gill reactions are usually weak. The ‘shock’ effect of such an operation is of course severe, and is probably even greater when an animal is bisected transversely. In this case both halves are reactive, but the amplitude of the responses is much decreased, the gill responses to touch being absent. The local ‘reflex’ character of the regional movements is fully substantiated by experi- ments to be described on subsequent pages. This condition is reflected in the nervous architecture of Chiton, the central nerv- 184 LESLIE B. AREY AND W. J. CROZIER ous apparatus being relatively unconcentrated and containing ganglion cells along the length of both pedal and pallial nerve strands. The large size of C. tuberculatus and the fact that it Fig. 10 Outline of a medium-sized Chiton tuberculatus, dorsal aspect. The detail drawing of a part of the girdle is magnified diameters. X 1. Fig. 11 The same, ventral aspect. X 1. will remain in an active condition out of water for a long time make it possible to study the local reactions in this species with considerable detail. THE SENSORY RESPONSES OF CHITON 185 2. Movements of the aniumal as a whole Probably the most striking general reactions of chiton are the suction-process, whereby the animal adheres tightly to the rock, and the curling up, armadillo-fashion, which it exhibits when detached. Locomotion has less frequently excited remark (Cooke, ’95, p. 400; Heath, ’99, p. 579; sep., p. 4). The suction power of chiton is well known to collectors (Dall, 07, p. 23). When the animal is disturbed, the girdle is applied to the substrate over its whole length, the shell plates are closely approximated, and suction is also exerted by the foot. The girdle is, however, the most important organ concerned in this protective response. Its efficiency is in part conditioned by its flexibility and by the fine riblets upon its ventral surface, but especially by the fact that it is morphologically differentiated into two concentric rings. This differentiation is exhibited in the coloration of the girdle, a narrow pale line being frequently located immediately inside the peripheral half of the girdle breadth. When firmly attached, a depression appears along this line, the more peripheral zone of the girdle being applied to the rock, and the inner zone being then sharply arched (figs. 10 and 11). Ona smooth glass surface a chiton may readily be pushed about from side to side; in this case the foot is not exerting any suction, although the animal seems to be as firmly attached as ever. The girdle is important not only for protection as a ‘holdfast,’ but also because it prevents the entrance of rain-water and of sand into the gill channels. Rain-water is quite toxic for Chiton, killing it in about four hours when the animal is placed upon its dorsal surface in a liter of such water. However, chitons will live for twelve hours or longer completely submerged in rain- water, provided the foot and girdle are free to come completely into contact with a solid surface; we have already noticed the completeness with which a foul solution can be excluded in this way. Chiton rarely frequents situations where it might be cov- ered with sand, but occasionally it is left by the receding tide with the girdle and more or less of the back so covered. The 186 LESLIE B. AREY AND W. J. CROZIER girdle is then closely applied to the substratum, and although, while under water, a respiratory current may be demonstrated, no sand grains gain admittance to the gills. The riblets and tiny channels normal to the girdle margin are important in this con- nection. If during creeping a lightly sanded spot is encountered, the girdle acts immediately as a plough, causing the sand to be pushed to one side. Although the tactile response of the girdle is thus very delicately adjusted, the commensal isopods (p. 167) are able to insinuate themselves beneath it without (usually) inducing any response. The ‘rolling up’ of the body is the activity of chiton most frequently mentioned in descriptions. The animal when de- tached from the rock, even in the case of the smallest specimens, usually bends the head end sharply ventralward, the curvature of the posterior end following, so that the body becomes ultimately rolled together, the anterior edge of the girdle sometimes being beneath the posterior extremity, at other times the two ends being simply in close contact. This response might conceivably be of significance in the life of chiton. When placed upon its dorsum on a smooth surface, it is impossible for C. tuberculatus to right itself. When rolled together, however, it could easily be moved by wave action to a location more favorable for righting. Moreover, the dorsal sur- face of the valves being sharply arched in the mid-line, the ani- mal automatically rolls over to one side. This results in righting behavior somewhat similar to that evidenced by Holothuria (Crozier, 715 b). That it is ever actually resorted to in nature is quite improbable. It seems merely that the ‘curling’ is an un- natural result of the tendency to maintain the foot in contact with the substratum, its protective appearance and functional value in righting being illusory. The flexibility of the body shown in ‘curling up’ is also evi- dent in other movements. Although the plates are closely ar- ticulated, some sidewise bending is nevertheless possible. The animal may also become arched dorsally to a considerable extent, as well as bent sharply in the ventral direction, at any level. This flexibility is rarely shown in the natural habitat of chiton THE SENSORY RESPONSES OF CHITON 187 except when it is creeping over a sharply curve surface. There seems to be a pronounced tendency for the avoidance of un- even tensions among the muscles. The normal place of resi- dence is upon a flat surface; the somewhat unexpected flexibility of the body is nevertheless important, since it enables the girdle and foot to remain, during creeping, in close contact with the substratum, even though the latter be quite irregular. The flexibility of the body is more evident in a form such as Ischnochiton purpurascens. This animal is long and narrow. It creeps with unexpected freedom, drops from one rock surface to another, when stimulated by light, and rights itself easily. 3. Locomotion The locomotor activities of Chiton demand a few words at this point, and we are able to add slightly to previous descriptions of its pedal movements. C. tuberculatus characteristically pro- gresses In an anterior direction. This is accomplished by means of pedal waves which are of a retrograde character, coursing from anterior to posterior as the chiton advances (Parker, ’11, 714); in this respect it resembles another placophoran studied by Vlés (07). As Parker observed, however, C. tuberculatus can also make backward movements of limited extent. Olmsted showed (17 a) that in these backward movements of chiton the retro- grade direction of the pedal wave is retained, as is also true in the Fisurella which Olmsted forced to creep posteriorly for a short distance; this we can confirm both for the pedal wave in chitons constrained to creep posteriorly, as in Olmsted’s experiment, by having merely a small part of the posterior region of the foot attached to a substratum, and also for the occasional backward movements which occur when the whole foot is attached. Lat- eral waves, or at any rate one lateral wave-like movement at a time, are produced on the foot when the animal is intensely stimulated on one side (Parker, ’14); in this case we find that the pedal wave courses from the unstimulated to the stimulated side (i.e., it is retrograde), but it not noticeably lifted from the sub- stratum in wave form. Similar movements appear at the ante- 188 LESLIE B. AREY AND W. J. CROZIER rior end of the foot during active creeping. The lateral wave produces an appreciable sideways shifting of the whole animal. Parker (’14) also noted that ‘“‘by swinging the anterior portion of the foot to one side and the posterior portion to the other; the animal can rotate its body with the middle of its foot as a pivot.” We have observed that in the case of these turning movements, which are frequently employed by chiton, the anterior end of the animal is the one primarily and principally concerned; the ante- rior end of the foot is, as a whole, pushed over to one side and diagonal retrograde waves bring the rest of the foot into the new position. The posterior end of the foot is pushed, as a whole, toward the side opposite the anterior one, but relatively not so far. During the turning maneuver the shell of the animal and the girdle are usually left behind, but after one or two pedal waves have passed, the foot (now straight) is held stationary, while the whole body of the chiton is swung slowly into the new position. We have spoken of diagonal waves upon the foot during turning; these waves are diagonal so far as the anterior end of the foot is concerned, but they usually become almost perfectly transverse before they reach the middle of the animal’s length. From the foregoing account it will be seen that there is, in comparison with most gastropods, a considerable degree of flexi- bility as to the use of the foot as a whole and as to the nature of the muscular coérdinations producing pedal waves upon its sur- face, although this flexibility does not by any means involve such complexity of movements as appears in the foot of the gastropod Cyprea (Olmsted, ’17 a). In the main Chiton tuber- culatus progresses anteriorly by means of retrograde pedal waves; these waves in their characteristic form run almost entirely lengthwise on the foot from the anterior end backward and are not free to course in all directions across the foot as they are in the pedal disc of sea anemones (Parker, ’17b). Undoubtedly, this difference in the character of the pedal waves in the two cases is determined by the nature of the nervous arrangements within the pedal organ, and in fact the disposition of the nervous system of chiton allows us to analyze the relation experimentally. THE SENSORY RESPONSES OF CHITON 189 During ordinary locomotion one or two waves appear upon the foot of Chiton; usually two waves are present when the animal is engaged in turning, but even in the absence of pivoting move- ments, one wave may appear at the anterior end before its prede- cessor has reached the posterior extremity of the foot. These waves are 5 to 7 mm. in anteroposterior extent, and involve the full breadth of the foot. They require fifteen to thirty seconds, usually about twenty-five seconds, to pass from one end of the foot to the other. The speed of progression of the pedal waves is less at lower temperature; it is identical in either sex, pro- vided the chitons are of the same size. At 27°C, the speed of propagation of the wave is usually about 12-15 em. per min- ute, being therefore faster than the rate of movement of the pedal wave in actinians (Parker, ’17 b, 1 to 3 em. per minute). The pedal wave is a region, occupying about one-tenth to two- tenths the area of the foot, which is temporarily lifted from the substratum (Olmsted, ’17 a) and locally moved forward by mus- cular contraction. In backward locomotion, which may readily be induced by partial illumination of the shell, the retrograde character of the pedal wave is retained. ‘This is especially evi- dent in Ischnochiton purpurascens, which creeps freely back- ward if stimulated by horizontal light striking the anterior end of the shell. In chiton there can sometimes be seen a distinct longitudinal depression running the full length of the foot in the midline, as if the foot were about to be folded together lengthwise. This is more easily seen in Ischnochiton. No trace of this activity is apparent in the pedal waves, however. Nevertheless, it can be shown that the foot is controlled in a bilateral manner. If an incision be made into the foot sufficiently deep to divide the con- nectives which join the pedal nerve strands, the lateral halves of the foot exhibit independent wave movements. If such an incision is made at the posterior end, a normal pedal wave may bifurcate when it reaches the anterior end of the incision, one half of it becoming obliterated while the other half may continue. ‘Stationary waves,’ sometimes opposite, sometimes unilateral, appear on a foot completely divided in this way; four or five such waves may be present at once, 6 to 7 mm. apart. 190 LESLIE B. AREY AND W. J. CROZIER That the essential nervous mechanism of progression is locally contained within the foot is shown by the fact that when com- pletely excised the foot will exhibit spontaneous wave motions; usually such a foot (which will live for two to three days in sea- water) does not actually creep for more than a centimeter. The isolated foot reacts locally at its margin and on its ventral sur- face to touch, in the latter case giving well-defined suction re- sponses. The pedal waves formed by the isolated foot are normal as to their speed of transmission; moreover, they appear one at a time, in succession, as in ordinary creeping; usually two or three waves exhaust the foot for half an hour. The foot of placophorans, as of gastropods, serves also as a holdfast (Parker, 711), either by means of slimy secretions or through the action of the foot as a sucker. Parker (’14) pointed out that in Chiton tuberculatus the foot sucks locally, so that “if to the foot of an inverted chiton a rigid body with an area 5 mm. square is applied, the animal can attach itself to this area with sufficient strength to allow its weight to be lifted.” As we shall point out subsequently in this paper, this 5x 5 mm. area is about the minimal surface to which the Chiton foot will react by attachment and suction, so the full physical efficiency of its suction cannot, perhaps, be measured in this way. A chiton of 8 em. length weighs approximately 50 grams, so in Parker’s experiment just cited the foot was probably exerting a suction pressure of not more than 2 grams per sq. mm.—considerably less than the almost perfect suction efficiency of the tubercles upon the column of Cribrina (Parker, ’17 e). These observations indicate that, although the chiton foot is employed as a hold-fast, the foot itself is not sufficient to account for the full suction power of these animals. The tenacity with which they adhere to a rock surface is sufficiently remarkable to have gained for them the local name ‘suck-rocks,’ and in a pre- ceding section we have shown how the girdle is of prime impor- tance in this connection. An individual from which the girdle has been completely removed may with relative ease be sepa- rated from a stone over which it has been creeping. This is also true if a chiton is caused to become attached to a glass plate in THE SENSORY RESPONSES OF CHITON 191 which there is a hole, provided the hole be situated beneath the gill channel. During its normal existence, however, the foot is of course the organ whereby the chiton maintains its position. The whole girdle is often, especially when under water, completely removed from contact with the substratum; the support of the animal depends, in fact, almost entirely upon the foot. From the ease with which the chitons preserve their position in places where ~ wave action is considerable, and upon the under surface of rocks, or (as has been noted through continuous observation) upon the relatively smooth vertical wall of a concrete wharf for periods of more than five months, it will be seen that the working power of the foot is, after all, adequate for the creature’s needs. Chiton gets the most possible out of this suction power of its foot by keeping its whole area closely pressed against the substratum. Since it commonly inhabits smooth rock surfaces, the foot usu- ally exhibits no great unevenness when the animals are freshly examined; but they are occasionally obtained creeping over bits of stone or groups of small Modiolus or barnacles, and if these individuals are inspected it is to be noted that the whole surface of the foot has been thrown into blebs and deep depressions cor- responding closely to the unevenness of the substratum. Some of the blebs produced under these circumstances clearly demon- strate the basic mechanical principle upon which the foot works, for they appear as thin-walled vesicles filled with fluid (in the females, orange in color like the coelomic juice). Similarly, if a Chiton be caused to creep over a small hole (4 to 5 mm. diameter) in a glass plate, the substance of the foot will be perceptibly pressed into the hole. Apparently the foot of chiton can exert suction only in a very local fashion, for if a portion of glass tubing of 8 mm. internal diameter, 10 mm. external diameter, corked at one end so as to provide a cylindrical chamber 4 mm. high, be used to test the sucking power of the foot, it is found that in most cases the chitons cannot become attached to the circular rim of the tube with sufficient force to bear their own weight’ in air. In this experiment, it should be noted, the total area available for direct contact with the foot is about 28 192 LESLIE B. AREY AND W. J. CROZIER sq. mm., agreeing with the minimal area for attachment as found by Parker and in our own tests. The local character of the suc- tion mechanism of the foot is further suggested by the minute depressions, usually long and narrow and more or less communi- cating with one another, which are to be found on the foot of a non-creeping Chiton attached for some hours to a glass plate in air. These local suctions are probably assisted by slime secre- tion, which, although small in actual amount, enables a chiton to remain rather firmly attached to a smooth surface (e.g., of a glass plate) after the animal has been allowed to die in air or after it has been killed by heat (44°C.) in water. They do re- main so fixed, even when the girdle is not in contact with the substratum, and the slime may therefore be important during the use of the foot in life, including early postlarval stages (Heath, 99, p. 640; separate, p. 65). IV. MECHANICAL EXCITATION 1. Tactile stimulation In testing the local sensitivity of Chiton to tactile excitation, use was made of a blunt-pointed dissecting needle, a glass rod, or a blunt pencil-lead. In some instances, also, minute air bubbles (formed at the end of a pipette) and several other means of stimulation were employed. The responses observed when different regions of the dorsal and ventral surface of Chiton were lightly touched with one or the other of these objects are de- scribed in the following summary. Attention was given to the possibility that the responses of Chiton might vary depending on whether the animal was submerged in water when tested or was out of water. There were discovered no differences in behavior which require consideration at this point when the reactions of chitons in these two situations were compared. For the study of the responses obtainable from the ventral surface, we have mostly employed animals in air, placed upon their dorsal sur- face. The inability of chiton to right itself, coupled with the relative insensitivity of the shell surface, allowed us to work in this way without introducing serious complicating disturbances. THE SENSORY RESPONSES OF CHITON 193 In the more critical experiments we made use of a method of graphic registration, subsequently described. A. Dorsal surface. a. The shell plates appear not to be sensi- tive to touch. No responses were obtained when the surface of the tegmenta was lightly touched. (This is further considered on a subsequent page.) b. The mantle between the tegmenta, 1. e., the tissue covering the insertion plates, may be somewhat exposed, when Chiton is at- tached and ‘at rest,’ by the separation of the shell plates through the extension of the body. When the mantle was touched in this region the plates immediately adjacent to the site of stimulation were quickly approximated, covering the mantle area which had been touched. c. The girdle. When Chiton is attached, the lateral extension of the mantle, known as the ‘girdle,’ which is flexible, is locally lifted from the substrate unless the animal be disturbed. Under water the girdle may be completely removed from contact with the rock or other surface, but in air this elevation is usually local and commonly takes the form of slight puckerings of, at most, a centimeter or so in length. .To asingle touch the girdle, where elevated, responds by local lowering to the substrate at the point of excitation. A more vigorous touch causes a greater extent of the elevated girdle to be lowered. Four or five moder- ate touches in succession affect a still greater length of the girdle, as much as one-quarter to one-third of the circumference, and the time elapsing before recovery to the original elevation is longer than that following a single touch. Even when the girdle has not perceptibly removed from contact with the sub- stratum, it responds by a detectable ‘tightening,’ or flattening. Several successive touches upon a ‘flattened’ region of the girdle induce near-by elevated parts to return to the substratum.’ Un- less the excitation is continued for nearly one minute, however, or is in the first place very vigorous, the response to touch is strictly homolateral. The anterior end of the girdle is more reactive than the middle or posterior parts, and its peripheral border is more sensitive than the rest of its dorsal surface. A chiton quietly creeping in water, with the girdle lifted, re- 194 LESLIE B. AREY AND W. J. CROZIER sponds instantly to a touch upon the anterior or posterior girdle by ceasing locomotion and adhering firmly to the substratum. B. Ventral parts. d. The ventral surface of the girdle. The serial arrangement of eight dorsal shell plates affords a con- venient means of dividing the surface of the animal into defi- nitely delimited areas for reference. We shall consider the ven- tral surface of the girdle in terms of four ‘quarters,’—an ‘ante- rior quarter,’ delimited by the posterior margin of the second shell plate, two ‘middle quarters,’ and a ‘posterior quarter’ correspondingly marked off by the transverse borders of each succeeding two shell plates. The end quarters of the ventral mantle are more reactive to tactile excitation than are the middle quarters. a. The end quarters. To the single stimulation of an end quarter, the response is a curling of the animal in that region, as if it were beginning to roll up; the bending process elevates the stimulated end about 2 to 5 mm., after which the animal straight- ens out again. When stimulated on the posterior quarter, the foot may be pushed caudad at the time the bending response occurs. When stimulated on the anterior quarter, the head may retract somewhat, and the buccal region may be slightly in- verted, the head and ‘palp’ tending to close over the mouth. Light touches, when several times repeated, elicit a much stronger response. The stimulated end reacts first, by bending, and then the opposite end bends also, though to a less extent. This is true whether the anterior or the posterior end is the one stimulated. The muscles of the midportion of the animal are not specially contracted, however, and the closure of the shell is incomplete. 6. The middle half. The reactions listed under a are pro- duced most clearly when the most anterior or the most posterior region of the ventral girdle surface is stimulated. The reponses obtained from the ‘middle quarters’ of this surface are qualita- tively identical over the whole anteroposterior extent of the ctenidia. In other words, the convenient descriptive division of the animal into ‘quarters’ does not afford a basis for the organic classification of responses, inasmuch as the ctenidia extend an- THE SENSORY RESPONSES OF CHITON 195 teriorly and posteriorly beyond the limits of our ‘middle half.’ This artificial subdivision into quarters is retained in our de- scription, however, since the responses we are considering are most characteristically displayed in the respective ‘quarters’ of the chiton’s surface, although there is some ‘overlapping’ and, as already stated, the subdivision is by no means an organic one. A single touch applied to the midventral surface of the girdle is followed by a local puckering of the girdle toward the source of irritation. The foot, in the region immediately adjacent to the level stimulated, is pushed laterad and dorsad, toward the mantle, tending thus to assist the girdle in covering the gills. This reaction of the foot is not evident when the dorsal surface of the girdle is lightly stimulated. Unless the tactile stimulation is severe or several times repeated the homolateral side only of the foot is involved in this response. Simultaneously with these movements of the girdle and foot, a contraction of the gill ele- ments occurs opposite the singly stimulated area. This involves five or six ctenidia anterior, and as many more posterior, to the point of excitation. In this reaction the ctenidia are elevated dorsally, the tips are drawn toward their bases (thus throwing each element into a more convex arch), and at the same time they are drawn somewhat anteriorly. The whole response involves a movement something like the fairly rapid closure of the fingers of one hand. The response spreads in both directions from the level of stimulation, although at ordinary temperatures the propagation wave is difficult to observe because of its rapidity. Successive stimulations of the girdle lead to a greater puckering in toward the source of excitation, and to a more pronounced rolling up of the whole body. The foot is locally brought slightly laterad toward the girdle and is drawn dorsad to a considerable extent. This response of the foot is at first confined to the stimulated side, but subsequently spreads to the other side, finally involving the whole substance of the foot at the level of stimulation. Successive touches, 1 to 1.5 seconds apart, lead to a tetanic contracture of the foot and ctenidia; during this phase the animal tends to roll up. ventral surface of girdle > dorsal surface of girdle. The extreme outer margin of the girdle is about as sensitive as its ventral surface. c. At the caudal end: End of foot = inner surface of mantle > ventral surface of girdle > dorsal surface of girdle. d. On the sides: Inside of mantle = ctenidia > edge of foot > ventral surface of girdle > sole of foot > dorsal surface of girdle > dorsal mantle between shell plates, the last judged by its effect upon the approximation of the shell plates. e. The shell plates are insensitive to touch. It will be seen that in a broad sense the capacity of response to THE SENSORY RESPONSES OF CHITON 199 tactile irritation is distributed upon the body of Chiton in a manner appropriately correlated with its structure and habits. The various responses obtained from contact with a small sur- face are such as would have a protective influence. Reference should be made at this point to the local closing together of the dorsal valves when the intertegmental mantle is irritated, and especially to the ventralward movement of the girdle, associated with local retraction of the ctenidia and a corresponding local movement of the foot, which follows a touch upon the dorsal or ventral surface of the girdle. The preservation of the ctenidia from injury, and more particularly the effective use of the girdle for the exclusion of foreign objects and as a hold-fast, are de- pendent upon responses such as we find the girdle to exhibit. Further detailed correlations of this character might be pointed out, but enough has been said to indicate the useful nature of the responses. The ‘rolling-up’ reaction has, when carried to com- pletion, a clearly ‘purposeful’ aspect, as already intimated. Yet the natural history of Chiton yields no evidence that this re- sponse is ever used. We consider that it is the inevitable out- come of maximal possible contraction in the chiton’s effort to produce suction, and that it is neither of specific protective sig- nificance nor of the nature of a ‘reflex.’ Confirmation of this view is found in the fact that sometimes a ‘rolled-up’ chiton will re- main for hours tightly curled, although placed in position pur- posely made favorable for reattachment should it unroll. On _the other hand, after a short time upon its back, a chiton may spontaneously uncoil itself and remain fully exposed for a long time, if unstimulated. Moreover, isolated parts of the animal give (or attempt to give, so far as their deficiencies permit) the ‘rolling-up’ response when they are activated. D. The tactile receptors. The superficial layer of the shell plates of chitons is traversed by numerous canals, occupied by specialized organs having the histological appearance of sensory receptors. These canals are more or less nearly perpendicular to the surface (at least peripherally), and the organs they con- tain, piercing the tegmentum, are described as projecting slightly beyond its general surface. The remarkable character of these 200 LESLIE B. AREY AND W. J. CROZIER structures is well known. Definite evidence as to their functional significance has been completely lacking. In addition to the ‘eyes’ (Moseley, ’85; Plate, ’99; Nowikoff, ’09); micra- and megalaesthete organs of varied form are present, and some of them seem so constructed as to be (?) serviceable as tactile re- ceptors. A function of this sort has in fact been somewhat doubtfully suggested for them (Kafka, ’14, p. 100). As already stated, however, the shell plates of adult chitons seemed insensitive to touch. A slight pressure, however, de- forms the tissue underlying the plate, and is sufficient to induce a more or less pronounced sucking reaction. We tested there- fore young C. tuberculatus, one to two years old, under the im- pression that in older individuals the aesthetes might be de- stroyed, as their cavities are exposed by the erosion of the cuticula. The result was again negative so far as tactile sensi- tivity of any part of the shell surface was concerned. We then employed a method which completely avoided me- chanical depression of the tissues beneath the shell plate. The free umbo (‘beak’) of the third or fourth valve was tightly gripped between the jaws of a haemostat. This did not involve damage to any of the soft parts. The forceps could then be clamped to an upright. By means of a small hole through the girdle or with the aid of cement attaching a thread to one of the anterior valves, the movements of the chiton could then be recorded graphically upona kymograph paper. When the surface of a valve rigidly held in this way was explored with a needle or with a larger object, no tactile responses were elicited. (Shad- ing must be avoided and rhythmic spontaneous contractions of the animal must be discounted.) Single touches and a moving point were alike without effect. There are no tactile receptors in the shell plates. The histological nature of the sense organs in the tegmentum varies considerably in different genera of chitons (Plate, ’99). Upon the shell of Ischnochiton there are minute, projecting ‘hairs.’ We find that the tegmentum of Ischnochiton pur- purascens is very sensitive to touch. These results allow us to state that in all probability the sen- THE SENSORY RESPONSES OF CHITON 201 sory structures in the shell plates of Chiton have no functional significance as touch-receptors. The sensitivity of the girdle will be considered separately. On the foot and other soft parts a moving point source of tac- tile irritation is very effective in inducing responses, its effect equaling that of several or many repeated single touches. On the soft surfaces, it would appear, there are many scattered tactile receptors. The amplitude of the reactions which they mediate depends, in any given region, upon the intensity of activation and upon the number of the receptors which may be involved. The sense of touch exhibits also a certain degree of discrimina- tion. Thus, to contact with small areas the foot reacts by local retraction, but to contact with larger areas it becomes promptly affixed. Furthermore, chitons in the field have been observed creeping in a horizontal direction along more or less vertical rock surfaces, just about at the level of the water at that time, so that wavelets of some force were hitting the animals roughly; they continued creeping quietly, the girdle being freely lifted, and made no response to the intermittent slaps of the water; but at the lightest touch possible with the finger upon the lateral or anterior edge of the girdle they instantly stopped moving and adhered firmly to the rock. That the tactile sense is served by distinct receptors upon the soft ventral parts of Chiton may be shown through the physio- logical isolation of the tactile responses. Thus, when the ven- tral parts have been exhausted for photic excitation, by repeated shadings (vide infra), the mantle and other parts are still fully reactive to touch. When the ventral surface of the girdle was repeatedly touched, the animal responded ‘by rolling up to the maximal extent obtainable with tactile stimulation; an addi- tional vigorous response can still be obtained from the middle half of the girdle upon the application of n/10 to n/40 HCl, much greater contraction resulting from the use of acid in this way than can possibly be obtained through touch alone. When immersed in sea-water at 43°C. there was a considerable tempo- rary augmentation of tactile responsiveness, but after a few minutes no responses to touch could be secured; a normal re- 202 LESLIE B. AREY AND W. J. CROZIER sponse to a small volume of n/10 HCl was, however, still ob- tainable. Taken in their entirety, these findings tend to indicate the physiological distinctness of the tactile receptors, although the methods employed in the tests are open to the objection common to all such experiments: we cannot be entirely sure that we are dealing with sources of stimulation which are quan- titatively comparable. Less objection can be taken to the result of experiments of the following kind. If the inner ventral girdle surface, under water, be repeatedly stimulated with small volumes of n/30 HCl in sea-water, four or five successive re- sponses may be obtained from the activation of one area. This area is then exhausted for stimulation in this way. It is, how- ever, still reactive to touch. Since the acid is usually regarded as a more powerful excitant than touch, the objection above referred to may thus be removed. 2. Vibratory stimuli The characteristic statocyst of gastropods is not present among amphineurans. Hence it would be interesting to learn—al- though the theory of the statocyst as a merely positional organ seems now well established (Baunacke, ’14)—whether or not Chiton reacts to vibratory stimuli such as sound waves. It proves not to respond to attempted stimulations of this kind. Chitons placed in a beaker of thin glass containing sea-water were watched while the lip of the beaker was tapped with a glass rod. No reactions followed this treatment. Sounds transmitted to the beaker from a vibrating saw blade had like- wise no effect. The table top supporting the beaker was sharply struck, jarred, or rubbed, with the same absence of response. Chitons placed ventral surface upward in shallow water, so that they were just covered, did not react to drops of sea-water falling on them from a distance of 10 to 15 cm. A chiton resting upon a glass surface, under water, in some cases responded to a tapping of the glass immediately under the ap- pressed mantle by raising the girdle in that region. If the girdle was already raised, in a few cases only was it lowered to the THE SENSORY RESPONSES OF CHITON 203 glass surface as a result of the tapping. In several instances chitons treated in this way began to creep, and in one case began to creep backward. There is no suggestion of the perception of vibratory stimuli in these results. Direct contact excitation is quite effective, but vibratory disturbances, such as sound waves, seem not to be reacted to. In collecting chitons it is advantageous to employ a large cold-chisel and a hammer; yet one finds that the removal of several members of a group, involving repeated and fairly heavy blows, does not usually result in near-by chitons becoming firmly attached to the rock, unless they have been directly affected by a deforming pressure. This deficiency cannot be attributed to the absence of a statocyst, however, since other mollusks, well provided with statocysts, are known not to react to vibratory disturbances. A chiton may be suspended in water by having the beak of one shell plate clamped in forceps attached to a support. Under these circumstances it does not react to vibratory disturbances transmitted through the water. 3. Thigmotaxis The surface of the foot of Chiton, if touched locally, draws away In a rather sharp pucker, especially if a sharp point be used. No attempt is made to attach the stimulated spot by suction, and if the activation is repeated the resulting movements of the foot are such as to cause its removal from the region of stimulation. If, on the other hand, a larger surface, such as the flat end of a pencil, be applied, the foot becomes firmly affixed to the foreign surface, and is pulled deeply down below the gen- eral level of the rest of the foot. The minimal area reacted to by attachment is about 5x 5 mm. for chitons 6 to 9 em long, as Parker found (14). It is important to note that when a pedal wave has formed and is traveling down the foot of a chiton lying on its back, the surface of the foot immediately involved in the wave can attach to a small point (e.g., the pointed end of a pencil) very firmly by suction. 204 LESLIE B. AREY AND W. J. CROZIER . When placed upon a glass plate the animal quickly puts its whole foot in contact with it. In doing this, waves are set up frequently and three or four may appear upon its surface at one time; or, if the foot is already in nearly one plane, the attach- ment may be almost simultaneous over its whole extent. In the latter case, the local areas of the sole which are not at first attached are brought down to the substratum. As noted by Olmsted (17 a), a chiton repeatedly forced to creep backward, by requiring one-fourth of the foot to attach to the lower edge of a glass plate held vertically in air, becomes after several trials exhausted, so that it creeps just sufficiently to enable the whole foot to be in contact with the plate. Thus, to contact with a small area, such as a needle point or the rounded point of a pencil, the resting foot of Chiton reacts negatively, but to larger surfaces the response is a positive one. There is a very pronounced tendency to keep the whole of the foot in contact with some foreign surface. Inno case has a chiton ever been seen in nature with any section of the foot or head completely removed from the substratum. This response is sufficient explanation of the behavior of Chiton in ‘righting’ itself. At no age is there a detectable tendency for chitons to preserve an upward orientation of their dorsal aspect, even when placed so that it is physically possible for the animal to reattach itself (wde supra, p. 200); undirected movements finally result in a portion of the foot, usually the anterior end, effecting con- tact with the substratum; complete reattachment is then rapidly brought about. It is of interest to inquire if this form of tactile discrimination, favoring attachment to a sufficiently large area of surface, is evidenced by parts other than the foot. The ventral surface of the girdle and the head region were therefore examined. Finer degrees of tactile discrimination seem to be absent on the head and foot. Chiton shows no preference, when placed in an aquarium, for surfaces such as those to which it has been accustomed. Provided the surface be firm and sufficiently large, it creeps indiscriminately over smooth stones sparsely sprinkled with sand, glass, or wet paper. It will not creep, however, THE SENSORY RESPONSES OF CHITON 205 upon sand, upon mud, or upon a rock surface minutely studded with sharp points; the negative reaction to surfaces of this character is effective in determining the local habitat of Chiton, since they do not occur upon muddy beaches nor upon sand, nor do they at any time creep up upon the sharply pitted shore rocks in the narrow zone whichis covered at spring tides but other- wise exposed to wind erosion. It may be noted in addition that the young chitons, up to three years of age or more, and espe- cially in very young stages, occur conspicuously upon smooth stones. This is not altogether an accidental consequence of the fact that the rock surfaces in the situations where their tropisms force them to reside are frequently of a smooth character, since the smallest specimens are found upon the under side of bottles (of dark glass) below mean-tide level in company with Ischno- chiton. 4. Rheotropism It was noted that a number of chitons escaped from a col- lecting pail, located at one end of a long aquarium table, and that they tended to accumulate in the shallow gutter which carried away the overflowing sea-water. A good number of these animals moved down the gutter, with the current, even though in so doing they traveled slightly down hill, against their negative geotropism. Further tests, made in this gutter, showed that, to currents of sufficient strength to produce any effect, a majority of the animals were negatively rheotropic. For a more refined test, chitons were completely submerged in a trough of sea-water (a wooden fish-hatchery trough) through which a current flowing at the rate of 5 to 10 em. per second was maintained. The rheotropic response was here less definite than it appeared in the first observations, but was undoubtedly negative. The rheotropism of Chiton is clearly a ‘laboratory phenome- non,’ and may owe its appearance to the mechanical deformation of the girdle by currents or to other tactile irritations, in either case inducing negative orientation, or it may be traceable to a deforming influence of the current upon the body as a whole, 206 LESLIE B. AREY AND W. J. CROZIER especially upon the base of the foot, such as we suggest in the case of geotropism. The effects of local currents were also tested. A current from a pipette directed under the girdle of a submerged chiton induces a depression of the girdle, provided the gills are disturbed. A current directed upoh the girdle causes it to be depressed; it can usually be seen, in this case, that the ‘scales’ upon the girdle are moved or that the girdle itself is mechanically bent. A current impinging upon the dorsal mantle between the shell plates produces usually no effect. The girdle is the most sensitive region. Negative reactions of the whole animal are readily induced by repeated applicationsof a pipette current to a part of the girdle. These reactions are also concerned, probably, in the orientation of Chiton in a vigorous stream of sea-water. 5. Geotropism Since the Amphineura lack the statocyst organs characteris- tically developed in other molluscan classes, the question of Chiton’s behavor with respect to the pull of gravity deserves special consideration. The positions in which they are commonly to be observed strongly suggest that they are negatively geo- tropic. The younger individuals, particularly, are found most abundantly at the upper limit of the tidal reach. Older animals occur over the whole intertidal zone, and even in some cases slightly below it, but these, too, are most frequently encoun- tered near the upper tidal limit. Furthermore it is very notice- able, more particularly among the larger individuals, that the great majority of the chitons taken from perpendicular rock faces are oriented with the anterior end upward, rarely indeed with this end directed downward, although in many cases they are more or less nearly horizontal. When kept in aquaria they rapidly creep to the water surface and sometimes thence out into the air; nevertheless, they sometimes curl over the top of an aquarium and creep downward on its outer wall (more espe- cially in the case of aquaria with a free general overflow at the THE SENSORY RESPONSES OF CHITON 207 top). During downward creeping their orientation is rarely such that the long axis is strictly vertical. The following specific experiments show that the orientation of the movement of Chiton is generally in an upward direction. 1. Five chitons were placed, with their long axes horizontal, on the walls of a flat-sided aquarium jar. They were located at about mid- way between the bottom and the water line. The jar was removed to a situation where the illumination was even and diffuse. Three individuals moved upward to the surface of the water, and nearly emerged; one oriented upward through an angle of 45°; the other one oriented downward through an angle of 60°. 2. Six chitons, placed as in the preceding test, were kept in the dark- room for 1.5 hours. At the end of this period four were found to have become oriented vertically, moving upward until almost entirely out of water, one was oriented upward at an angle of 45°, and one had moved upward to the water surface, being oriented upward at an angle of about 45° from the horizontal. Another similar test was made with eight animals, six of which oriented upward at various angles and two downward; in this experiment the animals were overcrowded and became piled upon one another. 3. A similar test with five chitons, kept overnight in the dark-room, gave four animals orienting upward and moving nearly out of water; the remaining one had moved downward, assuming an orientation 45° away from the horizontal. Of these twenty-four individual responses, twenty were clearly such as involved an upward orientation from the hori- zontal position. Since the animals continued creeping until almost entirely out of water, it is hardly probable that want of oxygen determined the observed behavior. This conclusion is confirmed by subsequent tests in which chitons were placed in dishes where anaerobic putrefactive processes had begun or were allowed subsequently to begin. Frequently they did not creep out of such dishes. Moreover, Chiton orients upward in a closed vessel completely filled with water or one in which oxygen- ated water enters from beneath. The negative orientation is evidenced by animals of all ages. It is most perfectly ex- pressed when the chiton is completely submerged. The hori- zontal position assumed at the water level is a special conse- quence of stimuli associated with the water level. The tendency to upward creeping is clearly evident upon 208 LESLIE B. AREY AND W. J. CROZIER surfaces inclined at any angle above 30° with the horizontal. For these tests the larger animals are best. Observations in the field and many tests in the laboratory show clearly that there is no tendency for Chiton to preserve a constant dorsoventral orientation. In rock crevices they occur ‘upside down’ with great frequency. Careful observation of the movements of a chiton during geo- tropic orientation affords a clue as to the nature of the determin- ing stimulus. Accurate outlines of a specimen orienting in this way are given in figure 12. Inspection of these outlines will show that the sequence of events in orientation is as follows: ah Z at (SIRDLE Free) ING JI A. it CSIRDLE PRESSED To GLASS) Fig. 12 Outlines of the successive positions assumed by a Chiton in ori- enting upward from a horizontal position, under water, on a vertical surface; ventral aspect. (Traced through glass, on thin paper.) X 3. The girdle becomes freed from the substratum, so that the ani- mal remains attached by the foot only; when in the horizontal position (fig. 12, II), the weight of the body causes it to fall slightly, producing an uneven tension in the muscles, those on the higher side being stretched. The animal swings until this unilateral tension is relieved. It turns anterior end up, probably because that end is the more sensitive. The tendency is for the animal to turn toward the stretched side; the tenser muscles are the ones which contract. With animals in the vertical position (fig. 12, V) the downward pull of the creature’s weight is exerted at the posterior end. THE SENSORY RESPONSES OF CHITON 209 The chief ethological importance of Chiton’s negative geo- tropism is not that it directs the animal upward, but that it keeps it from going down. The pallial nerve cords are easily cut by an incision from the ventral side. A chiton with both pallial nerve strands cut at the level of the cleft between proboscis and foot no longer orients upward; it creeps about slowly and aimlessly, almost invariably moving downward, but in a slanting direction. Sometimes a chiton so prepared turns upward, but in no case did one of the six animals used move upward, as normal individuals consist- ently do. When the pallial cord is cut on one side only, and the animal allowed to attach itself (after a rest) to a vertical glass plate, under water, the long axis of the chiton being horizontal, the characteristic effects are as follows: if the side on which the pallial nerve cord is cut is placed downward, the animal orients downward; if the cut side is uppermost, the chiton orients up- ward, but usually passes beyond ‘top center,’ and then fre- quently reverses, coming back to an approximation of its origi- nal position, but not quite so nearly horizontal. This reaction is not always clear cut. The final position, in fifteen tests upon twelve different animals, was in all but three cases such that the cut side was downward. In a number of cases there seemed a well-defined failure of the anterior portion of the foot to attach itself to the substratum, so that, the posterior region being fully attached, the anterior end became directed downward in a purely mechanical way owing to the pull of gravity. This might account for the fact that in the chiton with both pallial cords cut the anterior end is in a general way directed down- ward, but would not explain the fact that in most cases with the cut side placed upward the orientation was upward. While not conclusive, the result of these tests is for the most part in har- mony with the idea that the weight of the body, exerting tension on the pedal musculature, supplies the stimulus to geotropic contraction. In the case of an animal placed on a vertical sur- face with the long axis horizontal, the muscles on the stretched side are the ones which contract, as we might predict, and since the animal orients upward, we must suppose this to be in a gen- 210 LESLIE B. AREY AND W. J. CROZIER eral way true. If the nervous connection of one ctenidial wall of the foot mass by means of the pallial cord be severed, then the muscles on that side, passively stretched, or compressed, could not (or might not be able to) get readily into nervous communication with the rest of the body; hence we should under these conditions expect geotropic movements to be imperfect. No tests have yet been made to discover the effect of uni- or of bilateral section of the pallial nerve cord on horizontal creeping. 6. Summary The general surface of Chiton, aside from the tegmental sur- faces, is actively responsive to touch. The aesthetes upon the shell valves are not sensitive to tactile stimulation. The foot is negatively reactive to small surfaces, positively thigmotactic to large surfaces. Chiton is negatively geotropic, and also nega- tively rheotropic to currents of some strength; these two modes of response appear to depend respectively upon the develop- ment of unequal tensions in the musculature, owing to the weight of the body, and upon the local deforming influence of a water stream—they result from stimulation through deforming pressure. Although much has been written upon the morphological character of the sensory organs in Chiton and upon the general structure of the integument (Blumrich, ’91: Plate ’87—01 a), there has been very little in the way of experimental evidence bearing upon functional relations. Heath (’03) thought that the pro- boseis of Cryptochiton stelleri might exercise tactile as well as gustatory functions, and described some feeding experiments in support of this idea. From the foregoing account it will be seen that tactile (contact) irritability is a general integumentary function, leading to responses of an ‘advantageous’ character. The receptors concerned are apparently of several kinds: 1) those upon such surfaces as the foot, mouth area, and gills, and, 2) those associated with the tubercles and ‘hairs’ upon the girdle. The former are probably of a more generalized character than the latter, but, for some parts at least, the propo- THE SENSORY RESPONSES OF CHITON 2A sition is nevertheless defensible that they are tactile receptors and that the responses to which they lead are not aspects merely of the activity of generalized (‘universal’) sense organs. The positive response of the foot to contact with a sufficiently large area is significant upon this point, since this type of reaction is never obtained upon chemical stimulation of the foot. The tubercles and ‘hairs’ upon the girdle of most chitons are characteristically embedded in the cuticula of the integument and connected at their proximal ends with secretory and probably also with sensory cells. By means of movements of these stiff projections, relatively slight mechanical disturbances may be transmitted through the thick cuticula. We have shown that on the shell surfaces of Ischnochiton the projecting ‘hairs’ are open to tactile activation. In Acanthochites spiculosus the girdle is regularly beset with circularly arranged groups of long spicules. We find these spicules to exhibit an interesting reac- tion. If one of them be touched, ever so lightly, the rest of the spicules in that group react also, the whole bundle being spread widely apart. Usually all the bundles on one side respond if a single spicule is disturbed. As the spicules are 5 mm. long, in an animal 15 mm. long, a slight touch exerts a considerable lever- age upon the base of the spicule. At rest the spicules are directed posteriorly, but when disturbed the axis of each bundle is directed more or less perpendicularly to the surface of the girdle, the spicules themselves being spread wide apart; this results in a disposition of their sharp points which may have protective value. The ‘scales’ on the girdle of Chiton tuberculatus are homol- ogous to the spicules of Acanthochites (Plate, ’01 a), and their functional significance for tactile reception is of a similar kind. At the outer margin of the girdle are short, stiff ‘hairs,’ directed normally to the periphery, which also function in this way’ (Plate ’01 a, p. 497, believed the ‘hairs’ and ‘thorns’ of the chiton girdle to be sensory organs). 7 Heath (’99, p. 637; sep., p. 62) has described the use of the anterior flagella of the transforming larva as tactile organs. 212 LESLIE B. AREY AND W. J. CROZIER Chiton exhibits mouth movements similar to those figured by Heath (03) for Cryptochiton. It is questionable whether these movements originate normally from tactile excitations. On a glass surface they are never observed except in starved indi- viduals. The mouth opens widely, permitting the subradular organ to be pushed forward. In a starved animal these move- ments, which occur in rhythmic series, may be initiated by causing a bubble of air to become entrapped in the depression surrounded by the lips. The radula is never thrust forward sufficiently to come clearly into view. Full feeding movements are obtained, however, when a splinter of the intertidal rock surface is placed in contact with the proboscis. It is of course possible, as Heath suggests, that the rich supply of nerve endings on the surface of the subradular organ is in part of tactile significance. V. THERMAL EXCITATION 1. Behavior at different temperatures It is worthy of remark that very few marine invertebrates, if indeed any, seem to possess a well-developed temperature sense. Although as yet we know little of the integumentary senses in marine annelids, crustacea, or mollusks (Kafka, ’14), we may note that in echinoderms (Crozier, 715 b; Olmsted, 717 b) sensory discrimination for heat and cold is very weak, and that even in Amphioxus, where there are good indications of possibly both heat and cold receptors (Parker, ’08, p. 430), the responses of the animal to either heat or cold are by no means of that delicately sensitive character which we usually asso- ciate with photic, tactile, or chemical receptors. We believe this to indicate a poorly developed sensory mechanism for heat and cold reception. There is little reason to regard this con- dition as one of adaptation to, or correlation with, life in a situation where thermal receptivity would not be valuable, owing to the small range of temperature occurring in the habi- tat of the creatures in question. It would be equally sensible to consider that, if the animals concerned had developed deli- THE SENSORY RESPONSES OF CHITON 243 cate thermo-receptors, some ‘use’ would have been found for them. In either event the adaptationist rests intellectually satisfied. It is, nevertheless, a fact, although it may have little’bearing upon this matter, that the temperature variations endured by Chiton are rather wide. These variations are both diurnal and tidal, and their amplitude, in summer months, ex- tends during the twenty-four hours in some situations from 23° to 37°C.; this is especially the case on hard bare limestone rocks, where the heat of the midday sun at low tide is quite intense. At the time these experiments were made the surface tem- perature of the sea was 26° to 27°C., and this temperature was taken as the ‘normal’ in the tests that are here described. Chitons were immersed in sea-water maintained at the tem- perature noted, and their movements were carefully recorded. Behavior of chitons transferred from sea-water at 25° to 26°C. to sea-water at the temperatures indicated at the margin TEMPERATURE IN DEGREES CENTIGRADE 2° Ctenidia almost instantly contracted, and remained so for 3 min.; 5 min. from time of immersion, all were again expanded. Cte- nidia respond to tactile excitation by contraction, but weakly. No tactile responses obtainable from other regions. No spon- taneous movements; remaining as if anesthetized. 4° Ctenidia contracted; began to expand in2 min. At first, the foot partly curled, as if preparing to roll up, but soon became straight again. Tactile sensitivity soon abolished; after this had been ascertained the foot and mantle were tested with weak HNO; solution, but no reaction resulted. 5° Ctenidia contracted within 3.5 min.; after 2 to 4 min. began to expand; fully extended after 4 to 5 min. Ctenidia respond to touch, feebly in some cases. No spontaneous movements of the foot or animal. Responses of the body to touch absent after 15 Semin? 8° to 10° | Foot spontaneously thrown into smooth contractions that lasted 2 min. Ctenidia contracted; after 4 min. they relaxed one by one. After 10 min. ctenidia responded to touch. General tae- tile responses very poor. 10° Ctenidia contracted after 0.5 to 6.0 min.; expanded after 7 to 10 min.; after 10 to 11 min. reactions to touch returned, but very slowly. In several cases after 1 min. immersion the gills began THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 2 214 TEMPERATURE IN DEGREES €CENTIGRADE 15° 20° to 35° 38° 40° 42° 43° LESLIE B. AREY AND W. J. CROZIER to contract rhythmically at intervals of 5 to 8 sec., continuing in this way for 2.5 min.; the gills of the two sides did not beat synchronously, but the plumes of either side alone were at first more or less coérdinated, the unison of the ‘beats’ becoming less after 2 min. Foot exhibited few or no spontaneous contrac- tions, but was much extended, exposing the ctenidia. The ani- mals as a whole insensitive to touch. No contractions of the ctenidia; a few smooth contractions of the foot and slight movements of the palps for several min. Ani- mals normally extended. Tactile responses subnormal. All responses normal. General sensitivity (‘reactivity’) slightly increased during the first few minutes. No spontaneous movements of the foot or palp. Sensitivity to touch rather quickly decreased, but still present (feeble on the ctenidia) after 45 min. Animal could not close the shell when stimulated (note that at higher temperatures the shell upon immersion shows a tendency to open, if it had been closed, i.e., rolled up). If shell is rolled up, it opens. Spontaneous writhings of the foot either absent or lasting 3 to 5 min. Ctenidia in some cases contract for 1 min., irregularly. For 1 to 2 min., ctenidia and other parts are extremely reactive to touch. Tactile re- sponses gradually decrease; at first, a single stimulation of the foot (after spontaneous movements have ceased) induces sev- eral irregular contractions; after 30 min., foot alone is slightly responsive to single or repeated touches; but irregular con- tractions producing a welt result from moving a pointed rod over the surface of the foot. After 1 to 1.5 hour, still in same condition. Violent contractions of the foot for 1.5 min.; then ceased. After 3.5 min., all contractions ceased, and no tacile responses were obtainable. If shell was rolled up when immersed, it opened; few, and no suc- cessful, attempts to close the shell. Foot thrown into irregular contractions, originating as local puckerings, which spread rapidly, lasting for 2 to 2.5 min. These contractions not so con- vulsive as at 42°. After 1 to 5.5 min. (in one case following tests for tactile sensi- tivity) a second series of weak contractions of the foot appeared and lasted for 2 min. The shell plates moved slightly back and forth. In one instance there were a few irregular but widespread con-— tractions of the ctenidia, in which each filament appeared to act more or less independently. THE SENSORY RESPONSES OF CHITON 215 TEMPERATURE IN DEGREES CENTIGRADE No reactions to touch obtainable after 2.5 to 4 min., from either foot, palp, or ctenidia. After 15 min., the foot and mantle are much bloated. 44° No movements of the foot; no responses to stimulation. Chiton attached to a glass plate raised the mantle, except at either end, but remained passively attached, after death, for 10 min. Died in 2 min. 45° Chitons straightened out and the shell and foot became convex. No responses. Died at once. Animals attached to a glass plate showed slight writhings of the foot when first immersed, due probably to slow warming up through the glass. These tests show that sudden changes in temperature between 15° and 40°C. have little in the way of direct sensory effect upon Chiton when the whole animal, having previously been maintained at about 27°,is suddenly immersed in sea-water of any temperature between these limits. Below 15° an ‘anaesthetized’ condition is quickly arrived at (Matisse, ’10); above 40°, sensitivity quickly decreases. Temperatures of 44° to 45° are almost instantly fatal, although Chiton will survive for nearly two hours after sudden transference to a temperature of 40°. This is the highest temperature which they will successfully withstand for more than fifteen to twenty minutes, and is but a few degrees above the summer temperature sometimes experienced for a similar period in their natural habitat. The factor of safety is here, conse- quently, very small, as compared even with that of other littoral animals of the tropics, which as a group live in the upper zone of temperatures compatible with life (Mayer, ’14). The smallness of this safety factor, and the actual magnitude of the temperature quickly producing death, as compared with that for other forms frequenting near-by localities, is sufficient to show that there is little or no trace of adaptational modifications correlated with external thermal conditions. Thus, among animals which have been studied at Bermuda we find such facts as those set forth in table 2, where it will be seen that although there is an undoubted general correspondence in the upper temperature limits, or thermal death points, the correlation of these values with the 216 LESLIE B. AREY AND W. J. CROZIER normal life conditions of the several animals involved is by no means precise. The temperature producing rapid death in Chiton is a little higher than that found for many shallow-water species in tropical seas (Mayer, 14), but the maximal temperature successfully withstood for a short time (40°) is not noticeably greater than that for other animals which do not live upon sun- baked rocks. Shelford (16) has insisted upon the correlation between the survival time of organisms (of the same and of different species) at elevated temperatures and the character, and especially the depth below the surface, of the habitats which they severally frequent. Doubtless these correlations result, at least in part, from the gradual effects of temperature upon the composition of the body, since they can be determined experi- mentally (Loeb and Wasteneys, 12); although in Just what way they operate, we do not know. We are chiefly concerned, however, with evidence bearing upon the possibility of a thermal sense, or senses, in Chiton. The ‘spontaneous’ behavior of the gills is perhaps the most significant evidence upon this point, although the nature and variation of the tactile responses are also illuminating. When immersed in sea-water at temperatures of 15°C. or below (down to 8°) the foot of Chiton produces a few smooth contractive movements, which are usually not produced at temperatures between 15° and 38°. The labial palp also moved slightly. The intensity of these movements increased as lower and lower temperatures were employed, down to about 8°. The ctenidia contracted in sea-water at 10°, and subsequently expanded; there is no regular increase in the vigor or duration of this response with lower tem- peratures, but it continues clearly down to 2°. The abolition of all responses, more quickly the lower the temperature, possibly interferes with the production of other, slower movements which might otherwise result from sensory activation at the lowest temperatures used. With elevated temperatures, not until 40° is reached do we find even slight indications of movements, of both foot and ctenidia, resulting from immersion. Above 48° these movements did not appear in any form. THE SENSORY RESPONSES OF CHITON DAG. These findings are in close accord with the effects of temperature in producing movements among other marine invertebrates. In table 2 data taken from several sources show that the temper- atures inducing definite movements indicating response are in general about 15°C, and 35° to 40°C for ‘cold’ and ‘heat,’ re- spectively. This is true of even Amphioxus, where Parker (’08) was of the opinion that separate ‘cold’ and ‘heat’ senses are de- monstrable. In Ascidia thermal sensitivity is, however, compara- tively great (Hecht, 718). With the exception of Stylotella, the animals concerned in table 2 were studied at Bermuda, and at the same season of the year. Stylotella has been included in order to show that in a sponge living at a normal temperature corresponding to that of other animals with which it is com- pared response to a sufficiently high temperature—identical or nearly so with that inducing motor effects in Chiton, Holo- thuria, Amphioxus, etec.—is clearly produced, although sensory elements are not here (Parker, ’10) in any way concerned. The responses obtained at high temperatures do not, in our belief, necessarily demonstrate the operation of differentiated thermoreceptors; they result rather from the general effect of high temperature upon the superficial protoplasm of the animal concerned, leading to increased tactile irritability (Crozier, ’15b) and other effects, and may indeed be in some instances due to direct action upon muscle fibers. In Amphioxus the motor behavior of the whole animal in removing itself from a localized current of sea-water at 39° is quick and definite, but is not of different character from that to tactile excitation, although evidence from experiments upon differential sensory exhaustion (Parker, ’08, p. 440) show that the heat- and tactile-receptive mechanisms are here distinct. In Chiton, how- ever, the tests so far cited do not yield conclusive evidence of the presence of heat receptors. | On the low temperature side the results are more encouraging. ‘When the animal was immersed in water at about 12°C. or slightly below, the ctenidia of Chiton exhibited a definite contraction of brief duration, after which they expanded. This was true even at 2°, at which temperature tactile responses were very quickly CROZIER AREY AND W. J. LESLIE B. 218 IO1Z -019 pus Aaty o¥V (4 21.) PeSWO | o9F OF SF (q 9T,) ToIZorD é (q GT.) 1eTZ01p oOF (OT.) 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The response of the ctenidia to low temperatures is particularly definite and resembles the contrac- tion of the oral tentacles of holothurians (Crozier,’15 b; Olmstead, "17 b), appearing at about the same temperature. These re- sponses are more clearly indicative of thermal receptivity in the strict sense than are those to high temperatures, for below 15° the tactile sensitivity of Chiton quickly diminishes, whereas at 38° to 40° the tactile irritability of the ctenidia, foot, and palp, leading to responses identical with those obtained in the heat treatments, is greatly enhanced for several minutes subsequent to immersion; the augmentation in responsiveness to touch is greater at 40° than at 38°, but at both temperatures sensitivity to touch decreases to below normal after ten minutes and at higher temperatures it disappears more quickly still. So far, then, we believe that in Chiton there is evidence of something akin to cold reception, but that there is reason to regard the responses obtained upon immersing the animals in warmed sea-water as the result of increased tactile irritability or of some related, non-thermospecific type of irritability. 2. Local application of heat and cold There is reason to believe that the responses of many animals to chemical influence, for example, and possibly to heat, differ considerably when, 1) a small area of the integument is affected by the stimulation, and, 2) the whole surface is simultaneously exposed to activation. For this reason, and also with the purpose of locating the regions mainly concerned in thermal receptivity (if any should be found), it was necessary to carry out tests in which small portions of the surface of Chiton could be locally heated or cooled. ‘These tests were made in several ways, by the application of small volumes of sea-water at different tempera- tures or by the use of heated or cooled solid objects. Chiton is THE SENSORY RESPONSES OF CHITON 221 easily employed for such experiments because it can be tested in air, the disturbing influence of a sea-water medium being thus eliminated. These tests resulted, however, merely in the confirmation of those made by immersing the chitons in sea-water at different temper- atures. Even a red-hot needle directly applied to the surface elicited at most but a slight and very local reaction, on the foot and head; the mantle beneath the girdle, the dorsal surface of the girdle, the tegmenta, and the mantle between the plates were quite insensitive. The reactions of the foot and palp, although slight, followed the application quite promptly (within 0.4 sec- onds). No responses from any region were obtained when a red- hot glass rod was brought within 2 mm. of the animal’s surface. A cooled glass rod (at about 5°C.) induced no responses other than those attributable to tactile irritation. With care, a glass rod could be applied even to the gills without leading to a reaction; this was also true with a cooled rod. Similar results were obtained by the use of test-tubes contain- ing water at different temperatures. Sea-water adjusted to different temperatures was gently poured in 0.5 ee. volumes from a pipette over different regions of Chiton (in air). At 40°C., local movements of the palp and foot were induced, and a rather indefinite contraction of those ctenidia directly affected; when water at this temperature, or even at 37°, was applied to the dorsal mantle between the plates of an extended chiton, the two neighboring plates were approximated, just as with tactile irritation of this region. Water at 12° led to slight movements of the foot and palp; at 9° to 10°, to prompt local contractions of the ctenidia. Between 12° and 40° no activation was obtained from the general surface. Although these results are throughout consistent with those outlined in the preceding section, their interpretation presents some difficulties. An attempt was therefore made to separate, through differential exhaustion, the processes of thermal re- ception from those for tactile and chemical stimulation. The responses to heat and cold indicated that the reactivity of the 222 LESLIE B. AREY AND W. J. CROZIER various parts of Chiton under the action of these agents follows the relative orders: dorsal mantle > foot, palp, ctenidia, for heat; foot, palp > ctenidia, for cold. To tactile excitation, the order of reactiveness is slightly dif- ferent (see p. 199), but there exists no adequate criterion for the comparison of the relative sensitivity of the several parts to heat. Only in the case of the dorsal mantle, between the plates, does it appear that thermal sensitivity is relatively enhanced as compared with tactile, since the high-temperature threshold (37°) seems to be lower than for other regions (40°) which are superior to the inter- tegmental mantle in tactile reactivity. The amplitude and vigor of the responses from this region are comparatively slight, how- ever, and little emphasis can be put upon this result. On the basis of their relative distribution, thermal and tactile receptivity cannot be clearly separated. By differential exhaustion an apparent separation of this kind can, nevertheless, be effected. When water at 10°C. was poured, in 1 ce. portions, from a pipette several times in succession over the anterior ctenidia of a chiton in water at 24°, the animal ceased to respond after the fourth treatment; six applications of cool water were made at intervals of three minutes. Very weak tactile responses were then obtainable from the affected ctenidia, al- though they still did not respond to water at 10°C. In attempt- ing to differentiate between ‘heat’ and tactile responses, this method of attack fails completely, since, as we have described, when chiton is placed in water at 38°, its general tactile reactivity was perceptibly increased, and much more conspicuously so im- mediately after immersion in water at 40°, although tactile re- activity gradually decreases after a few minutes’ exposure to this latter temperature. As a consequence of this condition, we are not warranted in speaking either of the separateness or the sen- sory identity of ‘heat’ and tactile effects, even though the surface of the foot and the ctenidia did almost cease to respond to touch after they had been exposed to four local treatments, in air, with 1 to 2 cc. of water at 40° at intervals of two minutes. THE SENSORY RESPONSES OF CHITON 223 3. Summary The evidence we have presented relative to the existence of a temperature sense in Chiton shows that if specific thermoceptors of some sort do indeed occur upon the surface of the animal, they are of a very poorly developed kind. Responses to high tem- perature, under the various conditions of these tests, cannot be adequately distinguished from tactile effects or even from direct influences upon muscle. The minimal temperature (37° to 40°) eliciting a ‘heat response’ is very close to the maximal temperature which the chitons successfully withstand, and is even higher than that which induces a distinct effect upon the muscular ‘sphincter’ about the oscula of Stylotella (Parker, 710), where no receptor organs are involved. Although this temperature is identical with that producing heat responses in Amphioxus (Parker, ’08), it cannot be clearly shown by exhaustion tests—as apparently it can in Amphioxus—that ‘heat’ and tactile receptivity are in any way organically distinct. Only in the case of the intertegmental mantle is there a suggestion of special thermal sensitivity, and here the response elicited is not of a character favorable for analysis. With low temperatures, as with high, the limiting temperature producing perceptible responses in Chiton is prob- ably just outside the range of its normal thermal experience. The ‘cold’ responses, however, elicited at 12° to 15°, are of a definite character and may apparently be separated, through differential exhaustion, from purely tactile responses; that they are mediated by special sensory structures remains uncertain, but is possible. This matter of sensory differentiation is an exceedingly com- plex one. The fact that isolated cells of the metazoan body (e.g., chromatophores) are capable of excitation by heat, as well as by chemical agents, local pressure, and light (Spaeth, ’13), has of course no decisive weight as an argument for ‘generalized receptors’; yet the degree of heat (high temperature) effective as a stimulus is in such cases of an order of magnitude comparable to that found effective in the sensory activation of many inver- tebrates. In comparing the relative sensitivity of different 224. LESLIE B. AREY AND W. J. CROZIER species, account must be taken of the toughness of the tissue concerned (the delicacy of the respective cell surfaces). This may explain why the delicate, internal, protected surface of the oral siphon of Ascidia (Hecht, 718) exhibits a sensitivity to heat and to cold superior to that known for many other animals. VI. PHOTIC EXCITATION 1. Effects of light a. Behavior in an illuminated field. «a. Preliminary experiments. Chitons collected more or less at random and without much at- tention to size were tested in a qualitative way with reference to their photic behavior. At the bottom of one end of a wooden box, 29 em. long by 23 em. wide by 30 em. deep, there was cut a horizontal slot about 12 em. long and 1 em. high. This box was coated on the inside with lampblack suspended in turpentine, giving an approximately dead-black finish. A rectangular glass jar containing sea-water to a depth of several centimeters was placed inside the black box, within which it fitted closely. Chitons were put in the glass jar, the box covered, light admitted (or directed) through the slot, and the subsequent movements of the animals determined. With diffuse sunlight twenty-one experiments upon twenty individual chitons gave this result: 6 did not move at all during the course of the test (lasting about one hour); 1 oriented a few degrees away from the light, while 13 animals made definite progress toward the light, irrespective of their original orien- tation in relation to it. These chitons were probably all of average size or larger. In some instances they were allowed to become fixed to the bottom of the aquarium with their anterior ends toward the light, in other cases they were placed with long axis perpendicular to the light, in still others deliberately headed away from it or quite at random. The nature of the result in these experiments will be evident from the following record: THE SENSORY RESPONSES OF CHITON 225 Experiment 1. 11:15 a.m. A chiton placed with long axis parallel to the slot admitting light, distant 13 em. from it. 11:19 a.m. Oriented toward the light. 11:20 a.m. Began moving forward. At first moved in a diagonal di- rection, until the girdle touched the side wall of the container; it then turned further and moved directly toward the light. 11:24 a.m. Stopped, half-way toward the slot. 11:30 a.m. Began moving again. 11:34 a.m. Reached light end and began climbing end wall. 11:39 a.m. All except posterior quarter attached to end wall of con- tainer.. Stopped. 11:43 a.m. Began again and moved until all of body was on vertical end wall. Turned until body axis was parallel to water line, where, just submerged, it lay directly over the slot. Experiment 2 11:45 a.m. Same chiton as in experiment 1, placed transversely to the light, but with other side illuminated, and 26 em. from the light slot. 11:48 a.m. Began turning away from the light. 11:50 a.m. Had rotated away from the light,’ then back toward it, through an angle of more than 270°. 11:54 a.m. End had come in contact with side of container. Animal now began to climb. No forward progress toward the light. Experiments 3 and 4 In two further chitons tested in this way, orientation was: in one case direct, beginning almost at once; in the other it required 29 min. (involving a preliminary turn- ing through 45° away from the light). Both animals. made definite progress toward the light. These tests indicated in a general way the presence of a definite, though sluggish, positive phototropism, with reference to dif- fuse light. With direct sunlight, reflected horizontally from a mirror, three individuals oriented promptly and move directly toward the light, two oriented toward the light and then away from it, four individuals immediately oriented more or less away from the light, and two did not move at all. This result obviously required further analysis; it might have been the outcome of a general illumination of the whole aquarium or might have ref- § Note this apparent persistence of a turning tendency once established- 226 LESLIE B. AREY AND W. J. CROZIER erence more specifically to some definite peculiarity of behavior. These tests were made with chitons of relatively large size (6 to 8 cm.) in which the shell valves were probably more or less eroded, although at the time no special note was made of their condition. 8. Analyses of responses to general illumination. The fore- going section indicates the somewhat obscure relations, with respect to phototropism, discovered in random samples of the chiton population. The younger individuals, especially those less than 2 em. long, live in dark situations. When stones bearing them are turned over, the chitons creep rapidly to the under, dark side. Not until a length of 7 to 9 cm. is attained does Chiton occur with any frequency upon illuminated rocks. If chitons from habitats representing these two divergent ex- tremes are compared, it is found that in ordinary sunlight the larger ones are photopositive, the younger ones photonegative. Their orientation is precise, definite, and without ‘trial move- ments.’ There are, however, certain complications in the mode of orientation which will be fully considered on a later page. The foliowing test is typical :° LENGTH OF INDI- HABITAT PHOTIC BEHAVIOR VIDUAL cm, 1.0 | Under stone on a sandy beach, | Consistently photonegative to the south side Darrell Island. (No. weakest daylight used. VI, 119.B)® Del Same locality. (VI. 119.C) Photonegative to direct sunlight; photopositive to weak diffuse light, and to twilight. 3.9 | In a pocket at the mouth of a | Photonegative to direct sunlight; cave, north shore Long Island. photopositive to light from a (Vale 22-19) north window 10 ft. away. 5.0 Same locality (VI.128.9) Same 7.2 | On an approximately horizontal | Photopositive to diffuse daylight; rock, exposed in the sun, north photopositive to direct sunlight shore Marshall Island. (VI. from a cloudless sky 140.2) 8.3 | Same locality. (VI. 140.4) Same. book. ° The specific animals bear definite numbers given to them in the field note- In a subsequent report on the ethology of C. tuberculatus the necessity for this will be made apparent. THE SENSORY RESPONSES OF CHITON 27 These six specimens illustrate a correlation which is universal in our experience: the youngest chitons are found in dark- situations, the older ones in the light; these two groups are, respectively, photonegative and photopositive to ordinary sun- light; animals of intermediate size are photopositive to weak light, photonegative to stronger light, and the character of their normal photic environment (typically, in horizontal crevices near the mouth of caves, and in other non-brilliantly illuminated spots) is completely correlated with this behavior. There is usually a region of intensities of ordinary daylight within which an animal of intermediate size may be either photopositive or photonegative in different tests—a region of seeming indifference to light. The largest chitons are usually quite indifferent to weak, diffused light. The actual distribution of the chitons of different sizes in the field shows in a most convincing manner that this differentiation in photic responses is not a matter of adaptation to environmental circumstances, but is on the con- trary based upon structural changes determined with advancing age. Note, for example, the following instances in which an individual photonegative to sunlight (as found by test) occurred on a shore where no deep caves were available, nor any large stones under which it might hide. VI. 111. (Apl. 4, 1918). North shore of Hawkins Island; a more or less horizontal shelf of rock, 1 foot beneath high water mark; nine chitons in a closely compacted group, in the zone of Modiolus and barnacles. Eight of the chitons with eroded valves, forming a fairly close match with the color of the exposed rock; in sunlight; these chitons 6.5 to 8.8 em. in length. One chiton, however, was a <7, 4.5 em. in length, the valves greenish, very slightly eroded; it was located under another individual (o, 6.8 em. long), which completely con- cealed and sheltered it from the light. VI. 140 (Apl. 22, 1918). Northwest shore of Marshall Island. In a small pocket in the rock four chitons of 7.2 to 8.7 em. were found; of these the shells were eroded and bleached. Under one of them a fifth specimen, 2.8 em. long, blue-green in color, valves uneroded. b. Results of partial illumination of the body. «a. One side of the body illuminated. Chitons were adjusted in the apparatus shown diagrammatically in figure 13; they were so situated, directly under the vertical partition, that either the right or left 228 LESLIE B. AREY AND W. J. CROZIER side was in comparative darkness, the other side in the light. Diffused sunlight, employed with chitons of medium to large size, induced responses of a variable kind. Experiment 1. Two chitons introduced. One moved partly toward the dark side, and subsequently, in the course of 45 min., moved back into the center of the light compartment. The other one rotated through 180°, its anterior end passing through the dark side and then orienting into the light. Experiment 6. 'Two chitons used. One began to move at once; oriented 90° into the dark, and moved so that almost the whole of its body was in the dark compartment; after 5 min., it turned through 180° and moved straight out into the light. The other one within a | Fig. 13 Sectional view of apparatus for the partial illumination of a Chiton: a shallow pan fitting the bottom of a box with blackened walls; one half of box open to receive light, as indicated by the arrows, the other half covered and separated from first half by a blackened vertical partition extending into the sea-water, that nearly fills the pan; horizontal overhang from the lip of the pan exposed to light reduces reflection. few minutes oriented weakly into the light, halted 1 min., and then oriented anterior end back into the dark, so that half of its body was on either side of the partition. In 15 individual tests of this nature, 4 chitons turned and moved directly into the light, where they remained. 0 chiton turned and moved directly into the dark. 4 chitons turned and moved into the dark, then into the light. 0 chiton turned and moved into the light, then into the dark. 4 chitons turned directly into the light, but did not creep. 0 chiton turned directly into the dark, but did not creep. 1 chiton oriented into the light, then into the dark. 2 chitons gave no response during the time allowed for the ex- periment. THE SENSORY RESPONSES OF CHITON 229 8. One end of the body illuminated. Similar tests were made in which either the anterior or the posterior end of the animal was illuminated. Numerous experiments of this sort were also made in the field. In brief, chitons upon sunlit rocks were found to move into the light when either the anterior or the posterior end had been shaded. The locomotion subsequent to ilumina- tion of the posterior half only, by means of bright sunlight, was in a posterior direction; usually, before the chiton had moved completely into the light, it executed a turning movement. The readiness with which backward creeping, for distances of several centimeters, may be resorted to, is worthy of remark. y. Analysis. The predominating movement in these tests is photopositive. The peculiar variations observed remain to be explained. This can be done, as in the case of orientation by general illumination, through consideration of the size and habitat of the individuals and of the character of their periostracum and tegmenta. This analysis agrees in its results with that pre- viously given. The small chitons, less than 2 cm. in length, move into the shadow when their surface is half illuminated. They creep backward with greater readiness than do the large ones. These experiments show that with light approximately ver- tical in direction the effect of partial illumination of the body is such as to parallel completely the result in orientation to hori- zontal light. 2, Differential sensitivity a. Shading. An attached chiton, undisturbed and at rest, tends to lift the girdle from the substratum, either along its whole circumference or else in one or more local areas. If.a raised portion of the girdle be shaded, there results a relatively quick and smooth lowering of the girdle to the rock or other surface. This response occupies about two seconds; its vigor depends upon the original distance of the girdle from the sub- stratum. After a little time (about ten seconds) the part con- tracted is relaxed to its original condition. The speed of re- action and the time for recovery vary considerably in different animals. In some cases a single stimulation resulted in the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 2 230 LESLIE B. AREY AND W. J. CROZIER girdle being closely applied to the substratum for a long time (save at the anterior and posterior ends, involved in respiration). In other instances five to nine shadings and responses were obtained, after which the response grew weaker until it finally disappeared. When the response became very weak, or imper- ceptible to a single shading, two or more shadings in fairly rapid succession were still effective in producing a reaction from the girdle. Associated with depression of the girdle is a pronounced contraction of the gills in animals shaded dorsally. Chitons resting upon their dorsal surface and shaded ventrally gave also a pronounced response. Some individuals were very sensitive, coming from a fully extended condition to complete rolled-up closure as the result of a slight decrease in the illumi- nation; if already partially rolled up, a general shading induced still further, but still incomplete, curling of the body, after which the chiton returned to its original condition. The response became fatigued after seven to sixteen trials. In good reactions of this type, five seconds were required for the curling up of the shell, fifteen seconds for its recovery. To successive shadings the curling-up process becomes more complete; the time required for this effect varies directly with the size of the animal, as seen in the following notes (table 3); some animals are decidedly less reactive in this way than others, however. In Holothuria (Crozier, 14) conditions of similar import are known. The reaction to shading of the ventral surface comprises longitudinal bending, contractive movements of foot and head, inward curving of the edge of the girdle, and contractions of the gills. ‘The time occupied by the response of the ctenidia (‘reac- tion time’) to a single shading at the posterior end of the animal is also proportional to the length of the Chiton (table 3). The response to shading is due to a decrease in the intensity of light in the visible region of wave lengths. The interposition of a glass plate between a chiton and the source of light led to no reaction, and the shading response was easily elicited through a considerable thickness of glass. Glass ray filters transmitting restricted portions of the spectrum were placed between chitons and a source of sunlight (which induced shading responses), THE SENSORY RESPONSES OF CHITON 2a" and then the effect of cutting off the colored light was deter- mined. The transmitting regions of the spectrum for the color filters used are given in table 4. These values are of course not very precise. The red filter transmitted light that seemed about twice as bright as that coming through the blue. Nevertheless, the re- actions obtained upon shading chitons through these filters TABLE 3 RNS TTGUNE TIME REQUIRED TO EFFECT ROLLING UP OF ‘REACTION-TIME’ OF THE SHELL BY REPEATED] POSTERIOR CTENIDIA TO SHADINGS OF THE AN- SINGLE SHADING Number Length Se ae ee cm. seconds seconds 1 3.0 5) 2 5.5 10 3 9.0 15 4 3.0 1-2 5 6.0 2-3 6 8.9 4-5 TABLE 4 Regions of spectrum transmitted by certain color filters COLOR LIGHT at ND tio RANGE AA GC bare rR emcee cca ae, eee ace eae od See 690-634 56 ICU Wik ee seo eee eet ae DAY Oe ee, eee 690-605 85 GES TELY THAR SAU hots Cake LER SEIN T EG PN Bae eee 589-508 81 TS TVS 43 Sine eee SR Re OER O28 oe 523-450 + 73 were sufficiently clear cut to be of some use. Responses pro- duced by cutting off the light coming through the green or the blue screen were equal in amplitude to those obtained in sunlight; whereas the red and yellow lights, when occluded, led to quite obviously weaker responses. This is also the case in the shading reactions of the shore barnacle (Crozier, ’15 b, p. 273; the same light filters were used). Chitons of all sizes (ages) and from every type of habitat give 232 LESLIE B. AREY AND W. J. CROZIER precise responses of the character described when the light in- tensity is suddenly reduced. b. Increased light intensity. Chitons from medium to large size, under water in aquaria placed in direct sunlight, give also a response to increased illumination. This response is not evident except in bright sunlight. It consists in a depression of the girdle similar to that induced by shading. The response is, however, never of such quickness, vigor, or completeness as that to shad- ing. In smaller chitons, or with larger ones in diffuse light, the reaction to increased illumination (if present at all) is so slight as to escape detection. At its maximal development, it comprises a local depression of the girdle to the substratum, and does not involve a ‘suction reflex’ of the whole animal, such as is induced by a very slight decrease in illumination. The thresh- old of sensitivity for increased illumination is, indeed, very much higher than that for decreased. It should, however, be noted that if a large chiton is shaded dorsally the girdle may not be much elevated again for some time, provided the state of lowered intensity is allowed to continue. On removing the shadow, the girdle is again elevated. If the intensity be slightly decreased the girdle of a large chiton may be lowered rather slowly, so that after two seconds the shadow may be removed before the girdle has been completely depressed; the movement of the girdle may then cease abruptly with the incidence of the more intense light. A similar response is evidenced upon the ventral surface of the animal. If the light be suddenly increased (to direct: sunlight) about two seconds subsequent to the beginning of the ‘curling-up’ response induced by a shading, this response sometimes ceases abruptly; the chiton may or may not proceed then to straighten out. The effects of increased illumination are more conspicuous in lamplight at night. Clear indications have been obtained of a specifically higher photic sensitivity at night as compared with daylight hours. A discussion of this matter awaits further. investigation. THE SENSORY RESPONSES OF CHITON 233 3. Distribution and nature of photoreceptors a. For illumination. We have described thus far the responses obtained from chitons submitted to photic excitation involving the edge of the girdle, a large portion of the surface, or the whole surface of the animal. There remain to be considered the dis- tribution and variety of the photoreceptors. A chiton from which the girdle has been completely removed is still oriented by light. This fact, taken together with the known histological structure of the sensory organs in the shell plates, suggested that the surface of the shell might contain photosensitive organs. Chitons were rigidly clamped in the way already described in discussing tactile stimulation (p. 200), and allowed to write their contractions kymographically. Tested in air, the shell was found to be sensitive to moderate faradization. When so stimulated, the animal undergoes a pronounced general ‘curling up,’ which ceases when the current is interrupted. A similar result follows the application of a spot of light.!° By this method it can also be shown that the shell plates are sensitive to shading. They do not appear sensitive to increased illumination. These results, together with those obtained in orientation experiments, furnish adequate proof that the ‘shell eyes’ of chiton are indeed sensitive to light. We did not attempt to localize the minimal areas sensitive to light, but the illumination of one-eighth of the surface of an anterior or posterior valve with an intense spot of light is sufficient to induce a good response. In the genus Chiton ‘extrapigmental’ eyes are lacking (Moseley, 85; Plate, ’99; Nowikoff, ’09), the ‘intrapigmental’ eyes being, however, plentifully distributed over the surfaces of the valves according to a definite pattern; their locations are well shown in eroded shells. They are most abundant in the anterior and posterior valves, but occur on all the shell plates. The ‘eyes’ are not the only parts sensitive to light, however; the ventral edge and the dorsal surface of the girdle indicate by 10 This method of investigation permits quantitative work of a relatively precise kind; but in the present paper we are concerned mainly with qualitative effects. 234 LESLIE B. AREY AND W. J. CROZIER movements a local sensitivity to illumination, as likewise the anterior edge of the proboscis probably does. Heath (’99, p. 579; sep. p. 4) thought the proboscis of Ischnochiton to be sensitive to light. In the adult Chiton there are no detectable cephalic eyes, although in some small species the larval ‘eye’ may persist into adult life (Heath, ’04 b). The larva of Ischnochiton magdallensis is positively phototrophic (Heath, ’99, p. 637; sep. p. 62). b. For shading. The edge of the girdle and the tegmental surfaces of the valves are sensitive to decrease in light. In the former case the nature of the receptors is obscure; in the latter case they may or may not be identical with the organs sensitive to the constant action of light. The minimal area which must be shaded in order to effect a response is very small. The shadow of a fly 6 feet distant in moderately bright sunlight induces violent shading responses. An opaque spot 2 mm. in diameter on a glass plate at a distance of about 2 cm., casts sufficient shadow upon a valve to lead to pronounced reactions. The threshold of sensitivity is very low, an almost imperceptible decrease in light intensity being quite effective. The arrangement of the aesthetes in the tegmentum is such that many micraesthetes accompany each megalaesthete; it might be supposed that the micraesthetes are of different re- ceptive value, but no proof for this can be given. The physiological distinctness of the shading receptors on the ventral edge of the girdle is shown by the fact that their complete exhaustion by repeated activation does not interfere in the least with tactile responses. c. For increased illumination. The great sensitivity of chiton to shading and the poorly developed character of the responses to increased illumination make adequate experimentation very difficult. The edge of the girdle is the most sensitive part; probably the surface of the proboscis and palp are also sensitive. Nothing more can at present be said under this head. THE SENSORY RESPONSES OF CHITON 2a5 4. On the theory of phototropism a. The orienting stimulus. In a previous article (Crozier and Arey, ’18) we have given some discussion of this matter, and need refer here merely to the main conclusion. In young individuals the simultaneous exhibition of a precise reaction to shading, and no such response to increased illumination, is thoroughly incon- sistent with the idea that negative orientation by light is induced by stimulus derived from an increase of illumination, as such (Crozier, 714, ’715b). The further continued presence of this shading response in older chitons under conditions in which they are photopositive, together with the fact that any reaction to increased light intensity which these older chitons did exhibit was clearly of a negative character, is of the first importance theoretically. It completes the cycle of qualitative proof that the constant action of light, not change of intensity, is the causal stimulating agent in photic orientation. Two further types of experiment may be cited in this connection. A chiton of medium size orienting away from the light is purposely shaded on the side to which it is turning. Light falling at an angle of about 45° is used in the experiment. The chiton reacts by depressing the girdle, as usual, and swings somewhat away from the shaded side. By repeated successive shadings in this way, the chiton may be made to move for a time at an angle with the line of the incident light, although after it becomes exhausted for shading it completes its orienta- tion in the usual way. A chiton completely exhausted by shading orients without delay away from or toward the light, depending on the age of the animal. Both types of experiment result in behavior agreeing with the conclusion above stated. b. The determination of positive and of negative orientation. In an earlier section of this paper we have called attention to the fact that at a length of about 5.5 em. (for the Chiton population in Great Sound) the shell begins to show noticeable erosion. 236 LESLIE B. AREY AND W. J. CROZIER This erosion continues with increasing severity until natural death supervenes. By its ravages the canals containing the aesthetes are laid bare and the sense organs therein contained are destroyed. The empty canals are visible under a hand lens. This phenomenon was noted by Plate (Ola, p. 383) in connection with forms possessing large extra-pigmental eyes. At a length of 7 cm. the erosion is quite pronounced; the shell is also frequently covered with adventitious organisms. We have shown the organs of photoreception concerned in orientation to be located in the tegmenta of the valves. There is thus a pro- nounced correlation between the development of positive photo- tropism toward daylight and the erosion of the shell, for animals less than 7 cm. length are rarely found completely exposed to sunlight. There are a number of instances in which animals alter the sense of their phototropism depending upon the intensity of the light. It isin general the rule that this alteration is one from a photopositive condition in weak light to a photonegative con- dition in strong light, rarely the reverse. The general rule holds in the case of Chiton, and in other forms exhibiting general in- tegumentary sensitivity to light. The indifferent point dividing the range of light intensities into a lower range, exciting positive orientation, and a higher range, inducing negative phototropism, shifts in Chiton toward the upper limit as age (and erosion) advances. Hence it would appear that in this animal the excitation of a smaller number of sense organs (in the eroded chitons) is equiv- alent in stimulating power to the action of a lower intensity of light upon a larger number of sense organs. This is further evidence pointing to the constant action of light as the source of the orienting stimulus. No case of this kind has previously been examined. It will be interesting to make a further study of this matter, in relation to temperature, for example. ~¢. The method of orientation. In Chiton tuberculatus, es- pecially in older, unevenly eroded animals, there are to be found traces of a condition, more plainly evidenced in Ischnochiton purpurascens, which is at first sight anomalous. Sometimes an THE SENSORY RESPONSES OF CHITON 2 animal will orient precisely away from the light, and then move caudad toward the light for a distance of several centimeters. Close inspection usually makes it evident in these cases that the posterior valve is more deeply eroded than the anterior valves. Its sensitivity is therefore less. Similarly, a few animals were noted in which photopositive orientation was succeeded by backward creeping, i.e., away from the light, owing presumably to the higher sensitivity of the anterior valve. ‘There is evident in these reactions a type of local response, rather than of unitary behavior, which is highly instructive and deserves further study. In Ischnochiton the behavior above referred to is somewhat similar to that just described, as this amphineuran creeps poste- riorly with considerable freedom. Horizontal light parallel to the long axis, incident upon the anterior end, induces the animal frequently to creep backward for several centimeters. In chitons relatively uneroded or evenly eroded the method of orientation is direct, diagrammatic, as it is in most mollusks. In a large individual the process is so slowly carried out, unless very bright light is used, that its details may be carefully ex- amined. The results of such examination are not merely con- sistent with the theory of Loeb regarding animal phototropism, but furnish valuable evidence in support of this view. ‘The behavior of Ischnochiton and of unevenly eroded large individuals of chiton is of particular importance in this connection. 5. Bionomic correlations involving photic behavior Many writers have considered the photic reactions of animals in the light of their normal conditions of life, coming usually to the conclusion that these reactions are highly adaptive, and hence that they have been determined through natural selection (Mast, 711, chap. 13). In spite of its traditional sanctity, the presentation of this view involves an inversion of logic, a veri- table somersaulting, which is to the last degree unconvincing. That the modes of response which specific organisms are found to exhibit are individually correlated in an appropriate way with 238 LESLIE B. AREY AND W. J. CROZIER the conditions of existence, no one of experience will be prepared to deny; there are also, of course, modes of reaction under con- ditions artificially imposed which are important from this stand- point. It is rather the harmonious complexity of these indi- vidual responses which presents in reality the difficult problem and the one of importance. The behavior of Chiton is most illuminating in this respect. In an earlier chapter we have briefly touched upon the co6r- dinations between size, habitat, coloration, and photic orienta- tion in the Chiton population. The main fact appears to be that as Chiton gets older it moves out into more open situations. The other correlations follow automatically in the wake of the chang- ing sense of the animal’s phototropism. This change is due to a. gradual shifting of the ‘indifferent point’ of light intensity sepa- rating the region of lower intensities, leading to photopositive behavior, from the region of higher intensities, leading to photo- negative movements. We have shown that the principal factor involved in this alteration is the erosion of the tegmenta of the valves (bringing about the destruction of the photosensitive aesthetes). It remains to discover what it is that produces the erosion of the shell. Comparative studies of differing environments fre- quented by Chiton are now in progress and should ultimately afford a quantitative answer to this question. The erosion of the shell is general among the chitons of large size. Plate (’01 a, pp. 381-3) supposed it to result from ‘wave action’ and the mechanical effect of sand. In C. tuberculatus it would seem that the periostracum is in part eroded as a result of chemical action of the water, combined with the failure to continue its secretion upon older parts of the shell; as well as to the activity of barnacles, algae, and other organisms which settle upon the valves." In different localities it would seem that these two factors are of 11 Barnacles live upon the valves of a chiton until they have formed two or three distinct growth lines. The firmness with which they are attached to the shell plates of Chiton depends upon the degree of the original erosion. In chitons of medium size the barnacles usually are not very firmly attached; after death they drop off and leave no scar. On older chitons (seven to nine years) the dead remains of barnacles adhere for some time. THE SENSORY RESPONSES OF CHITON 239 varying relative importance. The general feature which is the most significant, however, is the correlation of slight exposure with incipient erosion. A study of over 1500 individuals has shown the invariable completeness of this correlation. The amount of exposure is, in part at least, determined in a purely mechanical way. The smallest chitons, dwelling at the very upper limit of the tide, under loosely piled small stones, become after a year or two too big to fit into the crevices there provided. The food supply is also insufficient. They therefore come to inhabit stations further below high-water mark. The occurrence of other organisms is here more abundant, and there are addi- tional factors making for more ready erosion. In this way the history of a chiton can be followed in a fashion which shows that although its habitat is determined by its be- havior, the reverse is not apparently the case. Hence the logical inversion to which reference was made in the first paragraph of this section. It is also evident that the modifications in Chiton’s behavior and appearance, its occurrence in groups, and the probably advantageous correlations in this way resulting, are determined ina catenary manner. The original position (habitat) of the young individual is determined by the tropisms of the larva, in their turn determined by the inherited chemical com- position of the egg. The lack of active wandering movements (p. 178; Plate, ’0la, p. 509) in the older individuals is important, because opportunity is in this way made for the full operation of the influences in particular circumscribed habitats, and thus for the development of homochromic elements in the coloration of the chitons and for concomittant phases of progressive erosion of the shell. The chitons as a whole are known to be photosensitive and to reside in general in dark situations (Cooke, ’95, p. 400). Heath (99) observed that a number of chitons were nocturnal in their habit, ‘‘withdrawing into some shaded position upon the approach of day,”’ some species remaining out on their feeding grounds ‘‘only when the day is foggy or dark.’”’ The larva of Ischno- chiton magdalenensis is positively heliotropic, the adult negative (Heath, 99). It would be of some interest to study a variety of 240 LESLIE B. AREY AND W. J. CROZIER species from the standpoint we have developed in this paper (Plate, ‘Ola, p. 509). Ischnochiton purpurascens, Acantho- chites spiculosus, and a species of Tonicia we find to be photo- negative to light of all intensities. The deep-sea solenogastres may also be photosensitive, although the only observation known to us on this point (Heath, ’04a, p. 461) is not decisive. VII. CHEMICAL EXCITATION 1. Reactions to various substances The surface of the soft parts of Chiton is sensitive to a wide variety of chemical excitants. We are concerned, in the first place, to discover something of the nature of the process of chemical excitation by electrolytes. For this purpose experi- ments are cited in which, unless otherwise stated, approximately 0.5 cc. of solution was locally applied to the surface of chiton, tactile stimulation on the part of the stream being avoided. The chitons were tested in air. After each response, the stimu- lating fluid was washed away by a gentle stream of sea-water before proceeding to any further tests. a. It was attempted to arrange certain salts in the relative order of their stimulating power. The alkaline cations K, N Hg, Li, and Na, and Ca and Mg, were compared through the effects of their chlorides, in 5/8 M solutions (made up in rain-water). The mouth, the sole and the edge of the foot, the gills, and the ventral surface of the girdle were stimulated in various indi- viduals. The comparative efficiency of the different chlorides was judged on the basis of their average effects upon all the parts activated. Since it may be supposed that an effectual subjective factor entered into these judgments, we will give rather full notes of the experiments. KCl: Very strong stimulation in all regions, even on the ventral surface of the girdle, which bends inward toward the source of excita- tion. All the responses very sharp. NH,Cl: Also gives pronounced contractive responses, but not so ex- cessively strong as with KCl, nor of such long after duration. No responses from the girdle. THE SENSORY RESPONSES OF CHITON 241 IiCl: Much as with NH.Cl, but reactions noticeably weaker; re- sponses not initiated so quickly, nor so completely carried out. NaCl: Responses in the mouth region extremely weak; on the edge and the sole of the foot, still weaker; the gill responses possibly as decided as with LiCl. The edge of the foot moves at first toward the stimulating liquid, then away. CaCl: No reactions from the gills; on the mouth region, and the edge and sole of the foot, stronger responses than with NaCl. MgCl.: The gills were raised toward the source of application; weak negative response from the sole of the foot; the edge of the foot at first pushed locally toward the fluid—as with NaCl—then away if the application were prolonged. In the mouth region, fairly strong contractions. From these findings it appears that the cation order of decreas- ing stimulating power is Ks Niki >. Nar Subsequent experiments with more dilute CaCl, and MgCl, solutions, isosmotic with sea-water, showed that to the former salt practically no responses were given except at the mouth, while to the latter no responses were given save the positive out-pushing of the ctenidia. These bivalent cations are mark- edly less efficient in sensory excitation than are the monovalent alkaline cations. The curious ‘positive’ reaction of the gills to MgCl, solutions is similar to that given by the podia of holo- thurians (Crozier, 715 b). b. The neutral salts of potassium—KCl, KBr, KNO;, and KI—were compared, at 5/8 M concentration as in the previous tests. The local reactions elicited were very violent in all parts of the body, but in no place were the animals stimu- lated to roll up. The ctenidia responded by contracting until curled in a tight circle against the dorsal wall of the gill cavity; at the same time the side of the foot was moved far away, later- ally, thus exposing the gill cavity more widely, while the girdle was rolled over and inward so as to cover the ctenidia. After a brief interval the foot relaxed. These responses were of variable duration, depending upon the amount of the stimulating solution applied. 242 LESLIE B. AREY AND W. J. CROZIER For purposes of comparison, ‘reaction times’ were measured, covering the interval from the contraction of the gills until the moment they were judged to be completely relaxed. The results obtained in this way may be illustrated by the following sets of data: TIME OCCUPIED BY THE GILL REACTION SALT SOLUTION Series I SERIEs II SERIES III minutes minutes seconds Cie 3.0 3,0 SIN @) ah eret ate tas n che aes reieee ce RON OE EUS one ed, 1 5s 30 TOR AR AE MONE REY Rie ei anehne ae Ree 1.0 1.0 35 o£) Las rR A MSE Se SRG CA ae 20 KCl was plainly more stimulating than the other salts. After numerous attempts to graduate the other three under various conditions as to quantity of solution and quickness of application, in the same and in different animals, the series KCl > KNO; > KBr > KI was chosen as the most satisfactory. c. Successive dilutions of several representative substances were employed to stimulate various regions of chiton’s surface, with the object of establishing the respective limiting dilutions effective in excitation. Sea-water was largely used as the sol- vent in these tests, since it is the normal fluid medium for chiton; moreover, as we shall see presently, the surface of the animal is reactive locally to osmotic conditions differing from the normal. KCl: To KCI solutions more concentrated than N/16, made up in sea-water, all of the ventral portions of Chiton are reactive. N/16 All portions respond except the girdle. N/32 No response from the sole of the foot. N/48 Good reactions from the mouth region; the gills some- times fail to react. N/64 Gills fail to respond. N/80 Mouth response good; edge of the foot weak. THE SENSORY RESPONSES OF CHITON 243 N/112 Edge of the foot fails; the palp is sensitive on its edge and bends first toward, then away from, the stimulus. N/160 ‘Faint responses from the mouth region. From one animal, considerably more sensitive than the rest, faint ‘mouth-region responses were also obtained at dilutions N/224 and N/256; this was very exceptional. KOH: The dilutions tabulated are here only approximate, owing to the precipitation of calcium and magnesium hydrates at concentra- tions above 0.012 N + KOH (Haas, ’16). N/10 Reactions produced on all parts except upon the ven- tral surface of the girdle (mantle edge). N/50 to N/125 Same. N/250 to N/375 The edge of the foot is but slightly sensitive. N/500 No responses from the foot; reaction at the mouth just perceptible; gill responses of fair intensity. HCl: N/10 No reaction from the ventral surface of the mantle edge. N/50 to N/425—responses from all parts, save the mantle edge. N/500 ~=No reactions from the foot; very faint responses from the mouth; gills respond more weakly still, but with fair sensitivity. Several organic acids were also tested. At M/10 concentration, in rain water, tannic, malic, lactic and acetic acids produced reactions from all parts, including the mantle edge. M/100 solutions of the same acids made up in sea-water also induced responses, but not from the mantle edge; these acids were compared by noting the ‘recovery time’ of the gills when stimulated with them (as in the case of salts); the measurements obtained were: Tannic, 35 sec. Malic, 20 sec. Lactic, 12 sec. Acetic, 8 sec. Picric acid: made up in sea-water, produced very powerful reactions ‘everywhere, at concentrations above M/150. M/250 Responses of the same character, but weaker. M/500 to M/750 The edge of the mantle fails. M/1000 The foot still sensitive, especially along its periphery. M/1200 Gills fail; foot and mouth give good reactions. M/1500 Foot fails; mouth alone responds, but smoothly and evenly. 244 LESLIE B. AREY AND W. J. CROZIER Cocaine hydrochloride, atropine sulphate, and ‘iicotine at M/100 concentration in sea-water stimulated very well the mouth, gills, and the sole and edge of the foot. Chloretone M/200 in sea-water also stimulated these parts; but Urethane under the same conditions did not. Curare in saturated sea-water solution, gave rise to good, but not pronounced responses. Urea. A M/10 solution, in rain water, did not lead to any clear response on the part of the foot, and gave but weak responses from mouth and gills. Ethyl and methyl alcohol solutions 10M, in rain-water, evoked reac- tions from all parts save the mantle edge. With the methyl alcohol the responses were slightly stronger, the gill retraction enduring about twice as long (about 20 sec., as compared with 8 to 10 sec. for the ethyl); this may have been due to impurities; the ethyl alcohol was especially pure. Full strength amyl alcohol induced responses as with the two previous types, but considerably stronger movements resulted, It has been of interest to inquire if the surface of Chiton is generally sensitive to sugars or to ‘sweet’ substances as a class. In view of the usually high value of the limiting stimulating concentration for sugars, it is necessary to consider the follow- ing observations in the light of the subsequent experiments upon osmotic excitation: Maltose: solutions M/3 or M/10, in sea-water, produced no clear responses from any part. A 1M solution in rain-water gave responses from mouth and gills. only. Sucrose: 1M, in rain-water, led to no responses from the mouth region, but gave good reactions from the ctenidia. M/3 in sea-water, gave general reactions from all parts. M/2, made by adding 1 volume of 1M in rain-water to 1 volume of sea-water, led to fair responses. M/3, made by adding 1 volume of 1M in rain-water to 2 volumes of sea-water, induced no reactions at all. Lactose: M/2 in rain-water gave strong responses everywhere except at the mantle edge. d. To a variety of more obvious ‘irritants,’ the whole soft ventral surface of Chiton reacts powerfully. Thus, H.O: (3 per cent) induces strong movements in all regions. Ether, chloro- form, carbon bisulphide, aniline oil, and oils of cassia, Juniper, cloves, pennyroyal, thyme, bergamot, and origanum, when ap- plied as a drop of the raw substance or in the form of a satu- THE SENSORY RESPONSES OF CHITON 245 rated extract in sea-water, induced good reactions from all parts, including the girdle, which reacted by bending and twisting away. The order of sensitivity of the several parts of the surface was made out to be: gills > head > edge and posterior end of foot > sole of foot > ventral girdle surface > dorsal girdle surface. e. Osmotic excitation of the general ventral surface of Chiton was investigated by means of dilutions of sea-water. Three parts sea-water + 1 part rain-water: gave responses only upon the lips. Two parts sea-water + 1 part rain-water: no response from the mantle edge, but other parts react weakly. One part sea-water + 1 part rain-water: no response from the mantle edge, good responses from other parts. Rain-water: sole of the foot puckers away strongly; gill response very active; even the girdle reacts, and the shell tends to roll up. Sea-water concentrated by evaporation to one half its original volume gave good reactions on all regions. The same upper limit of osmotic stimulation appears in the effects of glycerin solutions: Glycerin: in sea-water solution, Strong reactions everywhere, even from the mantle edge. 3M Mantle edge fails; pitting of the foot away from the fluid is deep and local. 2M Same, but weaker responses. 1M Ctenidia give a faint response, but only occasionally; mouth region still sensitive; very doubtful responses from the foot. M/2 Faint response from the lips; ctenidia doubtful. 2. The mode of excitation by solutions The observations above detailed require analysis from several points of view. We shall deal first, briefly, with the evidence they contain relative to the method of activation by solutions. a. It is clear that ‘osmotic sensitivity’ is possessed by the soft superficial tissues of chiton. Whether this depends upon the activation of ‘tactile’ or other end organs or upon the stimu- lation of chemoreceptors proper we cannot at first entirely THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 2 246 LESLIE B. AREY AND W. J. CROZIER decide. The comparative distribution of tactile and of osmotic sensitivity is nevertheless suggestive in this connection. We have seen that for stimulation by gentle contact the reactivity of the several regions of chiton’s surface was as follows: head = ctenidia > edge of foot > girdle > sole of foot; while for irritants, such as essential oils, the order of reactiveness for the same parts was: ctenidia > head > edge and posterior end of foot > sole of foot > girdle. To local osmotic disturbances the sensitivity of these areas ap- peared, on the basis of the experiments with dilutions of sea-water, to be related in the following sequence: head > ctenidia = foot > ventral edge of girdle. So far as these responses go, they indicate that the receptors concerned in osmotic excitation are distinct from those concerned in tactile reactions, from those concerned with responses to irritants, and (as seen in a following section) from those impli- cated in chemical excitation, but the evidence is not conclusive. The sensitivity of the proboscis (‘head’), especially of its peripheral edge, is probably concerned in determining the rela- tive immobility of chitons in exposed places at the period of low tide; the same, to a lesser degree, is perhaps true of the edge of the foot. In active creeping the anterior edge of the proboscis is kept in close contact with the substratum, as shown in figure 14; this organ undergoes ‘spontaneous’ local contractive move- ments, depending for their execution on the pressure of fluids contained in its interior spaces (Heath, ’05 b). b. The osmotic reactivity of Chiton’s soft surfaces is im- portant in connection with the question as to whether sugars are successful as activating agents for this animal. Sea-water of 36.5 per mille salinity (5/8 M) has at 27°C. an osmotic pressure of something more than 25 atmospheres, corresponding to a sucrose solution about 0.8 M. The limits within which various concentrations of sea-water do not stimulate were found to be 4/8 to 8/8 M (for the lips, the other regions being less sensitive) ; M/2 sucrose in 5/16 M sea-water gave fair responses from all THE SENSORY RESPONSES OF CHITON 247 parts, although 1 M sucrose in rain-water did not, except from the ctenidia. This total concentration (< 6/8 M) is well within the ‘osmotic limit,’ and indicates that sucrose may be mildly efficient in stimulation. The behavior of maltose also shows faint indications (at 1 M in rain-water) of some stimulating capacity. According to Brooks (716), sucrose penetrates (plant) protoplasm quite readily, and affects permeability after the manner of a monovalent kation, although not with special ra- pidity. The fact that in Holothuria (Crozier, 715 b) no evi- dence was obtained that sucrose could stimulate the integument —although maltose and glycerin apparently did, while in the experiments of Olmsted (’17 b) and of Hecht (’18) no sucrose effect was detected apart from that exerted through the osmotic pressure of its solutions—shows that some such factor as the gaa Fig. 14 Showing the manner in which the proboscis is kept in contact with the substratum during creeping. Anterior end, seen from the side: a, proboscis margin (‘palp’); 8, foot; y, girdle. Arrow shows.direction of creeping. time of exposure to the sugar solution (or the concentration of accompanying salts?) may be important in determining whether or not activation occurs. Sensitivity to sugars, even if present, is however, undoubtedly very low on the general soft surface of chiton. With Chromodoris zebra we have found no stimulation induced by maltose or sucrose 1 M in rain-water, applied to various parts of the animal’s surface in small volumes from a pipette, the animal being in sea-water. Similar tests with Balanoglossus (Crozier—unpublished experiments) resulted in no detection of activation by 1 M solutions of sucrose or of maltose. c. Independently of their osmotic sensitivity, the soft surfaces of Chiton are also open to activation by dissolved electrolytes, which are much more powerful as excitants than are non-elec- 248 LESLIE B. AREY AND W. J. CROZIER trolytes of the sugar type. The order of kation stimulating efficiency for the alkali chlorides, K,. > NAi > Li Na, is that found in the sensory activation of Balanoglossus (Cro- zier, ’15a), Ascidia (Hecht, ’18), Chromodoris (Arey), and other marine animals, and is in accord with the general order of action of these ions upon various protoplasmic processes. The anion order, Cl NO; > Br > 1, does not agree with that determined by Hecht (’18) for Ascidia; but the methods of experimentation were in the two cases quite different, in the tests with Ascidia the method of limiting effec- tive dilutions being employed and the salts being dissolved in sea-water. For Synaptula (Olmsted, ’17b), the order Cl < Br < I, as in Ascidia, was found, by the same general method. Each of these anion series has its counterpart in other salt actions (Hober, ’14), but why they should be reciprocal is not altogether clear, particularly since the kation orders obtained by these respectively different methods are in agreement. In Chiton, general chemical excitation is primarily an affair of the kation. The minimal concentrations of different electrolytes which are effective in the excitation of different regions of Chiton appear to be as follows (in sea-water solutions): The three organic acids we employed stimulated according to the following order of efficiency : Malic > lactic > acetic (M/100 in sea-water). This order indicates that for Chiton lactic acid is less efficient as a sensory excitant than malic, which is not the sequence shown (Crozier, 16a) by the earthworm’s reactions, nor in the penetration of tissues by these acids, but does correspond with the respective magnitudes of acid strength. Since the solutions were made up in sea-water, much emphasis cannot be placed on this point. The very general nature of Chiton’s sensitivity, involving excitation by a great variety of materials in solution, adheres nevertheless to the rules already available in the activation of THE SENSORY RESPONSES OF CHITON 249 numerous protoplasmic processes. The effects of salts, acids, alkalies, anaesthetics, and other substances in evoking reactions are best understood upon the assumption that cells of the external epithelium are acted upon by these substances after the manner of cells in general. Thus, with anaesthetics: chloretone (M/200) gave well-defined responses, whereas urethane (M/200) did not; the anaesthetic effect of these materials follows the same order (Crozier, 16a). Some form of union with the surface of the epithelial cells is undoubtedly involved in the process of excita- tion, but these experiments cannot be understood on the assump- tion that excitation is determined by an increase in cell perme- ability. The decreased permeability produced by bivalent cations (e.g., Ca) is a specific function of the cation; nevertheless, TABLE 5 The minimal concentrations of various substances effective in the stimulation of Chiton REGION KCl HCl KOH Picric acid PRS previ eet al ae ae ah IN ALGO N/10) (>N/10) M/600+ while CaCl, apparently does not serve as a sensory activator under the conditions of these tests, good reactions are elicited by Ca(NO3)2 (compare also the case in Balanoglossus, Crozier, ’15a). There is no specific parallelism between efficiency in sensory activation and permeability-increasing properties. (Compare, for acids and alkalies, Crozier, ’18 a.) 3. The chemoreceptors a. The ventral parts of Chiton exhibit a chemical:sensitivity which is essentially similar to that found for the general integu- ment of other marine invertebrates (table 6). The limiting dilutions of various substances effective in activation of various parts of the animal (table 6) follow an order which is, for each 250 LESLIE B. AREY AND W. J. CROZIER tissue, consistent among different substances. This suggests that—since the ‘thickness,’ ‘toughness,’ or density of these parts is in the same general order (lips > edge of foot > ctenidia > sole of foot > girdle), with the exception, perhaps, of the ctenidia—we are in reality dealing with a generalized form of sensitivity, the effectiveness in arousing reactions depending upon, 1) the ease with which the surface of the cells locally con- cerned may be actively penetrated by the excitant, and, 2) upon the relative richness with which these parts are respectively TABLE 6 Minimal concentrations of various substances effective in the sensory activation of various animals ANIMAL HCl pcr NaCl KCl QUININE PICRIC ACID AUTHORITY Vitara), fetes) ee M/1000| M/400|M/50 M/25,000 Parker (12) Ameiurus..... N/20 N/100} N /50 M/150 Parker (712) Amphioxus...| N/500 M/1,250 | Parker (’08) Ascidia!......| N/625 | N/100 N/4 | M/2,500 Hecht (18) Balanog- lossus......}| N/500 | N/400 N/200} M/1,000 Crozier (un- published) Chiton.......| N/500 | N/500 N/160 M/1,500 Chromodoris.} N/700 | N/200 N/10 M/10,000 | Crozier and Arey (un- published) Synaptula....| N/600 | N/200| N/4| N/40 | M/10,000 Olmsted GLb) Holothuria...| N/500 | N/500 N/500 Crozier (15 b) 1 “‘Uncorrected’’ concentrations; Hecht’s paper (18). innervated. In a previous chapter we have shown that the tactile reactivity of these regions of chiton’s surface follows approximately the order: head (palp, lips), ctenidia > edge of foot > girdle > sole of foot. There is, thus, a distinct inconsistency in the relative sensitivity of these parts to touch and to chemical excitation, which shows that differences in the method of excitation undoubtedly exist THE SENSORY RESPONSES OF CHITON Pes b and that probably the relative richness of general sensory inner- vation is not the sole factor determining the chemical sensitivity of any one region. Further evidence for the distinctness of the chemoreceptors will be considered in the following section. Through the comparison of the effectiveness in stimulation for different materials in Chiton and in other animals (table 6), it will be seen that the integumentary sensitivity of invertebrates corresponds in its general features (limiting effective dilutions) with that of taste in man rather than with the common chemical sense; there are, however, noteworthy differences from the physiology of taste excitation (Parker and Metcalf, ’07; Crozier, "15 b). The fact that the isolated substances considered are, as such, foreign to the daily experience of Chiton, has nothing to do with the information they give concerning the process of excita- tion. That we are not concerned with general ‘pain’ reactions in the Chiton experiments can be shown in this way: The ventral surface of the girdle of Chiton, although relatively the most insensitive region to chemical excitation, is nevertheless decidedly reactive to touch. This region is excitable by pure anaesthetics, saturated sea-water solutions of. essential oils, rain-water, by 10/8 M sea-water, and by 5 M glycerin, but is inexcitable (un- der the conditions of our experiments) by alkaline chlorides ‘(other than KCl at 5/8 M concentration in rain-water), by KCl (in sea-water) more dilute than M/16, by HCl or KOH more dilute than M/10, by picric acid more dilute than M/700, or by ethyl alcohol even in 10 M concentration. Hence it would appear that only under conditions of an excessively heterologous quality may the ventral surface of the girdle be excited by these chemical agents, under such conditions in fact that ‘pain,’ tac- tile, or any other form of sense organ might be activated. This result adds to the conviction that the general chemical sensitivity of Chiton’s soft tissues is distinct from any form of tactile irritability, and is not consistent with the view that here —as there may be in Balanoglossus (Crozier, ’15 a), or in Synap- tula (Olmsted, ’17b)—there are ‘generalized sense organs’ (Nagel). Some indications are afforded that a distinct general chemical sense is adequately represented in Chiton. This con- 252 LESLIE’ B. AREY AND W. J. CROZIER clusion may be tested through the attempted physiological isola- tion of chemical and tactile irritability. Such tests are, of course, open to several sources of serious error; the most critical results should be given by cases in which sensory exhaustion to chemical stimulation did not interfere with tactile irritability (Parker, ’08, p. 440). A result of this kind is free from the objec- tion that sensory fatigue may result in heightening the threshold to the more delicately acting forms of activation. Such a result is readily obtainable with Chiton: the ctenidia cease to respond to chemical activation by 5/8 N NaCl after about ten trials at brief intervals; they continue, however, to respond to touch. This finding strengthens the opinion already derived from the distributional study of tactile and chemical activation in chiton. The fact that the reactions induced by these modes of activation are in some cases qualitatively identical, involving similar muscular contractions, is no obstacle to this view. b. In the buccal cavity of chitons cup-shaped organs, with a suggested ‘gustatory function,’ have been described, as well as numerous nerve terminals in the subradular organ. We have nothing on this subject to add to Heath’s (’03) observations,. which we can confirm; these observations showed that positive food-taking responses are initiated by the excitation of the ex- ternal surface of the mouth region with the materials upon which chitons feed. We have seen that mouth movements are initiated by chemical activation. It will be of interest to dis- cover to what class of substances chitons react by food-taking responses. Some writers speak of an osphradium situated at the base of each ctenidium in the chitons (Burne, ’96). Pelseneer (’99, p. 13) has described and figured the two erectile ridges on the inner side of the posterior inner ventral border of the girdle (the ‘lateral lappets of the mantle fold” of Plate, ’97, pl. 2, fig. 15, llp.), immediately caudad of the gills (figs. 7 and 8); he con- siders these structures to be homologous with the osphradia of other mollusks. In some species these protuberances assume the shape of well-defined papillae. They are situated on the distal face of the pallial nerve cords, which supply them with a rich THE SENSORY RESPONSES OF CHITON 258 innervation, and constitute, in Pelseneer’s opinion, special sen- sory regions, each of them being protected by the ventral face of the papilla, which is sometimes spiculose. We have described how the respiratory water current impinges on the dorsal face of each papilla. The conditions are therefore favorable for the sit- uation of an organ ‘‘testing the quality of the water.’’ The papilla may be significant for egg-laying responses (i.e., in the reception of a stimulus provided by the spermatic fluids), as it seems more prominent in the females. This remains to be tested. Copeland (’18) has brought forward good evidence that the osphradium of carnivorous snails is concerned in the recep- tion of chemical excitation by dilute solutions of materials ema- nating from food; his further contention, that this organ is therefore an ‘olfactory organ,’ because the exciting agent is very dilute, is an unnecessary metaphor (cf. Arey, 718 b). VIII. THE NERVOUS SYSTEM AND SENSE ORGANS OF CHITON The main features of the foregoing account may now be briefly summarized. This report makes no claim to completeness; it does lay a solid foundation for further investigation in at least two directions: 1) the phenomena of seeming adaptation in the ethology of chiton, and, 2) the physiology of certain types of irritability. The sensory conditions are here unexpectedly com- plex. The major pathways of nervous transmission are, by contrast, unusually clear and well .defined. The manner in which sensory capacities and modes of reaction are involved in the complex determination of natural behavior can be followed in great detail. a. Tactile receptors are absent from the shell surfaces. The ‘scales’ and ‘hairs’ upon the girdle are important tactile organs. The ctenidia are also sensitive to touch, as are the proboscis, the foot, and the ventral surface of the girdle. The foot is posi- tively thigmotactic to large surfaces, but retracts locally when stimulated by a small surface. The tegmental aesthetes are photosensitive; they are activated by light of constant intensity and by sudden decrease in light 254 LESLIE B. AREY AND W. J. CROZIER intensity, not by an increase. The dorsal surface of the girdle (scales) is also sensitive to light—characteristically to a decrease of light intensity, also to the constant intensity of light, and to a sudden increase in light intensity, provided this intensity be great. The soft ventral surfaces are sensitive to light. The periphery of the girdle is the ventral part most sensitive to shading. The superficial soft tissues of Chiton are open to chemical activation, to stimulation by abnormal osmotic pressures, and by ‘irritants.’ Evidence has been secured, through the study especially of the topographic distribution of the various types of excitability, that tactile receptors, photic receptors, and chemoreceptors are physiologically distinct. There is no clear evidence of sensitivity to heat; that to cold is less doubtful. There is a pronounced tendency for the animal to come to rest in positions avoiding uneven tensions in the musculature. This is responsible for the precise negative geotropism exhibited by Chiton. This mollusk is not sensitive to vibratory mechanical disturbances. b. This brings us to the consideration of one of the most un- settled problems in sensory physiology: In what manner is differ- ential irritability determined? The immediate receptors of ex- citation in metazoans above the sponges are cells which function primarily as detectors and transmitters of disturbances in the energies of the environment. In a general way it is true that all forms of protoplasm are capable of being changed (activated) by light, heat, cold, pressure, chemical agents, and so forth. Con- siderations of this order, which hold also in certain cases upon the quantitative side (as in the action of chemical agents), have been responsible for the view that ‘irritability’ is a generalized property of living matter, best studied in uni- (or non-) cellular organisms. It does not seem probable that this conception can at present be of any great help; in spirit it is deductive, whereas the manifestations of irritability (e.g., in the diversified taste receptors of the human tongue) are manifold, specific, and must be investigated in a more purely inductive manner. Neverthe- THE SENSORY RESPONSES OF CHITON PAS) less, it is true that in those instances where photoreceptors, for example, may be isolated and distinguished, we frequently find that the sensory elements are removed from the external surface of the animal, protected from the action of environmental chemi- cal disturbances and deforming contacts. From this standpoint one of the factors determining differential receptivity is to be found in the degree of anatomical isolation of the receptor; another, in the morphology of the sensory cell or organ, as in the development of distal projections. These factors of form and position undoubtedly facilitate the respective operation of dif- ferent qualities of stimulation, and to that extent determine the functioning of differential irritability. But the problem is not in this way wholly solved. The evidence for ‘generalized sense organs’ is in some cases good, though perhaps not final. Even on the sole of the foot of Chiton physiological evidence of sen- sory separateness for photo-, tacto-, and chemo-reception is available. The skin of Amphioxus is fully as sensitive as that of other marine animals to touch and to chemical influences (Parker, ’08), but is insensitive to light. The additional factor is probably found in the possession by certain receptor cells of special substances which enter into excitation reactions. Even if in some cases it could be shown that the epithelial cells of an animal were open to sensory activation by a variety of means, it would not lead to the view that a ‘universal’ type of sense organ (Nagel) was that first developed in evolution, re- ceptors of special kinds being by some obscure metaphysical proc- ess subsequently derived from it. In coelenterates, so far as we know (Parker, ’17 a), tactile receptors and chemoreceptors are organically distinct. c. The reactions of Chiton to local stimulation are of a character consistent with the known distribution of the central nervous system. At the sides of the body, those parts innervated by the pallial strands are conspicuously homolateral in their responses. The codrdination of the pedal musculature for the production of locomotor waves depends upon a mechanism locally contained, and apparently upon the integrity of the pedal cross-connectives. The coérdination of the gill movements on one side of the body 256 LESLIE B. AREY AND W. J. CROZIER depends upon the intact condition of the pallial nerve strand on that side. The responses of isolated portions of an animal sec- tioned transversely are such as to show the absence of any strong centralization. This is in agreement with the known occurrence of ganglion cells throughout the whole length of the nerve strands. In Chiton nervous centralization is relatively at an incipient stage. d. Alteration in the behavior of Chiton toward light with ad- vancing age of the animal is the primary variable determining the exhibition of a very complex series of environmental inter- relations. The young Chiton is photonegative, the old Chiton photopositive, to sunlight. Chitons of intermediate age are _ positive to weak light, negative to strong. Photic orientation is direct, and is determined by the constant intensity of light, not by change of intensity. The progressive alteration in the sense of phototropism is determined by the erosive destruction of the photosensitive aesthetes, conditioning in older Chitons a lower specific stimulating power of the light. The erosion of the shell is in turn produced, in part, by, 1) normal growth effects; 2) the activity of organisms settling upon the shell plates. The homochromie coloration of Chiton is determined by the nature of its algal food and by the organisms living upon its dorsal surface. The older chitons are relatively stationary; therefore specific local environmental influences have oppor- tunity to affect the appearance of the chitons. The animals associate in groups, commonly of a certain average size and con- taining numbers of both sexes. Certain seemingly ‘adaptive’ consequences may reasonably be attributed to this mode of occurrence. A homochromically colored isopod is characteristi- cally associated with Chiton tuberculatus. These and other harmonious correlations, of which mention is made in the body of this report, follow automatically in the wake of the changing phototropism of Chiton. The animal’s habits determine the environment in which it dwells. The pre- cise and intricate bionomic correlations here briefly mentioned are an automatic consequence of its modes of reaction. Dyer Island, Bermuda, May, 1918 THE SENSORY RESPONSES OF CHITON 204 LITERATURE CITED Arxry, L. B. 1918a The sensory behavior of Chiton (abstr.). Anat. Rec., vol. 14, p. 95. 1918 b The multiple sensory activities of the so-called rhinophore of nudibranchs. Amer. Jour. Physiol., vol. 46, pp. 526-532. BaunackE, W. 1914 Studien zur Frage nach der Statocystenfunktion. II. Noch einmal die Geotaxis der Mollusken. Biol. Centralbl., Bd. 34, S. 371-385; 479-523. Buumricu, J. 1891 Das Integument der Chitonen. Zeit. f. wiss. Zool., Bd. 52, S. 404-476, Taf. 23-30. Cited in Schneider’s Lehrbuch, 1902. Brooks, 8. C. 1916 A study of permeability by the method of tissue tension. Amer. Jour. Bot., vol. 10, pp. 562-570. Burne, R. H. 1896 Notes on the anatomy of Hanleya abyssorum. Proc. Malacol. Soc. (Lond.), vol. 2, pp. 4-18. Cooks, A. H. 1895 Molluscs, in: The Cambridge Natural History, vol. 3, pp. i-xil, 1-459. CorrLanp, M. 1918 The olfactory reactions of the marine snails Alectrion obsoleta (Say) and Busycon canaliculatum (Linn.). Jour. Exp. Zooél., vol. 25, pp. 177-228. Crozier, W. J. 1914 The orientation of a holothurian by light. Amer. Jour. Physiol., vol. 26, pp. 8-20. 1915a The behavior of an enteropneust. Science, N.S&., vol. 41, ‘pp. 471-472. 1915b The sensory reactions of Holothuria surinamensis Ludw. Zool. Jahrb., Abt. f. Physiol., Bd. 35, S. 233-297. 1916a Cell penetration by acids. Jour. Biol. Chem., vol. 24, pp. 255-279. 1916b The rhythmic pulsation of the cloaca of holothurians. Jour. Exp. Zodl., vol. 20, pp. 297-356. 1917 The photoreceptors of Amphioxus. Anat. Rec., vol. 11, p. 520. 1918a Sensory activation by acids. I. Amer. Jour. Physiol., vol. 45, pp. 323-341. 1918b Growth of Chiton tuberculatus. Proc. Nat. Acad. Sci., vol. 4, p. 322. . 1919 Sex-correlated coloration in Chiton. Amer. Nat. (in press). Crozimr, W. J., AnD Anny, L. B. 1918 On the significance of the reaction to shading in Chiton. Amer. Jour. Physiol., vol. 46, pp. 487-492. Datt, W. H. 1907 Instructions for collecting mollusks, and other useful hints for the conchologist. Bull. U. 8. Nat. Mus., no. 39, pt. G, 56 pp. Datu, W. H., anp Stmpson, C. T. 1901 The mollusca of Porto Rico. Bull. U.S. Fish Comm. for 1900, vol. 1, pp. 351-524, 6 pls. Haas, A. R. 1916 The effect of the addition of alkali to sea-water upon the hydrogen ion concentration. Jour. Biol. Chem., vol. 26, pp. 515-517. Happon, A. C. 1886 Report on the Polyplacophora collected by H. M. S. Challenger during the years 1873-75. Results H. M. S. ‘‘Chall.,”’ Zo6l., pt. 43; vol. 15, 50 pp., 3 pls. 258 LESLIE B. AREY AND W. J. CROZIER Heatu, H. 1899 The development of Ischnochiton. Zool. Jahrb., Abt. Anat., Bd. 12, S. 576-656, 5 pl. 1903 The function of the chiton subradular organ. Anat. Anz., Bd. 23, S. 92-95. 1904 a The habits of a few solenogastres. Zool. Anz., Bd. 27, 8S. 457- 461. 1904 b The larval eye of chitons. Proc. Acad. Nat. Sci. Phila., 1904, p. 257-259. 1905 a ‘The morphology of a Solenogastre. Zool. Jahrb., Abt. Anat., Bd. 21, 8. 703-734, pls. 42, 43. 1905 b The excretory ma circulatory systems br Cryptochiton stel- leri Midd. Biol. Bull., vol. 9, pp. 213-225. 1905 c The breeding Tae of chitons of the Californian coast. Zool. Anz., Bd. 29, S. 390-393. 1907 The gonad in certain species of chitons. Zool. Anz., Bd. 32, S. 10-12. Hecut, 8. 1918 The physiology of Ascidia atra Lesueur. II. Sensory Physi- ology. Jour. Exp. Zodél., vol. 25, pp. 261-299. HeIuprin, A. 1889 The Bermuda Islands. Phila., 231 pp., 17 pls. Hoser, R. 1914 Physiologische Chemie der Zelle und der Gewebe. xxiv + 85 pp. Leipzig. Jennines, H. 8. 1907 Behavior of the starfish, Asterias forreri De Loriol. Univ. Calif. Pub., Zodél., vol.. 4, pp. 538-185. Jongs, J. M. 1888 The conchologist in Bermuda. Conchol. Exchange, vol. 2, p. 118. JoRDAN, H. 1913 Vergleichende Physiologie wirbelloser Tiere, Bd. I. Ernah- rung, xxli+ 738 pp. Jena. Karka, G. 1914 Einfiihrung in die Tierpsychologie, Bd. I: Die Sinne der Wirbellosen, xii + 593 pp. Leipzig. Lors, J., anD WastEeNEYs, H. 1912 On the adaptation of fish (Fundulus) to higher temperatures. Jour. Exp. Zoél., vol. 12, pp. 543-557. Mast, 8. O. 1911 Light and the behavior of organisms. xi+ 410 pp. New York. ; Matissz, G. 1910 Action de la chaleur et du froid sur l’activité motrice et la sensibilité de quelques invertebrés marins. Bull. Stat. Biol. d’Arca- chon, T. 13, pp. 1-52. Mayer, A. G. 1914 The effects of temperature upon tropical marine animals. Carnegie Instn. Wash., Publ. 183, pp. 1-24. Mercatr, M. M. 1892 Preliminary notes on the development of Chiton. Johns Hopkins Univ. Cire., vol. 11 (no. 97), pp. 79-80. 1893 Contributions to the embryology of Chiton. Studies Biol. Lab. Johns Hopkins Univy., vol. 5, pp. 249-267. Mosetey, H.N. 1885 On the presence of eyes in the shells of certain Chitonidae, and on the structure of these organs. Quart. Jour. Micr. Sci., vol. 25, pp. 37-80, pls. 4-6. Nowrxorr, M. 1909 Uber die intrapigmentiren Augen der Placophoren. Zeits. wiss. Zool., Bd. 93, 8. 668-680, 1 Taf. THE SENSORY RESPONSES OF CHITON 259 OumstEeD, J. M. D. 1917a Notes on the locomotion of certain Bermudian mollusks. Jour. Exp. Zodél., vol. 24, pp. 223-236. 1917 b The comparative physiology of Synaptula hydriformis (Lesueur). Jour. Exp. Zodél., pp. 333-380. Orton, J. H. 1914 Note on the feeding habits of Patella. Jour. Mar. Biol. Assn., N. S., vol. 10, p. 257. Parker, G.H. 1908 The sensory reactions of Amphioxus. Proc. Amer. Acad. Arts and Sci., vol. 48, pp. 418-455. 1910 The reactions of sponges, with a consideration of the origin of the nervous system. Jour. Exp. Zo6l., vol. 8, pp. 1-41. 1911 The mechanism of locomotion in Gastropods. Jour. Morph., vol. 22, pp. 155-170. 1912. The relation of smell, taste, and the common chemical sense in vertebrates. Jour. Acad. Nat. Sei. Phila., Ser. 2, vol. 15, pp. 221-234. 1914 The locomotion of chiton. Contrib. Bermuda Biol. Sta., vol. 3, no. 31, 2 pp. 1917 a Nervous transmission in the Actinians. Jour. Exp. Zodl., | vol. 22, pp. 87-94. 1917 b Pedal locomotion in Actinians. Ibid., pp. 111-124. 1917 ec Actinian behavior. Ibid., pp. 193-229. 1917 d The pedal locomotion of the sea-hare, Aplysia californica. Ibid., vol. 24, pp. 189-145. 1917 e The power of suction in the sea-anemone Cribrina. Ibid., pp. 219-222. PELSENEER, P. 1819 Recherches morphologiques et phylogénétiques sur les mollusques archaiques. Mem. Cour. Acad. Roy. Belg., T. 57, 113 pp., 24 pl. Puatse, L. 1897 Die Anatomie und Phylogenie der Chitonen. (Thiel A.) Zool. Jahrb., Supplement-Bd. 4 (Fauna Chilensis, Bd. 1), pp. 1-248, Taf. 1-12. 1899 Id., Fortsetzung. (Theil B.) Ibid., Supplement 4 (Fauna Chilensis, Bd. 2), pp. 15-216, Taf. 2-11. 1901a Id. (Theil C.) Ibid., Supplement V (Fauna Chilensis, Bd. 2), pp. 281-600, Taf. 12-16. 1901 b Uber einen einzelligen Zellparasiten (Chitonicium simplex) aus der Mantlehéhle von Chitonen. Ibid., 8. 601-606, Taf. 17. RicHarpson, Harriet 1902 The marine and terrestrial isopods of the Ber- mudas, with descriptions of new genera and species. Trans. Conn. Acad. Sci., vol. 11, pp. 277-310, 4 pls. 1905 Monograph on the isopods of North America. Bull. U. 8S. Nat. Mus., no. 54, lili + 727 pp., Washington (Smiths. Instn.). Scuneiper, K. C. 1902 Lehrbuch der vergleichenden Histologie der Tiere. xiv + 988 pp., Jena. [Amphineura, pp. 510-536.] SHELFORD, V. E. 1916 Physiological differences between marine animals from different depths. Puget Sd. Mar. Sta. Publ., vol. 1, pp. 157-174. SparTH, R. A. 1913 The physiology of the chromatophores of fishes. Jour. Exp. Zodl., vol. 15, pp. 527-485. 260 LESLIE B. AREY AND W. J. CROZIER Vuiis, F. 1907 Sur les ondes pédieuses des Mollusques reptateur. Compt- rend. Acad. Sci., Paris, T. 145, pp. 276-278. VeRRILL, A. E. 1902 The Bermuda Islands. x + 548 pp., 38 pls. New Haven. Watson, J. B. 1914 Behavior: An Introduction to Comparative Psychology. xii + 489 pp. New York. AUTHOR’S ABSTRACT OF THIS PAPER ISSURD BY THE BIBLIOGRAPHIC SERVICE, august ll SENSORY REACTIONS OF CHROMODORIS ZEBRA! W. J. CROZIER Bermuda Biological Station LESLIE B. AREY Northwestern University Medical School EIGHT FIGURES CONTENTS Mees DEOL UGE oso). t ioe scas Sha tae ON Sc SARS ne SEN ed alt ats was tad, Aa aneiala ah On MearMiechamienl excltatiOly. t3 tach aso eins wales Sa ae a ylale Wa ih aty cic tbie/s clayelahe CeO APE s tHe StI GOR hh eens Ae rte, ane Parola Saee Ge aicha reer eee SOO Horightinese eeotropinwd. 4. NT LE TERE ath dad UES acy Outen Tk on Se MEO UEO MISHA: Sana) s os kee ow whee nd Sicko dienes. cdeyelsjene see eToys» 278 AGBNICR WARES BELAUtIOUS otis, oie one Nig s ascaendueletead ein’ cot eae Oe aeRO NIB tICREXCLULLOMG cee uecied socinee tle vhereecstee cd ece eee cies Sucka Starerenere olavorets 288 PBUShnding cera Wess. she dyepllMegeegh ys dure fetet Ge. yeh pepe d eee SD AS Sse Crs ae ae OR oe Le ee eM VRE en TEER aS eraN ne 75: UPR OENIAIe METER OIE. ccs et ark eee ee els Bae nt he ci dnd i tees OE EEO We hemircahexcrtantOn oss. fires Pee G VS for TA Sah EL ae. Bee, (OM MlaSuainarys...0 oe. oh pis ont eee eorigGs. Needs ao LR ea eue AeiGersuIe GILG Othe: at hp Meee tepals We cue Walls uaie Us thea toloeteko Shoreslen ts. 1nd eibepahe 308 I. INTRODUCTORY With the objects of presenting data valuable for the compara- tive study of ‘animal behavior’ and of laying a foundation for the intelligent pursuit of certain inquiries in sensory physiology, this article aims to record and briefly to discuss some aspects of the sensory responses of the nudibranch Chromodoris zebra Heilprin.2. In 1884 Bergh wrote that almost nothing was known 1 Contributions from the Bermuda Biological Station for Research, no. 111, and from the Anatomical Laboratory of the Northwestern University Medical School, no. 68. 2 Data upon which this paper is based were in part secured by L. B. A. dur- ing the summer of 1914. These observations have been extended and the paper written by W. J. C. 261 262 W. J. CROZIER AND LESLIE B. AREY about the activities of the large exotic nudibranchs, and very little has since then been added to the subject. We shall deal with questions of natural history and behavior only in an inci- dental way, paying attention specifically to the manner in which Chromodoris responds when activated by various stimulating agents. It was desirable to undertake a study of this kind be- cause C. zebra has already provided material, of a very exceptional character, for the treatment of some questions in which sensory phenomena are implicated (Crozier, 15a, ’16a, ’16b, 717d, 18d). A knowledge of the sensory capacities and modes of re- sponse in Chromodoris affords, also, some information as to the comparative physiology of the nervous system in mollusks, about which, particularly in nudibranchs, very little is known. Chromodoris zebra (fig. 1) is a large species very common at Bermuda,’ with the form typical of the genus. An account of its morphology will be found in the papers of Smallwood (’10) and of Smallwood and Clark (’12). The body is elongated, es- pecially in creeping, and measures up to 18 cm. in length. The animal as a whole is very soft and contractile, and becomes read- ily bent or twisted under appropriate conditions. Throughout the year individuals of a variety of sizes are to be had by dredg- ing in depths down to 10 fathoms. From September to June great shoals of them, numbering thousands in all, crowd up at intervals into shallow water (Crozier, ’17b). They become notably concentrated in certain shallow mangrove creeks con- nected with Great Sound. The cycle of events which determines the shoreward flocking has not yet been fully established. Its coloration makes this nudibranch easy to distinguish upon the bottom (Crozier, 716 b), and the migratory movements of the species, owing to its lack of concealing behavior, may to some extent be followed in the field. The animal moves with a smooth, even, gliding motion over rock surfaces or on the muddy bottom, the entire surface of the 3 As with many other marine forms found at Bermuda, it is probable that the range of C. zebra is quite extensive, although so far it has been reported only from Bermuda (Heilprin, ’89; Smallwood, 710). I am informed by Prof. W. H. Longley that a few individuals were obtained by his collectors, in seining on grass-flats at Tortugas. W. J. C. SENSORY REACTIONS OF CHROMODORIS ZEBRA 263 foot being applied to the substratum. The whole body can, however, be supported by the use of a small part only of the foot; thus C. zebra has sometimes been observed to creep over the edge of a submerged rock, the body of the animal projecting horizontally beyond the edge, or its anterior part being even sharply elevated, until only a centimeter or so of the posterior region of the foot served as a hold-fast. Chromodoris can also swim attached to the surface film of quiet water, but has not been observed to do so in nature. (This behavior has been ob- served in another nudibranch, Facelina goslingi, at the time of its reproductive activity.) Fig. 1 Outline figure of Chromodoris zebra; dorsolateral view from the right side (after Crozier, 718 d). Especially in the mangrove creeks already spoken of, C. zebra is found in localities where eel-grass grows in great profusion. It creeps up the blades of eel-grass and is often found at the very tip. In connection with this habit it is important that the foot, although perfectly flat and without a trace of median division, when in contact with a flat surface, exhibits a most pronounced tendency to fold together, so that the lateral halves of the whole surface of the foot are in contact. This occurs whenever the whole or a part of the foot is removed from a substratum, and makes it possible for the nudibranch to climb the flat-bladed eel-grass. In C. roseapicta no such behavior of the foot is seen, and this species lives under stones along the shore, never coming into contact with eel-grass. 264 W. J. CROZIER AND LESLIE B. AREY According to Vlés (’07), no pedal waves are observable upon the foot of Aolis, at least in the form of color differences, al- though the direction of the pedal wave was said by him to be direct, on the basis of the rippling movement seen at the edge of the foot during creeping. In Doris, also, the pedal’ wave was said by Vlés to be direct. In Tectibranchs the direction of the pedal wave, well defined in this case, is retrograde (Parker, ’11, p. 156). The locomotion of Chromodoris is accomplished in the form of a smooth, planarian-like glide. No macroscopic waves have ever been seen on the foot, even when the animal was swimming attached to the surface film and therefore in a very favorable position for observation. Some unevenness of move- ment may usually be detected along the lateral margins of the foot, and, when fixed to the surface film, where locomotion is very slow, gentle ‘billowing’ movements can usually be detected. Hence it is possible, as Parker (’11) has suggested, that in this case the muscular activity of locomotion is not codrdinated in wave form, but is arhythmic. The lateral surfaces of the body and the surface of the foot are, however, richly ciliated. Much slime is laid down by the foot in creeping. The possibility, then, cannot be entirely excluded that progression is in part at least ciliary, especially since the cilia beat in the appropriate direction, namely from anterior to posterior. Small pieces cut from the mantle or from the foot continue to move for several days in a fixed direction about the bottom of a dish, owing to the beat of the cilia which they bear. According to Olmsted (17), cilia are the means of locomotion in Marginella, Haminea, and Bulla. Local muscular activity, however, shares in loco- motion, for the extreme lateral margins of the foot are the re- gions exerting suction when the foot serves as a hold-fast. This may be seen in the cup-like puckerings along the edge of the foot when ‘swimming’ along the surface film. These regions are not sticky, as if with slime, yet to a sufficiently large surface the out- line of the foot, save at its anterior end, becomes so firmly at- tached that it is with difficulty pushed loose at any given point, although it can be loosened instantly when the animal begins to creep. Local movements of this muscular rim of the pedal SENSORY REACTIONS OF CHROMODORIS ZEBRA 265 surface are visible during creeping. It is possible that the margin of the foot is alone concerned in active progression,’ at least in so far as this is independent of cilia. With an indi- vidual attached to the vertical wall of a glass aquarium, the axis of the animal being horizontal, the lower edge of the foot some- times becomes freed from contact with the glass, the creature then being suspended by the attachment of the upper margin of the foot; the median furrow is then plainly visible on the foot surface. On such a free lower pedal margin two or three dis- tinct waves may be made out at one time. These waves, retro- grade in direction (as in Tectibranchs), are confined to the outer margin of the foot. Similarly, in specimens resting in the angle made by the vertical wall of the aquarium with its bottom, the foot may be free anteriorly, being attached only at the posterior end. In these cases two or three waves were observed on each side of the foot at one time, the waves on the two sides having neither definitely ‘opposite’ nor ‘alternate’ relations, but ap- pearing to be quite independent of each other. The fact that copious slime secretion occurs along the margin of the pedal surface in animals anaesthetized with MgSO, or with chlore- tone enables a test to be made of the adhesive properties of this slime; it is not sufficiently sticky to cause the attachment of the foot to a glass rod, although in the unanaesthetized nudibranch such attachment is readily demonstrated. These several lines of evidence agree in pointing to the essentially muscular, non- ciliary, nature of the act of creeping in Chromodoris. It should be noted that the marginal pedal waves are retrograde, not direct, as Vlés states for Doris. The direction of progression is always anterior. On a smooth surface the rate of creeping in active, large animals (at 27°C.) is about 1 cm. in five to seven seconds; when swimming on the surface film, about half this rate. Although we are not concerned to give an: exhaustive account of the effector systems of Chromodoris, mention may be made of 4 In an unidentified species of Leptoplana I have observed locomotion essen- tially of this kind, obviously muscular, in which the outer edge of the body was the only part in contact with the substratum. W. J. C. 2.66 W. J. CROZIER AND LESLIE B. AREY ‘the fact that slime glands are important for the production of a slippery condition of the whole surface of the animal; and that repugnatorial glands, in part at least under nervous control (Crozier, ’16 b; ’17 a), are also involved in the creature’s effector equipment. In addition to ciliary activity and several types of gland secretion, muscular movements of some variety are evi- denced. Chromodoris has no hard supporting skeleton; its movements depend conspicuously upon the distribution, under muscular pressure, of the body fluids, and comprise: bending movements, twistings, contraction and extension of the whole body, and of its projecting outgrowths—tentacles, ‘rhinophores,’ and gills; protrusion and retraction of the proboscis, of the genital papilla, and of the oviduct; rhythmic contractions of the extended oviduct during egg laying, as well as local contractions of practically every part of the animal’s surface. C. zebra is functionally hermaphroditic, and reproduces at all times of the year (Smallwood, 710; Crozier, ’17b). The ani- mals employed in this work were for the most part collected in Fairyland Creek, near the laboratory of the Bermuda Biological Station, where a practically unlimited supply of material was available during spring and early summer, when these experi- ments were chiefly performed. This nudibranch is easily main- tained in aquaria (Crozier, ’18 d), contrary to Smallwood’s (10) belief, but freshly collected individuals were almost always used. The largest specimens collected in late spring are the least viable; at a length of 16 to 18 em. Chromodoris zebra undergoes natural death. It would be of interest to determine the growth rate of the animal, but this cannot as yet be attempted. A more de- tailed account of the natural history of C. zebra will be found in reports dealing with the phenomena of its breeding habits (Crozier, ’17 d, 718d) and of the coloration of the species (in course of publication; Crozier, ’16 b). SENSORY REACTIONS OF CHROMODORIS ZEBRA 267 Il. MECHANICAL EXCITATION 1. Tactile stimulation The oral tentacles are very sensitive to touch, especially at the tip. When the tip alone is very lightly touched with a fine glass hair, it is contracted and slightly introverted. To slightly more intense stimulation, however, and always when touched at the side or at the base, the tentacle is introverted at the base after the fashion of a glove-finger. To unilateral stimulation of one tentacle, even to sharp and repeated touches, that tentacle alone responds. But after a tentacle has been completely, ex- cessively, contracted, strong continued local mechanical stimula- tion of it (while remaining retracted) causes the opposite tentacle to be retracted. In this case the whole head region is more or less contracted, and it may be that the skin at the base of the retracted tentacle must be stimulated in order to result in a spreading of the response to the other one. The ‘rhinophore’ on the same side with a stimulated tentacle usually contracts slightly, by a twitch of the muscles at its base, synchronously with the activated tentacle itself. If the stimu- . lation is originally strong or if it is repeated, the opposite ‘rhino- phore’ also responds, but usually to a less degree. Stimulation of a tentacle also involves response from the head region gener- ally, causing it to retract; at the same time the buccal veil is drawn down so as to cover the whole mouth region, including the anterior edge of the foot. The anterior end of the body is under these circumstances contracted more strongly on the stimu- lated side, and after reextension the whole body is usually caused to bend in the opposite direction, away from the side originally stimulated. If the anterior part of the foot should not be in contact with the substratum, it also contracts, on the homolateral side, when a tentacle is touched. This general form of reaction is the common response when the nudibranch is stimulated any- where with sufficient severity. Further evidence for the neuro- muscular unity of the head region will be found in what follows. To a single light touch upon a tentacle, the general head _re- sponse is very slight, but is nevertheless evident. The full head 268 W. J. CROZIER AND LESLIE B. AREY reaction involves a deep insinking of the dorsum at the level of the ‘eye spots.’ This form of ‘reflex’ is seen also in other Dorids. (C. roseapicta, Lamellidoris, etc.). The tentacles do not easily become exhausted. After ten to fifteen successive applications of a glass rod, a tentacle is still reactive to light touch, although the resulting contraction is not. so complete. The ‘rhinophore’ of Chromodoris (Arey, 717, 718) is a some- what complex structure. Its extreme distal tip is usually pale blue or white, the rest of the organ deep blue or purple. On either side of an anterior, median line, which is plain and smooth, the ‘rhinophore’ bears a series of twenty-eight projecting leaves. To a light touch at the extreme tip, a ‘rhinophore’ responds by partial retraction; the anterior, unmodified, median line is less sensitive; the posterior and lateral surfaces are the most sensitive. Even moderate intensities of activation cause a ‘rhinophore’ to be retracted within its collar, suddenly and completely, then reextended, more slowly. In animals of average size (8 to 12 em. long) the ‘explosive’ type of response is the result of even light tactile stimulation. To a very delicate touch on the lat- eral or posterior face the retraction is only partial. The ‘rhino- phore’ is itself contractile, longitudinal contraction occurring locally along its length when lightly touched, and it is pulled within its collar by the operation of basilar muscles within the ‘rhinophoral’ pocket. The retraction of a ‘rhinophore’ involves the subsequent sphincter-like closure of its basal collar. A ‘rhinophore’ is not easily exhausted. When approximately the same spot on the side of the organ is touched fifty times at ten-second intervals, the amplitude of contraction decreases, but the ‘rhinophore’ is still reactive. Stimulation of a ‘rhinophore,’ even repeated stimulation, does not influence the homolateral tentacle. The ‘rhinophore’ reac- tion is itself characteristically homolateral, as was seen particu- larly in the case of abnormal variates in which the ‘rhinophores’ were found naturally fused in varying degrees (Crozier, ’17 e). A sharp tap administered to one ‘rhinophore’ results in the partial, less complete contraction of its mate, and also of the SENSORY REACTIONS OF CHROMODORIS ZEBRA 269 gill crown. Less vigorous stimulation has, no effect on gill contraction. The gill plumes are individually sensitive, and react sepa- rately to slight stimulation. More violent activation (e.g., a . sharp tap or gentle pinching) of a single plume spreads through the other plumes, according to its intensity. A single plume presents a smooth, narrow, distally tapering outer edge, a simi- lar inner edge, and running between them two broad blade-like faces from which jut out the thin gill plates. Tactile excitation of the outer or of the inner faces leads to similar reactions of about equal magnitudes. The gill-bearing faces of the plume are less sensitive; frequently, an individual gill may be bent back and forth without eliciting a response. Presumably this occurs naturally in tidal currents, and during the movements of the gill crown as a whole. To a light touch, the common form of response is a local constriction of the plume, usually not equal on the two sides, accompanied by local longitudinal contraction, so that a slight swaying movement of the plume results. The plume as a whole may or may not be pulled down at its base. ‘To stronger stimu- lation the characteristic response involves the following events: local constriction, spreading distally from the point of activation, leading to the collapse and ‘shriveling’ of the plume distal to the point of activation; this is succeeded by the retraction of the plume through the traction of muscles not intrinsic to the plume itself, but situated in the basal tissue of the gill crown. Still stronger activation leads to longtitudinal contraction of the gill plume, both distally and proximally to the site of touching. The reaction of the plume distal to the point of activation is nicely demonstrated by plumes which have acquired a branching or dichotomously divided form (Crozier, ’*17e). If one of the branches of such a plume be touched on the side, this branch alone, and only distal to the stimulated point, contracts, unless the stimulation be too strong. The type of polarity evidenced in the reactions of a gill plume is curiously akin to that seen in the responses of an actinian tentacle under similar conditions of local activation (Rand, ’09, 270 W. J. CROZIER AND LESLIE B. AREY 15; Parker, ’17)—with this important difference: in the actinian tentacle it is the part proximal to the point of activation which contracts. The polarization of the gill plume, which is a neuro- muscular matter since it disappears under magnesium sulphate anaesthesia, is further seen in the fact that the distal tip of a plume, when touched, gives rise to only a slight longitudinal contraction in the immediate region of the tip, although the basal contraction may lead to the retraction of the plume as a whole. Bionomically, the significance of the difference between the neuromuscular polarizations within the actinian tentacle, on the one hand, and the gill plume of Chromodoris on the other, lies in the fact that the actinian tentacle carries food to the animal’s lips, hence the part between the disk and the point of excitation is shortened; whereas the mode of retraction of the gill plume probably saves it somewhat from being bitten by fishes. The gill plumes are bitten at by fishes, and there is evi- dence to show that some of the structural variations which they present (Smallwood, ’10; Crozier, ’17 e) originate as the result of injury. The basal contraction of a plume spreads to other plumes in proportion to the intensity of the stimulus and to the nearness of its application to the base of the gill-plume; this is the reaction which is responsible for the retraction of the whole gill crown. Contraction at the base of a gill induces collapse of the whole plume. Any desired degree of contraction of a single plume or of the whole set may be induced by grading the intensity of the tactile stimulus. Also by stimulating single plumes weakly and one at a time, as many plumes as may be desired can be caused to contract; e.g., all but one may be made to contract. Under slightly stronger activation, especially in the case of the more anterodorsal gill plumes, it can be demonstrated that the suc- cessive stimulation of two adjacent gill plumes is much more effective for the production of retraction of the whole gill crown than is the equivalent stimulation of any single plume. Thus, if two adjacent plumes are touched in quick succession or simul- taneously the whole crown is retracted more or less completely; SENSORY REACTIONS OF CHROMODORIS ZEBRA 271 whereas if either one of them is itself touched twice in this way, even if on the opposite faces, it alone reacts, though more vigor- ously than to a single touch. It is best to use large animals, with gill plumes widely extended, in testing this point, as other- wise the plumes may stimulate each other through mutual con- tact, a single slight stimulation then sometimes inducing rela- tively complete gill retraction. A single gill plume will react as many as twenty-five times in succession, when repeatedly touched at its tip, without mechanically involving another plume and without leading to contraction of the gill crown. It would seem that the contraction of the whole gill crown when the gills are touched is a secondary phenomenon, depending upon the extent of the disturbance produced in the basal tissue as the result of the individual gill plume activation. Two gill plumes separated by two or three intervening members of the . series do not, when touched in succession, lead to retraction of the whole gill crown. The base of the branchial apparatus is also sensitive to touch. Stimulation of the brim of the anus, within the circlet of plumes, causes retraction of the gill plumes immediately adjacent to the stimulated site. To stronger stimulation of the anal brim, more and more of the plumes become involved in contraction. Acti- vation of the anal brim is more efficacious in causing retraction of the gill plumes than is stimulation of the plumes themselves. The surface of the branchial organ outside the base of the gill plumes is very sensitive to touch; a slight stimulation induces complete retraction of the plumes. It is difficult to fatigue the gill reaction. If the plumes are forced, through adequate stimulation, repeatedly to contract completely within the branchial collar, and the time required for subsequent expansion is noted, it is found that the time first shortens, then lengthens, as in this example (times in seconds): Shy G57°453030;/23; 1759155175028, 2223, 26,):30,.36; 34,150, 55; The relative rate of exhaustion of the phases of the gill response is seen in the following experiment: Ze W. J. CROZIER AND LESLIE B. AREY The anal brim was stimulated by touching it with a glass rod. After twenty-four applications, at successive intervals, the gill plumes still responded by contraction; although after the first twelve responses it demanded longer and harder stimulation to accomplish complete retraction of the gill crown. In another animal the same form of stimulation was used, but the stimuli were supplied in groups of three successive touches. The indi- vidual touches were throughout of about the same force. lst application of three touches—complete retraction within collar. 2nd application of three touches—incomplete retraction; not drawn within collar. 3rd application of three touches—about half the ‘normal’ response. Ath application of three touches—slight contraction only of the gills. 5th application of three touches—gills moved, but did not contract longitudinally. 6th application of three touches—gills moved slightly. The integument of the héad region is very reactive to touch. The anterior edge of the mantle fold (buccal veil) seems to act as the chief or immediately receptive part when the nudibranch during creeping meets obstacles raised above the general level of the substratum (fig. 2). A single stimulation of this part has a slight effect on the tentacles; repeated light touches cause first the homolateral tentacle, then the opposite one, to be fully retracted—or, if the median region of the mantle edge be touched, both simultaneously. The reaction of the ‘rhinophores’ when the buccal veil is touched is very marked. It tends to be homolateral, as in the case of the tentacles, but is much more pronounced. The ‘rhinophores’ react as fully and as quickly as when they them- selves are directly touched, although in the latter case the re- sponse is less easily fatigued. In some individuals this response of the ‘rhinophores’ is elicited by tactile activation of the dorsal integument as far back as the anterior level of the ‘eye-spots;’ in others, only as far back as the level of the ‘rhinophores’ them- selves. In the region behind this level the effect on the ‘rhino- phore’ becomes suddenly much weaker, and a response from the gill crown comes in, increasing in amplitude as places nearer the branchial collar are touched. The dorsum of Chromodoris is soft, flexible, and very easily stretched and distorted. Therefore the delimitation of the re- SENSORY REACTIONS OF CHROMODORIS ZEBRA 273 ceptive field for the ‘rhinophore’ reaction is best established with the aid of mild faradic stimulation. The electrodes can be placed in position without inducing local response and without leading to the reaction of distant parts, owing to mechanical deformation of the body wall. The receptive field of the ‘rhino- phore’ reaction, made out in this way, agrees precisely with that already given. It is much more clearly defined than in the case of the gill response, as we have indicated above. Stimulation of a ‘rhinophore’ does not ordinarily lead to a reaction from the gill plumes, unless the ‘rhinophore’ be sharply struck or pinched. The ‘rhinophoral’ collar reacts by constriction, sphincter-wise, when touched lightly, but first of all the ‘rhinophore’ is re- tracted. To a very light touch the stimulated part of the edge Fig. 2 Outline lateral view of C. zebra (anterior end) during active creep- ing, showing the manner in which the lips, tentacles, anterior part of the foot, -and the buccal veil are related to the substratum. of the collar contracts locally without inducing the sphincter-like constriction and without leading to movement of the ‘rhino- phore.’ When the ‘rhinophore’ is retracted as the result of being itself stimulated, the collar contracts over it. The branchial collar behaves in a precisely similar way. The projecting mantle-margin, if touched dorsally or ventrally, is locally depressed ventralward. This is also true of the caudal veil which carries on its ventral side the conspicuous mantle glands (Crozier, ’17 a). When the animal is generally disturbed by being handled, or is from any other cause much contracted, the margin of the mantle along the sides of the body is thrown into prominence laterally, owing to the forcing of fluid into its internal spaces; whereas the caudal veil continues to be bent ventralward, unless the point of application of the stimulating 274 W. J. CROZIER AND LESLIE B. AREY agent be on the caudal extremity of the foot or on the dorsal surface of the caudal veil itself (Crozier, ’*17 a). If strongly stimulated at one point on its dorsal surface, the projecting mantle margin is turned sharply dorsalward. Stimulation of the caudal veil leads to pronounced local contraction of the body musculature at that level, accompanied by contraction of the projecting ‘tail’ of the foot (analogous to the ‘head response’ at the anterior end of the animal). The peculiarities in the be- havior of the caudal veil are related to the functioning of the large mantle glands, of which mention has already been made. The peripheral edge of the foot is not very reactive to touch, excepting at its truncated anterior end. There, especially at the faintly projecting corners, corresponding to the place of origin of the ‘foot tentacles’ in other nudibranchs, it is very sensitive, a single touch causing head and tentacles to retract. Here again the response tends strongly to be homolateral. Stimulation of the lateral edge of the foot causes the foot to fold together lengthwise. This is also true of the sole of the foot (e.g., of an individual swimming at the surface film) ; a very distinct line, about 1 mm. broad, which owes its appearance to the vertical, dorsalward contraction of the foot muscles, makes itself apparent even when the foot may not fold together. Touching the margin of the ‘tail’ of the foot induces deep local puckering, and that part of the foot itself is pulled forward and upward. The pharynx, when extended, is found very sensitive to touch. The lips of the fully protruded proboscis are relatively insensitive, but the very faintest touch upon its lateral wall leads to violent retraction of the whole head region; the pharynx is itself also momentarily introverted. Here, again, the reac- tion tends to be homolateral, as in all the responses of the head region. The oral area in animals with retracted proboscis is not so sensitive to touch as are the tentacles. In normal creep- ing it usually happens (fig. 2) that the proboscis is partially everted, so that a portion of its surface, exceedingly sensitive to touch, is brought into immediate relations with the surface over which the animal crawls. One-sided stimulation of the pharynx causes the homolateral ‘rhinophore’ to retract; stimulation of a SENSORY REACTIONS OF CHROMODORIS ZEBRA 215 ‘rhinophore’ does not affect the pharynx, unless it be repeated several times. The genital papilla and the mouth of the oviduct, when everted, react locally to light touch, always contracting away from the point touched. They induce no general reactions of the whole body. Chromodoris is relatively insensitive to vibrational stimuli. Continued tapping of the wall of a thin glass dish containing the nudibranchs may cause near-by resting individuals to begin to move, owing apparently to tactile irritation of the foot. No effect whatever is produced on creeping individuals, and no reactions are given under any circumstances by either ‘rhino- phores’ or gill plumes. In nature the gills, ‘rhinophores,’ and mantle edge are moved about by tidal currents, and the body is by the same means caused to sway from side to side, with- out leading to noteworthy response, save in the case of the ‘rhinophores’ 2. Righting: geotropism When the foot is removed from contact with a substrate, Chromodoris contracts to one-third or one-half its normal length, then subsequently becomes extended. The foot folds together longitudinally. The head end, after the preliminary re-extension of the body, is twisted on the long axis until the anterior part of the foot can be attached. The body is quite flexible, and in righting it may be twisted 180° or even 270° about its long axis. The foot is attached progressively, beginning at the anterior end; nevertheless, as already described, when once attached the poste- rior end is so well fixed to the substrate that the animal may be fully supported by this end alone. The process of righting occupies about one minute, ten to twenty seconds of this time being taken up with the twisting of the body in the effort to secure contact by means of the anterior part of the foot. The anterior edge of the foot is the only part which becomes spontaneously attached in this way. Observa- tions on many animals in the field, as well as in the laboratory, have shown that there is no pronounced tendency for C. zebra THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 2 276 W. J. CROZIER AND LESLIE B. AREY to maintain an upright position of the body, with the foot ven- tral. The righting behavior is probably due merely to the stereoptropism of the foot, especially at its anterior extremity. A Chromodoris placed on its back will become attached to a glass plate appropriately held in contact with the foot, even though the body remain upside down. This is best tested in C. roseapicta, where the foot does not tend to become folded to- gether. The surface of the foot must be in contact with some- thing. When removed from a substratum the foot folds to- gether longitudinally so that the lateral halves of its surface are in mutual contact. The origin of the twisting movements is probably found in the mechanical excitations of the skin induced by placing the nudibranch on its side or back; the anterior edge of the foot also exhibits writhing movements when the animal is so stimulated, but upon getting into contact with a solid surface it reacts positively, by attachment and slime secretion, and righting is begun. Repeated tests have been made to discover good evidence of geotropic orientation in Chromodoris, but without a decisive result being always obtained. Many individuals, in the light or in the dark, creep upward to the water’s edge in an aquarium; but they also move downward, horizontally, or in any inter- mediate direction with perfect freedom. When situated on a glass plate which was tilted in various directions, they continued creeping ‘as they were,’ and could not be made to alter at the experimenter’s will the direction of their creeping. These ex- periments were made at temperatures of 17° to 27°C. ‘There seemed a somewhat more pronounced tendency to upward move- ment at 17° than at 25° to 27°, but the difference was not clear- cut and is perhaps fictitious. If C. zebra possesses statolyths (otoconia) mle to those known in other nudibranchs, and perhaps of general occurrence in the group, they are not conspicuously involved in determin- ing the direction of the animal’s movements in the laboratory, nor the posture of the body in nature. It is of course conceiv- able that a vaguer type of geotropism is really functional, which might be difficult to detect in laboratory experiments. The SENSORY REACTIONS OF CHROMODORIS ZEBRA 207 movements of the nudibranchs in nature are suggestive of this possibility (Crozier, ’17 b, and section III of the present paper). For reasons subsequently discussed (section III), it seemed ad- visable to test the possibility of a relation between geotropism and the temperature. The experiments already referred to were made at different seasons of the year, and the possibility was not lost sight of that the reproductive phase of a given individual might, through in- ternal secretions or otherwise, be instrumental in determining, or in helping to determine, geotropic behavior. The natural movements of C. zebra are of very considerable complexity, and the following statements cannot be applied to the total analysis of these movements. These statements are based upon experi- ence with many hundreds of C. zebra during the last five years. During the winter months, at an average laboratory tempera- ture (in the aquaria) of about 17°, C. zebra is notably geotropic, © orienting upward and tending to remain at the water surface, especially when about to deposit eggs. This behavior is also notable in the field. After the egg mass is laid, the animal may wander downward again. At 27°, in summer, the same be- havior is manifest, but less pronouncedly. Hence it is unlikely that the decided upward creeping in the first case is the result of oxygen-want. The effect of oxygen-want, or of some associated condition, may be tested in two ways: 1) by observing the behavior of ani- mals from which the branchial plumes have been removed or in | which these organs are prevented from functioning, and, 2) by observing the behavior of C. zebra on a vertical surface in a jar closed above, containing no air space, but communicating with oxygenated water at its lower end. That the gills are respira- tory organs is suggested not only by their blood circulation, but also by the fact that in sea-water of decreased alkalinity (p, = 7.95-8.00) the gill plumes become widely extended, the base of the gill crown being then inflated and protruded beyond the pro- tecting branchial collar. The result of such tests was as follows: Sexually ripe indi- viduals tend to move upward, even though this be away from the 278 W. J. CROZIER AND LESLIE B. AREY oxygen supply. ‘Spent’ individuals do not. When the branchial collar was sewed together so that the gill plumes could not be ex- truded, non-geotropic individuals did not tend to creep upward, but remained on the bottom. The correlation of egg-deposition with negative orientation was very marked. For example, a group of fifteen nudibranchs had been in the laboratory for four months; during the last three and a half months of this time they deposited no eggs and re- mained for the most part at the bottom of their aquarium; sud- denly, on the same morning, six pairs were formed, the animals crept up to the water edge, and deposited Cees, after which they wandered aimlessly. If the temperature be gradually increased in a vessel in which Chromodoris is creeping upward under diffuse light, the ante- rior part of the foot becomes detached from the substratum when the temperature reaches 29° to 30°C., and if this temperature is maintained the animal creeps or falls to the bottom. This is probably an indirect effect of temperature upon geotropism. It is possible that the reproductive mass, enlarged when ripe, acts as a statolyth; if this is correct, geotropic orientation may re- sult: from, 1) a general increased irritability accompanying sexual ripeness, plus, 2) the mechanical stimulation of the loosely anchored internal organs; on a vertical surface, the animal would then turn away from the side against which these organs pressed. This would result in negative geotropism, as found. Together with positive phototropism (vide infra) and a negative reaction to high temperature, geotropic behavior might, then, be im- portant for the determination of the vertical migrations of the species into shallow water at periods of breeding; it would never- theless be incorrect to say that the animal ‘‘moves into shallow water for the purpose of breeding.” 3. Rheotropism In some of the situations where C. zebra abounds, as, for example, in Fairyland Creek, the nudibranchs are well exposed to the possibly directive influence of tidal currents of considerable SENSORY REACTIONS OF CHROMODORIS ZEBRA 279 volume and velocity. The habitat of this animal is preemi- nently within the semienclosed lagoons or sounds at Bermuda, where tidal currents must frequently be encountered; it does not occur upon the reefs. It was important to discover the nature of the animal’s rheotropism, if it should be found to be oriented by water currents. Since the usefulness of information upon this point lay in its application to the natural movements of the ani- mal, the experimental work was done in the field. Laboratory tests, moreover, were found unsatisfactory because water cur- rents of sufficient volume could not be employed conveniently. KL Fig. 3 Chart of a portion of Fairyland Creek (F. C.), showing the situation (cross within a circle) for testing the behavior of C. zebra in tidal currents (see text). Ais drawn toa scale of 6” tol mi. B is an enlarged sketch of the region within the rectangular area surrounded by dashes in A. An appropriate situation was found at the western end, or mouth, of Fairyland Creek (fig. 3), in a locality where hundreds of the animals were living at the time the experiments were made, and involving, therefore, water currents normally encountered by the nudibranch. At the period of the falling tide the currents in the location selected were as shown in figure 3. The observations were at first confined to sunny days. When low water or high water occurred at about midday—no water then (for a short period) flowed across the channel indicated—the sun was sufficiently 280 W. J. CROZIER AND LESLIE B. AREY far south (in December) to cause the nudibranchs, not singly, but by the dozen, to migrate southward in this channel. During falling tide, with the current flowing northward, the nudibranch, moved with the current; but once out of the channel they tended to turn around so as to face the sun as soon as they were out of the current. In this way groups of fifty to sixty mdividuals were caused to collect just beyond the northern end of the little channel. For the study of rheotropic orientation a flat slab of rock was placed horizontally in this channel, and nudibranchs were placed up on it in various positions with reference to the current. In some cases the Chromodoris was allowed to become attached to the rock while surrounded by an inverted glass jar which temporarily protected it from the action of the current. So long as the current was of fair velocity, orientation was always precisely negative, the nudibranch moving with the current. Under these conditions, the whole body is swayed to one side or the other by the force of the current, the gill plumes are moved by it, the ‘rhinophores’ are bent backward or to one side, and the buccal veil of the mantle is irregularly distorted. The gill plumes and ‘rhinophores,’ in particular, are forcibly moved by a cur- rent too weak to noticeably affect the body as a whole in a grossly mechanical way, yet leading to precise orientation in the current. It was considered that some or all structures mechanically distorted by the water current might be responsible for the negative rheotropic orientation. Experiments were begun with the ‘rhinophores.’ When exposed to mild water currents of sufficient volume, as in the natural channel already considered, the ‘rhinophores’ are forced backward (fig. 5). When the current is stronger, the position assumed is as shown in figure 4. The ‘rhinophores’ are easily removed by seizing with forceps and cutting close to the collar. In one experiment six nudibranchs from which both ‘rhino- phores’ had been removed the previous day (they crept about in an entirely normal fashion, for removal of the ‘rhinophores’ has no seriously adverse effects) were found not to be oriented by a current in the natural channel, although a dozen or more individuals with intact ‘rhinophores’ oriented precisely. The SENSORY REACTIONS OF CHROMODORIS ZEBRA 281 rhinophoreless individuals assumed a position like that in figure 4, and the gills and buccal veil were forcibly distorted to a maximal extent by the current, but no orientation took place. A group of fifteen nudibranchs was then prepared, from nine of which the right ‘rhinophore,’ and from six the left, was removed. When the current was allowed to impinge on the anterior end of the nudibranch, parallel to the long axis of the animal, in almost every case orientation was prompt, and the bending of the body took place in such a way that the side contracted was the © one carrying the intact ‘rhinophore.’ Experiments were also made with more localized currents. A stream of sea-water flowing through a tube of 6 mm. bore at a speed of 4 to 5 em. per second was allowed to impinge horizontally Fig. 4 Showing the posture of the body in a Chromodoris exposed to a tidal current (see text). upon the anterior end of C. zebra. To this current normal indi- viduals became promptly oriented, the process occupying three to five minutes (at 17°C.). Animals without ‘rhinophores’ were conspicuously slow and unsuccessful in orienting away from this current, although the buccal veil and the gills were moved by the current to an equal extent in both cases. The negative orientation to the current did occur in some cases, but only after half an hour or longer. The ‘rhinophores’ are easily distorted by currents and do not retract when moved in this way. A current of small cross section, affecting only the ‘rhinophores’ (fig. 5), causes the animal to bend toward the unstimulated side. These results leave no doubt that to currents of adequate velocity the nudibranchs are negatively rheotropic and that the ‘rhinophores’ are the prime receptive organs for this kind of reaction. 282 W. J. CROZIER AND LESLIE B. AREY 4. Nervous relations 1. There are several very characteristic features about the sensory responses of Chromodoris; these are of considerable general significance. Yet the apparent variability of these responses has made it necessary to study them very carefully and in many individuals. To local excitation, not too intense, the response is local merely; to more vigorous stimulation, the response obtained involves more distant structures—at the anterior end, the general head contraction; at the posterior end, the caudal contraction; A B Fig. 5 Behavior of the ‘rhinophores’ in a water current; A showing the ‘positive’ reaction of the ‘rhinophore’ itself; B indicating the method of reaction to local current affecting directly only the ‘rhinophores.’ likewise at the anterior end the ‘reflex’ involvement of other structures than the one stimulated proceeds upon a conspicuously homolateral plan. In the case of the tentacles and ‘rhinophores,’ moreover, stimulation of a tentacle easily induces contraction of the homolateral ‘rhinophore,’ whereas the reverse operation is exceedingly diff cult. The manner in which, at the head end, additional structures become concerned in reaction to the activation of distant parts, corresponds precisely to the distribution of the main anterior nerve trunks; and the character of the responses, particularly in the apparently non-reciprocal nature of conduction between tentacle or pharynx and ‘rhinophore,’ is strongly suggestive of true reflex action. SENSORY REACTIONS OF CHROMODORIS ZEBRA 283 2. On the other hand, in the case of each of the projections from the body (tentacles, ‘rhinophores,’ particularly the branch- ial plumes, and perhaps the pharynx) the local reaction of each stimulated part has certain definite peculiarities, best studied in the gill plumes, but seemingly identical in all the parts enumerated. These peculiarities are: localized longitu- dinal contraction at the immediate side of activation; circular constriction and longitudinal contraction beyond (distal to) the level of activation; contraction at the base when the activation is sufficiently intense, in this case involving a spreading of the response to neighboring parts; a lesser reactivity when the tip of the organ is activated than when it is touched near its base; in the gill plumes, neuromuscular polarization such that the activation spreads distally from the point of excitation; slight fatiguability of the local reactions, whereas the heterolateral responses (e.g., in case of the ‘rhinophores’ and tentacles) are much more readily exhausted by repeated activation. The local responses are exhibited in pieces of the mantle removed from the body. When the central nervous ganglia, supra- and subcesophageal, have been completely extirpated, stimulation of the head region near a ‘rhinophoral’ collar causes that ‘rhinophore’ to be retracted, the collar closing over it, as normally stimulation of a tentacle leads to its reaction, but does not involve retraction of the homolateral ‘rhinophore.’ ‘Tactile excitation of a ‘rhinophore’ in a Chromodoris with the ganglia excised causes the ‘rhinophore’ to retract, after which it is slowly re-extended. The phenomena of local response to faradic stimulation in the excised gill plumes are also substantially similar to those of the individual plume in the intact nudibranch. Within fifteen minutes after amputation a gill plume becomes relaxed, though, like the excised tentacle of an actinian (Parker, 717), it is not so fully extended as it may be when attached to the animal, because no fluid is being forced into it. The relaxed, isolated gill plume is fully as sensitive to touch as when forming part of the normal nudibranch, the peculiarities of its reactions are identi- 284 W. J. CROZIER AND LESLIE B. AREY eal, although it is somewhat more quickly exhausted and only rarely responds at all to shading. The responses disappear under chloretone anaesthesia, but returnagaininsea-water. The neuromuscular polarization of the gill plume is therefore a local matter, conditioned by a self-contained nervous apparatus which conducts impulses more easily distalward than proximally. These facts speak unmistakably for the presence of local peripheral conducting paths, having the characteristics of true nerve nets. Similar nerve nets have already been identified in Octopus (Hofmann, ’07), and in Aplysia (Bethe, ’03). The body of Chromodoris may be laid open by a dorsal or a ventral incision, and the animal will live for a long time in sea- water. The nerves which originate from the ‘cerebral’ and sub- cesophageal ganglia and traverse the body cavity are readily employed for faradic stimulation experiments. ‘The results of such tests confirm Bethe’s (’03) description of the effects of nerve-trunk stimulation in Aplysia. Local responses, of no great magnitude, are induced; much more general effects are obtained, with the same stimulus intensity, when the integument is acti- vated directly. These experiments incidentally afforded infor- “mation relative to the old controversy as to whether the pro- jecting marginal ridge is an epipodium (Herdman, ’90; Herdman and Clubb, ’92) or a mantle structure proper (Pelseneer, ’94, p. 70). Pelseneer was undoubtedly correct, at least so far as our species is concerned, for the motor nerves to this region are pallial; not pedal. 3. The general result of these experiments is to suggest the probability that peripherally a true nerve net is concerned in local sensory responses, but that a reflex system involving cen- tral conducting paths is called into play by more intense acti- vation. We are able to offer in addition physiological proof of a different kind that the peripheral conducting systems are nerve nets, and that the central paths of nervous transmission are part of a synaptic system, to which the term ‘reflex’ may properly be applied. This proof is based upon the assumption that the effect of strychnine affords a good test of synaptic transmission. SENSORY REACTIONS OF CHROMODORIS ZEBRA 285 The following notes are derived from observations with eleven Chromodoris of medium size (10 to 14 em. in length) into which 1 ce. of half-saturated strychnine hydrosulphate in sea-water had been injected. This quantity was found by other tests not to be fatal and to be the optimal concentration for our purpose. The injection was made into the region of the heart, on the dorsal surface. The behavior of each animal was studied individually, before injection, during the action of the strychnine, and after its effects had worn off. As a control, each individual was studied in comparison with an animal into which 1 ce. of sea- water had been injected. The latter operation had no detectable consequences of any kind. ‘Tactile activation was mostly used. The results herein summarized are to be compared with those given in the first section (p. 267). Following strychnine injection, the body remains for some minutes much contracted, its surface being ‘wrinkled’ and thrown into edematous blebs; the genital papilla is protruded, and the posterior mantle glands are made prominent, owing to the forcing of fluid into the spaces surrounding them. These effects appear under any conditions leading to pronounced general contraction of the body muscles. The gill collar, however, is strongly con- tracted in a peculiar way, its edge being rolled outward. The foot is folded together lengthwise and does not attach to the sub- stratum. The gill plumes remain half contracted within the branchial collar. The plumes tend to exhibit more or less rhyth- mic contractions, followed by rapid but incomplete expansion; perhaps this is in some way mechanically induced by the beat of the heart, which distorts the neighboring dorsal integument. The reaction of the gill plumes to shading is not apparent. After the lapse of half an hour to an hour in different indi- viduals, the body is less strongly contracted, the gill plumes more fully extended. The reactions of the plumes to touch are curious and important at this point: to a single touch, a plume reacts precisely as in non-strychninized individuals; but when two successive touches are administered to adjacent plumes, the reaction is of unexpected violence (fig. 6). A reaction of this amplitude is obtainable in normal animals only by six or seven 286 W. J. CROZIER AND LESLIE B. AREY repeated proddings of the gill crown, but relatively slight taps of adjacent plumes will produce this effect under strychnine. The ‘rhinophores’ are not retracted under these circumstances; whereas, if the ‘rhinophores’ themselves are touched, the gill plumes do contract. In some individuals the ‘rhinophores’ were found to retract noticeably, but not completely, when a bit of graphite or the end of a glass rod or of an aluminum wire was brought near them (within 2 to 3 mm., but not touching). Presumably this repre- sents a heightened tactile irritability such as that seen in some teleosts (Crozier, ’18 c) after the removal of the eyes (i.e., when the central reflex interference of optic impulses has been re- Fig. 6 Outline of Chromodoris to show gill-crown reaction under strychnine. moved). This type of irritability is not apparent in the non- strychninized animal. After one hour, the dorsum is still wrinkled, but the aieg) attempts to creep, usually falling over to one side after such an attempt has endured for two to three minutes. The main body is no longer forcibly contracted, but usually assumes a gentle spiral form about the long axis, the head pointing downward on one side, the ‘tail’ of the foot pointing upward and to the opposite side; the surface of the foot is for the most part longitudinally folded. Touching a ‘rhinophore’ causes both it and its mate to retract; sometimes the opposite ‘rhinophore’ contracts before the stimulated one, and usually the gill plumes contract also. The lightest touch applied to a tentacle causes the homolateral ‘rhinophore’ to be fully retracted. When a ‘rhinophoral’ collar is touched, it contracts, sphincter-wise, so quickly and so forcibly, SENSORY REACTIONS OF CHROMODORIS ZEBRA 287 that the ‘rhinophore’ has difficulty in being itself retracted within its pocket; this behavior is never seen normally. If the anterior edge of the buccal veil is touched at one side, there results, as in the normal nudibranch, a homolateral ‘rhinophore’ retraction, and also a retraction of the gill crown, which is rarely seen except under strychnine. After one and one-half to two hours, the pharynx invariably becomes extended; if touched at the side, an exceedingly violent homolateral head response is the result. If the lips be touched, however lightly, both ‘rhinophores,’ as well as the proboscis, are violently retracted. On reextension, the ‘rhinophores’ both retract when either one is touched, but the pharynx (extended) does not contract at all. Nor at any other time does ‘rhinophore’ activation induce retraction of the pharynx. In all of these reactions, for example, when both ‘rhinophores’ retract as the result of one of them being touched, it is very difficult, if not impossible, to secure the double response for five to ten minutes subsequent to the reaction, although each one responds to local activation readily enough after a 1- or 2-minute refractory period; this is true of the double response independ- ently of whether the ‘rhinophore’ first activated or the opposite one is the one subsequently stimulated. A precisely similar relation appears in the other responses studied. This period is usually succeeded by one (two and one-half to three hours after injection) during which a light tactile stimula- tion of one ‘rhinophore’ causes both the opposite one and the pharynx (still extended) as well as the tentacles and gill plumes, to be retracted. These effects of strychnine injection become obliterated after the lapse of three and one-half to four hours, under the conditions used in the experiments, and the animals return to an essentially normal state so far as their reactions are concerned. At no time is the response of the gill crown to shading (vide infra) in any way enhanced. 4. If these results are compared with those given for normal ani- mals (section 1), it will be seen that strychnine has a pronounced effect upon those responses involving irreciprocal conduction, 288 W. J. CROZIER AND LESLIE B. AREY and upon the reactions shown by experiments, with the central ganglia eliminated, to involve central transmission tracts. The progressive development of the strychnine effect, in point of time after injection, upon the several responses investigated; the pronounced refractory phase following each response involving the strychnine effect, and the enhancing nature of the effect itself, all point to the reflex nature of the nervous transmission concerned, since these effects are precisely those which strychnine exerts in decreasing synaptic resistance. On the other hand, the local responses, as seen particularly in the gill plumes, are not materially affected. Strychnine does not exert these effects upon de-ganglionated Chromodoris. Consequently we may assume, although we have not inquired as to the specific character of the strychnine effect, that peripherally, and in the outgrowths of the body wall (tentacles, pharynx, ‘rhinophores,’ gill plumes) there are local nerve-nets concerned with local responses, that these nets are characteristically polarized, and that they are domi- nated by the central nervous system of the nudibranch, the latter being essentially a synaptic system. III; PHOTIC EXCITATION 1. Light Chromodoris is sensitive to light. Most of the many indi- viduals tested oriented directly toward a source of sunlight or of diffuse daylight. The tests were frequently made in a rectangular glass dish, enclosed in a covered dark chamber admitting light through a slot in the bottom of one end. Most of the indi- viduals moved immediately toward the light aperture and re- mained for along time pressed against this end of the aquarium. Some continued to creep around the side of the dish when they came into a shadow, but ultimately, on coming again into the light, oriented toward it. Orientation is direct, without trial movements, and the anterior end does not react to shading; neither does it respond to increase of light intensity as such. In the great number of individuals which we have handled at dif- ferent times, no exceptions were ever found to the occurrence of SENSORY REACTIONS OF CHROMODORIS ZEBRA 289 positive prototropism, although many individuals may for a time be inactive and may appear insensitive to light. This is notably true when two or more individuals are together in a dish, in which case, if they be ready for pairing, they may stimulate one another to conjugation. Even here, however, photic irritability is evi- denced in one respect, for in the dark (in well aerated water) the gill crown is almost invariably contracted; upon illumination, even by the light of a match, the gills become extended; this occurs also during the progress of copulation in the dark. The directive effect of light is manifest not only in horizontal creeping, but also when the illumination is from above. Sunlight reflected from out-of-doors was caught by a second mirror and reflected vertically downward into a vessel containing Chromo- doris. Active locomotion ensued, and many animals on reaching the side of the jar climbed it until they met the water surface, continuing then along the water line; whereas, on creeping outside the path of the light beam, the nudibranchs tended to return to the bottom of the dish. In successive tests the animals could be forced to creep upward when illuminated, going downward again during intervals of shade. /The effect of illumination is distinctly a kinetic one. In a shaded dish the nudibranchs become quiet, but are set into activ- ity at once if light be thrown on the dish. In spite of their positive phototropism, these nudibranchs tend under some con- ditions to collect in the shade. In a shallow dish well shaded on one-half, the dish being in a chamber admitting direct sun- light from above on one-half, there seemed to be a decided ten- dency for the Chromodoris to collect in the shade; whereas when diffuse light was used the light compartment was the one more frequented. ‘This is explained by the fact that strong light in- duces greater activity, leading automatically to wandering move- ments, which become less pronounced in the shade, while with diffuse light the photopositive behavior is not interfered with by photokinetic effects. If originally placed in the dark com- partment, the Chromodoris wanders into the light. Even with direct sunlight, falling vertically into the light compartment, no reaction other than increased speed of creeping is detected. 290 W. J. CROZIER AND LESLIE B. AREY The eyes of Chromodoris are small and are situated beneath the skin. They are in close relation with the supraoesophageal ganglionic mass, being connected therewith by means of short slender nerves running to small optic ganglia (Smallwood and Clark, 712). Externally, the region of their location in the nor- mally extended animal is indicated by two clear-blue areas immediately posterior to the ‘rhinophores’ (fig. 1). In nudi- branchs found by previous test to be actively photopositive, these regions were cauterized with a hot needle. Subsequent experi- mentation showed that the photopositive behavior of these in- dividuals was in no way affected by the operation. A scar-like formation was produced, accompanied by some local puckering, together with a deep dorsal constriction and insinking of the body at the level of the burn. The movements and general behavior of the animal are in no way altered. It is doubtful if operations of this type really interfere with the possible functioning of the eyes. Nevertheless, when tested with small areas of illumination, even when the light was very intense, the normal Chromodoris was found to be reactive at the anterior end, in the region of the eyes; the posterior end was non-reactive to sunlight concentrated with the aid of a lens of 30 em. focal length when the heat rays had been eliminated. We are not in a position to decide as to whether the eyes are photosensitive (for they are not easily approached for excision tests), nor whether there are anterior integumentary receptors independent of the eyes. The ‘rhinophores’ are not sensitive to light. The gill plumes, however,—more or less completely retracted in the dark, as already described,—become fully expanded when the region of the branchial collar alone is illuminated. They do not become extended, it would appear, when only the anterior end of the animal is illuminated. The latter 'test is difficult to make because anterior illumination induces active creeping. 6 The region of the skin bearing the eye spots has also been removed in a number of cases, exposing the body cavity. Animals so prepared are photo- positive; they live for a week or more and seem in no way greatly incommoded by the operation. Through such a window in the skin it was attempted to stimulate the eyes directly, with a small spot-light. It seemed that the region of the eyes was sensitive to light, but the experiment should be repeated. SENSORY REACTIONS OF CHROMODORIS ZEBRA 291 There is, at any rate, an instructive correlation in these re- sponses: illumination induces creeping, and also induces extension of the branchial apparatus; it is to be presumed that increased oxidation necessary for locomotion is in this way assisted. The photopositive behavior of Chromodoris is not accompanied by any reactions to changes of light intensity. It is an example of phototropism in the strict sense, in which trial movements do not appear. Long-continued observation of Chromodoris in the field has shown that the positive phototropism of this nudibranch is of great bionomic importance (Crozier, 716 b, ’18 ¢), a preponderat- ing element in natural behavior. On days when the sky is overcast, relatively few of them are to be found. The brighter the day, other things equal, the more of these nudibranchs one can collect in shallow areas. In suitable spots they can be observed to follow with precision the direction of the sinking sun, whether it leads up-hill or down, according to the nature of the bottom. In this photopositive behavior C. zebra agrees with some Red Sea chromodorids, as described by Crossland (711), and differs sharply with the behavior of C. roseapicta at Bermuda and with tropical chromodorids in general as indicated by Eliot’s (04, 710) experience. In any one locality where they abound, more of the niielie branchs are obtainable in mid-afternoon, on a sunny day, than in the morning. But this is true only during the cooler months of the year (October to May). During the summer, none, or a few only, are so obtainable in shallow water (e.g., in the mangrove creeks or on shallow grass flats), though search among the densely packed eelgrass usually shows that in June and in July a certain number are still there. If one collects at early morning, before sunrise, in Fairyland Creek, relatively large numbers of C. zebra can be had in June and in early July. As the sun rises, the nudi- branchs creep downward on the blades of eelgrass and turtle- grass, and by the time the sun has risen several hours, practically none are to be seen. In view of the positive phototropism of C. zebra, consistently exhibited by individuals of every size, this phenomenon was very THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 2 292 W. J. CROZIER AND LESLIE B. AREY puzzling—especially since it was found that nudibranchs ob- tained (in Fairyland Creek) before sunrise, and tested immediately with lateral light, were without exception photopositive. No reversal of the customary phototropism occurs, under these conditions, at the time of sunrise. Nor is C. zebra photonega- ° tive immediately after long exposure to the dark; but even if this should be true, it would not explain the natural behavior described. Several possibilities were considered, among others the possible reversion of phototropism by rise in temperature. The tempera- ture of the water in Fairyland Creek was 24° to 25°C. just before sunrise. The water is very shallow, and is rapidly heated by the sun’s rays so that it quickly reaches a temperature of 27° to 28° as the sun rises. But the phototropism of C. zebra is not altered at any temperature between 17° and 31°; at the higher tempera- tures orientation is quicker, and still toward the light. Other possibilities are dealt with in a preceding section (p. 277). The positive phototropism of C. zebra is not affected by pro- longed starvation (four months; Crozier, 718 c), and is the same in sense with animals dredged at various depths down to 8 fathoms; nor does it vary with the reproductive condition of the animal. 2. Shading The gill crown of C. zebra reacts to shading, after a detectable latent interval. No other portion of the animal’s surface is sensitive in this respect. The gill plumes must themselves be shaded in order to produce a response. The reaction in question is in the form of an incomplete retraction of the gill crown, ac- companied by longitudinal contraction of the individual plumes. The responses are exceedingly variable. The first reaction ob- tained from an animal which has for some time been undisturbed, in the light, is likely to be the most pronounced. ‘This is not always true. Subsequent successive shadings commonly evoke a faint contraction of the plumes, the crown as a whole being little if at all retracted. This reaction is precisely similar to that which may be induced by tactile irritation of the gills, but the SENSORY REACTIONS OF CHROMODORIS ZEBRA 293 time occupied by the contraction process is less in the case of a shading response (0.6 to 1.0 seconds) than in a tactile reaction involving about the same degree of muscular activity. The duration of the shadow must be appreciable to have a detectable effect. The duration increases with the size of the animal. For nudibranchs 10 to 12 em. long, an opaque body, such as one’s hand, moving at about 50 to 60 em. a second between the gills and the sun provides very nearly the threshold of acti- vation. With the removal of the shading the gill plumes expand; they tend to remain contracted in the dark, provided the whole animal or its posterior end is shaded. If, while the plumes them- selves are contracting as the result of shading, the light be sud- denly or slowly increased again, the retraction of the gill crown is inhibited and protrusion begins. It was previously shown that the total response of the gill crown involves two reactions: contraction of each gill plume and the retraction of the whole branchial apparatus. These are quite distinct things. The retraction and extension of the gill crown is brought about by muscles at its base. This is determined by the acting light intensity, owing apparently to the fact that bright light decreases the tonus of the muscles in this region, allowing fluid to accumulate there, under pressure from other portions of the body. The reflex contraction of basal muscles causes the crown to be retracted. The contraction of the individual plumes, however, is determined by shading as such; because, after the initial twitch or longitudinal contraction of the plumes they become relaxed even if the state of decreased light intensity continues. ‘The plumes do not react to suddenly increased illu- mination. The degree of contraction of the gill plumes when shaded determines whether the whole crown shall be retracted or not. The relations here are very similar to those previously discussed under the head of the tactile activation of the plumes. If the self-contraction of the plumes be sufficiently violent, more or less complete contraction of the whole crown ensues; in this case the reextension of the gill crown commences within a few seconds, as under tactile activation, even though the shading re- mains constant and the crown may not be fully reextended. The 294 W. J. CROZIER AND LESLIE B. AREY shading response is therefore to be sharply distinguished from gill-crown retraction; the former is a local matter, involving the plumes individually, through their local and probably non-syn- aptic nerve nets (since strychnine has no effect whatever in increasing the shading response); the latter is a reflex effect (compare strychnine experiments cited in section I), so far as con- traction under shading is concerned, and when not secondarily involved, owing to the gill plume reactions, is governed solely by the constant intensity of the light (granted optimal conditions of oxygen supply). The extent of the gill-plume reaction to shading is very vari- able in different individuals, and it has not been possible to control this variability. Sometimes every animal in a dish was found markedly sensitive, in other cases only one or two gave detectable reactions. Subsequent investigation showed that strands of slime connecting one individual with another occasionally caused one sensitive animal to stimulate others confined with it (Parker, ’08, for a not dissimilar instance in the behavior of Amphioxus). Even when studied in individual aquaria, however, great vari- ability was found. Sensitivity to shading was not enhanced by confinement in the dark or in the light even for lengthy periods. Successive shadings at 30-second intervals elicit responses growing rapidly more feeble, commonly failing after the third or fourth. Here again, however, variability is very great; one individual gave twenty such successive reactions. Sunlight, diffuse daylight, light from an incandescent bulb, were each efficient for the production of shading reactions. The visible region of wave lengths is concerned, since responses are obtained on shading through several thicknesses of glass. Tests with ray filters showed that the cutting off of light passing through a blue filter (\ 523-450) produced good reactions, whereas that through a red filter (A 690-634) most often failed. Experi- ments with green and yellow filters gave no clear result. This agrees with results obtained in similar experiments, employing the same ray filters, with a barnacle (Crozier, 715 a, p. 273) and with Chiton (Arey and Crozier, 719). SENSORY REACTIONS OF CHROMODORIS ZEBRA 295 Some other nudibranchs, as Hermaea and certain NH, Lli>WNa. The stimulation here is primarily a matter of the cation. KCl, KB, KI, and KNO; lead to equally strong reactions from all SENSORY REACTIONS OF CHROMODORIS ZEBRA 303 parts. CaCl, and MgSO, (0.625 N) did not activate; MgCl, gave fairly good responses over the whole surface of the nudibranch. The limiting dilutions of different substances effective for acti- vation are likewise characteristic. For any one substance the distribution of limiting effective concentrations for the different parts of the body affords a measure of their respective recep- tivities. In the following summary it will be seen that the several concentrations, especially for the oral region, are of the orders of magnitude found in the stimulation of other animals: Picric Acid, dissolved in sea-water. M/150 Good, strong reactions everywhere. M/200 Gill plumes respond weakly, or merely flatten out against the body wall. M/500 : M ote Good responses everywhere, except from the gills. M/4,000 Oral tentacles still very sensitive ‘Rhinophores’ less sensi- tive: they may merely bend before the stream of acid, and not distinctly contract. The dorsal skin is also slightly sensitive M/8,000 Same as M/4,000, but weaker. M/10,000 The mantle edge fails to respond; the edge of the foot, espe- cially its anterior part, is still sensitive. Response from the oral tentacles is more constant than from the ‘rhinophores.’ This solution is distinctly bitter to human taste. KCl, in rain-water. M/700 Gill-plume reaction weak. M/1,000 No response from the gills. No responses distinguishable from those to an equal volume of rain-water were obtainable with weaker concentrations. In sea-water solution the reactions from the different regions of the body, at M/400 concentration, indicated the following regional order of decreasing irrita- bility (Crozier, ’16 a, p. 272): anterior tentacles, ‘rhinophores’ > base of the gill > crown > buccal “mantle > posterior mantle veil > lateral mantle edge (ventral surface) > edge of foot (at the sides) > dorsal integument. KOH, in rain-water. M/300 Gill plumes commonly fail to react. KCl, in rain-water. M/3 Responses from all parts. M/16 No responses from gill plumes. M/50 Doubtful if the responses are distinct from those to rain-water in sea-water. M/15 Gill plume reactions fail. M/20 No reactions from any part. 304 W. J. CROZIER AND LESLIE B. AREY c. From the comparative reactivities of different parts of the body and from the relative limiting concentrations of each sub- stance required for the activation of these parts, the distribution of general chemical sensitivity can be made out over the body surface of Chromodoris. These two criteria lead to mutually concordant results, as an inspection of the preceding paragraphs will disclose. The gill plumes are distinctly the least sensitive of the out- growths from the body; the oral tentacles probably the most sensitive; the ‘rhinophores’ almost as sensitive as the oral ten- tacles, but occupying apparently an intermediate position. On the ground of distribution, it would appear that chemoreception is served by distinct receptors, for the gill plumes are reactive to shading, touch, ete., as already described, in a way which indicates their possession of delicate receptive mechanisms for these sources of activation, yet their chemical reactivity is slight. Evidence of similar import is afforded by comparing the responses of the oral tentacles and of the ‘rhinophores.’ At elevated temperatures (38°C.), tactile responsiveness is quickly destroyed on all parts, but sensitivity to KCl solution (0.625 M) is pre- served. After complete exhaustion to shading, the gills are fully responsive to KCl, M/1,000 picrie acid, ete. The genus Chromodoris is characterized by the fact that many, or most, of its members tend to develop a blue or purple pigmentation of the skin. This pigment is a delicate indicator of acidity (Crozier, 714, ’?16a), turning pink with acids. This color change is not indicative of an alkaline reaction in the cell interior (Simroth, 714, p. 484), because, although the cell contents are more acid than sea-water, the pigment is still blue under faintly acid conditions (Crozier, ’16a). This natural indicator offers an exceedingly favorable opportunity for studies on the penetrability of cells for acids, leading to the possibility of in- vestigating the nature of the reaction between acid and tissue in the process of stimulation (Crozier ’18a). In the case of neutral salts, we must also suppose that stimulation is due to some chemical influence of the salt upon the surface of the receptive elements, possibly owing to the fact that the applied SENSORY REACTIONS OF CHROMODORIS ZEBRA 305 salt influences the ionization of protein salts located at these surfaces. The very beautiful experiments of Loeb (’18 a, b) open a way to precise interpretation of this matter. The relative effectiveness of various ions of neutral salts follows an order familiar in many cases of physiological action, frequently regarded as evidence of action upon the colloidal, as distinct from simply chemical, properties of tissue proteins. These effects cannot be interpreted in terms of ‘permeability,’ since, according to Oster- hout’s (16) exact experiments, the influence of neutral salts upon permeability does not follow this plan. Neither for salts nor for acids can stimulation be regarded as due to increased per- meability of the cell surface. d. Those reactions of Chromodoris which concern its ‘behavior’ in the larger sense have to do with feeding and with copulation. The generally accepted idea that the ‘rhinophores’ are specialized chemoreceptive organs concerned with olfaction has been already disproved (Arey, 17,718). C. zebra does, however, give evidence of being activated by low concentrations of materials secreted by its companions. These reactions are chemopositive, they are of several kinds, and they are important for conjugation. It is also probable that chemoreception enters into food taking, for it is only when creeping upon algae that the radula is brought into operation. When several sexually ripe nudibranchs are placed in a dish, they very soon protrude the genital papilla, and move toward one another. In fresh sea-water, as, e.g., in an aquarium with running water, conjugation is quickly effected.® : These nudibranchs produce constantly, when undisturbed, more concentratedly if irritated (Crozier, ’16 b), a curiously pene- trating ‘spicy’ odor. This odor is evident in sea-water with which they have been in contact. If the water is unchanged it assumes a blue color owing to the secretion of pigment. In stagnant water a Chromodoris may, but usually does not, protrude the genital 6 According to Pelseneer (’11), and as we also have observed, many smaller nudibranchs deposit several egg masses following each insemination. In Chro- modoris this is not true, a single egg mass being the consequence of each insemi- nation (Crozier, ’18°). 306 W. J. CROZIER AND LESLIE B. AREY papilla when isolated by itself. In stagnant (i.e., non-circulating) water copulation is interfered with (Crozier, ’18 d), although the nudibranchs may be active and healthy. These facts indicate a chemopositive response to low concentrations of some secretion, which is inhibited by higher concentrations. Mating behavior remains the same after the ‘rhinophores’ have been amputated. Therefore the ‘rhinophores’ are not of special importance for this response. Not only is the genital papilla protruded, but the pharynx as well is everted, even before two conjugating individuals come into contact. It is very difficult to experiment with the sensi- tivity of the pharynx, because it is seldom extruded in a position Fig. 8 The ‘mouthing’ behavior of Chromodoris preparatory to copulation favorable for observations, but it is undoubtedly very sensitive both to touch and to chemical activation. To KCl, M/20 in sea-water, the lips of the fully protruded pharynx were found more sensitive than its outer surface, whereas the walls of the organ (section I) were more reactive than the lips to touch. It usually happens that before becoming mutually adjusted for copulation (Crozier, 718d), the nudibranchs pass the end of the pharynx over each other’s surface, moving closer together the while (fig. 8). It seems hardly doubtful that the lips are the chief chemore- ceptive regions in this case, because they may be the only parts in contact with the other animal. This form of chemical attrac- tion is curious, because ‘sexual’ secretions can hardly be involved in a functioning hermaphrodite when reciprocal fertilization SENSORY REACTIONS OF CHROMODORIS ZEBRA 307 takes place (Crozier, ’18 d); still, it is possible, for one of the indi- viduals is commonly more active than the other in the maneuvers preliminary to copulation. VI. SUMMARY 1. Physiological evidence is adduced for the existence in Chro- modoris zebra of differentiated receptive mechanisms mediating reactions to tactile, chemical, and shading stimulation, to the constant intensity of light, and perhaps to heat. 2. Locally, responses of the general integument and all of the outgrowths of the body, gill plumes, ‘rhinophores,’ tentacles, pharynx, depend upon locally contained, peripheral, non-synaptic networks. In the gill plumes, and probably in the other pro- jecting parts, these nerve nets are polarized. 3. Reactions involving parts distant from the site of activation depend upon central, ganglionic, transmission. The peculi- arities of heterolateral response; of irreciprocal conduction be- tween the several homolateral parts; and of behavior following strychnine injection, show this central nervous system to be essentially synaptic. . 4. The nudibranch is positively phototropic, the chief recep- tive organs probably being the eyes. The branchial collar is also sensitive to light, which causes the gill plumes to be ex- panded. The gill plumes react by contraction when they are shaded; this response is very variable. When sexually ripe, Chromodoris is negatively geotropic. It is negatively rheotropic to strong water currents, the directive organs being the ‘rhino- phores.’ Vibrations transmitted through the water are not responded to. Temperatures of 31° to 32°C. induce negative reactions. Chemotropic reactions to body secretions of other individuals lead to conjugation; ‘olfactory’ stimulation (which . does not essentially involve the ‘rhinophores’) as well as ‘gusta- tory’ stimulation (of the lips) are concerned in this behavior. 5. The locomotion of Chromodoris is primarily muscular, not ciliary, the active part being the outer lateral margins of the foot, which suck locally. Progression is strongly polarized in THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 2 s 308 W. J. CROZIER AND LESLIE B. AREY the anterior direction. The foot is positively stereotropic, and when removed from a surface folds together laterally. These latter peculiarities enable the animal to creep upon narrow blades of eel grass where it feeds. The stereotropism of the anterior end of the foot is responsible for righting behavior; there is no apparent statolythic control of dorsoventral body orientation. Dyer Island, Bermuda, June, 1918. LITERATURE CITED Arey, L. B. 1917 The sensory potentialities of the nudibranch ‘rhinophore.’ Anat. Rec., vol. 11, pp. 514-516. - 1918 The multiple sensory activities of the so-called rhinophore of nudibranchs. Amer. Jour. Physiol., vol. 46, no. 5, pp. 526-532. \Arry, L. B., anp Crozier, W. J. 1919 The sensory responses of Chiton. Jour. Exp. Zoél., vol. 29, pp. 157-260. Bera, R.- 1884 Report on the nudibranchiata collected by H. M. 8. Chal- lenger, during the years 1873 to 1876. Rept. Sci. Res. Voy. Chall., Zool., vol. 10, pp. 1-154. 14 pl. Betur, A. 1903 Allgemeine Anatomie und Physiologie des Nervensystems. Leipzig, vii + 487 pp. CrossLtanp, C. 1911 Warning coloration in a nudibranch molluse and in a chameleon. Proce. Zoél. Soc. Lond., 1911, pp. 1062-1067. Crozier, W. J. 1914 Note on the pigment of a Bermuda nudibranch, Chromo- doris zebra Heilprin. Jour. Physiol., vol. 47, pp. 491-492. 1915a The sensory reactions of Holothuria surinamensis Ludwig. Zool. Jahrb., Abt. Physiol., Bd. 35, pp. 233-297. 1915 b On cell penetration by acids. Science, N.S., vol. 42, pp. 739-136. 1915 ¢ Regarding the existence of the ‘common chemical sense’ in vertebrates. Jour. Comp. Neur., vol. 26, pp. 1-8. 1916 a Cell penetration by acids. [I]. Jour. Biol. Chem., vol. 24, pp. 255-279. 1916 b On the immunity coloration of some nudibranchs. Proe. “ Nat. Acad. Sci., vol. 2, pp. 672-675. 1917 a The nature of the conical bodies on the mantle of certain nudibranéhs. Nautilus, vol. 30, pp. 103-106. 1917 b On the periodic shoreward migration of tropical nudibranchs. Amer, Nat., vol. 51, pp. 377-382. 1917.¢ Some structural variations in Chromodoris zebra. Nautilus, vol. 30, pp. 140-142. 1917 d Evidence of assortive mating in a nudibranch. Proc. Nat. Acad. Sci:, vol. 3, pp. 519-522. 1917 e Fusion of ‘rhinophores’ in Chromodoris. Amer. Nat., vol. 51, pp. 756-758. SENSORY REACTIONS OF CHROMODORIS ZEBRA 309 Crozier, W. J. 1918 a Sensory activation by acids, I. Amer. Jour. Physiol., vol. 45, pp. 323-341. 1918 b Cell penetration by acids, 1V. Jour. Biol. Chem., vol. 33, pp. 463-470. 1918 ¢ On tactile responses of the de-eyed hamlet (Epinephelus stria- tis). Jour. Comp. Neur., vol. 29, pp. 163-173 1918 d_ Assortive mating in a nudibranch, Chromodoris zebra Heil- prin. Jour. Exp. Zodl., vol. 27, pp. 247-292. Exror, C. 1904 On some nudibranchs from East Africa and Zanzibar. Part V. Proc. Zodl. Soc. Lond., 1904, vol. 2, pp. 83-105, pl. 3-5. 1910 A monograph of the British nudibranchiate mollusca, Pt. VIII (Suppl.). London, Ray Soc., 198 pp. Garstane, W. 1890 A complete list of the opisthobranchiate mollusca found at Plymouth; with further observations on their morphology, colours, and natural history. Jour. Mar. Biol. Assn., N.S., vol. 1, pp. 399-457. HeILprin, A. 1889 The Bermuda Islands. Phila., vi + 231 pp. Herpman, W. A. 1890 On the structure and functions of the cerata or dorsal papillae in some nudibranchiate mollusca, Quart. Jour. Micros. Sci., N.S., vol. 31, pp. 41-63, pl. 6-10. HerpMan, W. A., AND CiusB, J. A. 1892 On the innervation of the cerata of some nudibranchiata. Quart. Jour. Micros. Sci., vol. 33, pp. 541- 558. pl. 32-34. Horrman. F. B. 1907 Gibt es in der Muskulatur der Mollusken periphere, kontinuierlich leitende Nervennetze bei Abwesenheit von Ganglion- zellen? I. Untersuchungen an Cephalopoden. Arch. ges. Physiol., Bd. 118, pp. 375-412. Lors, J. 1918a The stoichiometrical character of the action of neutral salts upon the swelling of gelatin. Jour. Biol. Chem., vol. 34, pp. 77-95. 1918 b The influence of neutral salts upon the viscosity of gelatin solutions. Jour. Biol. Chem., vol. 34, pp. 395-4138. OumstED, J. M. D. 1917 Notes on the locomotion of certain Bermudian mol- lusks. Jour. Exp. Zodl., vol. 24, pp. 223-236. OstERHOUT, W. J. V.. 1916 Permeability and viscosity. Science, N.S., vol. 43, pp. 857-859. Parker, G. H. 1908 The sensory reactions of amphioxus. Proc. Amer. Acad. Arts and Sci., vol. 48, pp. 418-455. 1911 The mechanism of locomotion in gastropods. Jour. Morph.., vol. 22, pp. 155-170. 1917 The movements of the tentacles in actinians. Jour. Exp. Zodl., vol. 22, pp. 95-110. PELSENEER, P. 1894 Recherches sur divers Opisthobranches. Mém. couron. Acad. roy. Belgique, T. 53, 157 pp., 25 pl. 1911 Recherches sur l’embryologie des Gastropodes. Mém. Acad. , roy. Belgique, 2™ Sér., T. 3, 167 pp., 22 pl. Ranp, H. W. 1909 Wound reparation and polarity in tentacles of actinians. ‘Jour. Exp. Zo6él., vol. 7, pp. 189-238. 1915 Wound closure in actinian tentacles with reference to the prob- lem of organization. Arch. f. Entw.-mech. Org., Bd. 41, 8. 159-214. 310 W. J. CROZIER AND LESLIE B. AREY Srmrotu, H. 1914 Untersuchungen an marinen Gastropoden. Pigment, Loko- motion, Phylogenetisches. Arch. f. Entw.-mech.¥Org., Bd. 39, S. 457-515, Taf. 21. SmaLtLwoop, W.M. 1910 Notes on the hydroids and nudibranchs of Bermuda. Proc. Zoél., Soc. Lond., 1910 (1), pp. 137-145. SmaLLwoop, W. M., anp CuarxK, E. G. 1912 Chromodoris zebra Heilprin: a distinct species. Jour. Morph., vol. 23, pp. 625-636. Vurs, F. 1907 Sur les ondes pédieuses des mollusques reptateurs. Compt. Rend. Acad. Sci. Paris, T. 145, pp. 276-278. WHOLE A | Pvahy ; ah ri - a at ‘ Mia f rf Resumen por el autor, Matsuziro Takenouchi. Instituto Wistar de Anatomia y Biologia. Estudios sobre la supuesta funcién endocrina de la glandula timo (rata albina). El suero procedente de conejos immunizados con la substancia del timo de la rata albina presenta una reaccién de precipitina positiva con el extracto del timo, pero esta reacci6n no es estricta- mente especifica. El suero anti-timico de conejo no produce ninguna accién hemolitica positiva con los corptisculos san- guineos de la rata cuando se usa como complemento el normal del conejillo de indias o el suero de la rata. Los sueros anti- timicos inyectados en ratas no producen sintomas de “‘anafilaxia primaria’”’ ni tampoco afectan al crecimiento de estas. El autor no ha observado modificaciones en las visceras. Ha observado casi los mismos resultados con el suero anti-testicular de conejos inyectados con emulsién de testiculo de rata. Ha intentado provocar la produccién de hemolisinas en los conejos mediante la inyeccién de corptisculos sanguineos de la rata lava- dos, pero el suero normal de la rata con su complemento no puede activar la hemolisina contra los corptisculos sanguineos de ésta, a causa de la presencia de una substancia inhibidora. El pollo no es animal adecuado para la produccién de hemolisina capaz de actuar sobre los corptsculos sanguineos de la rata. Nuestro intento de producir suero anti-timico enérgico en el conejo mediante inyecciones de timo de rata fracas6é probable- mente por la produccién de anticuerpos en el conejo y porque las células del timo de la rata estan protejidas. No podemos admitir hasta el presente la existencia en el timo de una funcién endocrina bien establecida. Translation by José F. Nonidez Carnegie Institution of Washington AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, AuGusT ll STUDIES ON THE REPUTED ENDOCRINE FUNCTION OF THE THYMUS GLAND (ALBINO RAT) MATSUZIRO TAKENOUCHI The Wistar Institute of Anatomy and Biology TWO CHARTS CONTENTS 1. Introduction. . : MEN ae AE SG CORO ARE Toe OO Let RST MS 1d 2. General plan ea Pecnntcaer 4 ole 3. Immunization of the rabbit i the aheree end oF fie Api: oe a figs 317 4. Immunization of the rabbit with testes of the albino rat. LPS, Jaan 5. Immunization of the rabbit with the red corpuscles of phe alba rat. . 330 6. Immunization of the chicken with the red corpuscles of the albino Aig, . 335 em TERA NCIS CUSTOM Acne ee opr e ecw. Gini ary ine ene tems ¢ 2, erate sertay evel cus Ae cra Ree oS ewncsunie ancseomelusionsy sl IT Ie ue) AUR ad ee Pe OP eee, ee eooe 1. INTRODUCTION The various theories of the control of sexual development by the thymus, mostly founded upon the correlation in man of the time of thymus involution and sexual differentiation, do not seem as yet to be firmly established. On the one houd Klose and Vogt (’10), Lucien and Parisot (10), Paton (11), and other authors report that following thymus removal in birds and mammals various effects are found, such as interference with skeleton growth, including rachitic changes, together with adi- posity or emaciation, injury to the thyroid and degeneration of the testes; while on the other, Pappenheimer (’14), Park (17), and others state that if the experiments are carefully performed and carefully controlled, the thymus can be removed without producing any harmful effect whatever. In feeding experiments, Gudernatsch (’14) found that thymus- fed larvae delayed their metamorphosis, although the animals grew on this diet. Romeis (715) and Abderhalden (’15) were 311 312 MATSUZIRO TAKENOUCHI able to verify these findings in part, but Swingle (’17), Uhlenhuth (’18), and Hoskins (’16) report negative results. Uhlenhuth in his latest paper (’19), however, states that the metamorphosis of the salamander larva is retarded when thymus gland is fed. The theory of the relation of the thymus to general metabo- lism also lacks definite proof (Jackson, 15, ’715.a; Stewart, ’18), and the theory that rachitic changes in children are caused by disturbances of the thymus cannot be proved by any known facts. The cells which make up thymic tissue belong to the vascular system (Danchakoff, ’16), and Adami (’14) states on page 563 of his text-book that ‘‘To all intents and purposes it (the thymus) is a lymphoid organ,” and the formation of an internal secretion is no more likely to be the function of the thymic cells than of the cells of similar appearance in other lymphoid tissues. Sum- ming up all the physiological, anatomical, and experimental facts, E. R. Hoskins (’18) says that whatever be the real function of the thymus, certain it is that the production of an internal secretion by it has not been proved. Among other experiments with the thymus which cannot be placed under the headings, extirpation or administration of thy- mic tissue or extracts, there are two; one, which aims to destroy the thymic tissue in vivo by x-ray irradiation, while the other seeks to do the same by some serological method. ‘To the first category belong the experiments of Regaud and Crémieu (’12). They made the thymus atrophic, especially the cortical portion of it, by x-ray irradiation, but they did not observe any abnor- mality in the health and growth of the animals so treated. Hewer (16), however, reports that injury to the thymus by x-ray irradiation results in injury to the function of the testes. “Since complete removal of the thymus has no such effect, and since unhealthy animals never breed well, Hewer must prove that her treatment did more than injure the health of her animals’ (Hoskins, 718). Under the second category, namely, the action of thymotoxic or thymolytic serum, there are many reports on record. Gilberti ENDOCRINE FUNCTION OF THE THYMUS GLAND 313 (11) used rabbits for immunization to obtain a thymotoxic serum, with positive results when dog thymus extract was em- ployed as the antigen. ‘‘Weymersch (08) sah nach Injektion von thymotoxischen Serum eine weitgehende Atrophie und Sklerosierung der Thymusdriise, eine abnorme Verteilung der Leucocyten und exzessive Wachstum der Thiere’’ (cited from Shimizu’s paper, p. 262). Ritchie (08) obtained a serum from ducks by injection of the thymus of guinea-pigs, but observed no specific action of that serum, when injected, on the thymus of guinea-pigs. He states, however, that in the presence of this immune serum from ducks, guinea-pig complement becomes fixed to the guinea-pig thymus, lymph glands, bone marrow, and spleen. Ritchie used in his experiment a hemolytic system con- sisting of ox blood and anti-ox rabbit serum. He concludes from this experiment that the serum obtained from ducks which had been treated with the thymus glands of guinea-pigs contained a ‘leucophilic immune body,’ and not a specific thymolytic one, and that the structural changes in the thymus of the guinea-pigs following the injection of the serum were due to its ‘leucolytic action,’ and are not specific. Moorhead (’05), however, found that the serum of rabbits which had been injected with guinea-pig’s thymus glands had no recognizable leucolytie action, did not “agglutinate emulsi- fied thymus gland in vitro,” and had no constant action upon the animals into which it was injected. Recently Shimizu (’13) reports his success in obtaining a very strong thymolytic serum, which he says resulted, after injection in young dogs, in a marked retardation of bone growth, together with a strong atrophy of the medullary portion of the thymus, and a proliferation of connective tissue. These responses occurred in two animals among fifteen so treated, while there were in all the other individuals more or less pronounced toxic symptoms corresponding to the so-called primary anaphylaxis. In the summary of his paper he says: Bekanntlich ist die Verinderung der Thymus bei Inanition haupt- sichlich die Involution der Rindensubstanz, und das Mark bleibt dabei wohlbehalten. Thymusatrophie bei der Réntgendurchstrahlung 314 MATSUZIRO TAKENOUCHI betrifft auch die Rinde. Die bei den Ernahrungsstérungen und akuten Infektionskrankheiten der Sauglinge vorkommende Veradnderung der ~ Thymus ist auch eine Rindenatrophie. Wie Hammer betont hat, sieht man eigentlich nur die Rindenatrophie sowohl bei der Alters—, als auch bei der durch Hunger, toxische Einfliisse etc. hervorgerufenen akzidentellen Involution. . . Mein Thymolysin ruft haupt- sichlich deutliche Atrophie des “Marks hervor, und die behandelte Tiere zeigen dieselbe kérperlichen und geistigen Entwicklungen, die von den friiheren Autoren bei Thymektomie beobachtet worden sind. From the result of his experiments he concludes that the medullary portion of the thymus has biologically a distinctly different function from the cortical portion, and that the endo- crine function of this gland with its influence upon the growth of animals, must be ascribed to the medullary portion only. He does not give definite proof for any endocrine function whatever of the thymus, at least in his first paper,! and he made his con- clusion with the complete acceptance of the theory of the thy- mus-skeleton relationship, which had already been firmly estab- lished in the minds of many. As is easily imagined, it would be one of the best methods for the study of the function of the thymus gland to destroy it alone in vivo by some other than surgical means, for instance, by some serological procedure, and observe the symptoms or pathological alterations which might follow. Shimizu does not give any definite proof for the presence of his thymolysin in vitro. Also he does not give any histological de- scription of the lymphoid tissues of dogs injected with his thy- . molysin. This is important, because the cells which make up the thymic tissue belong to the vascular system, according to Danchakoff (’16), and therefore we might expect the lymphoid tissues to respond in some way or other to the thymolysin. Originally I hoped to repeat in full Shimizu’s experiments, using albino rats instead of dogs, and thus to get further infor- mation regarding the function of the thymus in the albino rat. ‘It is said that Shimizu has published his second paper regarding the same problem, and some other Japanese investigators have done work in Japan along this line and published their results. We are, unfortunately, not able to obtain these original reports in time, therefore this paper is written without the knowledge » of their publication. ENDOCRINE FUNCTION OF THE THYMUS GLAND 315 The first thing needed is to verify the fact that, according to Shimizu, the thymolysis in vivo can effect more than the total removal of the thymus gland which, if carefully done, can be accomplished without any harmful effect whatever Pappen- heimer, 714, and Park, 717). 2. GENERAL PLAN AND TECHNIQUE For the study of any cytolysin the first condition is to obtain a good antibody (amboceptor, cytolysin) of high potency, and the second condition is that the cytolysin thus obtained should show its action clearly in the body of the same kind of animal as that from which the antigen for the immunization has been ob- tained, utilizing the complement of that body, or at least it should be able to combine with the complement of some animal in vitro, to show its action clearly. To choose a proper combination of species (one animal for the source of antigen, the other into which the antigen is to be in- jected to obtain a cytolytic serum of high potency), is sometimes difficult without preliminary experiments, because the ease with which different species produce strong antibodies after injection of a given antigen is very variable. For the second condition mentioned it should be kept in mind, as Bordet (’06) says, ‘‘que la valeur hémo- ou bacteriolytique des alexines varie d’une espéce 4 l’autre, rein de plus admissible, les d’espéces differente n’étant pas entiérment identique.”’ In general for hemolysis, fresh guinea-pig serum is very potent in activating many sensitized blood-cell complexes, but weak in activating sensitized guinea-pig corpuscles. Often we find that the complement from an animal is entirely impotent or capable of produeing only a weak hemolysis of the sensitized cells of its own species, though this is not a general rule (Zinsser). The usual technique was employed. For the immunization of the rabbit with thymus we used the thymus from albino rats taken from the stock colony of The Wistar Institute. Rats were chosen between eighty to ninety days, which, according to Hatai (14), is the period when the thymus gland is largest in the albino rat. 316 MATSUZIRO TAKENOUCHI Under ether narcosis the blood of the rats was taken from the arteria carotis and the thymus gland was removed under aseptic precautions, avoiding the mixing of blood as completely as pos- sible. After being weighed, the glands were washed in sterile saline solution to free them from visible traces of blood and then were ground with sterile salt solution into an emulsion, which was strained through sterile cotton gauze and injected into the rabbit intraperitoneally. The injection was repeated three or four times with increasing doses of the thymus emulsion, at about one week’s interval, and seven to ten days after the last injection the blood was taken under ether narcosis from the arteria carotis. Autopsy findings were noted. The separated serum was carbolized (0.5 per cent) and kept in the ice-box. | The normal serum which was used in the control experiments was taken from the ear vein of a normal rabbit without narcosis, carbolized as above, and also kept in the ice-box. For the detection of antibodies in vitro, we used the precipitin reaction with the extracts of several organs from normal rats. The technique used in the preparation of the extracts will be given later. | For the determination of antibodies in vivo, we injected the antithymus serum repeatedly into albino rats, using generally 0.3 ce. to 0.5 ee., Injecting subcutaneously on the back near the tail, and observing any marked symptoms which followed. Gen- erally one half of one litter of rats was used as the test animals, and the other half as controls. All the rats were fed with ordi- nary laboratory diet. The growth curve was obtained by weighing each rat separately. The examination of the test and control rats followed: at differ- ent intervals after the last injection of the serum. Under ether narcosis, blood was collected, each organ removed separately and weighed carefully and, directly after the weighing, all the organs were fixed in Bouin’s solution for histological study. For the comparative study on the specificity of cytolytic serum, I also immunized rabbits with testes from albino rats, using almost the same technique as in the immunization with ENDOCRINE FUNCTION OF THE THYMUS GLAND Ole thymus tissue. The serum thus obtained was tested in vitro as well as in vivo. For the further serological studies, a preparation of hemolytic rabbit serum against the red corpuscles of the albino rat was attempted, and, furthermore, chickens were immunized against rat blood, and the action of the normal rat complement carefully examined in these cases, using the ordinary serological technique, which will be given later. 3. IMMUNIZATION OF THE RABBIT WITH THE THYMUS GLAND OF THE ALBINO RAT (RABBIT, GROUP A) a. Process of immunization A large, healthy female rabbit was immunized with the thy- mus substance of albino rats by intraperitoneal. injections. Rabbit, Group A, No. 1 Date of injection Amount of thymus emulsion injected PUMICE Aas MONS mementos ase te eel mek dhe sind ous Delo Prams OF thymiIs mbna te aN OU 5 ae ee aad Stra ae a ee ae od ck oe Op erema Or oh ys 5 SVU UO ae ete as elie ch lence rtanioh fairs Sedo, SERING Ol MEINE: Slo lOO h Aaa neato eos te a 4) prams Of thymus, Ue iegl OLS ee aac oleae wire Atel aad cee Laeeblood was'taken. The autopsy findings were as follows: Spleen somewhat smaller than usual, without marked macroscopical alteration, liver normal; kidneys both normal. On the right side, in the middle part of the abdomen, directly inside of the peritoneum, there was a small fibrinous body about 12 mm. in length, 6 mm. in breadth, which is nothing else than an abscess enclosed in a strong fibrinous membrane hanging between the convolutions of the intestine. This abscess was most probably caused by the injection of the thymus emulsion, which though strained, had in it some small particlés of connective tissue which were dis- solved with difficulty. | The separated serum was tested for the presence of the anti- bodies by the precipitin and hemolytic reactions and then in- jected into rats to determine the action in vivo. 318 MATSUZIRO TAKENOUCHI b. Test of the antithymus serum in vitro 1. Precipitin reaction. For the precipitin reaction, we followed the procedure given by Ricketts and Rothstein (’03) for the action of neurotoxic serum. They used for their precipitin reaction an emulsion of nervous tissue as a precipitinogen (antigen). Different precipitinogens (antigens) from different kinds of tissue were prepared in the following manner: Weighed tissue, first washed with saline solution, was ground thoroughly, emulsified with sterile saline solution and shaken six hours at room temperature, with preliminary carbolization for the purpose of avoiding bacterial contamination and alterations in composition. Then it was strained through sterile cotton gauze. The strained emulsion was centrifuged for a long time at high speed to free the supernatant fluid from macroscopic particles. The supernatant fluid was diluted again with sterile saline solu- tion and carbolized. The following tissues were prepared in this way. TABLE 1 TISSUES Kidney] Spleen | Testes | Brain Thy, Weight ‘(grams)< 5.323.086 caacst odes cee eetege| eat OL OS42|) 05742) aL a0Ol eile Salt solution added in grinding (ce.)..........| 15.0 | 5.0 | 5.0 {10.0 | 5.0 Salt solution added to the supernatant fluid (GOaospee idles Oe. Ty ety Perera Feed Hhossets| 308041050 fy MOO ce 20-0) celica To six test-tubes each containing 1 ec. of the diluted extract there were added from one to five drops of serum, the sixth test- tube being held as control. The quantity of the liquid in each test-tube was made the same with saline solution. The tubes were then put into the ice-box for sixteen to eighteen hours, avoiding any evaporation. The first reading was made after four to six hours and the second sixteen to eighteen hours later (table 2). Control tests with normal rabbit serum give a little precipi- tation in some tubes, but this is slight and inconspicuous In others. ENDOCRINE FUNCTION OF THE THYMUS GLAND 319 TABLE 2 Showing the precipitin reaction of the antithymus serum with the extracts of different rat organs TEST-TUBE 1 2 3 4 5 6 SUT UA GOR) ats ans oe ee Oe ee eae OAL OM AO EO ep OU eG Antithymus serum in drops............. 1 2 3 4 5 First reading (4 to 6 hours) TCICHO EN eee a SORBErS ie Sabai he PERE S oon ola

> Trail length, cm. 21.9 20.95 hy. Vig. 3 Record of bee no. 123 in non-directive light. angular deflections will give a result equivalent to the amount of continuous turning required to carry the animal from the start- ing point to the end of its course. Thus, in figure 3, the direction of locomotion at a makes with the direction at e an angle of 2% <.3605.— 2 3607 onji23) < 3607. Knowing the distance traveled in centimeters and the amount of turning in degrees, the average degrees turned per centimeter is easily computed. Denoting this average deflection, as I shall call it, by D, we have for the trail in figure 3, PHOTIC REACTIONS OF HONEY-BEE Save 21 x 360° 26.95 em. X16 D= == eel en. It is to be emphasized that the value + 5.01°/em., does not sig- nify that the animal turned only toward the functional eye. It merely shows that the algebraic sum of all its deflections aver- ages 5.01°/cm. toward the functional eye. Animal Apis metlifine Experiment; No>3|3)) | ns oe No. of Animal 5 oa Date %0f, L Time { 4 \ a Eye black Lop <” Light 95Tme. \ No. dx. loops a % be No. sn. loops a # b ¥ Wc les Trail length,em. a j2.3 bu iaeah Sage 12.25 eu Tl Ae Fig. 4 Two trails of bee no. 135 in non-directive light. A record is shown in figure 4 which represents two trials taken in rapid succession. This was necessitated by the animal’s en- countering the side wall of the light chamber so quickly that the first trial was shorter than usual. The deflections in these trails are estimated as previously described. It will be noted, however, that in the trail marked a, the angular deflection between m and n does not amount to quite 3 of a sinistral loop, although it is so counted. In such instances the angle was always estimated THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 3 358 DWIGHT E. MINNICH to the nearer § of a circumference, no attempt being made to discriminate differences of less than 45°. In this record it is desirable to combine both trails into a single computation. Pro- ceeding as before, (ES Ghia 2) 008 (12.25 + 7.1 em.) 6 D = = — 1.16°/cm. The negative sign of the average deflection here obviously indi- cates that the bee turned more toward the covered eye than toward the functional eye in these trials. In the course of experimentation, records of normal bees were also made in non-directive light. Since in such individuals neither eye was blackened, the positive sign was arbitrarily applied to the direction of greater angular deflection in each set of trials. Other- wise the computations for normal bees were performed in the same manner as those for bees with one eye blackened. These various examples will illustrate the method employed in all quantitative determinations. Upon the results thus obtained the chief conclusions of the present paper are based. IV. MATERIAL 1. General care of animals The bees used in all quantitative experiments were thoroughly active workers taken from the flowers of a near-by garden, and were, for the most part, individuals from a single large hive situ- ated there. The animals were trapped by simply inverting a long glass tumbler over the flower, and then transferring them to a small screen fly-trap. In some experiments, however, which were performed too late in the fall to obtain bees in this way, animals were used from a single comb of workers confined in an observation hive. The exit of the hive was kept securely screened, for such a colony quickly disintegrates if its members are per- mitted to leave the hive freely. Bees kept in this way remained in reasonably good condition, for at least a month. Bees destined to undergo experimentation were first subjected to having their wings clipped, an operation easily executed when PHOTIC REACTIONS OF HONEY-BEE 359 the animals were feeding. Each wingless individual was then confined in a small cylindrical cage of screen wire, the bottom of which was formed by a layer of tissue-paper over cotton to pre- vent injury in case of falling. In the same cage were also placed two friendly winged workers to counteract any possible effects of isolation. The cages of bees were kept in a darkened box when not directly under experimentation, since the influence of light often caused the animals to maintain a restless activity which appeared, in some eases, to shorten life considerably. In the dark, however, they usually remained more quiet. Each cage was supplied with water by a small wad of saturated cotton placed on its top. Small quantities of honey were also supplied on short wooden sticks stuck to the side of the cage. Early in the morning, at noon, and in the evening the cages were cleaned by removing excess honey, etc., and fresh honey and water were provided. Such operations were carried out at least a half-hour before any trials were made on the animals. The temperature of the laboratory in most of the experiments was kept above 20° to 21°C. This was found to be an important consideration, since at lower temperatures bees became torpid and inactive. In collecting the animals even, an attempt was made to take them on warm, sunny days which had, in general, been preceded by warm weather. It was found that bees brought in after a brief period of cold, wet weather were apt to be either unresponsive or extremely variable in their behavior. 2. Blackening the eye Any technic for blackening the eye of a wingless bee requires, of course, the use of an anaesthetic. In the present experiments ether was used exclusively. Care was taken to administer it rather slowly and in minimal doses. When completely anaes- thetized, the bee was placed on one side, on a small cork pinning board. Here it was fastened down securely by the use of insect pins, with which the thorax, abdomen, and legs were securely braced against the cork. The blackening was then applied to the eye, the entire surface being covered with as thick a coat as pos- 360 DWIGHT E. MINNICH sible. Two kinds of blackening material were used, viz., lamp- black in shellac and a dead black paint known commercially as ‘Jap-a-lac.’ The latter proved the more satisfactory and was used throughout the majority of experiments. Although bees under ether often began to recover in five to ten minutes, they were not removed from the pinning board for twenty to twenty-five minutes, when the covering of the eye was well hardened. Recovery from anaesthesia was usually com- plete in an hour and often much less. As a rule, however, opera- tions were carried out in the evening, and the bees were not sub- jected to further experiment until the following day. Ample time was thus allowed for the animals to recover as much as possible from the effects of the operation. V. BEHAVIOR OF NORMAL BEES 1. Kinetic effect of light The remarkable sensitivity of the honey-bee to photic stimu- lation must have long been patent to students of its behavior. Bethe (98, p. 83) says, ‘“‘Das Licht ist bei diesen Tagthieren [bees, flies, etc.] der auslésende Reiz zum Fliegen; in einer dunklen Schachtel fliegt keine Biene auf, auch nicht, wenn man sie reizt. Das Licht gibt die Regulirung beim Fluge ab.” This observa- tion was repeatedly confirmed in the present experiments. When collecting bees from flowers, fifteen to twenty individuals were confined in a single cage, which was then placed in a closed box. Although at the height of activity when captured, a few minutes in the darkness of the box seldom failed to reduce these animals to a state of quiescence. If a little light was admitted to the box, however, by even partially removing the lid, there was a sudden resumption of activity. Precisely the same behavior was exhibited by wingless bees. If confined in a dark box, they were, as a rule, reduced to com- parative inactivity. A brief exposure to light, however, was usually sufficient to excite vigorous locomotion, and continued exposure not infrequently resulted in the maintenance of an intense activity for extended intervals of time. PHOTIC REACTIONS OF HONEY-BEE 361 Individuals which had been subjected to operations of remov- ing the wings and blackening the eye frequently responded some- what more slowly to this photic activation than did normal bees. In the former, locomotion was preceded by a more or less prolonged sequence of cleaning operations. ‘The proboscis was extended and stroked with the fore legs. The eyes, particularly the covered one, were the objects of repeated and vigorous scrap- ings, responses no doubt largely attributable to the irritation of the blackening material. The abdomen was meanwhile bent from side to side, while the middle or hind legs were rubbed to- gether, or the hind legs assiduously stroked the dorsum of the abdomen. ‘These movements became more and more intense until at length they culminated in active creeping. Light, then, exerts a strong activating or kinetic influence upon the honey-bee, while darkness has the opposite effect. Es- sentially similar phenomena have been reported by Loeb (’90) for the plant louse, Carpenter (’05) for the pomace fly, and Turner (12) for the mason wasp. Stockard (08) has reported the case of Aplopus, the ‘walking-stick,’ which also falls into this category of behavior. In Aplopus, however, light inhibits ac- tivity, while darkness induces it. Hence the ‘walking-stick’ is nocturnal, whereas the plant louse, the pomace fly, the mason wasp, and the hive bee are diurnal. In diurnal animals this response is apparently due to the con- tinued action of light rather than a sudden change in it. Thus, while many bees respond almost, if not quite, at once to the presence of light, others may respond only after some minutes of exposure. According to Turner (12, p. 360), the same is true of the mason wasp. 2. Directive light Not only does light induce locomotion in the honey-bee, but directive light regulates the course of locomotion. Bees brought into the laboratory direct from their foraging activities out of doors seldom failed to exhibit a most striking phototropism. Such insects when liberated in the laboratory flew almost im- mediately to the nearest window, where they remained fluttering 362 DWIGHT E. MINNICH against the glass. Or, if escaping in a darkened room, they not infrequently flew directly into the flame of the nearest gas jet. Observations of this sort were long ago reported by Lubbock (82, pp. 278, 279, 284). A few years later, Graber (’84) demon- strated the same thing experimentally by confining forty to sixty bees in a small box, one half of which was illuminated by direct sunlight, the other half being shaded, with the result that the majority of the bees soon collected in the illuminated end. More recently, Hess (13 a, 713 b, ’17) has repeated this and a variety of other experiments. As a result of these he has been able to show that in the presence of several sources of photic stimulation, which differ in color and intensity, bees always orient toward the one which to a totally color-blind person appears brightest. The positive phototropism of the honey-bee is thus demonstrable in a variety of ways. In the experiments just cited, winged bees were used exclu- sively. My own experiments, on the contrary, were confined en- tirely to workers from which the wings had been clipped. Such bees when creeping in the directive light area exhibited an orien- tation which was striking in three respects, viz., its rapidity, its precision, and its constancy. An individual to be tested was removed from the dark box and exposed to light for a few minutes until it was thoroughly active. It was then allowed to creep from its screen cage to a small, rectangular piece of black paper, and on this it was transferred to the edge of the directive light area. An effort was made to start the animal creeping at a right angle to the direction of the light rays by turning the paper just before it crept off. The rapidity of orientation was so great, however, that the intervening centi- meter or so was frequently sufficient to allow the animal to reorient © perfectly. Since the velocity at which bees creep averages 3 to 6 em. per second, orientation in these cases occurred in considera- bly less than one second. I have also tried leading a bee by moving the light, now in this direction, now in that, with varying degrees of curvature. Always the animal followed, orienting rap- idly to even slight movements of the lamp. PHOTIC REACTIONS OF HONEY-BEE ese The precision with which orientation was maintained was no less conspicuous. Once oriented, the animal generally moved in a nearly straight line toward the source of light. In figure 5 are shown two records of each of six bees in the directive light area. Of a large number of animals tested in the course of ex- perimentation, considerably over 25 per cent maintained their orientation as precisely as did bee no. 66. The deviations of / \ Nie eX / x 110 | QI a 36 Fig. 5 Two trails of each of six normal bees in directive light. In this, as in subsequent figures of records in directive light, the clear circle represents the light source, and the straight lines from it, the direction of the rays. most of the animals would, moreover, easily fall within the lati- tude of that exhibited by bees nos. 66, 33, and 23. Results sim- ilar to those shown for animals nos. 110 and 21 were, on the con- trary, less frequently encountered, while trails such as those of bee no. 36 were seldom or never found among normal, healthy bees. The response to directive light is very constant in the bee. The oncoming of death itself seems often to intensify rather than to weaken this phase of its behavior. Bees occasionally escaped 364 DWIGHT E. MINNICH in the laboratory. Such individuals rarely survived the lack of food for more than a day or so. Yet it was not an infrequent occurrence to observe one of these starved animals, so weak that it was barely able to creep, slowly emerging from a hidden corner in a final struggle toward the light. Nevertheless, bees were discovered which in a few instances failed to exhibit the usual positive reaction to directive light. Such cases, however, are not to be construed as a total absence ae SS Fig. 6 Three successive records of a normal bee in directive light, showing a failure to orient in two cases. of phototropism, but rather its momentary suppression by other factors of behavior. This is well illustrated by the following example. Seven cages of bees were prepared from the stock in the observation hive, Oct. 30, at 2:45 p.m. When tested about an hour later in the directive light area, six of the seven animals exhibited the usual positive response. One animal, however, gave the records reproduced in figure 6. This bee when given its first trial at 4:06 (fig. 6, 7) did not ori- ent toward the light source. Instead it pursued a devious course PHOTIC REACTIONS OF HONEY-BEE 365 looping now to the right, now to the left, and finally turning al- most directly away from the light. In a second trial at 4:14, it exhibited a somewhat similar response (fig. 6, 2). One minute later, the animal was subjected to still a third trial, being started on this occasion some 30 em. nearer the light. This time it ori- ented and moved in a fairly direct course toward the source of illumination (fig. 6, 3). What the temporary, inhibiting factors were which produced these very atypical responses could not be ascertained. In all other respects this bee was quite indistin- guishable from the other individuals in the experiment. This example, however, shows that even the constant response of the bee to directive illumination is not free from abrupt and appar- ently inexplicable departures. 3. Non-directive light The behavior of bees in non-directive light is no less charac- teristic than that in directive illumination. Since all quantita- tive experiments on circus movements were conducted in non- directive light, an intimate acquaintance with the behavior of normal animals under the same conditions was necessary. Every bee was, therefore, subjected to several trials in non-directive light before having one eye blackened. It was a matter of continual observation that a bee creeping in the directive light area ceased to move in a straight course upon reaching the area near and immediately beneath the lamp. Here, where the illumination was essentially non-directive, the animal deflected from its former, precise path and began to loop in a constant or varying direction (fig. 1). In other words, the bee was trapped; for directly it crept away sufficiently for the light to become directive again, it was forced to turn back. Thus the animal continued to creep round and round in a limited area, occasionally rearing on its hind. legs in an abortive attempt at flight, or finally ceasing locomotion to begin cleaning operations. In the non-directive light apparatus (fig. 2), the same tend- ency to loop was manifested, only on a much more extensive scale. Here the bee seldom crept in a straight line for any great 366 DWIGHT E. MINNICH distance. Each animal was subjected to two sets of trials, an hour or so apart. Usually a single trial only constituted a set. In case the bee quickly encountered the side wall of the light chamber, however, or exhibited unusual variability in its be- havior, additional trials were made. The aggregate duration of the trials of each set varied considerably, even in the same animal. Sometimes they were as short as thirty seconds; again, as long as two minutes. The average was in the neighborhood of thirty to sixty seconds. Preliminary to each trial, the bee was exposed to light until aroused to active creeping. The illumination used throughout in experimenting with normal bees was 957 mce.* The average deflection to the right or to the left has been computed for each set of records thus obtained, and the results of these computations presented in table 2 (appendix), columns B and C. On the basis of these data, the fifty-two bees experi- mented upon may be classified into three groups: 1. Bees whose average deflection in both sets of trials was over 2°/em. and in the same direction. 2. Bees whose average deflection in both sets of trials was small. 3. Bees whose average deflection in the two sets of trials varied widely, either in magnitude or direction, or in both. The first class is composed of animals which exhibited a more or less pronounced tendency to turn in a constant direction (right or left). These animals, 29 in number, comprised 56 per cent of the total 52 bees. Fourteen of these were chiefly right-handed in their turns; 15, left-handed. In 14 of the 29 bees, or 27 per cent of the total number, the average deflections exceeded 4°/cm., while in 6 individuals, or 12 per cent, it rose to over 8°/em. Sim- ilar right and left-handed tendencies of locomotion in non-direct- ive light have been reported by Walter (’07) for planarians and by Patten (’14) for the blowfly larva. A typical example of this behavior in bees is illustrated in figure 7, bee no. 101. In its first trial (fig. 7, 101, a), this animal showed an average deflection of 7.11°/em. to the left, and in the second trial (fig. 7, 101, 6), a sim- ilar deflection of 6.10°/em. Since these records were made nearly an hour apart the left-handed tendency was not the result of a PHOTIC REACTIONS OF HONEY-BEE 367 101 36 < Left — Right Left — Right \ | a ( Left Right | \ Fig. 7 Records of normal bees in non-directive light. In this, as in subse- quent figures of records in non-directive light, a solid circle is used to indicate the center of the floor of the non-directive light apparatus. a, records of the first set of trials; b, records of the second set of trials. Bee no. 101 deflected constantly toward the left. Bee no. 36 varied its deflection in the course of single trials. 368 DWIGHT E. MINNICH brief, temporary condition, but was probably a more or less permanent feature of this animal’s behavior. The second class of animals includes those whose average de- flections were small in both directions. The results obtained here are attributable to either of two causes: a. The bee varied its turning from right to left, so that on an average, one tendency nearly or quite balanced the other. b. The bee exhibited little or no tendency to turn either to the right or to the left. An example of the first type is seen in the records of bee no. 36, figure 7. In its first set of trials (a, 1, 2) this animal turned sometimes to the left, sometimes to the right, so that the result- ant average deflection was but 0.79°/cm. to the left. Similarly in the second set of trails (b, 1, 2), the average deflection amounted to only 1.94°/em. to the right. The second type of this class is illustrated by bee no. 134, figure 8. This animal showed no pro- nounced tendency to turn either to the right or left. The aver- age deflection for each set of records was, therefore, small, being only 1.52°/cm. to the left for the first set (a, 1, 2,3) and 1.22°/em. to the left for the second set (b, 1, 2, 3, 4). In the third class of bees are to be found those which, although they exhibited fairly uniform behavior in a single set of trials, varied widely in different sets. For example, bee no. 82, in its first record (fig. 8, 82, a) showed a pronounced deflection which averaged 5.81°/cm. to the left. In its second set of ‘trials (fig. 8, 82, b, 1, 2, 3, 4), on the contrary, it showed little tendency to turn, and the average deflection was but 0.65°/em. to the left. An even more striking case of variation, however, was afforded by bee no. 63. In asingle record of fifty-three seconds’ duration (fig. 8, 63, a) this animal deflected, on the average, 5.58°/cem. to the left. Approximately two hours later, in a record of sixty seconds’ duration (fig. 8, 63, b), the same animal exhibited an even greater average deflection in the opposite direction, viz., 7.50°/em. to the right. The range of variation presented by these two records is no less than 13.08°/em. In a uniform, non-directive light field, therefore, many bees exhibit a fairly constant tendency to turn toward a given side, PHOTIC REACTIONS OF HONEY-BEE 369 134 a ij 2 Fig. 8 Records of normal bees in non-directive light. a, records of the first set of trials; b, records of the second set of trials. Bee no. 134 exhibited little tendency to deflect in either direction. Bees nos. 82 and 63 varied widely in their average deflections in the two sets of trials. 370 DWIGHT E. MINNICH others display little or no such tendency, while still others vary widely in their deflections from time to time. Since the animal moves in a uniform environment, the conspicuous asymmetry of response so frequently noted must be attributed to internal factors. Such factors are, for the most part, probably quite in- dependent of light. A more detailed discussion of these will be presented in a subsequent section of this paper. 4. Total darkness If internal factors are responsible for the asymmetric responses of bees in non-directive illumination, a similar behavior should be exhibited in the total absence of photic stimulation. Such was indeed the case. Animals creeping on smoked paper, in total darkness, showed the same conspicuous tendencies to loop and turn as did animals in non-directive light (fig. 16). The data here referred to were taken in connection with experiments conducted for a different purpose. They are, therefore, not sufficiently extensive to establish more than a similarity to the behavior exhibited in non-directive light. Responses essentially like those of bees in total darkness have also been described by Pouchet (’72) for the larvae of Lucilia caesar, Davenport (’97) for the amoeba, and Frandsen (’01) for the garden slug Limax. Frandsen’s observations in particular bear a striking resemblance to those which I have just described for the honey-bee in non-directive light. Thus he found that while most of his animals looped in a fairly constant fashion to the right or left, a few were extremely variable, while still a few others moved in rather straight courses. The responses of creep- ing bees in the total absence of photic stimulation are, therefore, very similar to those observed for other animals under the same conditions. 5. Summary In the preceding pages certain responses of normal bees have been described in considerable detail, but only as a prerequisite to an adequate understanding of the behavior of the animals PHOTIC REACTIONS OF HONEY-BEE atl when one eye is blackened. The features of behavior which are important in this connection may be summarized as follows: 1. In the honey-bee, light tends to induce activity; darkness, to inhibit it. This response is dependent upon the continuous action of photic stimulation. 2. Isolated worker bees in an active condition exhibit strong positive phototropism when flying or creeping. ‘Temporary sup- pressions of this response may occur, however. 3. Normal bees when creeping in non-directive light usually exhibit pronounced asymmetrical responses of constant or vari- able index. Since essentially the same responses occur in total darkness they are not fundamentally dependent upon photic stimulation. They are probably, therefore, conditioned largely by internal factors. VI. BEHAVIOR OF BEES WITH ONE EYE BLACKENED 1. Directive light ‘The previous investigations of circus movements have pointed unmistakably to the generality of these responses among photo- tropic arthropods. Positive animals with one eye covered tend to circle toward the functional eye; negative animals, under the same conditions, tend to circle away from the functional eye. The honey-bee exhibits a striking positive phototropism. When one eye is blackened, therefore, we should expect the bee to circle toward the remaining functional eye. Such is indeed the case, as Axenfeld (’99, p. 374) has previously shown. In my own experiments, bees thus operated upon were no longer able to creep in a straight course toward a source of il- lumination. Instead, their progress thither was marked by re- peated loops. If the right eye was blackened, the bee looped to the left; if the left eye was blackened, it looped to the right. Moreover, it was possible by blackening one eye, then removing the black and blacking the other eye, to cause a single individual to perform circus movements first in one direction, then in the opposite direction. ore DWIGHT E. MINNICH The above experiment was carried out on five bees. In gen- eral, all of these animals looped more or less markedly toward the functional eye as they crept toward the light source. This tend- ency, moreover, was not confined to the period immediately subsequent to the operation of blackening the eye, as the experi- ment clearly demonstrates. The first records of these bees with their left eyes blackened were taken in the evening between 6 and 7 p.m. No further tests were made until the following day at 11:30 a.m. Yet the behavior at the end of this seventeen- hour period was practically the same as it had been before. One bee, it is true, showed considerable improvement. In the other four animals, however, the two sets of records were indistinguish- able. In the absence of experience, therefore, the performance of circus movements remains a permanent feature of behavior. Of the five bees tested, the most pronounced and uniform exhi- bition of circus movements was displayed by bee no. 5. Its records are almost diagrammatic in their close approximation to the theoretical expectation. Records of this animal are repro- duced in figure 9. Not all of these animals, however, yielded such striking results. Some individuals were found which manifested little or no tend- ency to deviate toward the functional eye, except in the area immediately beneath the lamp, where the illumination was es- sentially non-directive. Thus, bee no. 4, with its right eye black- ened, circled toward the left in the usual manner. But a few hours later, when the black had been removed from the right eye and the left eye painted over, it exhibited little or no tend- ency to circle toward the right (fig. 10, B). The explanation at once suggests itself, that in such cases the eye was imperfectly covered, and hence not absolutely free from stimulation. This may be correct. As will be shown later, however, there are also a variety of other circumstances which might account for such behavior. The tendency to circle toward the blackened eye was not fre- quently encountered in the reactions of bees to directive light. No instance of it occurred in the experiment described above, al- though it was occasionally met with in other experiments. A PHOTIC REACTIONS OF HONEY-BEE ole ° y / Normal | % Right Eye Black I i. Left Eye Black a 5 - \e y Fig. 9 Consecutive records of a bee in directive light, showing the effect of blackening first one eye, then the other. Left Eye Black THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 3 374 DWIGHT E. MINNICH single record of this kind is shown in figure 10, A. This was ob- tained from an animal which subsequently became wholly unre- active. Its aberrant tendencies may, therefore, have been due to an abnormal condition. In any case it is significant that, al- though the bee looped toward the covered eye, yet it progressed toward the light source. Consequently, this was not a case of reversal of phototropism. Instances somewhat similar to the one last mentioned have been described by Dolley (’16, p. 373) for the butterfly Vanessa. —_—_— | slack iE Fig. 10 A. Record of a bee in directive light, showing loops toward the blackened eye. B. Two records of a bee in directive light which showed no - deflection, although the left eye was blackened. Although positive to light, this insect with one eye blackened occasionally turned toward the covered eye instead of toward the functional eye. Possibly results of this sort are to be attributed to the effect of contact stimulus afforded by the covering of the eye, as indicated by Dolley (’16, pp. 394-399). This will be dis- cussed more fully in a subsequent portion of the paper. 2. Non-directive light a. Amount of turning. In non-directive light, bees with one eye blackened tended, in general, to turn more or less pronouncedly toward the functional eye. As was to be expected, the course PHOTIC REACTIONS OF HONEY-BEE 375 taken by the animals under these conditions assumed no specific direction. They either continued to circle in a fairly limited area or proceeded in a looping fashion in any direction whatever. The variability of this response, moreover, was much greater than in the case of the response to directive light. Thus, a num- ber of animals circled almost continuously toward the covered eye in non-directive light, while still others varied, circling some- times toward the covered eye, sometimes toward the functional eye. This was doubtless true for much the same reasons that normal bees also exhibited a greater variability of response in non-directive light. Circus movements attendant upon the elimination of one pho- toreceptor undoubtedly represent the orienting process of an asymmetric animal. The specific photic stimulus, therefore, which produces these reactions must be identical with that which effects orientation in the normal animal. Whatever the nature of this stimulus be, moreover, it is afforded by both directive and non-directive light, since cireus movements occur in either situation. What is the nature of this orienting stimulus? Per- haps the best method of demonstrating the dependence of a par- ticular response upon a certain stimulus is to show that the in- tensity of the response varies with the intensity of the stimulus in question. It seemed possible to attack this problem, therefore, through a study of the relationship existent between the amount of turning displayed by an animal with one eye blackened and the intensity of the illumination to which it was subjected. Non-directive illumination was chosen in preference to direc- tive illumination because of the simpler experimental conditions which the former affords. In directive light, every movement of the entire animal is accompanied by more or less complicated changes not only in the intensity of the stimulation received, but also in the area of the eye stimulated. Asan animal with one eye covered moves toward a light source, the stimulation of the functional eye steadily increases. As it loops toward this eye, however, this steadily increasing stimulus is subjected to rapid and transitory fluctuations. When the animal begins to loop, the functional eye is first turned away from the light, resulting 376 DWIGHT E. MINNICH in a rapid decrease of photic stimulation. As the loop is com- pleted, the photoreceptor in turn experiences an increase in stimulation. In non-directive illumination such as was employed in the present experiments, however, these complications are largely avoided. Photic stimulation here is maintained at a fairly uniform and constant intensity over the entire surface of the compound eye. Two slightly different types of experiment were performed. The procedure in the first type was as follows. Bees were col- lected from flowers in the morning between 8 and 10 o’clock, brought into the laboratory and prepared for experimentation. One and two hours later, respectively, they were given single trials in the directive light area. On a basis of these records, individuals of abnormal tendencies were discarded, and those evinc- ing the greatest accuracy of orientation were selected. An hour or so later, the selected bees were tested in non- directive light of 957 me. Two sets of records, about one hour apart, were made of each animal. Each set was composed of one to several records, the aggregate duration of which, in gen- eral, was between thirty and sixty seconds. An examination of the records showed clearly whether the individual was normally right-handed, left-handed, or variable in its deflection in non- directive light. These results determined which eye should be blackened. If, for example, a bee normally circled to the right, the right eye was covered. Whatever influence was exerted by photic stimulation, therefore, would tend to force the animal toward the left. In this manner, responses which might other- wise have been mistakenly attributed to photic stimulation were to some extent eliminated. The operations of blackening the eye were carried out in the late afternoon of the first day of experimentation, in accordance with the technic previously described. On the following morn- ing, before resuming experimentation, it was not infrequently necessary to discard a few additional animals either because of extreme weakness or occasionally death as a result of the operation. The majority of bees usually appeared quite normal, however, and were subjected to several series of trials in non-directive PHOTIC REACTIONS OF HONEY-BEE BL light. Throughout a single series of consecutive trials, or, as I shall call it, a determination, one intensity of light only was employed. But in the total number of determinations the more intense illumination of 957 me. and the less intense of 24 mc. were used an equal number of times. The animal to be tested was first removed from the dark box and exposed from half a minute to several minutes in the intensity of light in which it was to be tried. This was usually sufficient to activate the animal thor- oughly, and several records were then made in the non-directive light chamber. In case the bee failed to respond to photic acti- vation, recourse was had to mechanical stimulation. ‘The cage was tapped or even shaken fairly vigorously until locomotion was induced. This procedure seldom failed to elicit activity. When it did fail, it was usually necessary to discard the animal altogether. The number and duration of the records comprising a single series or determination varied widely even in the same animal. If the bee quickly encountered the side wall of the light chamber, records were short, and a number had to be taken. If, on the contrary, the animal kept well toward the center of the floor of the apparatus, one or two records were quite sufficient. In cases of great variability of response or unusual departures from the general, expected behavior, additional trials were made, on the assumption that a greater number would more accurately ex- press the average tendency of the animal. Single trials seldom exceeded thirty seconds, and were often much shorter. Occa- sionally, however, records of forty-five seconds, sixty seconds, or even slightly greater durations were taken. The aggregate duration of the trials comprising a single determination, for one intensity of light, was usually in the neighborhood of thirty or sixty or ninety seconds. The adoption of any more uniform period for all animals, at all times, was quite impossible. Upon completion of a series of trials in one intensity of light, the bee was returned to the dark box. Here it was allowed to remain for a period of about fifteen minutes to one hour. In the earlier experiments the longer period was practiced; in subsequent experiments, the shorter. After this period in the dark, the ani- 378 DWIGHT E. MINNICH mal was subjected to a second set of trials of the same aggregate duration as the first, but in the other of the two light intensities. The order in which the two intensities of illumination were em- ployed was varied from time to time. Sometimes the first de- termination was made in the more intense light; the second, in the less intense. Sometimes the reverse order was observed. A single series of records in one intensity of non-directive light together with the corresponding series in the other intensity con- stitute what I shall term a pair of determinations. The protocol of such a pair of determinations on bee no. 42 is given in table 3. Four or five pairs of determinations were usually made on each in- dividual of an experiment in the course of a day, beginning TABLE 3 DETERMINATION FOR NON-DIRECTIVE LIGHT DETERMINATION FOR NON-DIRECTIVE LIGHT or 24 mc. OF 957 Mc. Number of Duration of Number of Duration of racord Hour of record arraerail anon Hour of record cecord seconds seconds 4 1:47 p.m. 30 i 1:32 p.m. 31 5 1:473 p.m. 30 2 1:322 p.m. 30 6 1:48 p.m. 30 3 1:332 p:m- 30 POtAl sweet: asa ee teie a 90 91 between 8 and 9 o’clock in the morning and concluding between 4 and 5 in the afternoon. The bees often seemed to become slug- gish in the late afternoon. Whether this was due to fatigue or a natural rhythm of activity from day to night, I am unable to say. This phenomenon, however, led me to abandon any attempt to continue experimentation much after 5 o’clock. On the third and concluding day of the experiment, the scheme of the second day was again carried out as far as possible. Bees usually survived the first two days of experimentation, and in case they did not, the data on them were discarded. A number of individuals, however, failed to survive in fit condition for the trials of the third day, and still others had to be discarded in the course of the day, although in both cases the results were counted. PHOTIC REACTIONS OF HONEY-BEE 379 Some of the more vigorous animals survived not only a third day of experimentation, but lived on for three or four days, and in a few instances even longer. Although no further trials were made with such bees, they were kept and, as far as possible, records of their subsequent longevity taken. Having described the first type of non-directive light experi- ment in considerable detail, the second type may be described very briefly. It differed from the first only in the method of making pairs of determinations. In this case, the two determi- nations of a pair were made during the same period of time, TABLE 4 DETERMINATION FOR NON-DIRECTIVE LIGHT DETERMINATION FOR NON-DIRECTIVE LIGHT oF 957 Mc. Number of Duration of Number of Duration of record Hour of record record record Hour of record record seconds seconds 1 1:41 p.m. 30 2 1:43 p.m. il 3 1:48 p.m. 44 + 1:51 p.m. 23 5 1:53 p.m. 40 6 1:56 p.m 30 i 1:59 p.m. 30 Ye | ae a eS | 104 | 104 instead of an appreciable interval apart. The bee was first tested in one intensity of light, then within a minute or so in the other intensity, then again in the first, and so on until a series of one to five records had been completed for each intensity. Care was exercised, however, even with this rapid alternation of intensi- ties, always to expose the animal for thirty to sixty seconds in a given intensity before subjecting it to a trialin the same. The following protocol from bee no. 83 (table 4) will illustrate this method of making determinations. In both types of experiment, there were obtained for each bee a number of pairs of determinations, usually four to ten, depend- ing upon the longevity of the individual. The records of each 380 DWIGHT E. MINNICH determination have been computed collectively in the manner already described. Single values have thus been derived which express the average deflection, or tendency to turn, exhibited by the animal in each determination. When the turning was chiefly toward the blackened eye, the sign of these values is negative; when chiefly toward the functional eye, it is positive. If, now, the value of each determination in 24 me. light be subtracted from the corresponding one in 957 me. light, differences will be obtained which should answer conclusively the question of rela- tionship between the amount of turning and the intensity of photic stimulation. The differences obtained in the manner just described I shall designate as d._ A given value of d may be negative or positive. If it be negative, it signifies one of the two following possibilities: 1. The bee turned more toward the blackened eye in an illumina- tion of 957 me. than it did in one of 24 me. For example, in the second pair of determinations on bee no. 32 (table 2), 957 me. 24 me. —13.28°/em. — (—9.22°/em.) = —4.06°/cm. 2. The bee turned less toward the functional eye in an illumi- nation of 957 me. than it did in one of 24 me. For example in the second pair of determinations on bee no. 23, 957 me. 24 me. +6.44°/em. — (+8.92°/em.) = —2.48°/cm. If, however, the value of d be positive, it signifies one of the two following possibilities: 1. The bee turned less toward the blackened eye in an wllumina- tion of 957 me. than wt did in one of 24 me. For example, in the first pair of determinations on bee no. 31, 957 me. 24 me. —3.87°/em. — (—7.14°/em.) = +3.27°/em. 2. The bee turned more toward the functional eye in an tllumi- nation of 957 me. than in one of 24 me. PHOTIC REACTIONS OF HONEY-BEE o81 For example, in the first pair of determinations on bee no. 21, 957 me. 24 me. +29.41°/em. — (+15.84°/em.) = +13.57°/cem. If the great majority of d values are of the first category, viz., negative, we may conclude that the animal experiences a greater impulse to turn toward the functional eye in an illumination of 24 me. than it does in one of 957 me. If equal numbers of nega- tive and positive values occur, there is no relation between the intensities of photic stimulation employed and the amount of turning. If, however, d is generally positive, we may conclude that the tendency to turn toward the functional eye increases if the intensity of photic stimulation is sufficiently increased. Experimentation soon demonstrated that the only satisfactory solution of the problem was to be had through a statistical treat- ment of large numbers of data. Even the more constant animals often varied widely from one pair of determinations to another without any apparent external cause. Therefore, a large num- ber of bees were experimented upon and each individual was sub- jected to many tests, the averages of which were relied upon to indicate the general trend of behavior. In table 2 (appendix) are presented the results obtained from a careful measurement and computation of over two thousand records taken on fifty-two bees. On some individuals as few as sixteen records were taken; on others as many as seventy-four. This difference was due in part to varying longevity of individuals and in part to the fact that more favorable animals were frequently experimented with longer than less favorable ones. The determinations of approxi- mately the first half of the animals were made on the plan of the first type of experiment, while the remainder were carried out according to the scheme used in the second type. From the figures presented in columns F and G of table 2, it is evident at once that there is a marked preponderance of the positive d values over the negative. The ratio of the two is strikingly shown in the frequency polygon (fig. 11) Since the number of d values varied considerably with the individual, due 382 DWIGHT E. MINNICH to the causes noted above, I have included in the polygon only the first four values for each bee, thus giving equal weight to every animal. Of the total two hundred and seven values rep- resented in the polygon, 81.16 per cent are positive, whereas- only 18.84 per cent are negative, a ratio of over 4 to 1. AV EEE AW ZZ AW, 16.14) — 19) —|0)— 8-64 ao (O pip at. 4 6 810° 12) 14-16" 18> 20) 22) 24) 96 Degrees per cm ; Fig. 11 Frequency polygon of the first four d values of fifty-two bees. The negative values are represented by the shaded areas; the positive values, by the clear areas. 6 In the case of one bee it was possible to include only three values for d, because of a missing record. See Table 2 (Appendix), bee no. 45. PHOTIC REACTIONS OF HONEY-BEE 383 The objection might be raised that although the majority of values of d are positive, a number of them are too small to be of any significance. It is true that differences of the order of 1°/em. or less might easily be attributed to slight errors in tracing the course of a bee. Errors in recording, however, are as likely to occur in one direction as the other. Such is not the case with these small values. The class of d values betwen 0 and —2°/cm. contains but fifteen, while the class of 0 to +2°/em. numbers twenty-nine—nearly twice as many. Moreover, the mode of the curve, +2°/em. to +4°/em., lies well beyond these small values. Differences of this magnitude are readily detected in the records of bees which circled constantly toward the functional eye, as the pair of determinations in figure 12 demonstrate. Each record shown in the figure was of exactly thirty seconds’ duration. The first two were taken three min- utes apart in 24 me. illumination, while the third and fourth were taken about twenty minutes later in 957 me. illumination, one minute apart. The value of d in this case is +2.98°/em.— about the average modal value. In bees exhibiting considerable variation in their deflections, however, d values, or of the modal class of even greater magni- tude, are not so easily recognized without the accompanying fig- ures. To illustrate this, I have selected a pair of determinations (fig. 13) approximating the mean value of the frequency polygon, which is +4.35°/em. As attested by the data given in connec- tion with the figure, this animal was extremely variable in its direction of turning. All four records were taken in the brief period of eight minutes, nos. 1 and 2 being of twenty-eight sec- onds’ duration each; nos. 3 and 4, of thirty seconds. The value of dis +4.09°/em.—a fact which does not become apparent until the records are submitted to a careful scrutiny. The data presented in table 2 and figure 11 show clearly that bees with one eye blackened tend to turn more toward the func- tional eye in a non-directive illumination of 957 me. than they do in one of 24 me. The validity of this conclusion is confirmed by still another line of evidence. A certain number of animals were found to exhibit a very constant tendency to turn toward 384 DWIGHT E. MINNICH the functional eye, which was always much more pronounced in the intense than in the weak illumination. In other words, these individuals always yielded positive values of d of rather high mag- nitude. Bees nos. 73, 83, 95, 105, and to a lesser extent numer- ous others afforded striking examples of such behavior. These 24 mc. Ss = vA 4 cae @ 957 mc ie Fig. 12 A pair of determinations of bee no. 42, left eye black, non-directive light. The records are numbered in the order in which they were taken. 24 mc. Light 957 mc. Light Number +Degrees — Degrees Number + Degrees — Degrees o turned turned os turned turned resord urne urne second urne urne 1 900 0 3 1440 0 Pe 945 0 4 1485 0 Average deflection, +9.49°/cm. Average deflection, +12.47°/cm. d = +2.98°/cem. animals were all thoroughly vigorous individuals, surviving not only the three days of experimentation, but living on for at least two days thereafter. Two of these bees, in fact, survived no less than four days after the conclusion of the experiment. In figures 14 and 15 are shown pairs of determinations from two of these animals. The eight records of bee no. 73 (fig. 14) were taken in the course of 28+ minutes, while the six records of PHOTIC REACTIONS OF HONEY-BEE 385 24 me. i / =~ 2 957 me. Se We Dae @ A s- I 3 Fig. 13 light. 24 me. Light ber + Degrees — Degrees record turned turned 2 495 1170 4 270 450 Average deflection, —3.53°/cem. ol = A pair of determinations of bee no. 123, left eye black, non-directive The records are numbered in the order in which they were taken. 957 mc. Light Number + Degrees — Degrees eo 4 turned turned 1 630 0 3 995 720 Average deflection, +0.56° /em. +4.09° /em. 386 DWIGHT E. MINNICH 2 a 6 8 Fig. 14 A pair of determinations of bee no. 73, left eye black, non-directive light. The records are numbered in the order in which they were taken. 24 me, Light 957 me. Light N eer + Degrees — Degrees nes +Degrees — Degrees aay turned turned record turned turned 1 360 90 2 1485 0 3 45 0 Ce 990 0 5 2115 0 6 3510 0 a 2070 225 8 4005 0 Average deflection, +6.07° /em. Average deflection, +13.82° /cm. d = +7.75°/em. PHOTIC REACTIONS OF HONEY-BEE 387 bee no. 105 (fig. 15) required only fifteen minutes. In both figures, each record of the top row is of exactly the same duration as the corresponding one of the lower row, except records 1 and 2 of bee no. 73, which differ by one second. It would be difficult to 24 mc. SS >= | 3 5 957 mc. GO XS : 2 4 § Fig. 15 A pair of determinations of bee no. 105, right eye black, non-directive light. The records are numbered in the order in which they were taken. 24 mc. Light 957 mc. Light Number +Degrees — Degrees Rumiber + Degrees — Degrees record turned turned eartil turned turned 1 1260 45 2 19385 0 3 855 0 4 2430 0 5 1305 90 6 2520 0 Average deflection, +6.74° /em. Average deflection, +14.83°/cm. d = +8.09° /em. imagine more conclusive results than those afforded by these two bees. It might be supposed that animals would be found which would exhibit the opposite of the condition just described. Such, how- ever, was not the case. I failed to find any individuals which continually circled more toward the functional eye in an illumi- 388 DWIGHT E. MINNICH nation of 24 me. than they did in one of 957 me. Bees presenting a number of negative d values, such as nos. 22, 25, 34, 41, 43, 55, 56, 62, 66, 85, 126, and 135, with one exception, showed an equal or greater number of positive values. ‘The exception noted was bee no. 56. Three of the four pairs of determinations obtained on this animal not only yielded negative differences, but differ- ences of large magnitude as well. Bee no. 56, like a number of other individuals presenting a considerable number of negative: d values, varied considerably in its behavior and turned chiefly toward the blackened eye. How far the disturbing factors thus evidenced account for the results is not absolutely certain, since: a number of bees of apparently similar tendencies yielded posi- tive values of d. Certainly, however, there are a number of factors, particularly in the type of experiment under considera- tion, which do interfere with the effect of photic stimulation. Some of these serve to intensify the response, while others tend to counteract or even completely annul it. Without attempting to minimize the significance of these negative data in the least, I believe some of them, probably all of them, find their explana- tion in such factors. If this be correct, the negative results ob- tained lie well within the range of variation which might be expected in experimental work of this sort. A more extended discussion of the factors responsible for variability of behavior in. the present experiments is presented in the next section of this paper. The evidence in general, therefore, seems to warrant the con- clusion that bees with one eye blackened tend to turn more toward the functional eye in an illumination of 957 me. than in one of 24me. This tendency may result in the animal’s actually turn- ing more toward the functional eye, or in its turning less toward the covered eye, depending upon the idiosyncrasies of the indi- vidual. In either case, however, with increased photic stimula- tion, there is an increased tendency toward the functional eye. The nature of the stimulus afforded by the apparatus employed was continuous and of almost uniform intensity, and since the circus movements of the honey-bee vary with the intensity of such stimulation, they must be dependent upon it. ‘These con- PHOTIC REACTIONS OF HONEY-BEE 389 clusions are the exact antitheses of those reached by Dolley (’16) in his work on Vanessa. He says (p. 417): ‘‘Vanessas with one eye blackened do not move in smaller circles in strong light than they do in weak light, unless it is extremely low. On the con- trary, the evidence seems to indicate that the stronger the light is the larger the circles are. These results also are not in har- mony with those demanded by the ‘continuous action theory.’ ”’ I shall return later to a more complete consideration of the theo- retical significance of the results afforded by the honey-bee. b. Rate of locomotion. Although bees with one eye blackened tend to turn more toward the functional eye in a non-directive light of 957 me. than they do in one of 24 me., there is no differ- ence in the rate of locomotion in the two intensities. In table 5 are given the average velocities of thirty-four bees for each of the two light intensities employed. These figures show a con- siderable range of individual variation, from as low as 3.49 cm. per second to as high as 6.77 em. per second. There is, however, no consistent difference which might be attributed to the effect of light. Eighteen of the animals showed a greater velocity in 957 me. illumination; sixteen, in 24 me. illumination. Unilateral photic stimulation of the intensities employed is, therefore, without effect upon the rate of locomotion. 3. Summary 1. Bees with one eye blackened usually loop toward the func- tional eye as they creep toward a source of light. Some indi- viduals are encountered however, which display little tendency to loop, and occasionally an animal will be found which loops toward the covered eye. In the absence of experience, the tendency to loop toward the functional eye remains a permanent feature of behavior. 2. In non-directive light, bees with one eye blackened gener- ally circle toward the functional eye, although a number are found which circle more or less toward the covered eye. 3. In a uniform non-directive illumination of 957 me., the tendency to turn toward the functional eye is greater than it is in a similar illumination of 24 me. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, No. 3 390 DWIGHT E. MINNICH 4. Since the amount of turning varies directly with the intensity of continuous photic stimulation, the turning is produced by this stimulus. 5. Unilateral photic stimulation of the intensities employed has no effect upon the rate of locomotion. TABLE 5 A B Cc D Numperorsen | V8N0oauanr | 957 MC.LIGHT c-B 21 6.10 6.22 +0.12 22 5.10 5.12 +0 .02 23 5.02 5.03 +0.01 24 4.95 9.18 +0.23 25 4.67 4.53 —0.14 31 3.77 4.23 +0.46 32 4.20 4.74 +0.54 30 5.27 aye —0.08 34 4.78 4.99 0-21 36 3.49 3.63 +0.14 41 4.45 4.29 —0.16 42 4.76 4.30 —0.46 43 5.59 5.76 =\-O. 27 44 4.94 4.90 —0.04 45 4.36 4.99 +0.63 ol 4.51 4.40 Sol 52 5.09 5.08 —0.01 53 6.77 6.53 —0.24 54 5.53 5.17 —0.36 50 6.09 5.21 —0.88 56 5.32 5.53 +0.21 62 4.82 4.59 —0.23 63 5.80 5.72 —0.08 66 5.10 5.15 +0.05 68 5.14 5.34 +0.20 72 4.69 4.87 +0.18 73 4.97 5.44 +0.47 77 4.26 3.99 O27 81 4.41 4.32 —0.09 $2 5.25 9.29 +0.04 83 5.22 4.93 —0 20 85 4.91 4.79 —0.12 91 4.48 4.64 +0.16 92 4.49 4.51 +0 02 PHOTIC REACTIONS OF HONEY-BEE 391 VII. VARIABILITY OF PHOTIC RESPONSE The honey-bee is remarkably constant in the strong positive phototropism which it evinces. The course of individuals creep- ing in directive light is a straight path toward the source. Yet, as has been shown, occasional departures from this behavior do occur. In non-directive light, moreover, the responses of normal bees are frequently extremely variable. The animal may turn markedly toward a given side in one trial, and in the next, turn quite as markedly toward the opposite side. Again, the direc- tion of turning may be completely changed several times in the course of a single trial. It is not surprising, therefore, that bees with one eye blackened also exhibit considerable variability of response in both directive and non-directive illumination. In non-directive light particu- larly, there were a number of cases in which animals turned little or not at all toward the functional eye, while there were still others in which they circled chiefly toward the covered eye. In fact, over 25 per cent of the first four pairs of determinations on the fifty-two bees, when averaged, gave negative values. These departures from the more usual tendency, to turn toward the functional eye, sometimes characterized the entire behavior of an individual; again, they appeared only spasmodically. Thinking that some of the results above mentioned might be attributed to a loss of phototropism, either permanent or extend- ing over a considerable interval of time, I frequently subjected the animals exhibiting them to one or more immediate trials in direc- tive light. This, however, failed to show anything which might be construed as a loss of phototropism. The variations of response noted, therefore, must be referred to external and internal factors which are capable of modifying, in a more or less profound way, the dominating effect of unilateral photic stimulation. Such factors are of two sorts, those which are continuously effec- tive and those which are not continuously effective, but fluctuate from time to time. Both types played so considerable a réle in the experiments previously described, that I have felt they mer- ited the somewhat extended analysis presented in the following pages. 392 DWIGHT E. MINNICH 1. Continuous factors a. Temperature and humidity. Of the continuously operative factors, none are more important than the general conditions of temperature and humidity. These profoundly affect the activity of bees. McIndoo (14, p. 279) says: ‘‘Climatie conditions per- ceptibly affect the activity of bees. When it is extremely warm, they are most active and are rarely quiet even for a few seconds. When it is moderately warm, they are less restless, and when rather cool, bees do not move freely.’’ Again he says: ‘‘ During cool weather their movements are quite sluggish, and when the humidity is high they are much less active and respond to vari- ous odors more slowly than when there is low humidity.” Precisely the same effects were noted in the present experi- ments. So serious did they become on several occasions that the experiment had to be abandoned. On cool, damp days bees were apt to be quite unreactive, and prolonged exposure to light often failed to induce locomotion. Considerable mechanical stim- ulation might call forth creeping, but it was of desultory kind and was apparently unaffected by photic stimulation. There can be no doubt, therefore, that general weather conditions consid- erably affect the behavior of bees toward light. Since some experiments were continued under less favorable weather con- ditions, it is quite probable that they account for some of the aberrancies observed. b. After-effects. In making quantitative determinations in non-directive light, trials were frequently made in the two inten- sities in rapid succession. Although the bee was always exposed for at least thirty seconds to a given intensity before making a test, there still existed the possibility that an after effect of the first intensity might influence the behavior of the animal in the sec- ond. Thus Herms (’11, p. 215) has demonstrated an after-effect of photic stimulation in the blow-fly larva, which may manifest itself in the continued orientation of the animal for as much as fifteen to twenty seconds after the cessation of the stimulus. That such is not the case for bees, however, is very clearly dem- onstrated by the following experiment. PHOTIC REACTIONS OF HONEY-BEE 393 Normal bees were allowed to creep on a strip of smoked paper toward the 80-c.p. incandescent lamp. When, after creeping a distance of about 60 em., the bee reached a point 40 em. from the source of light, the lamp was extinguished. Not only was the current turned off, but a screen was simultaneously placed before the lamp, so that even the slight after-glow of the filaments was eliminated. After ten seconds of total darkness the light was turned on and the bee removed. ‘Two to four records were made of each of eleven animals in this manner. In not one case did the bee fail to lose its orientation within at most a couple of seconds after the extinction of the light. Often the loss of ori- entation was almost, if not quite, simultaneous with the cessa- tion of the stimulus. The deviation from the former orienta- tion was sometimes marked by a pronounced tendency to turn toward one side, with the result that the animal crept in circles in the dark. At other times the bee merely wandered, turning first in one direction, then another. Typical records of two ani- mals are reproduced in figure 16. There is, therefore, no after- effect of photic stimulation in the honey-bee, and this does not account for any of the irregularities observed. c. Failure to eliminate photoreceptor. The difficulties in manipu- lating bees necessitate relying upon a single operation to elimi- nate completely the function of the compound eye. Although in this operation great care was exercised to cover the eye com- pletely with a heavy coat of paint, there isa possibility that in a few cases this was not accomplished. Moreover, from the moment the animal began to recover from the anaesthetic, the covered eye was repeatedly subjected to vigorous scrapings by the front leg of the same side. While the examination of a number of animals after experimentation showed that this seldom resulted in a removal of any of the eye covering, it probably succeeded in doing so in a few cases. Occasionally, also, the varnish cracked somewhat on drying. These unavoidable failures to keep the compound eye entirely free of light, undoubtedly modified, to a greater or less extent, not a few of the results obtained. Beside the failure to cover the eye, there is the possibility that the three ocelli of the honey-bee are concerned in its photic 394 DWIGHT E. MINNICH Fig. 16 Smoked-paper records of two normal bees creeping toward a light source (upward in the figure). As the animals reached the line indi sated by the letter P the light was extinguished, Note the extremely rapid loss of orientation. PHOTIC REACTIONS OF HONEY-BEE 395 behavior. In blackening the compound eye I made no attempt to cover the ocelli, but afterward in examining the specimens used, I found that sometimes all, sometimes only one or two, at other times none of these organs had been covered. If the ocelli do exercise any considerable function, therefore, there was involved here a variable of no small magnitude. It is also possible that in bees other portions of the body, or even the entire integument, may be photosensitive. Photoder- matic sensitivity is not unknown among arthropods. It has been reported by Graber (’84) for the cockroach, by Plateau (’87) for two species of blind myriapods, and by Stockard (08) for the walking-stick. In order to find out if these several possibilities were affecting results, I conducted several experiments with bees both eyes of which had been carefully covered with a thick coat of ‘Jap-a-lae.’ On the morning after the operation, each bee was placed in a separate cage in non-directive illumination of 957 me. Although with one or two exceptions the bees were quiet when first exposed to the light, within fifteen minutes all had become thoroughly active, showing clearly that they were not free from the activat- ing effect of the light. At the conclusion of the above test the same bees were individu- ally subjected to trials in the directive light area. Here they looped and turned in a variety of ways, some circling more or less constantly toward a given side, not unlike bees with only one eye blackened. Despite most devious courses, however, they sooner or later managed to work their way to the general region of the source of light. It is quite clear, therefore, that photore- ception had not been entirely abolished, although both com- pound eyes had been covered as carefully as possible. Which of the several explanations advanced accounts for these results, is not certain. I am disposed to believe that the failure to eliminate the compound eyes completely was chiefly, perhaps solely, responsible. However that may be, it is certain that in this, as well as in other experiments, the incomplete suppression of photic stimulation was the source of a large amount of varia- tion in the results obtained. 396 DWIGHT E. MINNICH d. Kffect of contact stimulus. ‘The effect of the contact stimu- lus afforded by the blackening material on the eye and the adja- cent parts of the head must also be recognized as a considerable factor in modification of photic behavior. The influence of this stimulus has been clearly demonstrated by Dolley (’16, pp. 394— 397) on Vanessa antiopa. With one eye blackened, this butter- fly, when creeping in total darkness, turned, with few exceptions, continuously toward the blinded eye. The tendency to circle was often quite pronounced, and showed little or no modification from day to day. The effect of contact stimulus on the covered eye was, therefore, antagonistic to that produced by light on the opposite, functional eye. I tried experiments with the honey-bee similar to those car- ried out by Dolley on Vanessa. Bees with one eye blackened were allowed to creep on smoked paper in total darkness. Un- fortunately, the trials were of such duration that the bee recrossed its course many times. This made it quite impossible to decipher the records, and I have not since had an opportunity to repeat the experiments. It is not unlikely, however, that the effect of con- tact stimulus on the eye of the bee is similar to that demonstrated for Vanessa. If such be the case, it probably accounts for much of the circling toward the covered eye, which was evinced by numerous individuals particularly in non-directive light. e. Asymmetry of the animal. As was previously pointed out, normal bees frequently exhibited a marked tendency to turn more or less constantly toward the right or left when creeping in non- directive ight. Such tendencies are doubtless due to a lack of perfect symmetry on the part of the animal. The asymmetry may be physiological; it may be anatomical. It may consist of a differential sensitivity of the photoreceptors on the two sides of the body, as Patten (14, p. 259) has suggested, or it may be due to an inequality of any two symmetrically located elements of the neuromuscular mechanism. Under the influence of directive light, these idiosyneracies are, as a rule, continually corrected. In non-directive illumination or total darkness, however, they at once assert themselves. Since the eye to be blackened was always chosen so that the effect of photic stimulation would be opposite PHOTIC REACTIONS OF HONEY-BEE 397 to that exerted by natural asymmetry, many failures to turn toward the functional eye are probably thus accounted for. f. Modifiability through experience. The work of Axenfeld, Holmes, Brundin, and Dolley has shown that a number of ar- thropods are able to modify their photic behavior through expe- rience. ‘The same is true of the honey-bee, at least in directive light, as the following experiment shows. Each of a number of normal bees, selected on the basis of the accuracy with which they oriented to directive light, had one eye blackened. On the fol- lowing day those animals which exhibited a more or less pro- nounced tendency to loop toward the functional eye were sub- jected to trials (twenty to twenty-five in number) in the directive light area. Bees which displayed little or no tendency to loop were given several trials to ascertain if their behavior was constant, and then discarded. These animals may have been able to modify their behavior almost immediately, or their failure to exhibit circus movements may have been due to an imperfect covering of the eye, or to the effect of contact stimulus. Of those bees which did perform circus movements, records were taken about every ten to twenty minutes from 9 a.m. to 5 p.M., with the exception of about an hour at noon. Ten bees were thus tested. Four of these animals displayed a steady and marked improvement in the course of the trials. Two others showed some improvement, although considerably less than the first four. Two more of the ten improved for a time, only to regress again, so that while a number of trials near the middle of the series were somewhat modified, those at either end were much alike in the number of loops performed. The last two bees showed practically no improvement, although in one of the ani- mals the tendency to circle was at no time very pronounced. It is quite certain, therefore, that at least some bees are able to mod- ify their responses to directive light through experience. . The records shown in figure 17 afford a striking example of this modifiability. Although in its first trials the animal looped re- peatedly as it crept toward the light, it was subsequently able to reach the light by a nearly straight course. This animal, how- 398 DWIGHT E. MINNICH | ee I t ; } | 5 pi | 9 x I ~ i . ew ieee / 13 | 16 17 \ 19 | Qt is | Fig. 17 Records of a bee with the left eye blackened, in directive light, show- ing modifiability of behavior through experience. Alternate records from a series of twenty-six are shown in the figure. 25 PHOTIC REACTIONS OF HONEY-BEE 399 ever, as well as the others, usually circled again toward the func- tional eye upon reaching the non-directive region near and directly beneath the lamp. The records shown in the figure do not include this region. Dolley (16, p. 402) states that he observed some modification from day to day in the behavior of Vanessa in non-directive light. The following evidence seems to indicate that the same is true, to at least some extent, for the honey-bee also. In directive light, individuals with one eye covered were sometimes observed to begin to swerve toward the functional eye, only to check them- selves by a sharp turn in the opposite direction. Correcting movements of this sort sometimes occurred repeatedly in a single trial, with the result that the animal reached the source of light by a much more direct course than it would have otherwise been able to follow. Precisely the same sharp turns away from the functional eye were occasionally seen in non-directive light also. It seems probable, therefore, that modifiability through experi- ence affects the behavior of bees in non-directive as well as in directive hght. 2. Fluctuating factors The variables which have thus far been discussed are those which continuously or progressively affect the behavior of bees throughout an experiment. They probably account in a large measure for such phenomena as the persistant turning toward the covered eye in certain individuals or the apparent lack of any tendency to turn at all in still others. They do not, however, explain the many sudden changes cf behavior which were ob- served. Such variations are dependent upon factors of behavior which fluctuate from time to time. om w or for) oO © won OoOnrnN for) on _ OOo ~I si SH Ooonwn nN HK KH & 420 DWIGHT E. MINNICH XI. APPENDIX (TABLE 2)—Continued A B Cc D E F G. H I NORMAL | SCHEMA ONE EYE ONE EYE DURATION| DURATION seaten | ren cx, | "ren Ok. eee ott” [ann chy [ ore >| ore =>] OMNI | OnDe IN 957 uc. | 957 wc, |TCRNED 24 |ruRNep 957, ord | ord | “y4\yc. | 957 mc. LIGHT LIGHT , y LIGHT LIGHT 77 4.22 PEO Al | ae eae) a7) 57 75 7.46 + 9.62] +16.59| 6.97 8s 65 | S218 52 8439 ee. 86 48 40 “1 243) ee 4 ye (et 104 80 58: 4h) (id 402) (Ab 6.47 90 80 +11.64 | +16.89 | 5.25 120 130 +12.60 | +15.38| 2.78 75 70 +15.29 | +17.98 | 2.69 40 35 S157) S236) te 6.78 80 80 81 2.09 3491) | TONGO! lt 6.89 114 117 2.59 + 1.03 | + 3.98 2.95 53 53 S26. 03) Peo b a7) Melero 116 106 + 5.76| + 5.86] 0.10 108 101 oS 30) esas) nea 05 70 65 +459|}+ 9.89] 5.30 56 61 SB N03) ene On len 2 4G 63 63 et ANO 1) Se 3e5 9: he Beal 57 80 a2 76) easel narod 110 122 a2 3) 71) Ae 12860! sees 67 67 82 Bret |) 2553104) Goss: peaivel 124 119 0265 [22556377 (8501 Or 36 110 123 = 10-30 eG r4st ee oes 60 60 S46 105 tbe et eee) ye 2507 59 59 =) 2-50) Ree b1e4o: ee AO 66 64 £03020) Gaus 7s yer 74 85 #1706) | W882 46h 9252 54 56 2516140) eo 20n| elect 70 80 =*)/0'7S0)| a5 waz: 4.67 | 108 105 83 3.59 + 5.62] +15.93 | 10.31 81 76 5.85 Seales ee ree 90 90 a2 19980) |02-10-53. (Oe 64 104 104 + 6.93 | +11.49| 4.56 71 ma +12.84 | +21.53.| 8.69 58 60 39-300 04-17 -47 || 8508 60 60 + 4.93 | +16.39 | 11.46 94. 90 He 4153 02-18 50) | 797 41 33 42°3.52) | 2414642" | 10°00 133 128 PHOTIC REACTIONS OF HONEY-BEE 421 XI. APPENDIX (TABLE 2)—Continwed A B c D E F G H I NORMAL NORMAL DURATION] DURATION wonsen | ben tn. | ponsn, [BEACK Av.‘|anack Av.‘[+ vanoms|— vauopa| Oxo" | Guoatin oy eae Bh eae OE ace TURNED 24 |TURNED 957 ord ord oot ak Snitcoe LIGHT LIGHT MOSULCHy WE TARGHEE LIGHT LIGHT. 85 0.84 | + 6.46] +12.52] 6.06 47 46 01930) =e dealers 7a 781g. 37 113 117 +.7.08 | + 3.74 3.34 75 82 + 6.87 | + 6.42 0.45 80 108 + 4.34] + 3.54 0-80) 2Gaa| | 23 + 1.73] + 3.63| 1.90 64 74 AG) 48.19 |) 3.58 137 123 PEO eevee || Sales 94 94 Ae A. G8 FA 7.99. 3.31 71 58 91 0.48 =yD3 42) fat 4098 2.56 | 125 125 6.99 105059); 04. 00; |) | Gs05ql0- 85 80 6.44 P= Grn) 6039 95 95 27.33. | 3305, | 4.28 81 81 EAS ety 3152, \h)) tas 110 131 + 0.73} + 6.69] 5.96 64 64 +10.19 | +27.54] 17.35 30 30 92 105 664| 404042, W577 | 4.35 113 104 8.67 | + 4.28] + 9.86] 5.58 58 62 +7.41|/+9.19| 1.78 94 85 + 9.64 | + 7.64 2.00 63 61 + 7.49 | +17.34| 9.85 52 52 S74 19 |) 4223182 |) 19.63 60 60 93 0221) Se eleoih ee) Gr4en ie 487 120 120 2.44 Seen A2. een o23 99 99 2587 e046 3.33 63 63 + 2.37 | + 2.92 | 0.55 98 87 95 220) Ped. st As CON 76, 118 123 6:52 |) =F 113 | 23:07 |) 1.94 100 109 0.00 | + 5.67] 5.67 58 46 + 0.32} + 6.58] 6.26 71 80 =f 11,29) = aise WP 13.53 70 78 + 0.93 | +11.94| 11.01 34 30 2.39 ERTS +4. 79 69 74 13:67 | eigetl | 3.44 - 66 67 + 0:85] + 6.35] 5.50 82 75 APAO: Dk Peer OS ale -Al)-72 61 60 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO.3 422 DWIGHT E. MINNICH XI. APPENDIX (TABLE 2)—Continued A B Cc D E F G H I NORMAL NORMAL DURATION |DURATION womenn | eee AY. | BBE AY. lnzack av."|Buack av.°|+ vations |—vanuus| OF EEC | OF REC. OF BEE |To RIGHT] To LeFT | PER CM: PERCM. | OFE— D | OFE—D | gsmconps | SECONDS ON ce. || ahtaro. || SUR 2 ERNE” O57 (ict Rit s Aa ce | Meee LIGHT LIGHT : a LIGHT LIGHT 96 2.76 | +10.46 | +19.08 8.62 90 92 DRO4 + 9.29 | + 8.50 0.79 195 192 +11.89 | +15.99 4.10 60 60 +14.09 | 117.84 3.75 60 60 + 5.33 | +10.15 4.82 35 29 101 7 5 AE NV — 52255, 2.92 62 63 6.10 | — 1.67 | — 2.07 0.40 88 79 — 2.09 | — 0.61 1.48 60 59 — 1.02} — 1.00 0.02 67 70 — 3.04, 14.— 1-58 1.76 46 49 102 3.67 — 1.58 | +14.37 15.95 49 49 5.30 + 2.98 | +15.02 12.04 50 54 + 3.75 | +11.08 Tse 71 78 — 2.23 | + 1.45 3.68 . 62 66 — 0.32 | + 6.46 6.78 64 53 — 3.38 | +11.11 14.49 Or 58 — 1.98 | + 4.52 6.50 41 60 — 2.23 | +10.19 119422 88 88 + 3.55 | +13.35 9.80 95 95 — 1.28 | +12.23 1B} ck 70 60 103 0.00 — 2.46 | +13.48 15.94 Yi Lf 6.34 — 4.29 | +11.25 15.54 Te 60 + 0.16 | + 8.49 8.33 60 69 — 1.26 | +11.36 12.62 61 61 — 0.44 | + 2.15 2.59 84 84 — 3.75 | +11.04 14.79 60 60 + 3.89 | + 6.47 2.58 60 65 + 2.27 | + 6.99 ASD 63 68 — 0.18 | + 0.22 0.40 105 104 + 0.86 | + 4.22 3.36 62 64 105 5.93 + 8.46 | +14.64 6.18 68 60 8.81 + 9.19 | +13.78 4.59 50 60 + 6.89 | +14.10 eal 80 80 + 6.55 | +15.20 8.65 60 60 + 6.74 | +14.83 8.09 93 93 + 6.21 | +12.73 6.52 60 60 +10.34 | +16.05 evil 60 60 PHOTIC REACTIONS OF HONEY-BEE 423 XI. APPENDIX (TABLE 2)—Continued A B c D E F G H I NORMAL NORMAL ONE EYE ONE EYE DURATION| DURATION wuper | renew, | Penca, |2EACK AV."| BLACK Av.°/+ VALUB|~ VALUES) Qooeiw | OnDs IN OR ae Pome. 037 ue. TURNED 24 |TURNED 957} ord ord gree OT ae LIGHT LIGHT BeAtle LEME Est PAE LEANER LIGHT LIGHT 105 410.03 | +17.05 | 7.02 60 60 + 9.04 | 11.01 | 1.97 100 | 100 746 | E1268 | 10 5.22 163 | 163 106 | 6.43 23118 ee EAS | 2, 67 70 68 1.71 +E OAL 8.22 | 9.2.78 81 79 + 2.79 | + 6.72] 3.98 41 41 — 1.49} + 5.19] 6.68 35 35 + 0.59} — 5.49] 4.90 ie 77 + 0.53 | + 5.33] 4.80 103 | 115 fel |) -as6i + 2.96 | +24.04] 21.08 60 60 6.87 | -F11.57,| £19.71 |): 8.14 60 60 + 3.13 | 415.11 | 11.98 92 88 + 1.91 | 412.12] 10.21 87 91 — 4.71 | 417.50 | 22.21 60 60 oe ee 2e3t + 1.88 | + 8.96] 7.08 69 73 2.93 + 3.65|+3.77| 0.12 18.) |) sik + 3.25 | + 1.99 1.26] 90 85 — 4.39 | + 6.50 | 10.89 56 55 123 17.86 | + 7.16 | +18.55 | 11.39 54 60 3560) eet ou) et 4.00| 65 69 —10.76 | + 0.59 | 11.35 69 67 = 8.08 | + 2.29 | 10.37 79 79 Ss e6t | = 2 ak 4g 60 60 =) 3:46 | “ 1.12)|) 4.58 140 | 135 — 3.25| + 5.69] 8.94 60 58 — 3.53] +0.56| 4.09 58 58 Sr tere |) Ve 90 | 101 124 2.59 | + 3.88) +11:18| 7.30 73 68 3.00| + 3.94) + 6.45] 2.51 53 59 + 2.54 + 8.38] 5.84 79 71 410.36 | +11.19| 0.83 80 80 +11.04 | +17.61 6.57 90 90 +10.39 | +17.41 7.02 60 60 4+ 5.81 | 412.54] 6.73 58 58 + 6.55 | + 8.95] 2.40 74 60 + 7.67 | +10.19 2.52 60 60 424 DWIGHT E. MINNICH XI. APPENDIX (TABLE 2)—Continued A B (o D E F G H I NORMAL NORMAL - DURATION |DURATION sompnn | DEEAY,” | BEEAY” | tack av.°|tace av.°[+ vanues | vanes | OF REC | OF REC. OF BEE | TORIGHT| TOLEFT | »oawep 24 [roRNED $57/ ond | ond | SECONDS | SECONDS LIGHT LIGHT. WAG EINES BIC -7LIGHE, LIGHT LIGHT 126 6.51 |. S0a%30) 22 sti | a4350 105 100 1.19, |) 4051 |. Sfs0 7.40 48 50 TS | GL 14.) Oa 78 77 SAA 93 37 | oA eT 45 45 1063) @P 4 41] SLOSS 107 107 + 4.57 | + 3.65 0.92] 148 153 5h | Se Age 1.19 72 72 = 2.98 | = 2.26] 0.72 83 83 Aikég | 4-873 | eHiog 25 28 133 2.82 9/99 12-— 3s 4) tartaric > oxalic = malonic, while at 0.0001 N the order is tartaric > malonic > formic > oxalic. This change in order is indicated by intersection of the curves. There is a similar though less important change at the next concentration, where the curve for HCl crosses that of salicylic and the tartaric curve crosses those of malonic and formic. With Euplotes change in concentration produces less variation in order, but instead a considerable variation in the degree of difference existing between the acids. Thus at 0.0005 N HCl formic and benzoie acids kill in 14, 23, and 3 minutes, re- spectively, while at 0.0001 N the relative times are 9, 40, and 35 minutes. Obviously, if conclusions were based solely upon the results for a single concentration, 0.0005 N for instance, they would not be valid, since the results at other concentrations are in so many respects different. This conclusion agrees with the results pbained by Crozier (’16) in studying the rate of penetration of acids into the mantle cells of Stichopus, which contain a natural indicator. He found that the relative order obtaining at a concentration of 0.01 N was in many respects unlike the order at 0.001 N, and that the order of penetration found by Harvey (’14) and by Haas (’16) at 0.01 N with different material closely resembled his results at 0.01 N, but not his results at other concentrations. If other KEY TO GRAPH SYMBOLS 1. Hydrochloric ———— 8. Citrie ——|—— 2. Formic —|-|——|-|— Oe Maloney see sce 3. Acetic ———<— 10; Lartaric———o10— 4. Propionie —-|——<|— Ws TONG 5. Butyric ——~—-— 1A, Elon oe he) >} ===> 6. Lactic —— - —— 13. Salicylic — — — — — 7. Oxalic o—— TOXICITY OF ACIDS TO CILIATE INFUSORIA 447 Nx 163 50 20 10 7 Graph 1, A, B_ Toxicity of equinormal acids at 20°C. (Euplotes above, Paramoecium below.) Should be N-* and not N-3, 448 M. HE. COLLETT TABLE 1 Effect of temperature on toxicity of equinormal acids. Time in minutes N/ 12° Paramoecium NOE ea aekt es Pe 20° Pein Speech ase S 70 Aver Aa Un 20° Stylonichia Euplotes Lect aee > cea —- 0.001 0.0005 0.0002 0.0001 0.00008 0.001 0.0005 0.0002 0.0001 0.00008 0.001 0.0005 0.0002 0.0001 0.00008 0.0002 0.0001 0.00008 0.0002 0.0001 0.00008 0.001 0.0005 0.0002 0.0001 0.00008 0.001 0.0005 0.0002 0.0001 — noe ®e OXALIC No) 2 Hi 10 40 TARTARIC 14 oo MALONIC 11 20 iil 90 1 2 FORMIC 12 3l 22 BUTYRICc! 30 33 e | 4 8 3 7\| <3 HO) 3 lay, || até! 5 20 70 | 10 48 3 | <3 1a) || 2 35 7s 40 | 25 8 15 6 17 5 o) || 14} 63 23] <3 10 12 Bo 4 60 | 25 8 2 45 3 100 | 22 TOXICITY OF ACIDS TO CILIATE INFUSORIA 449 TABLE 1—Continued a Se | eof enclue, vam ieee tA) rl eed me i aE Mi lla co ° a = iS < 2 a ai (| 0.001 10 15° 0.0005 | 11 129) 17) O20) ot) keh FS eet ea ReL main | iA 60 le ne 0.0001 | 45 21 (| 0.001 tien 2 O000S aa Om Ia MIs! 5 babe 5S || 5 | Para- moecium caudatum > Euplotes charon > Stylonichia pustu- lata > Urostyla grandis > Euplotes patella > Vorticella. Additional evidence of specific differences is presented in the sections following. Although several experiments upon the cilia of other organ- isms have been reported previously, it is almost impossible to compare them, for they were made by different observers under widely varying conditions. Thus in Barratt’s experiments (’04, 452 M. E. COLLETT 705) upon Paramoecium aurelia, the acids were not used consist- ently at the same concentrations, the temperature varied from 16° to 22°, and the method of washing injured the organisms. Weinland (94) studied the oral epithelium of the frog at 18°, but made up his solutions at 0.001 M in physiological NaCl. Harvey (14), in studying the cilia of the giant clam, used acids made up to 0.01 N in van’t Hoff’s solution and worked at a temperature of about 27°. We do not know in these cases whether the salts antagonize the acids (as was found by Loeb, 12, and by Osterhout, ’14) or whether they reinforce its action (as found by Paul et al., ’10), and consequently are not sure how far these experiments are comparable with other experi- ments in which the acids were made up in distilled water. Neither are Koltzoff’s studies (14) upon the cilia of Carchesium available for comparison, for, unlike the previously mentioned experiments, they deal with concentrations which do not kill the cilia, but only increase their viscidity. There are, however, reports upon other material capable of showing specific differences clearly. The experiments of Land- steiner (713) on acid agglutination of blood corpuscles and of Walbum and of Cunningham (’16) on haemolysis give clear evidence of specific differences in resistance. Ritter (712) and Clark (96) found striking differences in resistance among vari- ous mold spores, as did Heald (’96) with seedlings. Taylor (17) found that the concentration of any acid necessary to clear a wound of bacteria varied with the organisms concerned and that the toxic order likewise varied. This is precisely the condi- tion observed in the present experiments. Similar results have been obtained by Kopazcewski (’14) with enzymes. His obser- vation that the optimal Ps was not the same for his usual prepa- ration of maltose and for a well-dialyzed preparation indicates the importance of salts in determining acid resistance and may perhaps lead to an explanation of certain specific differences. TOXICITY OF ACIDS TO CILIATE INFUSORIA 453 C. Temperature In order to find out something of the nature of the toxic action, acids were tried at several temperatures between 10° and 30° as is indicated in table 1. The conditions are more readily seen in the curves shown below. In almost every case an increase above room temperature produces an. increase in toxicity both to Paramoecium and to Euplotes and a decrease below room temperature generally produces a decrease in toxicity. If the length of life at, let us say, 10° is divided by the length of life at 20°, the figure so obtained (the temperature coefficient for 10°) can be used as a measure of the degree of influence exerted by these temperatures upon the toxicity of the acid. Table 2 gives the coefficients found for Paramoecium and Euplotes. It will be observed that the coefficient for any one acid is not the same at all temperatures nor at all dilutions. For example, benzoic for the range 20° to 30° has coefficients of 2 at 0.0002 'N and 3.88 at 0.0001 N, and for the range 10° to 20°, coefficients of 3 and 2.1, respectively. Often the more dilute solutions have higher coefficients than the more concentrated. Itis also remark- able that the temperature coefficient for any given range is not the same for different acids even at equinormal concentra- tions. Thus at 0.0002 N the coefficients for the range 10° to 20° are salicylic 1.5, hydrochloric 2.2, benzoic 3, malonic 2.5, etc. All this would indicate that the mode of action of dif- ferent acids is by no means the same and that the action of any one is markedly influenced by concentration and by temperature. Neither do the various acids affect Paramoecium and Euplotes in precisely the same way; if the coefficients for the two organ- isms be compared at corresponding temperatures and concen- trations of the same acids, one finds that they are almost. never the same. It is impossible at present to interpret these results fully, but some few conclusions suggest themselves. As is well known, a coefficient of from 2 to 3 for every increase of 10° generally indicates a chemical reacticn, while a coefficient of less than 2 or over 4 1s frequently associated with physical processes. Most THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 3 454 M. E. COLLETT TABLE 2 Temperature coefficients PARAMOECIUM EUDPLOTES N/ 12-20% | 15-25° | 20-30° | 12-20° | 15-25° | 20-30° 0.0005 | 4.3 . D7 ey 0.0002 | 2.7 1oselpas66 1.9 paar, ii ue ise cakes O,0001" | 259) 92:50) 18) 1.0) Abele 0.00008 22 18 Gene {| 0.0005 | 3.3 val 12 CORLL eae accents Bie | 0.0002 1.9 1.0 0.0005 | 2.8 12° |) 13" eG ee 0.0002 |2°2" Ors Was7) | 10! Biola eso HUGE a7 aoa eeanmeeanylPortad (ar iae ely o 0.00008 6.0 0.0005 | 2.4 1.38 1.62 Malone) 208. Se ee 2s || 02000227 | 322) UO za) A Oel LHOF re atia OL0OOL iE sho 25. lel en 0.0005 | 1.8 24 pe ee 00002" )$S2270) is aie 4 IO onan Prt EIS natal hordionaallioks ae 0.00008 2.5 0.001 | 2.5 2.0 | 0.41 mes Ncabicvey bets Tt. tele tes | OP OOO r lgout 0.55 2.2 0.0002 | 2.2 OL000 7 Fl F788 0.36 3.1 BUttyrie we Ratcas thas ee oe 0.0005 | 1.88 0.60 22 0.0002 | 2 0.0005 | 2.8 1.6 gales 0.0002} /3¥9° S25) 20 SOs ais sae 0.00008 2.88 0.0002 | 1.6 12 Salicylic icq ae 00002: 2. de les Ob AO a TONG eae || 0.00008 3.84 ' Coefficients for 10° calculated from observations at 12° and 20°, except in the case of acetic and butyric acids, which were used at 10° and 20°. TOXICITY OF ACIDS TO CILIATE INFUSORIA 455 of the coefficients for a change of 10° obtained with Paramoe- cium lie between 2 and 3, whether the temperature range is 10° to 20° or 20° to 30°, so chemical reactions would seem to be im- portant. Euplotes also has coefficients between 2 and 3 for the range 20° to 30° which are to be explained in the same way. At lower temperatures, however, the condition for Euplotes is different. Over therange 15° to 25° and 10° to 20° the coefficients are generally little more than unity. A few coefficients of less than 2 were observed over this range for Paramoecium, but were decidedly the exception, instead of being the rule as with Euplotes. These low coefficients suggest complicating physical Temp: °C. 20 Minutes. 10 20 30 40 Graph 3, D Effect of temperature upon the toxicity of hydrochloric and of butyric acids to Euplotes. A. HCl, 0.0005 N; B. HCl, 0.0002 N; C. Butyric, 0.001 N; D. Butyric, 0.0005 N. factors perhaps masking a chemical reaction. Especially remark- able are the results obtained with Euplotes in acetic and butyric acids. LENGTH OF LIFE IN MINUTES COEFFICIENTS 10° tbe 20° 30° 10-20° 20-30° eee OROOUIN( Coco oss 7 10 17 6 0.41 2.8 d N@LOOO5 Noo. si2 224). 25 20 45 20 0.55 IoD tenets OROOMRIN Gree oss. 9 2 2h 8 0.36 Sil tr OTOOUS NS 2... peas 35 55 25 0.60 pi. 456 M. E. COLLETT In the other acids (formic, tartaric, HCl, ete.) lowering the temperature either lessens the toxic action slightly or leaves it quite unaffected. Butyric and acetic, however, are more toxic at 15° than at 20° and still more toxic at 10°. Indeed, they are almost as toxic at 10° as at 30°. If they consistently become more toxic with falling temperature throughout the range 30° to 10°, this might be explained as due to rate of adsorption, since this is a process favored by low and diminished by high temperatures. But this cannot be the case, since at 20° they are less toxic than at 30°. Probably some further processes are involved, such as solubility, etc. A striking example of this sort has been described by Hans Meyer (quoted by Lillie, 716). The solubility of ethyl alcohol and chloral hydrate is increased by rise in temperature, while the solubility of salicylamid and monoacetin is decreased, and corresponding with this difference in solubility is the fact that the narcotic power of the former group is increased by rise in temperature, that of the latter decreased. Such conditions apparently complicate my results. It appears from these experiments that temperature defi- nitely influences the toxic action of acids. The degree of influ- ence, however, varies with the acid, its concentration, the organ- ism, and the temperature chosen. These irregularities show that the action is by no means simple and that it probably involves both physical and chemical factors.’ D. Importance of the H ion The general order of toxicity obtaining in equinormal solu- tions gives some indication of the mode of action. Full data are given in table 1 and graph 1. The order runs: HCl > dibasic 1 Lillie (17) found that long exposure to sea-water at 2° to 6° would suffice to initiate development in starfish eggs, and that the rate of activation in butyric acid is greater at 6° than at 8° to 10°. These results are not strictly parallel with those obtained in the present experiments upon cilia, in which the lower temperatures (10° to 15°) increase the effectiveness of butyric acid, although alone they are not effective; but in both series of experiments Doctor Lillie’s explanation seems applicable, viz., that a change in the physical condition of the structural colloids due to cold may alter their permeability or other properties and so affect their resistance to certain reagents. TOXICITY OF ACIDS TO CILIATE INFUSORIA 457 and hydroxy acids > fatty acids except formic. This is roughly the same as the order of dissociation and indicates that the H ion is important. But if the H ion is the sole factor in deter- mining toxicity, the order of toxicity and the order of dissocia- tion should agree perfectly, which, as the following table shows, is not the case. Toxicity and dissociation Toxicity AT 0.0002 n DISSOCIATION CONSTANTS (SCUDDER) Paramoecium Stylonichia Euplotes Salicylic Salicylic Salicylic HCl HCl HCl HCl Salicylic Axe lOme Formic Tartaric Formic Oxalic 30) S¢ MOF Lactic Lactic Citric Lactic (ray SX lOm4 ee ae Oxalic Formic 1.96 X 10-4 Tartaric Formic Benzoic Tartaric eo. { Malonic Lactic Malonic 1.64 X 10-3 Benzoic \ Oxalic Phthalic Benzoic 6.64 XK 10- Acetic Phthalic Tartaric Citric Spo Ome Phthalic Acetic Malonic Acetic 1.85) xX 10m® Citric Citric Acetic Propionic 1.42 X 10> Butyric Butyric “Prop Butyric Weal x< lor Valeric Valeric Valeric Valeric We S< Ore Still clearer evidence should be given by solutions of the vari- ous acids at the same H ion concentration, for if the H ion is the only toxic factor, they must all be equally toxic. Solutions were therefore made (without buffers) of all the acids in the series at four of the following concentrations, viz., Pu 3.5, 3.85, 4.0, 4.15, 4.75. The concentrations were determined by means of indica- tors tetrabromphenolsulphonphthalein and methyl red (Clark and Lubs, 717) in the usual way by comparison with acetic acid Na acetate standards. The method is not perfectly exact, but upon determining the Px of a serie& of equinormal acids (0.0005, 0.0002, 0.0001 N) both colorimetrically and by calculation from the dissociation constants the indicators prov BE to be accurate to within about 0.1 Pa. The results obtained by this method are indicated in table 3 and graph 4. It will be seen at a glance that the toxicity of different acids of the same Ps is by no means identical. ‘This 458 M. E. COLLETT L P 3.5 385 4,0 415 474 Graph 4, E, F Toxicity of acids of equal Py at 20°C. (Euplotes above, Paramoecium below. ‘Time given in minutes.) TOXICITY OF ACIDS TO CILIATE INFUSORIA 459 TABLE 3 Toxicity of solutions of equal Py at 20°C. Time in minutes ° e a 1) oD 1) 2 g SHzabe ke | 2.) ven] 2 log .| Brew saich asl [ieee || Stall th] a a 5 8 a allel Pulses Se | So laa ek | aol eal eales (ia do) aleozitele 5h So | 22°)" 29 | ) eis et salicylic > formic > dibasic and hydroxy acids > acetic and butyric; but in solutions of equal Ps acetic and butyric become equal in toxicity to the cyclic acids, and the dibasic and hydroxy acids together with hydrochloric become least toxic of all. Formic, which in equinormal solution is more toxic than the other fatty acids, probably because of its greater dissociation, becomes less toxic than the rest when the solutions have the same Ps. Somewhat the same conditions hold with Euplotes. In solutions of equal Ps salicylie retains 460 M. E. COLLETT Order of toxicity PARAMOECIUM PARAMOECIUM 0.0005 n 0.0001 n Px 3.5 4.0 Salicylic Salicylic Valeric Propionic Hydrochloric Hydrochloric Butyric ee Formic Tartaric Propionic Benzoic (tea eee Acetic eee Benzoic Malonie Formic | Butyric lOvatie Formic Oxalic Valeric Malonic Oxalic Hydrochloric Formic Acetic eae faearee Hydrochloric Butyrie Butyric Malonic Tartaric eet: Oxalic EUPLOTES EUPLOTES 0.0005 n 0.0002 n PH 3.5 4.0 Salicylic Salicylic Salicylic ae eee ee HCl Butyric Benzoic Benzoic Formic beret Butyric HCl Oxalic Acetic Propionic Oxalic Benzoic Formic Acetic Tartaric Tartaric Malonic . HCl Malonic Malonic (aa oe E Acetic Acetic HCl | Citric Butyric Butyric Citric Formic Oxalic Malonic its position at the top of the list, the other cyclic and the fatty acids (except formic) stand next, while the hydroxy acids and hydrochloric are much less toxic than the rest. Evidence of like nature has been obtained in experiments upon very different sorts of material. In every case the relative efficiency of equi- normal solutions failed to parallel their H ion concentration exactly, and solutions of equal Px were always unequal in effect. Only one conclusion is possible. The H ion is an important factor in the physiological effects of acids, but at least in organic acids some other factor or factors are involved which makes them more effective than their Px alone would lead one to expect. TOXICITY OF ACIDS TO CILIATE INFUSORIA 461 These experiments with solutions of equal Ps also give evi- dence of specific differences in resistance. The order of resist- ance to every acid 1s Paramoecium > Stylonichia > Euplotes > Vorticella. There is considerable evidence of specific difference in resistance to H+ among other organisms, such as bacteria (Michealis, ’14, 715; Kemper, 716) it is noteworthy, however, that here the limit of tolerance of a particular species is not the same in all acids (Wyeth, 718). This is true also of Paramoe- cium; thus, a concentration of Ps 3.5 kills Paramoecium in four minutes in HCl, five minutes in tartaric, less than one minute in valeric, etc., and so with the other organisms. The order of P, G G. 4.75 Minutes. 10 2050 $30) 9401) 50) 60! 7770 80 Graph 5, C Relative resistance of Paramoecium, Stylonichia, Euplotes, and Vorticella to equal Px of hydrochloric acid. Temperature 20°C.; time in minutes. toxicity of the different acids at the same Px also is slightly dif- ferent for each organism (table 3) as is the case with equinormal solutions. It will be remembered that Kopacweski (714) found the same condition in enzymes. E. Importance of anion and molecule Although there is ample proof from many sources that the effect of an acid is not determined solely by its H ion concentra- tion, there is very little evidence indicating exactly the part played by anion and molecule. One way of approaching the matter is to compare the relative toxicity of a series of acids 462 M. E. COLLETT with the relative toxicity of their salts. I have not experimented with salts, but there are interesting data to be had from other experiments. The order of toxicity to Lupinus seedlings (Kah- lenberg and True and True, ’96) is as follows: Acids equinormal, salts equimolecular. Acids: HCl > benzoic > salicylic > formic = propionic > butyric. Salts: Na— benzoate > salicylate > formate > propionate > butyrate > chloride. The order for equinormal acids is almost the same as that obtained in my experiments: that is, HCl is more toxic than benzoic, and benzoic in turn is more toxic than the fatty acids. Since there is little difference in dissociation among the salts, their order, unlike that of the acids, is a measure of the relative toxicity of the anions. Salts of the cyclic and of the fatty acids are more toxic than NaCl and must therefore have more toxic — anions. ‘These are salts of the very acids which in solutions of equal ionic concentration proved to be most toxic to cilia. Practically the same conditions hold in the haemolysis of blood corpuscles (Fiihner and Neubauer, ’07, and Hoeber, 710). Acids: HCl > formie > acetic > propionic. Salts: Na—Salicylate > benzoate > formate > acetate > butyrate. The similarity between these experiments with equimolar salts and my experiments with acids in solutions of equal Ps makes it seem probable that the acids which are most toxic in these solutions owe their effectiveness to the anions as well as to the Hion. The fact that a different order of toxicity obtains in normal solutions may perhaps be explained as follows: If an acid is not highly dissociated, even though it has a slightly toxic anion, it is léss toxic than a more highly dissociated acid with a non-toxic anion (for instance, butyric as compared with HCl). If, however, the anion is very toxic, as is the case with salicylic, the acid in spite of its slighter ionization may equal or even exceed a more completely ionized acid such as HCl in toxicity. Another method of investigating this point has been suggested by Kloeman (14). To various concentrations of a weak acid a TOXICITY OF ACIDS TO CILIATE INFUSORIA 463 fixed amount of HCl is added; this depresses the dissociation of the weaker acid and produces a solution containing chiefly the . organic acid molecule, Ht and Cl-. If the anion of the organic acid is toxic, this mixture should be less toxic than the original solution, provided the H ion concentration is not greatly altered. Klocman found that the acetic anion was toxic, but did not try the method with other acids. In the present experiments the method was somewhat modified and was applied to a num- ber of organic acids of the same concentration (0.0002 N). The P, of each solution was first determined by the use of indicators, then enough HCl was added to increase the Px slightly, by a known amount when the toxicity of each of these mixtures had been determined HCl was again added, increasing the Ps decid- edly. With each addition the ionization of the organic acid was depressed further, until finally most of it was in molecular form and the H ions were derived chiefly from the HCl. By deter- mining the toxicity of the acids alone and again after each addi- tion of HCl it was possible to follow the effect of the decreasing dissociation of the acids and at every stage to compare them with HCl of the same H ion concentration. The figures obtained in this experiment are recorded in table 4 and expressed graphi- cally in graph 4. It will be seen at once that some of the acids become less toxic as the H ion concentration is increased, formic lactic and acetic, for example, in the experiments with Paramoecium. This means that as the anions decrease in number the acid be- comes less harmful. In other cases, addition of HCl increases the toxicity, but only until it is equal to that of HCl of the same Ps. Thus phthalic by itself is more toxic than HCl, but as ionization is depressed approaches it more closely; that is, In graphic form, the curves are at first distinct, but as Ps is increased through the addition of HCl the phthalic approaches the HCl curve and finally coincides with it. The same is true of oxalic, benzoic, citric, acetic, propionic, butyric, valeric. In these cases, too, the anion must be toxic, since depression of ionization does away with the greater toxicity of the organic acid as compared with HCl of the same Pu. Since these acids 464 M. E. COLLETT 35 385 4.0 4-75 Minotes 5 10 15 20 25 30 35 Min. 40 50 60, 70 80° 60 100 Graph 6, H,I Effect upon toxicity at 20°C. of depressing ionization. (Para- moecium above and Euplotes below.) TOXICITY OF ACIDS TO CILIATE INFUSORIA 465 when in molecular form are not more toxic than HCl of the same Pu, it seems probable that their molecules do not exert a toxic action. . The curves for Euplotes present a slightly different picture. Certain acids (oxalic, malonic, tartaric) become more toxic as ionization is depressed. It will be noted that these are all dibasic acids. They are, however, so nearly like HCl that it is difficult to draw any definite conclusions from the results. With TABLE 4 Effect of depressing ionization Pe LENGTH OF LIFE IN MINUTES ACID Alone Plus HCl Paramoecium Euplotes I II III ity 8 arf) iar I II Iil Hormichees ess s568 3.95 | 3.9 3.87 54 3 10 25 Pale ||. 555 INCE LICA I aber ews tld 3.97 | 3.85 22 23 14 60 90 | . 60 I2MOVOMOMIOS ne cone eo 4.7 3.97 | 3.6 30 21 6 60 100 12 IBTUN AIG se hae do eis ol) Cee Be MN adakets) 30 743) 13 70 110 Vallericees es sel AAG Set |] Bitsy 45 25 13 70 105 60 Salicyligeje se o.siu we 3.6 3.49 2 L 3 14 BONANG. déscocnueanll GOs || Bre I Ss ian 12 11 45 70 40 phGhalivekes ss 4.6 3.95 | 3.85 22 18 14 70 60 80 EV CUIG Acree cee Ooo 3.87 | 3.87 7 20 50 60 OS Gai ee ea ee se) 3.6 9 8 40 15 Mialoniciaes casas: 3.95 3.85 10 11 85 65 ITENAUENENOS co oooc eae ol] Oey 1 Be) 3.87 9 9 9 60 45 ( CHTLTE CS a aeaerenaas Yd: Sa | 3.95 | 3.6 25 20 6 60 100 25 the first addition of HCl the toxicity of many of the other acids is decreased (lactic, formic, acetic, propionic, butyric, valeric, citric, benzoic), proving that their anions must be toxic. Indeed the decrease proceeds so far that the mixtures of these acids with HCl become much less toxic than HCl of the same Px alone. Phthalic does something of the same sort, viz., first becomes equal to HCl and then falls far below it in toxicity. When the second lot of HCl is added to some of the acids (ben- zoic, citric, acetic-valeric) a curious result is obtained. The toxicity, instead of being decreased as by the first addition, is 466 M. E. COLLETT sharply increased, until the mixture again becomes equal to pure HCl. This is indicated though much less clearly in the Paramoecium curves for citric and acetic, propionic, ete., but not for benzoic. Until further work is done I cannot with assur- ance offer a definite explanation of this condition, but the results so far suggest that the molecule is to some extent capable of antagonizing the action of the H ion. Thus as dissociation is depressed and the anion is removed from the sphere of action, antagonism between molecule and H ion may cause the mixture to become less toxic than HCl of the same Ps, and this antag- onism may cease to be important only when the H ions largely out-number the molecules present. From this series of experiments we find that the following anions are toxic. ‘To Paramoecium: formic, acetic, propionic, butyric, valeric, benzoic, phthalic, lactic, oxalic, malonic, tar- taric, citric. To Euplotes, all of these except oxalic, tartaric, © lactic, and possibly malonic. These findings agree with the conclusions suggested by the previous experiments, viz., that the anions as well as the H ions are sometimes toxic, and that the same acid need not act upon different organisms in exactly the same way. F. Nature of the toxic action At higher concentrations (0.0005 N) Paramoecium discharges trichocysts, and as it coagulates turns rapidly opaque without great swelling, but at 0.0002 N the swelling is pronounced and no trichocysts are discharged. At death the protoplasm be- comes granular and the nucleus stands out sharply. Vorticella and Euplotes, too, become granular and in all the vacuoles grow large and rigid before death. The cilia themselves swell and become sticky, the beat grows irregular and slows until finally the cilia dissolve (see also Koltzoff on Carchesium). Stylonichia swells and some of its vacuoles increase in size, then anteriorly, at a point near the edge, the protoplasm dissolves and releases large apparently insoluble droplets. The cilia stop only when the body is completely disintegrated and must there- fore be more resistant than the rest of the cell. Careful obser- TOXICITY OF ACIDS TO CILIATE INFUSORIA 467 vations under high power, such as Greeley made, of the changes induced in the physical state of the protoplasm might throw light upon the nature of the effect of various acids upon the tissue of different organisms. 7 It is certain that the H ion is exceedingly important in the swelling of muscle (Loeb, ’98, no swelling in hypotonic salt solution unless acid is also present) as well as in the swelling of other hydrophilous colloids, such as fibrin, gelatin, etc. (Loeb, 19; Proctor, ’16; Fischer, 718). It seems probable that the H ion is also important in the swelling of cilia, perhaps the specific differences observed are due in part to differences in the colloids present. Another factor seems to be surface tension. Working with salts, Clowes (16) found that slight changes in surface tension produce great changes in the physical state of oil-soap emul- sions, and he suggested that the physiological effects of these salts were due to changes induced in the protoplasmic emulsion. There is evidence in other experiments of profound changes in the surface tension of protoplasm induced by acids. Ham- burger (13) found that ingestion of India ink or charcoal par- ticles by phagocytes was depressed in too high concentrations of fatty acids, but was stimulated in more dilute solutions, and this stimulating action he attributed to changes in surface tension. Koltzoff (14) used Carchesium (a colonial relative of Vorti- cella) for the same purpose, and found that at some concentra- tions acids increased the ingestion of ink particles, and at shghtly higher concentrations produced visible evidence of change in surface tension in the accumulation of ink particles on the cilia. The optimum concentration for this softening effect as well as for stimulation of phagocytosis varied: with the acid used. Another factor in toxicity is lipoid solubility. Benzoic acid is much more lipoid-soluble than the fatty acids and should therefore penetrate a lipoid rich membrane and attack the cell contents more rapidly. The fact that benzoic is more toxic than valeric to Euplotes but not to Paramoecium suggests the pres- ence of some lipoid in Euplotes which is not present in the same 468 M. E. COLLETT concentration in Paramoecium. In many experiments upon other material benzoic has been found very effective, and simi- lar specific differences in susceptibility have been observed which suggest differences in lipoid content (Harvey, ’14; Crozier, 16; Haas;:716zshboebs/13)e Peters and Burres (’09) conclude from their experiments upon the toxicity of Cu salts to P. aurelia that toxic effect is due to injury to an essential enzyme and not to direct chemical injury of the protoplasm. If the normal metabolic processes of the cell are interrupted, as they would be by the failure of an im- portant enzyme, it is obvious that the chemical and physical balance of the whole cell would be affected. Possibly something of the sort may be involved in the toxic action of acids upon the cilia of Paramoecium, Euplotes, etc., and may account for the specific differences involved. This explanation is of course purely hypothetical at present. There are many other possible factors in toxicity, but conclusions are difficult and uncertain the moment one ventures beyond very simple and obvious comparisons. . SUMMARY It is found that the relative toxicity of a series of acids varies decidedly with the concentration, and therefore it is unwise to base conclusions as to mode of action upon results obtained with only one concentration. The fact that power of penetra- tion also varies greatly with concentration makes it probable that the same is true of many of the physiological effects of acids. ‘There are also great differences in the effects of acids upon different species: even organisms so closely related as the infusoria used in these experiments show characteristic differ- ences. When tested in the same solution, one species may be two, four, or even twenty times as resistant as another; and in addition the order of toxicity of the series of acids is somewhat different for each species. Another factor in determining rela- tive toxicity is temperature. Ordinarily toxicity increases with increase in temperature and decreases with decrease in tem- perature, but the degrees of influence exerted by temperature TOXICITY OF ACIDS TO CILIATE INFUSORIA 469 varies with several factors—the species used, the acid, and the concentration. The acids are unequally affected by different temperatures. The coefficients for Paramoecium and Euplotes are scarcely alike at any point. Most of the coefficient of 10° for Paramoecium lie between two and-three and suggest a chemi- cal reaction. The coefficients for Euplotes, however, are rarely so high and agree better with the idea of a physical factor. Indeed, with butyric and acetic, though not with any of the other acids tried, a decrease in temperature below 20° accelerates the toxic action upon Euplotes, so that their toxicity at 10° is nearly as great as at 30° and much greater than at 20°. A comparison of the acids at various equinormal concentra- tions shows that a rough parallelism exists between toxicity and degree of dissociation, as would be expected if the H ion is the most important factor in toxicity. When, however, the acids are compared in solutions of equal Px, it is evident that other factors must enter, for the toxicity of the various acids is mark- edly different. The order of toxicity, unlike that of equinormal acids, is closely similar to the toxic order of salts of these acids. This suggests that the anions of at least some must be toxic. Additional evidence of the toxic action of certain anions (mainly of the fatty and the cyclic acids) is afforded by the action of acid mixtures, in which the ionization of the organic acid is progres- sively diminished by addition of a strong acid, HCl. All the acids used bring about swelling followed by precipita- tion of part of the cell contents and by solution of the cilia. There is clear evidence of change in surface tension, for the cilia always become sticky before they stop beating. It seems prob- able that the toxic effect of the various acids depends upon their solubility in the tissue as well as upon capillary activity and the changes in colloidal state wrought by the H ion. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 3 470 M. E. COLLETT CONCLUSIONS 1. The order of toxicity of a series of acids varies with the concentration, the temperature, and the species. The action is therefore not simple. 2. The H ion is an important factor, for the toxie order of equinormal solutions is roughly parallel with the order of dissociation. 3. The H ion is not the only factor in toxicity, for in solutions of equal Px the acids are not equal in toxicity. 4. Depression of ionization shows that the anions of certain acids are toxic to both Paramoecium and Euplotes, viz., formic, acetic, propionic, butyric, valeric, citric, benzoic, phthalic, salicylic. The anions of oxalic, tartaric, lactic, and malonic are toxic to Paramoecium, but not to Euplotes. 5. The temperature coefficients indicate that both chemical and physical reactions are probably concerned in the toxic effect of acids. 6. A most marked irregularity is shown by acetic and butyric acids, in that their toxicity to Euplotes (though not to Para- moecium) is greatly increased by temperatures below as well as above 20°C. TOXICITY OF ACIDS TO CILIATE INFUSORIA 471 BIBLIOGRAPHY Barratt 1904 Die Wirkung von Saéuren und Basen auf Par. aurelia. JZ. Allgem. Physiol., Bd. 5. 1905 Die Addition von Séuren und Alkalien durch lebendiges Proto- plasms. Z. Allgem. Physiol., Bd. 5. Cuark, J. F. 1899 Toxic effect of deleterious agents. Bot. Gaz., vol. 28. CuarK-Lusss 1917 Colorimetric determination of H*t concentration. J. Bacteriol., vol. 2. Ciowres 1916 The protoplasmic equilibrium. J. Physical Chem., vol. 20. Crozier 1916 Cell penetration by acids. J. Biol. Chem., vol. 24. 1917 Sensory activation by acids. Am. J. Physiol., vol. 45. Cummina 1916 Specific differences in haemolysis with acids and alkalies. dj, Lsabis IDWS sills ake} Date aND Mines 1911 Action of acids upon skeletal muscle. J. Physiol., vol. 42. FiscHeR AND Hooker 1918 Swelling of gelatine in acids. J. Am. Chem. Soc., vol. 40. FUHNER UND NEUBAUER 1907 Haemolyse durch Substanzen homologer Reihe. Arch. Exp. Pathol. u. Pharm., Bd. 56. GREELEY 1904 Paramoecium and electrolytes. Biol. Bull., vol. 7. Haas 1916 Permeability of plant cells to acids. J. Biol. Chem., vol. 27. HAMBURGER UND DE Haan 1914 Wirkung von Fettsiuren auf die Phagozytose. Cent. Physiol., Bd. 29. Harvey 1914 Permeability of cells to acids. Intern. Z. phys.-chem. Biol., Bdeis Heratp 1896 Toxic effect of dilute solutions of salts and acids. Bot. Gaz., vol. 22. Horser 1910 Der Einfluss einiger organischen Alkalisalze auf Muskeln. Pfliiger’s Arch., Bd. 134. KAHLENBERGER AND TRUE 1896 Toxic action of dissolved salts and acids. Bot Gaz., vol. 22. Kemrer 1916 Precipitation of bacteria by a definite Py. J. Inf.-Dis., vol. 18. Kiocman 1911 Kinetik der Giftwirkung von organischen und unorganischen Saiuren. Dissertation, Miinchen. Kourzorr 1914 Phagozytose von Charchesium. Intern. Z. phys.-chem. Biol., Bd. 1. Kopaczpewski 1914 Die Affinitiitsreihe und biologische Wirkung der Sduren. Intern. Z. phys.-chem. Biol., Bd. 1. LANDSTEINER UND PraAsek 1913 Siureflockung der Blutstromata. Z. Immun., Bd. 20. Lituin, R. 8. 1916 Theories of anaesthesia. Biol. Bull., vol. 30. 1917 Temperature coefficients in the activation of starfish eggs by butyric acid. Biol. Bull., vol. 32. Lorn, J. 1898 Physiol. Untersuchungen iiber Ionenwirkung. Pfliiger’s Archiv, Bd. 69, 71. 1913 Artificial parthenogenesis. Chicago. 1917 Membrane permeability to electrolytes. J. Biol. Chem., vol. 32. 1919 Amphoteric colloids. J. Gen. Physiol., vol. 1. 472 M. E. COLLETT Lops unp WasTENEYs 1912 Entgiftung von Saéuren durch Salze. Biochem. Z., Bd. 39. Micuartis 1914 Wasserstoffionenkonzentration. Berlin. 1915 Siureagglutination von B. typhosus. Deutsch. med. Wochen- schrift, Bd. 41. OsteRHOUT 1914 Effect of acid upon permeability. J. Biol. Chem., vol. 19. Pav, BrrnsTein, Reuss 1910 Kinetik der Giftwirkung geldéster Stoffe. Biochem. Z., Bd. 29. Peters AND Burres 1909 The diastatic enzyme of Paramoecium and the killing concentration of CuSO, J. Biol. Chem., vol. 6. Proctor AND Witson 1916 The acid-gelatin equilibrium. J. Am. Chem. Soc., vol. 109. Rirrer, G. E. 1912 Die giftige und formative Wirkung der Sauren auf die Mucoraceen. J. wissenschaftliche Botanik, Bd. 52. Roar AnD WurtLey 1906 Action of acid and alkali on the tadpoles of R. tem- poraria. Biochem. J., vol. 1. Scupprer 1914 Conductivity and ionization constants of organic compounds. Van Nostrand. Szirr 1906 Sdureintoxikation. Pfliiger’s Arch., Bd. 115. 1909 Vergiftigung mit anorganischen und. organischen Sdauren. Pfliiger’s Arch., Bd. 130. Taytor 1917 Specificity of antiseptics. Lancet, vol. I. Trur 1900 Toxic action of acids and Na salts. Am. J. Sc., vol. 9. Watpum 1914 Bedeutung d. Py fiir Haemolyse. Biochem. Z., Bd. 63. WEINLAND 1894 Chemische Reizung des Flimmerepithels. Pfliiger’s Arch.,: Bd. 58. WrnocrRADOFF 1911 Beitrige zur Kenntnis der physiologischen Wirkung der organischen Sduren. Dissertation, Miinchen. Wreru 1918 Effect of acids on B. coli. Biochem. J., vol. 12. Uncer 1916 Toxic action of acids and alkalies on fish. Chem. abstr. 10. Ath. « 4104 h d | i ? av ry sy piawade me hale L i T eee) eocelatvitels . oe tet ee aI B A[IIey oT Idyrep erclzZ ©) (© Oar Ia[[Vug ® SSO ® oe l-ze 7 |‘woe| «cP CP Se aw) ecce 91 . I store @ shah seta ameeateperatetersi i) geeemeteh tee VI Té 9} 0€ “9C| 62°98 | 8 °99q) 1z ‘20q 9¢ “99d $6 2M | FZ OEM |" 99 | ZZ 99] 1 00d] OZ ‘20q LL. 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J. 496 8& 913 OL 0» OL += OCI “448 auoN OG | 0 GEG 02, |= O01 O) <8 = OF J ‘uve OG ‘FI “448 Hol 1 O OL + O8T “448 ~1d Op O &1 + OST (qyoBe Apoq Te 2°q@| 0s eG Lt + LUE Gl "348 ‘Id 0% Ov + 001 (O) |! = 006 66 99d 8 § I! 8 OL +886 | OPE |FOSE |FO0E | 0G | E9T Apoq ‘ [ sur (peop -UIB{U00 Auvul) Ou or!! +81 +0Z OL |+*09 +OV |G “448 ‘id OT | “49% Oks SHOAL TO eo tO) 1h = 06 +06 |*OOI |*06 +09 | 98 “448 448 “el || Sail ey Ons OM O& +OOT | +O0OT |+*O0T |+O00T SAVE) sei OG Oks + OE +08 |*08 |+*0S +09. (87 8299 | 12°98 | 9599) GzP) FZ °C | E490 STIVWINY 40 HAaWON LV 9T 8 v 8 V GT V OT V G6 (99 | TS “99M "erst s" STBQIUUBD [BJO], -IUUB)-UOU d[SUIS [BJO], GUOLTAO ssvw Z 2190} U2 SD SpIW]ap LaYyIQ, “pappv som wnrpau ysaif pun fiywp fo unvup som asay wnypowu ay J, °g ainqynagns fo qe pun ‘pg ‘Te ‘VE saunqjna sspw apys-uorssaidap fo fisojsry] € HIaVL AN AMICRONUCLEATE OXYTRICHA 497 supplied with fresh culture medium or not. From this time on, however, since no fresh medium was added, both the supply of food became scanty and the excretion products of the animals accumulated, a balanced relation of the animals and their envi- ronment was reached, and little or no further multiplication by fission took place. Just at the time this condition was reached, pairs were observed sticking together and cannibals began to appear. In all cases pairs and cannibals were isolated as soon as observed. In the second subculture cannibals appeared on the same day. It will be seen that the appearance of cannibals in both the first and second subcultures is due essentially to the same cause, since in the latter case the medium was changed daily and the removal of waste products plus the added food supply gave a more rapid increase of the animals. This rapid increase, which in turn tended to bring about a state of balance rapidly by the using up of the food supply and the increase of excretion prod- ucts, is most marked in mass cultures 2B and 2C. In mass culture 2A an accident on the seventh day led to the loss of over half the animals. The sudden drop in division rate in mass culture 2D I am at a loss to explain. Mass cultures 2B and 2C were most instructive. In these cultures there was an initial rapid multiplication. Just after this period of multiplication had reached its climax, but before a state of equilibrium had been reached, for numerous divisions by fission were still taking place, cannibalism occurred and the animals showed also a strong tendency to become united in pairs. Addition of fresh culture medium prevented a permanent balanced state such as that in the first set of cultures with the result that cannibal formation proceeded steadily until, as in the case of 2B, 20 per cent of the animals on the slide were cannibals. The divison rate for subcultures 3 (see curve on p. 501) and 4 show that in the former set for the first five-day period repro- duction took place more rapidly. This seemed somewhat sur- prising in view of the results obtained by Woodruff (11) in studying the effect of excretion products on the division rate of Paramecium. Evidently in the present study excretion products 498 J. A. DAWSON did not become a factor influencing the divison rate until after the first five days. During the second and third five-day period the divison rate of subculture 3 is distinctly lower than that of subculture 4. Here, no doubt, the increasing excretion products had a depressing effect. In each line of subculture 4 the animals seemed at all times perfectly typical. In mass culture of subculture 3, however, on December 26th, two animals were present on the slide when it was examined. One, as usual, was removed. On the 27th, upon examination it was found that this animal had divided to give two and that these were united peristome to peristome. On the 29th they were more firmly joined and died that night. In this case, since all animals but one had been removed daily it would hardly seem that lack of food had brought on this ‘mis- cible’ state, since animals in set 1 were much more numerous on the depression slides and were still dividing, though slowly, but rather that the excretion products of the animals themselves were the underlying factor. This experiment indicates clearly that cannibalism does not take place while the culture medium is comparatively fresh and also that the greatest amount of cannibalism does not occur in a medium in which the food supply is much depleted, but in one in which the scarcity of food is just beginning to be felt. Thus it seems probable that the accumulation of excretion products plays a part in inciting cannibalism. Jennings (’10) stated that the cause of conjugation was ‘‘a decline in the nutritive conditions after a period of exceptional richness that has in- duced rapid growth and multiplication.”” These conditions have in this case been duplicated with the result that instead of conjugation cannibalism occurred. 4. The physiological effect of cannibalism On December 10, 1917, three apparently typical animals were isolated from three separate stock cultures coming from the original culture A. On December 11th, each animal had divided | twice, giving four animals, and these were isolated to form the AN AMICRONUCLEATE OXYTRICHA 499 four lines of subcultures, 1A, 2A, and 3A. These subcultures were carried with daily isolations until December 26th. On this date two new subcultures, C2 and C3, of four lines each were begun from three cannibals isolated from five-day-old stock cultures of 2A and 3A, respectively. On December 31st two cultures, Cl and CN1, of four lines each were added. The ancestors of these cultures were a cannibal and a non-cannibal, respectively, isolated on the preceding day from a six-day-old stock culture of subculture 1A. As the curves, averaged for five-day periods, showing the division rates of the non-cannibal subcultures (1A, 2A, and 8A) and of the cannibal subcultures (C1, C2, and C3) were essentially identical in each set, they have been combined in figure 2. The curve shows that the division rate of non-cannibal subcultures (1A, 2A, and 3A) was highest during the first three five-day periods and that there was a considerable drop in the division rate, i.e., from 2.4 divi- sions to 1.4 divisions per day, during the next period, while the average division rate for the cannibal subcultures was con- siderably higher, i.e., 2.2 divisions. These cannibal subcul- tures maintained a consistently higher division rate until Janu- ary 20th to 25th, when it dropped below that of the non-can- nibal subcultures for the first time. From this time on till the end of the cultures the division rates are very similar. It should be noted that 3A and 2A died out on February 18th and February 23rd, respectively, whereas subcultures C1, C2, C3, and 3A lived until shortly after March Ist. Subcultures CN1 between January 10th and January 20th had a higher division rate than any of the others, but this rapidly dropped until February 9th, when the subculture died. The result of this experiment on cannibalism seemed plainly to indicate that animals descended from cannibal ancestors have a higher initial division rate as compared with animals descended from non-cannibal ancestors. This experiment also indicated the possibility, since their life in cultures was some- what longer, that cannibal progeny are more hardy than prog- eny of non-cannibal animals. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 29, NO. 3 500 J. A. DAWSON A second experiment was carried out in a somewhat similar manner. On January 1, 1918, from the three existing lines (3A, 3C, 3D) of subculture 3 (see former history of this sub- culture on pp 494, 497) were spread out to form three new sub- cultures, 3A, 3C and 3D, of four lines each and, similarly, from the four lines of subculture 4 (pp. 494, 497) four new sub- cultures, 4A, 4B, 4C, and 4D, were begun. These subcultures were carried on with daily isolations until January 22nd. The division rates are shown graphically in figure 3. No cannibalism had occurred in any of the lines of subcultures 3 and 4 of which the subcultures now being considered are lineal Fig. 2. Comparison of cannibal (broken line) and non-cannibal (continuous heavy and light lines) subcultures. December 12, 1917, to March 1, 1918. Con- tinuous heavy line is a combination curve showing, in five-day periods, the average division rate for non-cannibal subcultures (1 A, 2 A, and3 A). Dotted line shows a similar curve for cannibal subcultures (C1, C2, and C3) beginning December 26, 1917. Light full line shows the division rate, averaged for five- day periods, for the four lines of the non-cannibal subculture CN1 which died on February 4, 1918. At A the four lines of subculture C3 died (February 19). At B (February 23) the four lines of subculture C2 died. Methods of plotting same as in figure 1. AN AMICRONUCLEATE OXYTRICHA 501 descendants, and it did not occur on any of the slides of the present subcultures in which daily isolations were made. Can- nibalism did occur, however, in stock slides of four or more days standing in each of the twenty-eight lines of these sub- cultures. deem weenene 0 Fig. 8 Comparisons of cannibal (broken line) and non-cannibal (continuous line) subcultures begin at points B and C in this figure, which represents graphi- cally the complete history (December 20, 1917, to February 26, 1918) of sub- cultures 3 and 4 (section 3) and of all subcultures of the second experiment in this section. The curves in this figure, with the exception of those drawn with light continuous and broken lines, show combined division rates of several subeultures. The broken line from beginning to A (January 2, 1918) represents the division rate of subculture 3; from A to B (January 22, 1918), the averaged division rates of subcultures 3A, 3C, and 38D; from B to the end of the curve, the division rates of the cannibal subcultures 4A—-C, 4A-1C, 4B-C, and 3C-C. The continuous line to A shows division rates of subcultures 4; from A to B, the averaged division rates of subcultures 4A, 4B, 4C, and 4D; from B to the end of the curve the same for the non-cannibal subcultures 44—N, 4A-1N, 4B-N, and 3C-N. The broken line (light) from C (February 9, 1918) to the end of the curve gives the average division rate for subculture 4A—C1, while the continuous line (light) from C to the end of the curve shows the same for subculture 4A-N1. Methods of plotting the same as in figure 1. 502 J. A. DAWSON For the purpose now of making a comparison of cultures of cannibal and non-cannibal animals, eight new subcultures were begun from these subcultures as follows. On January 22nd from stock slides of four days standing four cannibals were selected—two from subculture 4A to begin subculture 4A-C and 44-1C; one from subculture 4B to begin subculture 4B-C, and one from subculture 3C to begin subculture 3C-C. In all cases as soon as the animal used to begin the respective subcultures had divided twice, four lines in each subculture were estab- lished. Non-cannibal animals, from the same slides from which the cannibals were taken, were selected to form the four lines each of subcultures 4A-N, 4A-1N, 4B-N, and 3C-N. The curve shows that the average division rate for the cannibal cultures (heavy dotted line beginning at B) was higher, 1.40 as compared with .93, than the average division rate for the non- ‘annibal cultures (continuous line beginning at B) for the first five-day period, but that otherwise little difference can be noted. On February 9th (continuous and dotted lines beginning at pt. C. in fig. 3) two subcultures, 4A-C1 and 4A-N1, were begun from a cannibal and a non-cannibal, respectively, selected on the previous day from a six-day-old stock culture of 4A-C. The difference in division rate here is not so striking as in the sarlier part of the experiment, but the cannibal line is slightly higher during the first full five-day period, and this difference increases in the favor of the cannibal line during the rest of the life of the culture. On February 26th these series of subcultures were discontinued. The general result of these experiments shows that the effect of cannibalism is to produce an initial higher division rate in the ‘cannibal lines. This higher fission rate may be due merely to the extra nutrition supplied by the ingested animals, in view of the fact that Joukowsky (98) also found a distinctly higher divi- sion rate in cultures of Pleurotricha fed on the ciliate, Uronema, in hay infusion, than in those kept in infusions of hay, flour, or albumin alone. Since, in this case, however, animals of the same species formed the food in question, there is a possibility, which should not be overlooked, that a more fundamental meta- AN AMICRONUCLEATE OXYTRICHA 503 bolic change occurs than is implied by mere nutrition, consider- ing that, in the present case, there is an addition of elements derived from similar cytoplasmic and nuclear constituents. 5. DISCUSSION The most recent researches have seemed to show conclusively that, though ciliates may exist in an apparent amicronucleate state at certain periods of their life-history, sooner or later a micronucleus or chromatin equivalent to that contained by a micronucleus takes on a definitive form. Studies on the ciliates, Opalina, Trachelocerca, Ichthyophthirius, and Blepharisma, have revealed that their amicronucleate condition exists only during part of the life-history. However, in all the Hypo- trichida studied to date a micronucleus has invariably been found as a normal cell constituent, although both temporary or per- manent disappearance of this organelle have been described under certain conditions. Thus Maupas (’88) stated that the micronucleus in pathological cultures of Onychodromus grandis disappeared entirely. R. Hertwig (’89) questioned this state- ment and suggested that Maupas might possibly have over- looked the presence of the micronucleus, since he himself had often found great difficulty in observing micronuclei in Paramecium. Calkins (’02) made the positive statement, based on his study of Paramecium, that he believed the micronucleus was present at all times in the hypotrichous ciliates. Woodruff (05) found that in a culture of Oxytricha one of the two micronuclei—the posterior—was not present in indiviuals from the 361st to the 369th generation. On the other hand, Popff (’07), working with Stylonychia, found that during depression periods the micronuclei increased in number. Lewin (711), working with the same form in regeneration, also found an increase in the number of micronuclei. Baitsell (’12) carried a culture of Sty- lonychia for 572 generations and found that micronuclei were present in non-conjugants, conjugants, and ex-conjugants at . all stages. 504 J. A. DAWSON The production of an amicronucleate race of Paramecium has been claimed by Lewin (710), who obtained, by cutting, an amicronucleate fragment which regenerated and produced the ‘amicronucleate race.’ This race reproduced by fission for nearly two months and was normal in every respect except for the absence of a micronucleus. It is also interesting to note that LeDantee (97) stated that, in the case of a ciliate (un- named, with an elongated macronucleus and a single micro- nucleus, a merozoite containing a portion of the macronucleus only regenerated a micronucleus. The results of both these investigators were published in short preliminary papers and, pending more substantial proof or confirmation by other workers, must be regarded as interesting possibilities rather than as established facts. The form described in this paper differs from all previously described hypotrichous ciliates in that it has never at any stage in its life-history, since it has been under observation, contained a definitive micronucleus. This statement is made as a result of long-continued and careful cytological study of many preparations of this form throughout the various stages of its life-history during a period of over two years. As already stated, in other groups than the hypotrichs in which no micronucleus is visible during the vegetative stages it was invariably, during sexual phases of the life-history, found to be present in more or less close connection with the macro- nucleus, so that the usual dimorphic nucleus was represented apparently during the vegetative condition by a macronucleus alone, i.e., the macronucleus thus was an amphinucleus contain- ing both tropho- and idiochromatin. In this form, therefore, since obvious but abortive attempts to conjugate occurred fre- quently, there is ground for considering that the nucleus is an amphinucleus, representing both the tropho- and idiochromatic phases. It is believed by the recent investigators (Woodruff, ’05, 713; _ Baitsell, ’14) who have done extensive experimental work on the life-history of the hypotrichida, that these forms will live indefinitely without conjugation or artificial stimulation, pro- AN AMICRONUCLEATE OXYTRICHA 505 vided an entirely suitable environment be secured. The amicro- nucleate species, Oxytricha hymenostoma, has lived continu- ously under laboratory conditions for over two years. During a considerable portion of this time (July 10, 1917, to April 30, 1918) it has been under continuous observation either on depres- sion slides or in small mass cultures in Petri dishes with the possibility of unobserved conjugation or cannibalism precluded. At the present time (April 30, 1918) the animals in these cul- tures give every evidence of being able to live indefinitely. The conclusion, therefore, in this case again, is that the only requisites for the continued existence of this form are favorable environmental conditions. The same conclusion is reached from experiments in which every effort was made to induce conjugation. Although no true process of conjugation has been obtained, there is every reason to believe that the organisms have frequently been in a general physiological condition similar to that of conjugat- ing animals. The entire absence of any of the usual nuclear phenomena attendant upon conjugation has confirmed the belief that this form not only possesses no micronucleus, but also is apparently lacking in the chromatinic material necessary for carrying out the process of syngamy. The continued existence of such an organism indicates, therefore, that conjugation though usually taking place in all other hypotrichous forms, may be entirely dispensed with without loss of viability. In cases where syngamy has not been observed there are three abstract possibilities (Minchin, 712): first, that it occurs, but has not been seen; secondly, that it is in abeyance; thirdly, that it is primarily absent, i.e., has-never occurred in the life- history of the form. The fact that syngamy is of such general occurrence in the protozoa renders the first of these possibilities the most probable. In this case, however, there is every reason to believe that syngamy has been repeatedly attempted, but has never been carried out for the obvious reason that this form lacks the nuclear constitution necessary for the process. The fact that periodic attempts to conjugate occur indicates the probability that it has occurred in the past history of this organ- 506 J. A. DAWSON ism. It is probable that this form at some time in its history contained a micronucleus, though it is of little value to speculate concerning the manner in which the present amicronucleate condition arose, since there are no data at hand. Such a con- dition might cohceivably arise by some abnormal process during conjugation which resulted in suppression of differentiation between macro- and micronuclear material. Although the significance of the process of conjugation has been exhaustively studied since Maupas’ time, a complete solu- tion of the problem has not as yet been obtained. The existence of a form which not only apparently may live indefinitely without conjugation, autogamy, or endomixis (assuming the possibility of the latter phenomenon in an hypotrichous form), but also apparently does not possess the ability to undergo any of these phenomena, brings to light an entirely new possibility in the life-history of ciliates. It has been proved quite conclusively (Woodruff, 714) that in forms which ordinarily conjugate, the continued prevention of this process brings about no loss of . viability if a favorable environment be provided. However, in the organism under consideration there is apparently no possi- bility not only of conjugation or endomixis, but also of autogamy, and thus we have from another source crucial evidence that none of these phenomena is an indispensable factor in the life-history of this hypotrichous form. 6. SUMMARY 1. A pedigreed culture of Oxytricha hymenostoma has been carried for 289 generations, from July 10, 1917, to November 17, 1917, and since that time to date (April 30, 1918) by means of small mass cultures in petri dishes. 2. A micronucleus has not been seen at any time in any of the animals during the history of the cultures. 3. Syngamy has not occurred during the course of the experi- ments, although it is believed that the animal has frequently been in a physical state similar to that in which syngamy takes place. AN AMICRONUCLEATE OXYTRICHA 507 4, While in this state, a) animals fuse in pairs in a manner similar to that of conjugating animals; b) cannibalism takes place. 5. Animals, fused as described in 4a, either remain fused until death occurs or separate. In the latter case the organisms continue to reproduce and give no signs of being in a depressed condition. 6. When cannibalism has occurred, digestion of the ingested animals proceeds rapidly and a return to the typical size and structure soon takes place. 7. Cannibalism has the effect of raising the division rate somewhat for a short time. 8. This amicronucleate race of Oxytricha hymenostoma appar- ently can live indefinitely under favorable environmental con- ditions without conjugation, autogamy, or endomixis. Indeed its amicronucleate condition seems to preclude the possibility of the occurrence of these phenomena. LITERATURE CITED BarttseLut, G. A. 1912 Experiments on the reproduction of the hypotrichous Infusoria. I. Conjugation between closely related individuals of Stylonychia pustulata. Jour. Exp. Zodél., vol. 13. 1914 Experiments on the reproduction of the hypotrichous Infusoria. Il. A study of the so-called life cycle in Oxytricha fallax and Pleuro- tricha lanceolata. Ibid., vol. 16. BuscukiEL, A. L. 1911 Beitrage zur Kenntnis des Ichthyophthirius multi- filiis Fouquet. Archiv f. Protistenk., Bd. 21. Cauxins, G.N. 1902 Studies on the life-history of Protozoa. I. The life-cycle of Paramecium caudatum. Arch. f. Entw. Mech., Bd. 15. 1912 The paedogamous conjugation of Blepharisma undulans St. Jour. Morph., vol. 23. Cauxins, G. N., AnD Cutt, 8. W. 1907 The conjugation of Paramecium aurelia (caudatum). Archiv f. Protistenk., Bd. 10. Enriques, P. 1903 Sulla cosi detta degenerazione senile dei Protozoi. Moni- tore Zool. ital., T. 14. 1905 a Ancora della degenerazione senile negli Infusori. R. C. Accad. Lincei., T. 14. 1909 a ‘‘Autorreferat”’ in Arch. f. Entw.-Mech., Bd. 27. Fermor, X. 1913 Die Bedeutung der Encystierung bei Stylonychia pustulata Ehrbg. Zool. Anz., Bd. 42. Haime, J. 1853 Observations sur les métamorphoses et l’organisation de la Trichoda lynceus. Annal. d. Sci. Nat., 3S, T. 19. 508 J. A. DAWSON Hertwic, R. 1889 Uber die Conjugation der Infusorien. Abhandl. d. k. bayer. Akad. Wiss. Miinchen, Bd. 17, Abth. 1. Jennincs, H.S. 1906 Behavior of the lower organisms. Columbia University Press. 1913 The effect of conjugation in Paramecium. Jour. Exp. Zodl., vol. 14. 1910 What conditions induce conjugation in Paramecium? Ibid., vol. 9. Jouxowsky, D. 1898 Beitrige zur Frage nach den Bedingungen der Vermehr- ung und des Eintritts der Konjugation bei den Ciliaten. Verhandl. naturhist.-Med. Ver. Heidelberg, Bd. 6. Luspepew, W. 1908 Uber Trachelocerca phenicopterus Cohn. Archiv f. Protistenk., Bd. 13. LeDantec, F. 1897 La régénération du micro-nucleus chez quelques Infusoires Ciliés. C. R. Acad. Sci. Paris, T. 125. Lewin, K. R. 1910 Nuclear relations of Paramecium caudatum during the asexual period. Proce. Camb. Phil. Soc., vol. 16. 1911 The behavior of the infusorian micronucleus in regeneration. Proc. Roy. Soc. Lond., ser. B, vol. 84. Mavpas, E. 1888 Recherches expérimentales sur la multiplication des Infu- soires ciliés. Arch. d. zool. exp. et gen., 28., T. 6. 1889 Le rajeunissement karyogamique chez les ciliés. Ibid., 2 S., AD (Gy Mertcatr, M. 1909 Opalina. Its anatomy and reproduction with a descrip- tion of infection experiments and a chronological review of the lit- erature. Arch. f. Protistenk., Bd. 138. Mincurn, E. A. 1912 An introduction to the study of the Protozoa. London. NerEesHEIMER, E. 1907 Die Fortpflanzung der Opalinen. Arch. f. Prot. Suppl. 1. 1908 Fortpflanzung eines parasitischen Infusors (Ichthyophthirius). Sitz.-Ber. d. Ges. f. Morph. u. Phys. Miinchen., Bd. 23. Pororr, M. 1907 Depression der Protozoenzelle und der Geschlechtzellen der Metozoen. Archiv f. Protistenk., Suppl. Bd. 1. Sroxrs, A. C. 1888 A preliminary contribution toward a history of the fresh- water Infusoria of the United States. Jour. Trenton Nat. Hist. Soc., vol. 1. Wooprurr, L. L. 1905 An experimental study on the life history of hypotri- chous Infusoria. Jour. Exp. Zodél., vol. 2. 1908 The life cycle of Paramecium when subjected to a varied environ- ment. Amer. Nat., vol. 42. 1911 The effect of excretion products of Paramecium on its rate of reproduction. Jour. Exp. Zodl., vol. 10. 1913 Cell size, nuclear size and the nucleo-cytoplasmic relation dur- ing the life of a pedigreed race of Oxytricha fallax. Ibid., vol. 15. 1914 So-called conjugating and non-conjugating races of Paramecium. Ibid., vol. 16. Wooprurr, L. L., anp Erpmann, Ru. 1914 A normal periodic reorganization process without cell fusion in Paramecium. Jour. Exp. Zodl., vol. 17. PLATES All of the figures in plates 1 and 2 are microphotographs of permanent prepa- rations (total mounts) stained with Delafield’s hematoxylin and in most ¢ases counterstained with tetra-brom-fluorescic acid. The use of other counterstains is denoted in the explanation of figures. The same magnification (525 diameters) was used in all cases with the exception of figures 8, 15, 18, and 19, which are magnified 375 diameters. 509 PLATE 1 EXPLANATION OF FIGURES 4 A typical normal individual from culture A in the fiftieth generation. July 24, 1917 (dorsal view). 5 Typical pair from stock of culture A fused in the peristomial regions. 6 Typical pair from stock of culture A. Appearance closely simulating that of conjugating animals (Del. hem.). 7 Pathological specimen from stock of culture A in 283rd generation, Novem- ber 6, 1917. Preparation made at the end of the fourteenth day without division. 8 ALE-producing stimulus through sea- M water. Studies on sex in the hermaph- rodite molluse Crepidula plana. III. Transference of the........ JoseuEenic Boos tle) Minnicu, Dwicut E. The photic reactions of the honey-bee, Apis mellifera L Mollusc Crepidula plana. III. Transference of the male-producing stimulus through sea-water. Studies on sex in the her- MAP NTOOUGE nse atda etal rlel sie dalle weitere 113 XYTRICHA. I. Study of the normal animal, with an account of cannibalism. An experimental study of an amicronu- ClEHBE SS .G cconte ns the naan hasnt monte 473 516 HOTIC reactions of the honey-bee, Apis melliteraws: «The. jaa. ccoae sheets 343 AT. Studies on inbreeding. IV. A fur- ther study of the effects of inbreeding on the growth and variability in the body weight of the albino.............. 71 Rat). Studies on the reputed endocrine func- tion of the thymus gland (albino......... 311 Reactions of Chromodoris zebra. Sensory... 261 Reactions of the honey-bee, Apis mellifera L. "Phe photic stewie + se cycrs itestoneiecal ass eee 343 Renewal of vitality through conjugation. Uroleptus mobilis Engelm. 121 Responses of Chiton. The sensory....... 157 ENSORY reactions of Chromodoris zebra 261 Sensory responses of Chiton. The...... 157 Sex in the hermaphrodite mollusc Crepidula plana. III. Transference of the male- producing stimulus through sea-water. Studies ones. ist oats ee ee ates 113 Stimulus through sea-water. Studies on sex in the hermaphrodite mollusc Crepidula plana. III. Transference of the male- TOC GIN ay eit cieiel foie clererciereiele olole aletelets a7areke 113 INDEX AKENOUCHI, Marsuziro. Studies on the reputed endocrine function of the thymus gland (albino rat).............. 311 Thymus gland (albino rat). Studies on the reputed endocrine function of the........ 311 Thyroidectomy. Growth and development of Amphibia as affected by............... Toxicity of acids to ciliate Infusoria. The... 443 ROLEPTUS mobilis Engelm. II. Re- newal of vitality through conjugation.. 121 ARIABILITY in the body weight of the albinorat. Studies oninbreeding. IV. A further study of the effects of inbreed- ing on the/gzrowth’and®)).-u.)..-14-) arnt 71 Vitality through conjugation. Uroleptus mo- bilis Engelm. II. Renewal of ............ 121 EIGHT of the albino rat. Studies on inbreeding. IV. 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