Vist S Seca fe ate ~ ees CoS at es THE JOURNAL EXPERIMENTAL ZOOLOGY WILLIAM E, CASTLE Harvard University Epwin G. CoNKLIN Princeton University CHARLES B. DAVENPORT Carnegie Institution Horacr JAYNE The Wistar Institute HERBERT S. JENNINGS Johns Hopkins University , OF EDITED BY FRANK R. LILuIE University of Chicago Jacques LorB Rockefeller Institute H. Morean Columbia University THOMAS H. PARKER Harvard University GEORGE CHARLES O. WHITMAN University of Chicago EpmuNpD B. WILSON, Columbia University and Ross G. HARRISON, Yale University VOLUME 10 Managing Editor Ome THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. CONTENTS 1911 No. 1. JANUARY * G. H. Parker. The olfactory reactions of the common killifish, Fundulus hepe co clintsel CMTS) ae cpr yer koe oot. (ale = oe 5 eae ore ae at ae 1 Sreraius Moreuuis. Contributions to the physiology of regeneration. III. Huruhers experniments.on lodarke Obscura... saeien sso. s oes oes nes NI - Georaina B. Spooner. Embryological studies with the centrifuge. Thir- {USSU NNYELUT EIS SD oer cs di Sate eo ei ex BBN oc RMT: 3 orc ORS RACE ROI ich ene the = 23 RaurpyH EF. Saetpon. The sense of smell in Selachians..................-.:.- 51 Montrose T. Burrows. The growth of tissues of the chick embryo outside the animal body, with special reference to the nervous system. Fourteen MOUIG CSO ene site ish Aiken SU Se Ja, Lye cite Te ante cei eked ec 63 Marian L. SHorey. A study of the differentiation of neuroblasts in artificial CUkiumevnrediiae ee nwhie UTES ees... Ss aA enema cis fo) fe ae ee ait 85 No. 2. FEBRUARY Gary N. Cauxins. Regeneration and cell division in Uronychia. Fifteen fa CULE GOR Rrra Ace ee ERR cin 2 | Ju MR) Sn Coe or! al 95 A. FRANKLIN SHULL. Studies in the life cycle of Hydatinasenta. IJ. The role of temperature, of the chemical composition of the medium, and of internal factors upon the ratio of parthenogenetic to sexual forms...... 117 Witu1AmM Bropseck Herms. The photic reactions of sarcophagid flies, espe- cially Lucilia caesar Linn, and Calliphora vomitoria Linn. Twenty-five SMUT RES s oct Ss SRE RS Oe os 5 SER RS Er er eas one 8 167 Mary ©. McGinnis. Reactions of Branchipus serratus to light, heat and TATE AVAU A atcs's Osc ote I CNC UE CEN LE RIE. SCSo MI See GN ae ne en 227 Nor 3. APRIG H. D. Goopate. Studies on hybrid ducks. Nine figures: two plates....... 241 Hans PrzipramM. Experiments on asymmetrical forms as affording a clue to the problem of bilaterality. Eleven figures: one plate...........:.....: 255 ©.M.Cninp. Studies on the dynamics of morphogenesis and inheritance in experimental reproduction. JI. The axial gradient in Planaria doroto- cephala as a limiting factor in regulation. Forty-one figures........... 265 Sereius Moreuuis. Contributions to the physiology of regeneration. IV. Regulation of the water content in regeneration. Seven figures......... 321 Now. NEA Y G. Harotp Drew. Experimental metaplasia. J. The formation of colum- nar ciliated epithelium from fibroblasts in Pecten. Three plates........ 349 HELEN Dean Kine. The effects of semi-spaying and of semi-castration on - the sex ratio of the albino rat (Mus norvegicus albinus)................. 381 Epwin G. Conxern. The organization of the egg and ’’ e development of single blastomeres of Phallusia mamillata. Fourt PHAROS ins cece 393 Francis B. SumNER. The adjustment of flatfish to various backgrounds. A study of adaptive color change. Thirteen plates..............7........ 409 E. Newton Harvey. Studies on the permeability of cells. Three figures.. 507 LorANDE Loss Wooprurr. The effect of excretion products of Paramaecium OMMistrate oh reproductions ws Levene OUne susan aren eee eee 557 CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE E. L. Marx, Director, No. 214. THE OLFACTORY REACTIONS OF THE COMMON KILLIFISH, FUNDULUS HETEROCLITUS (LINN.) G. H. PARKER In a paper on the olfactory reactions of fishes published in the eighth volume of the Journal of Experimental Zoélogy (1910), I have attempted to show that the olfactory organs of catfishes are stimulated by minute amounts of substance emanating from materials that can. >rve these fishes as food; in other words, that these organs are distance-receptors by which the fishes can scent out their food much as land animals do. } isl Yen} aaa = SS OD sH sH MA mm NNNNN AN OD NNNNANATN AN OD OD OD OD ANNNANANNAN & OD be} } nD G 5 ik 5 Ble oe Aine 6z Aine youqut g s pe7eqideoap. SUIIO AA peyeqideooep SUIIO AA SpBvoy IIA SUIIOM 3 "P Sa1ag “g saulag "2 Saray n 0 : A 14 SERGIUS MORGULIS decapitation upon posterior regeneration from the very beginning of the process. As will be seen from table 5, in which are given the results of this experiment, the number of worms proliferating new tissue two days after the operation (August 6) is twice as great in the control as in the case of the decapitated worms, being 82 per cent of the former and only 42 per cent of the latter. Four days after the operation (August 8) the detrimental influ- ence of the extra operation upon the regeneration of the tail is still apparent, but after a week’s time the difference between control and decapitated worms disappears almost entirely. To sum up the results of the above investigation, it may be said that the additional mutilation of the head in Podarke obscura causes a depressing effect upon the tail regeneration, which is expressed either in a smaller number of regenerated segments or in a greater frequency of regenerated tails with few segments. The effect, however, wears off as the time of regeneration is pro- tracted, and is the more pronounced the more anterior the level, 7.e. the shorter the moiety of the worm from which regeneration TABLE 5 August 4—September 2 Conpr- | | Sine | DECAPITATED | Heaps InvTact SEGMENTS REGENERATED i REGENERATED Date Aug. | Aug. | Aug. | Aug. Aug. Aug. | Aug | Aug. | Aug.| Aug. | Aug. | Aug. 6 8 10 | 12 | 14 18 6 8 10 1 14 18 | Gee em eT Cy) Oma memmbees| lae 3 [2k 3) eel ee 2 : 2 | 3 3 3 3 | & 9. | Bre 1) ay al eee 3 Sit | anaes 4 4 Agel ©-c5.'| 98: Aen eh ae a 4. | aS | bode 3 4 4 Ave gees | © 220 | yay al) A a ee bie ove baal nes 4 4 a (ere me we| 5 oe hei |) sige rs eee ee eS ea 1g) SE ieridg ines Anas ea Baal SE | o Oatanee) 55) SGC $, 165, pad | 4 | | Spee e ee bug | bre alles 9 | &8)|bud| 4 4 | 5 po | So) | sot | 5) Saas 100 | eh Be Nea a | OS eRe a ec sage "| 95) aes 11 | ,o8 | 2 | 4 Be nb 6 | .o8 | 93) 4 | 6 | ines 12s | PAR We ie (Meera a UE et) 633 iS 2 | | | | PHYSIOLOGY OF REGENERATION 15 proceeds. The detrimental influence of the extra injury is spe- cially strong during the first few days following the operation, owing probably to the fact that a greater claim is then made upon the organism’s reserve formative energy; but if the new tail has already got a sufficient start in regeneration its further progress can not be impeded by an additional mutilation of the organism. B. RELATION BETWEEN FREQUENCY OF INJURY AND RATE OF REGENERATION Next in importance to the problem of the relation between the rate of regeneration and degree of injury stands the problem of its relation to the frequency of injury. In earlier studies (Morgu- lis, ’08) I have shown that the regenerative rate decreases with each repeated operation. Similar conditions were also observed in Podarke (’09b), although it was there pointed out that the worms tend to regain their original rate of regeneration. The inference of real importance to be drawn from those experiments is that within a given period of time an organism generates more tissue the more often it has been operated upon. In 1908 Zeleny suggested that after successive operations the rate of regeneration increases, but his data at that time were quite inconclusive. Lately (09a) he has brought forth additional evidence, obtained with great precaution against any possibility of error, and that investigation has led him to formulate his opinion more care- fully, as follows: “The data as a whole make it highly probable that the pure effect of successive removal is either no change in rate of regeneration or an increase in rate” (’09a, p. 508) I performed a few experiments on Podarke to verify my former observation that the rate of regeneration decreases after repeated mutilations, and in what follows I shall present the outcome of those experiments. It should be recalled that in experimenting with worms one does not encounter such perplexing difficulties in the way of controlling conditions as the crustacea, for instance, offer. Other factors, too, are easily controllable. The experi- ment consisted simply in allowing some worms to regenerate continuously for several weeks, while on other worms from the 16 SERGIUS MORGULIS same lot the operation was executed twice during a similar period of time. On looking through table 6, it will be noticed that in the course of four weeks the worms have regenerated on the average 8.4 segments (5 to 14 segments). From previous studies on Podarke (Morgulis, ’09b), it is well known that the largest number of segments was regenerated within the first two weeks after the operation. TABLE 6 JULY pe 30 [ NUMBER a SEGMENTS AVBRAGE Old segments...........| 20 | 17 | 16 | 17 | 7/161 15/15!16|20/18| 17 New segments.......... 11 | 5 | a | 7 | 8 | 9 | 8 | AS) 9-758) 8 | ane Table 7 contains the records of an experiment where the worms were operated on twice during the four weeks, the second opera- tion having been executed at the end of two weeks. At that time the average number of regenerated segments was 6.2, the number ranging from 5 to 8 segments in different individuals. The stage of greatest regenerative rate had thus been passed at the time the regenerated tails were cut off, and the worms were left to regen- erate anew for another two weeks. The average number of seg- ments regenerated for the second period of two weeks is only 5.4, ranging from 4 to 7 segments, showing very definitely that the rate of regeneration following the second operation was slower. There is no significance in arguing that the decrease in the rate of regeneration following an operation once repeated is no decrease at all, but the outcome of a “general decline of the physiological TABLE 7 JULY 2—JuULY 16 NUMBER OF SEGMENTS AVERAGE Old segments......... 18 21 19 18 18] 17| 18} 17| 16) 16) 14) 18] 19) 17) 14; 17.3 New segments........ Soe bien! Ct) Deol G\scco) Gees iG GlaRol) SiGe | | | i JULY 16—sULY 30 REGENERATED SEGEMENTS REMOVED JULY 16 is — 3 | =a _ Old segments......... 16 18 18, 15, 14 16 16] 15 13) 19] 15 dead 16 New segments........ 4 7 7 7 7 4) «5} 4) 4 6] 4 5.4 PHYSIOLOGY OF REGENERATION Fe activity of the organism,”’ for what else can this fact signify except that the physiological activities are lessened? The argumenta- tion might justly and with equal pertinency be turned against the opposite proposition, viz., that an increase of the regenera- tive rate after successive injuries is the result of an acceleration of the physiological activities; but nothing could be gained by such an argument, which simply translates a tangible fact into an elusive conjecture, and in neither case leads anywhere. The fact which commands our attention is that, while the regen- erative process after the first operation is already declining, a new operation will cause a new output of regenerative energy exceed- ing the possible output where there had been no other injury in the meantime. In fact the worms operated on twice at intervals of two weeks regenerated during the space of four weeks an average of 11.6 segments, whereas after a single operation, but within a similar period of four weeks, only 8.4 segments regenerated. In TABLE 8 JULY 2—JULY 238 NUMBER OF SEGMEN1S AVERAGE Oldiscomentisawen. sak 17| 18) 15) 16) 16) 18) 16) 17) 16) 16) 14) 16) 17) 19) 16.5 New segments........... Ol 47). 7) Si Siieedieeeteeop LO 7) s5). 291) 19) “a 86 JULY 25—avuGusST 13 REGENERATED SEGMENTS REMOVED JULY 23 Oldtseamentsey ese 15) 17) 16) 15) 15) 14 ee | dead New segments........... Oi 6) St blew 6.1 other words, there was an average excess of over 3 segments caused by the repeated operation, even though the rate of regen- eration the second time was somewhat decreased. From table 8 it will likewise be seen that, while in three weeks the worms regenerated an average of 7.6 segments (5 to 11 seg- ments), the average number of regenerated segments for three weeks following a repeated. mutilation decreased to 6.1 (5 to 9 segments). THE JOURNAL oF EXPERIMENTAL ZoGétoey, Vou. 10, No. 1. 18 SERGIUS MORGULIS C. RELATION BETWEEN SEX AND RATE OF REGENERATION In studying the rate of regeneration, it is urgent to guard against several sources of possible error, since the conclusions are based almost entirely upon comparisons. In dealing with worms one is doubtless spared many pit-falls arising from the discontinuous method of growth or progressive developmental changes in the experimental animals. Temperature conditions, as well as the factor of size (age?) of the animal and of the level of the cut, can be regulated without much difficulty, and due weight has been given to all these matters in the previous experiments with Podarke. There was, however, one factor still uncontrolled, viz., the influence of the sex of the animal upon its regenerative rate. Generally it is not an easy matter to determine the sex, but I TABLE 9 Sex or Worm MALE FEMALE SEGMENTS | OLD REGENERATED | OLD REGENERATED Date July 7 July 21 July 26 July 7 July 21 July 26 if iG 6 5 20 8 5 2 16 a 5 il7/ a 6 3 19 6 5 20 8 6 4 19 9 6 19 6 6 5 15 4 6 i 18 8 7 6 16 5 Ta (10 7 7 7 18 7 i | 15 8 ‘i 8 16 6 ai £18 8 7 9 16 4 Thal 16 6 7 10 18 5 ee 7 Fh u 16 7 rl ame 5 7 12 18 8 8 | 17 9 8 13 15 8) 8 | 19 9 8 14 17 9 Ove 17 7 9 15 18 8 9 14 7 9 16 Le Oe 4 10 || 16 6 9 17 16 | 8 10 | 20 6 10 18 18 6 10 14 6 10 19 15 di 19 8 10 > Camm tsa bey eG 19 6 10 aoa 18 4 PHYSIOLOGY OF REGENERATION 19 have availed myself of an opportunity to investigate this factor when a number of specimens were found at the breeding season whose sex could readily be distinguished by the presence of eggs or of sperms within the body. Twenty worms of each sex were cut in two about the middle of the body, and left to regenerate new tails. As will be seen from table 9, both the males and the females regenerated from 4 to 9 segments in two weeks, and from 5 to 10 segments in twenty days. It is quite evident from this that the sex of the animal has no influence upon its regenerative power, and this factor, though not considered in earlier experiments (CO9b), probably had no effect upon the results. D. RELATION OF THE AMOUNT OF REMOVED TO THE AMOUNT OF REGENERATED TISSUE In a previous publication (Morgulis ’09b) it was maintained that in Podarke the regenerated tail does not reach its original length when it grows from about the middle of the body, although the full number of lost segments is usually restored when only a small part of the tail has been detached. About the same time a paper was published (Ellis, ’09) in which this matter of the rela- tion between the amount of removed and of regenerated tissue was treated more fully, the results leading to a similar conclu- sion, that “regeneration ceases before the part removed has been completely regenerated” (p. 444). Since I stated the matter at first more as an opinion than as a fact, I have now undertaken to verify the conclusion by a special experiment in which record has been kept of both the removed TABLE 10 JULY 6—AUGUST 28 | NUMBER OF SEGMENTS | AVERAGE Removed...... 20) 20 24 24 24) 26) 26) 26 26 28| 28, 29) 29) 30| 30) 31/ 32) 27 | | August 6. Net \iC8 |} 8} 8 8 9} 9/9 10) 10) 10) 11) 12) 14)’ 9. August 28./ 8 | 9 | 10 9 yi leeiha wil | 2 a a ISA cit | eeelhaebLO Regenerated 20 SERGIUS MORGULIS and the regenerated segments. The results of examinations made 31 and 53 days after the operation are given in table 10. The average number of removed segments was 27, or a little over one-half of the worm’s body. The first examination of the regen- erated tails showed that on the average after 31 days only 9.2 segments, or practically one-third of the number lost had regen- erated. Fifty-three days after the operation 11 segments on the average (8 to 16 segments) had regenerated, or about 0.4 of the amount removed. The worms were not examined at later peri- ods, but with the knowledge we now possess concerning the phases of regeneration, it may be said with certainty that the regenera- tive process had already reached practically a standstill, and that very few segments were added subsequently. The shortening of the tail thus effected by regeneration does not, however, destroy the proportions of the worm, as the whole organism apparently undergoes a corresponding reduction in dimensions. The result of the regeneration of about one-half of the animal is, therefore, to produce smaller worms, but such as are otherwise perfectly normal. CONCLUSIONS Among numerous perplexing problems with which it is the biologist’s lot to deal, the cessation of the growth of an organ- ism when it has attained a certain form is a matter of no small difficulty to understand. Attempts to explain the phenomenon on mechanical principles are conspicuously inadequate. Inves- tigations on regeneration show that, whereas growth may already have been brought to its normal termination, the capacity to grow is still unabated, and that there is sufficient potentiality in reserve to make good a substantial part of any lost portion. Of course, even formative growth on the part of an organism is never reduced to absolute zero, and while the organism maintains itself at a more or less definite status quo, separate parts or organs, as for instance the skin, may still retain the power to grow. Experiments have proven conclusively that regeneration may be repeated several, and in some cases even many, times in suc- PHYSIOLOGY OF REGENERATION 21 cession, an enormous amount of growth energy being thus uti- lized, which would otherwise have remained dormant in the organ- ism. The evidence of economy with which this inherent power of growth of the organism is used to compensate for a mutila- tion is a problem which we cannot go into at this moment. The interesting facts brought out by most of the recent researches are, that the lost portion is not fully restored, and, as I have also observed in Podarke, that regeneration produces smaller individ- uals but such as have normal proportions, the new growth being apparently brought to a termination when a definite form has been established. It should be emphasized here that the cessa- tion of regenerative growth does not imply an exhaustion of the regenerative energy, for, as experiments on regeneration after successive injuries show, a repeated stimulation will overcome the inertia of the organism and set its formative forces into activ- ity once more. Whether or not a repetition of the injury causes an acceleration of the regenerative process—and apparently either condition may exist—it causes an additional output of regenerative energy, and the quantity of tissue generated after several operations greatly exceeds that produced after a single operation. Likewise, a greater degree of injury—whether increas- ing or leaving unaffected the regenerative power or even decreas- ing it, as in the case of Podarke—results in a larger output of regenerative energy. The old problem of the cessation of growth recurs under a new aspect: Why does the regeneration of an organism cease? And why does it cease before the original size relations have been restored, notwithstanding the reserve potentiality to further re- generation? These are questions which must still await a solution. Euuis, M. EMMEL, V. MorGututs, SERGIUS MORGULIS BIBLIOGRAPHY M. 1907 The influence of the amount of injury upon the rate and amount of regeneration in Mancasellus macrourus (Garman). Biol. Bull., vol. 18, pp. 107-113. 1909 The relation of the amount of tail regenerated to the amount removed in tadpoles of Rana clamitans. Jour. Exp. Zodl., vol. 7, pp. 421-455. EK. 1906 The relation of regeneration to the moulting process in the lobster. 36 Annual Rept. of Inland Fisheries of R. I., pp. 258-313, pls. 40, 41, charts 8-10. 1907 Relations between regeneration, the degree of injury, and moulting of young lobsters. Science, vol. 25, p. 785. 8S. 1908 The effect of alkaloids on regeneration in the searlet-run- ner bean. Ohio Naturalist, vol. 9, pp. 404-412. 1909a Regeneration in the brittle-star Ophiocoma pumila, with reference to the influence of the nervous system. Proc. Amer. Acad. Arts and Sci., vol. 44, no. 23, pp. 655-659, 1 pl. 1909b Contributions to the physiology of regeneration. I. Expe- riments on Podarke obscura. Jour. Exp. Zodl., vol. 7, pp. 595-642. 1909¢ Contributions to the physiology of regeneration. II. Experiments on Lumbriculus. Arch. f. Entwicklungsm., Bd., 28, pp. 396-439. Scorr, G. G. 1907 Further notes on the regeneration of the fins of Fundulus STOCKARD, ZELENY, C. heteroclitus. Biol. Bull., vol. 12, pp. 385-400. C. R. 1909 Studies of tissue growth. II. Functional activity, form regulation, level of the cut, and degree of injury as factors in deter- mining the rate of regeneration. The reaction of regenerating tis- sue on the old body. Jour. Exp. Zodl., vol. 6, pp. 433-470, 1 pl. 1903 A study of the rate of regeneration of the arms in the brittle- star Ophioglypha lacertosa. Biol. Bull., vol. 6, pp. 12-17. 1905 The relation of the degree of injury to the rate of regener- ation. Jour. Exp. Zodél., vol. 2, pp. 347-369. 1907 The effect of degree of injury, successive injury, and func- tional activity upon regeneration in the scyphomedusan Cassio- pea xamachana. Jour. Exp. Zodl., vol. 5, pp. 265-274. 1909a The effect of successive removal upon the rate of regenera- tion. Jour. Exp. Zodél., vol. 7, pp. 477-512. 1909b The relation between degree of injury and rate of regenera- tion. Additional observations and general discussion. Jour. Exp. Zo6l., vol. 7, pp. 513-561. EMBRYOLOGICAL STUDIES WITH THE CENTRIFUGE GEORGINA B. SPOONER Leland Stanford University, California THIRTEEN FIGURES The following experiments with Cyclops fimbriatus were begun at Columbia University in the spring of 1908 and those with Arbacia, at Wood’s Hole in the summer of 1909. In respect to stratification of materials, direction of cleavage and develop- ment of normal embryos from centrifuged eggs Cyclops gave essentially the same results as others had obtained with Arbacia. The materials of the centrifuged eggs are separated into three layers, oil, protoplasm and yolk. Normal embryos develop from the eggs, whether centrifuged at the stage when the segmentation nucleus is present or after the cleavage spindle has formed. The eggs do not orient themselves in the machine, nevertheless the first cleavage is always perpendicular to the induced stratifica- tion. An examination showed that the segmentation nucleus is easily driven into the protoplasmic band and that the cleavage spindle may also be driven into the protoplasmic layer at any stage in its development but it takes a stronger force to displace the spindle than to drive the segmentation nucleus. The mate- rials of the egg also are harder to separate by centrifuging as the time of first cleavage approaches. There is apparently some increased tension in the egg substance but the karyokinetic fig- ure is not so rigid as to be prevented from its normal functioning by the rearrangement of the materials of the egg. Some light was thrown on the structure of the karyokinetic figure by the experiments upon Cyclops. The chromosomes, spindle fibers and the centers of the asters together comprise a unit system which bends, but is not torn apart, by the passage 24 GEORGINA B. SPOONER of the large yolk spheres around it. All appearance of astral rays is lost after centrifuging. Nothing remains but a disc of basic granules, marking the center of the aster. This shows that in Cyclops the astral rays are not fibers. The spindle “fibers,’’ in contrast to the astral rays, not only have the appearance of con- tinuous threads but behave as such, being in some cases bent. A separation of the materials composing the center of the aster results from centrifuging. In the normal egg the aster is composed apparently only of acid substance, but after centrifuging its reaction is basic, showing that in the normal egg the basic is also present together with the acid substance. The cleavage spindle of Arbacia may also be pushed out of its normal position as a result of centrifuging. Its subsequent appear- ance 1s in every way normal and the cleavages that follow are normal. In eggs killed immediately after centrifuging the astral rays extend radially into the protoplasm and are perfectly straight as in the normal egg, despite the rearrangement of materials. This seems to be opposed to a theory which supposes the astral rays in the sea-urchin to be true fibers since it is hard to conceive of a radial system of fibers remaining undistorted under such conditions. In the remaining experiments made with Arbacia several points were taken up which had not been sufficiently studied in previous work. cell is full-sized and nor- mal. Even at this period however, the power to regenerate is very limited as shown in section 8. Here, out of ten cases in which both parts were retained, only two resulted in complete REGENERATION IN URONYCHIA 13 regeneration of both parts, and in one of these (no. 37, the smaller one died in 48 hours, so that only one (no. 21) gave two cells that continued to live and in this case the organism was 24 hours old when cut and probably about ready to divide. In all of the other cases one part only regenerated normally. It is almost impossible to cut these normal vegetative cells in such a way that no macro- nucleus is retained, but the micronucleus, being single, is invari- ably absent in one of the fragments. The power to regenerate therefore, is only feebly developed during the vegetative period. Out of 24 cases cut after the division period, only two gave com- plete regeneration of both parts.. In striking contrast to this result are the results of cutting dur- ing the period of division, counting as the period of division the time from the first external evidence (precocious formation of the new cirri) until final separation. In all there were 37 experiments on cells at this time, 7 of which were in the early stages, 14 in the mid stages, 8 in the last stages, while 8 were multiple cuts. Analyzing these separately we find that of the seven cases cut immediately before division, six continued the division in the origi- nal plane forming two perfect cells in each case while the third part, or portion cut off, formed a perfect cell in five cases. In the majority of cases therefore, the original cell was made to form three perfect cells so far as general form and motile apparatus are con- cerned, and one of these parts in each case was without a micro- nucleus and must have regenerated with only a portion of the macronucleus. Of the fourteen cases cut during the mid phase of division, only six formed three perfect cells each. Of the others, sometimes one, sometimes the other fragment failed to regenerate, although macro- nucleus material was present (in three cases the cut was directly through the plane of division so that three cells were not formed; in each of these two perfect organisms resulted.) No general rule was observed in regard to the regeneration of cells cut during the late phases of division. When regeneration occurred, vitality of the regenerated parts seemed low and the life of the organism was short. Thus in experiment 10 both cells were perfect in 4 hours, but one portion (B) soon became abnormal and when killed after 72 hours showed a degenerated macronu- 114 GARY N. CALKINS cleus. Se ee RSA rena een Petes a8 oy 117 Rroblempandemet hodeeen ie neeee css. o> ee A ESC. Sic cic oe ho Eo Oe eae 118 Hixpenmentsmwltlmeexveria lita CbOLSi as. -1..0..c: crue rene oto elsiec ile 120 Influence of temperature on the percentage of male-producers............ 120 Influence of various undetermined constituents of feces on the percentage Groin Ae= producers vein eA aki: 25 x. 3 «ok a eee a eeaiert nates hte. Ane le 123 Influence of alkalinity on the percentage of male-producers.............. 129 Influence of urea on the percentage of male-producers.................... 132 Influence of ammonium compounds on the percentage of male-producers.. 132 Influence of beef-extract on the percentage of male-producers........... 135 Influence of creatin on the percentage of male-producers................. 137 Bixpenimnentswikh intern ale factors... s.:... «epee eee SRA e rcte sok areleeals « 140 IDIEYOWEIS OIL Tou b dino 2b Chou. 0 CO OER TERRE cicmnigic o oC Clo os ae LOE aes aaa ne 152 SUTIN Ay ae Pe eee TOTES SEAS ees on 2 cee ON ues Shoe ah acs 164 JEVlovirayiad?) ol shi7 ba Seema 2 cg Alba sa el PS oc 8 Sis Gis i ee 165 INTRODUCTION In two earlier papers (Shull, 10a and ’10b) I have discussed the influence of environment upon the transition from the parthe- nogenetic to the sexual mode of reproduction in Hydatina? senta. 1 [ desire to thank Prof. E. B. Wilson for the use of laboratory facilities at Cold Spring Harbor, Long Island, where much of the work described in this and my former paper was done. * It has been pointed out tome by several zodlogists independently that the name Hydatina can not stand as a genus of rotifers, that name having heen previously used for a mollusk. 118 A. FRANKLIN SHULL The life cycle of this rotifer and the substance of the previous literature on the subject were given in the second paper. Experi- ments were there described from which the conclusion was drawn that the mode of reproduction was not directly influenced by starvation, nor probably by temperature differences; but that the chemical content of the water in which the rotifers lived had a strong influence upon the proportion of sexual females (male- producers). In the present paper the nature of the substances involved is more definitely stated. I shall name a few single substances which have been found to influence the mode of reproduction, and give the evidence for my conclusion. Among these substances are several ammonium compounds, creatin, probably urea, and perhaps the degree of alkalinity. Certain internal factors have likewise been found to influence the proportion of sexual females, and these have been variously combined in crosses. This work has been continued with helpful suggestions from Prof. T. H. Morgan. I am also indebted to Prof. William J. Gies for suggestions regarding the chemical part of the work. PROBLEM AND METHOD In my earlier paper (Shull, ’710b) no evidence relating to the influence of temperature differences on the proportion of sexual females was given, because further experiments were in progress at that time. These experiments are now completed. The prin- cipal problem, however, was to discover the effect of various sub- stances in the water upon the proportion of sexual females; for it had been found that if the rotifers were bred in a fairly concen- trated solution of horse manure, sexual females might be wholly prevented from appearing. Such a manure solution contains a large number of substances, but it was not known which of these substances affected the life cycle. It also seemed important to search for internal factors which might likewise influence the proportion of sexual females, for some students of this and other similar life cycles believe that internal factors play a leading role in the production of the cycle. Punnett LIFE CYCLE OF HYDATINA SENTA 119 TABLE 17 Showing number of male-producers and female-producers in the progeny of two sister individuals of Hydatina senta, the one line being reared at a temperature of 18° to 23°C., the other at 23° to 26°C. Male-producers are designated 3 9 , female- producers @ Q. 18° To 23°C. | 23° TO 26°C. DATE OF | NO. OF DATE OF | amwenation | sumer | NGG | NOG" | cungra | rinem | NO.OF | Ng: oF Il aero etre ee Octans 37 2 1 Oi, a 0 13 2 10 11 34 2 9 1 6 Dh A 12 13 32 | 10, 3 9 1, eae 2 14 | 2 12 | 3 11 | 6 20 5 15 | 0 31 4 12 3 28 6 17 0 35 | 5 1a) 19 13 ii 18 | 0 48 6 15 1 15 Seen s 20; 10 4 7 17 1 6 9 2a 0 17 8 18 4 14 ei a tae 23 | 4 27 9 20N\y 18 12 VLE aa ea | 25 9 34 20 | Dey i230 1S Ae 278 2 17 10 7A ea Wh 11 (5). Le eRe ee 285 gol 13 11 22 7 16 14 i 30 6 32 12 24 ae: 15 mu 31 0 14 13 26 7 18 | Nov. 1 | 8 20 14 27 14% 11) ‘910 NOE ais. Sled 5, 2 1 23 15 30, 3 30 Yn 3 0 43 16 S15 4 15 Seer 5 4 33 ie Nowe 2 0 3 19... 8 Dew 14 3 Onda si2 20he. 10 2 5 18 4 | 2 4 12 | 2 9 4 | 5 5 21h. 12 2 Ga ie 1 5 0 7 13 | 0 19 20.01 7 2 24 OL ae a 15 0 32 21 8 | 0 21 230k 18 1 a1 | 22 10 5 25 | 18 | 2 0 23 11 els) | 24 13 Oe ey 25 Aa 14 ed ere st) 26 16 158 iloay ae 16 2 35 | 27 18 Ie My 290) | 28 19 6 26 THateaL 5. Mae ete 159 592 | | 143 526 Percentage of J @..... 21.1 | | 21.3 120 A. FRANKLIN SHULL (06) was inclined, in fact, to believe that external agents have no influence on the cycle of Hydatina, and that many of the phenom- ena observed by him were attributable to internal differences in distinct pure lines. It was shown in my earlier paper, cited above, that many phenomena are not due to internal differences; but it was left an open question whether internal differences might not explain other results. If distinct pure lines could be found, it was proposed to test the nature of their internal differences by appropriate crosses. The method of conducting the experiments has been in all essen- tial respects the same as that described in the previous article. EXPERIMENTS WITH EXTERNAL FACTORS Influence of temperature on the percentage of male-producers Experiment XV. On October 6, 1909, a female was taken from the pure line recorded in Experiment I of my former paper, which was being reared at room temperature (18° to 23° C.), and was placed in a dish in a closed box kept on or near a large water bath. The temperature in this box was noted frequently, and found to vary between 23° and 26°C. ae 22.| 0 7 | | 23 0 | 1a | D5 4th ch Abe a | Daye Me rey | DG ara x Nee | 26| 11 42 | | De ee BaP 26 | Pirate nent |Mar. 1) 9 | 43] | | DOU ANE tae Bil v6x |) 639 | S00 Pte Aa eG) Sia | | Biss eae at 5| 4 | 44] | | Had Janet 7 | 1 | 43 | | | | | |e Sack | BA So | [aS ee Re ee) wee STP ET Pall eet MTotnie aie: | 1 “1080. a | | 28 | 755 | 3.5 LIFE CYCLE OF HYDATINA SENTA 125 thepresent section. The manure solution was boiled; it was evapo- rated to dryness and redissolved; it was evaporated to dryness and extracted with ether and alcohol; and it was decolorized by boil- ing with animal charcoal. The following experiments show the results of these operations in detail. Experiment XIX. Boiling the manure solution. After the manure solution from an old food culture, made up with spring water, had been filtered through a Berkefeld filter, part of the filtrate was boiled gently for four minutes. The loss of volume by evaporation was restored with distilled water. The remainder of TABLE 21 Showing the number of male- and female-producers in the progeny of three sister individuals of Hydatina senta, one line being bred in boiled filtrate of old food cultures, one in unboiled filtrate, the third in spring water. | Spring WATER | UNBOILED FILTRATE | BorLeD FILTRATE | Pea | | aay ae cBNT OF | Nao or a cEND. OF | one ng yor cBNT OF 1 Pail 0 7 0 42 23 27 0 Oe | 0 51 5 46 0 4 | 0) 47 7 42 0 2 0 47 1 | 34 | 0 57 Ge) 92 | 0 26 | 0 42 37 live | ¥73 Oe (140 OxONa ee 219) | 020 the filtrate was not treated. The boiled filtrate was kept in stock and was boiled every day until exhausted, chiefly to prevent the development of bacteria. The unboiled filtrate was secured daily just before using. Three pure lines of rotifers derived from sisters were reared in April, 1910, one in boiled filtrate, one in unboiled filtrate, the third in spring water. The results are given in table 21. The boiled filtrate has precisely the same effect as the un- boiled. The substance in feces which prevents male-producers from appearing is not, therefore, an enzyme, nor bacteria, nor any other substance destroyed by boiling temperature. 126 A. FRANKLIN SHULL Experiment XX. Drying the manure solution. A portion of the filtrate obtained from an old food culture, made of spring water as in the preceding experiment, was evaporated to dryness. The residue was redissolved in distilled water equal in volume to the original filtrate, and then boiled. One line of rotifers, bred from a female collected at Grantwood, N. J., in April, 1910, was reared in this redissolved filtrate. A second line, from a sister to the par- ent of the other line, was reared in the filtrate that had been simply boiled. Another line was bred in spring water. The data of the three lines are given in table 22. The substance responsible for repressing the male-producers is not destroyed by drying and re- TABLE 22 Showing number of male- and female-producers in the progeny of three sister indi- viduals of Hydatina senta, one line being reared in the filtrate of old food cultures that had been dried and redissolved, one in boiled filtrate, and one in spring water. SPRING WATER | BoitEp FILTRATE } DRIED, REDISSOLVED FILTRATE | | | 5 ; NO. OF 0. OF PER | NO. 2 : ee | " . | . PER ae? ote | Se a | ae, of NO ae ee | ae on “8 2 | Se 0 16 | 0 6 | | 30 3 3 it | Fest, i a 0; |) aaa 0 oa | 0 13 | 0 39 15 20 | Ne giat) 2) | 0 43 it 18 ie 0 16 0 iy 0 | ul | = EE |. 19 74") 420-4 || Go) amaaen| “00 0 | 127 | 0.0 dissolving, for the redissolved filtrate has precisely the same effect as the merely boiled filtrate. Experiment XXI. Ether- and alcohol-soluble parts of manure solution. From an old food culture, made as in the preceding experiments from spring water, 125 cc. was filtered through a Berkefeld filter, and the filtrate exaporated to dryness. The residue was extracted with ether for twelve hours, after which the ‘ether was filtered through paper and the solution evaporated to dryness. Less than 0,01 gram of ether-soluble substances was thus obtained. It did not seem likely that so small a quantity LIFE CYCLE OF HYDATINA SENTA 127 would have any noticeable effect on the proportion of male-pro- ducers, but the experiment was nevertheless made. This ether- soluble residue was dissolved in 125 ce. of distilled water, giving a clear solution, and a line of rotifers was reared through four generations in it. The residue after ether-extraction was like- wise dissolved in 125 cc. of distilled water and boiled, making a brown solution not apparently different from the original manure culture, and a sister line of rotifers was reared in the solution. A third line was reared as control in spring water. Table 23 showsthe result. + TABLE 23 Showing the number of male- and female-producers in the progeny of three sister indi- viduals of Hydatina senta, one line being bred in a solution of the ether-soluble part of an old food culture, one line in the part insoluble in ether, the third in spring water. Serine WATER ETHER SOLUBLE Not ErHerR SOLUBLE | PER | | PER | PER NO. OF NO NO. OF NO. OF NO. OF NO. OF | NT O ‘ED ENT OF we Nakot eae (cen a RIG eee we ee] : 0 16 0 29 0 te 3 11 4 16 0 | 24 0 9 unites 44 en 20 B15 20 1 12 0 14 1 18 | 0 | 43 19 bs 7 awe ee al he 101 9.0 0 | 105 0.0 The experiment was so brief that the difference in the propor- tion of male-producers between the line in spring water and that in the ether-soluble part of the filtrate may mean nothing. The chance of obtaining any result from such a minute ether-soluble residue did not seem to warrant a more extensive experiment, especially since the part of the manure solution not soluble in ether had the same effect as the entire manure solution had in other experiments. An experiment was started, in which the alcohol-soluble portion of the manure solution was obtained in a manner similar to the ether-extraction above. The portion soluble in absolute alcohol was smaller than that soluble in ether. The experiment was dis- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 2 128 A. FRANKLIN SHULL continued in the middle of the second generation, hence the de- tailed results are not of value. It may be said, however, that the first family reared in the alcohol-soluble portion contained a considerable number of male-producers. Experiment XXII. Decolorized filtrate. It was noticed in the preceding experiments of this section that those residues which produced the same effect as the unaltered manure solution were brown like the original; those that had no effect were colorless. To determine whether the substance producing the brown color has any effect on the life cycle of the rotifers, a portion of the filtrate from old food culture was decolorized with animal char- coal. An excess of the charcoal was added to the filtrate, and the mixture boiled eight or ten minutes. It was then filtered through paper, and the volume of water lost by evaporation was restored, the added water being passed through the filter. More charcoal was added to the filtrate, and the boiling repeated. After three or four boilings the filtrate was practically colorless. It was not to be expected that the colored substance was the only one removed by this process for animal charcoal probably carries down most substances to some extent. But if the colored sub- stance is wholly responsible for the non-occurrence of male- producers in a manure solution, such a decolorized solution should yield the same proportion of male-producers as pure water. Whether it does or not may be seen from table 24. The experi- ment was performed in May, 1910, with rotifers descended from a winter egg collected in Grantwood, N. J., in April, and kept in an ice-chest for a month. Even the decolorized filtrate greatly reduces the proportion of male-producers, though not as much as the merely boiled filtrate. Whether the 7 per cent difference between the decolorized filtrate and the boiled filtrate is due to total removal of the colored sub- stance or to partial removal of some other substances, can not be decided from this experiment alone. That the latter is the case, at least in part, will appear later, when it is shown that cer- tain other substances which are presumably present in the manure, and which were probably carried down mechanically by the char- coal, do reduce the percentage of male-producers. This does not LIFE CYCLE OF HYDATINA SENTA 129 show, however, that the colored substance may not also have the same effect to a slight extent. The one male-producer in the first generation bred in the boiled filtrate is the only one I have ever obtained in undiluted filtrate of old food cultures. This one individual need scarcely surprise us if we note the very high percentage of male-producers in the first two generations bred in spring water. Manure solu- tion does not rigidly exclude male-producers. It is important to TABLE 24 Showing the number of male- and female-producers in the progeny of three sister individuals of Hydatina senta, one line being reared in boiled filtrate of old food cultures, one in filtrate decolorized with animal charcoal, the third in spring water. SPRING WATER | DECOLORIZED FILTRATE BoiLep FILTRATE NO. OF NO. OF PER 0. OF NO. OF PER NO. OF NO. OF PER ve 2Q CENT OF ae 99 CENT OF ave 99 CENT OF 31 6 3 39 1 42 20 5 3 29 0 9 7 21 0 6 0 14 6 23 8 Pall 0 22 6 39 2 337 0 14 16 14 1 30 0 38 30 10 4 45 0 1" 0 41 0 48 0 20 0 5 116 | 159 AD Me ei sil 261 7.4 1 27 0.5 — | = - — — remember this in the consideration of other experiments where various substances do not wholly prevent male-producers from appearing. ‘ Influence of alkalinity on the percentage of male-producers Experiment XXIII. It was found impracticable to rear the rotifers in water that was even faintly acid, so several degrees of alkalinity were used. A » solution of NaOH in distilled water was kept in stock in a tightly stoppered bottle. In making up this solution, no further precaution was taken to make the nor- mality exact than to weigh the solid hydroxid carefully, exposed 130 A. FRANKLIN SHULL to the air. A X solution of HCl was made up by titration against the NaOH solution immediately after the latter was prepared, and was kept in stock. Three grades of alkalinity were used in the experiment, the solutions being prepared as follows: Great Bear spring water, which is itself alkaline, was used unaltered in one line; for the second line, the 4; NaOH solution was diluted to ten times its volume with Great Bear water; for the third line, the *, HCl solution was diluted to forty times its volume with Great Bear water. The alkalinity of the Great Bear water was not TABLE 25 Showing number of male- and female-producers in the progeny of three sister indi- viduals of Hydatina senta, one line being bred in Great Bear spring water, one in water less alkaline, and the third in water more alkaline, than Great Bear water. Lower ALKALINITY GREAT BEAR WATER HiGgHEerR ALKALINITY “Gg” |*SST| cawror | “QPP [ERE] carr | SOF “xo. oF CENT OF | 0 31 7 24 | ee eye 0 ial 0 21 3 31 14 16 | Oy eas 0 39 | 0 16 OES (Valls preety 5 18 | Oy) 1 5 0 26 if 4 | eat 15 0 3 0 32 0 24 0 10 0 40 0 10 0 27 ri 40 eT at | 6 27 1 25 One aa 1 9 | | | 26 210 11.0 Oot 10.2 | | accurately determined, but was less than ,, so that addition of ® NaOH increased its alkalinity; neither was the alkalinity of the diluted solutions known. As it was necessary to expose the NaOH solution to the air while it was being used, the dilute solution was made up daily. The data from this experiment are given in table 25, where the alkalinity increases from left to right. From this experiment it would seem that the greater the alka- linity the fewer the male-producers, but the differences are too small to draw a safe conclusion from one experiment. LIFE CYCLE OF HYDATINA SENTA tS Experiment XXIV. The stock solutions in this experiment were produced in essentially the same manner as in the preceding experi- ment, but in diluting them for daily use distilled water was used instead of Great Bear water. The alkalinity of the final solutions was therefore more accurately known. It could not be exactly known, however, for the food cultures were of varying alkalinity, and, because the protozoa in them were not always equally abun- dant, a variable quantity of the cultures was necessarily used. TABLE 26 Showing number of male- and female-producers in the progeny of three sister indi- N viduals of Hydatina senta, one line being reared in —~ NaOH, one in distilled - 200 water, and one in —— HCl (but see text). 300 7 N rz i Pe 5 5 ee 300 HC DIsTILLED WATER | 500 NaOH “Ge (GT) omgzor | GET |NGRT ommtor | MG QT MBG cagtor 1 Lesiry 0 47 3 28 0 23 1 32 3 52 1 10 5 47 6 46 13 38. || 3 42 4 48 5 20 | f 22 0 48 0 15 0 55 0 49 1 21 4 34 1 25 0 32 0 48 4 34 | 5 | 40 | a eee Seas SES SS 21 194 | 9.7 | 19 [367 | 4.9 | 2 | 330 | 5.9 Before adding food to the dishes, the three solutions used were respectively :3, NaOH, neutral (distilled water), and 37; HCl. After adding food, the acid solution was practically neutral or faintly alkaline, while the alkalinity of the other two solutions was slightly altered. In table 26 the columns are designated according to the acidity or alkalinity of the solutions before add- ing food, but it should be remembered that an actually acid solu- tion was never used. eZ A. FRANKLIN SHULL While the highest percentage of male-producers is found as before in the lowest degree of alkalinity, there is not the regular decrease in the proportion of male-producers with increase of alkalinity, such as was seen in the preceding experiment. As it would not be practicable to rear the rotifers in much more alka- line water, all that we may safely conclude is that if alkalinity influences the proportion of male-producers it does so only toa small extent. Influence of urea on the percentage of male-producers Experiment XXV. One line of rotifers was bred in a zi5 solu- tion of urea, a control line derived from a sister individual was reared in spring water. The experiment was performed twice, A and B, in table 27. In A, the parents of both lines had beenin spring water before the beginning of the experiment; in B, both had been in urea. This experiment seems to indicate that urea in the water tends to reduce the proportion of male-producers. Influence of ammonium compounds on the percentage of male- producers Experiment XXVI. Ammonium salts. Four sister individuals became the parents of the four lines in this experiment, which was performed in June, 1910. One line was reared in spring water, the others in ammonium salts of the following strengths: =%5 NH.Cl, .*, NH.NO;, and ammonium carbonate 1 gram to 7500 cc. of the solution. The carbonate was not computed in terms of molecular solution, because the ¢c.p. salt was not used. The sub- stance used was probably what is known as the sesquicarbonate, a mixture of the bicarbonate and the carbamate. Table 28 gives the details of each line. All of these ammonium salts reduced the proportion of male- producers, two of them to one-half the proportion obtained in spring water, the other to one-third. The consistent results from the three salts seemed to make a repetition of the experiment with any one of them unnecessary. LIFE CYCLE OF HYDATINA SENTA 133 TABLE 27 Showing number of male- and female-producers in the progeny of two sister indi- viduals of Hydatina senta, one line being bred in spring water, the other in a solu- tion of urea. A and B are separate experiments. Serine WATER ! = UREA EXPERIMENT | NO. OF | | PER | NO. OF | PER ae ee more, || oa i CaN ee Seca Sey 1 0 2 1 0 4 Be ol 50% sh 944) | 2 Io} 428 Sve ee Sul 3 0 5 Ae Ome 4. -| 0| 4 5 Ba By OW} 10 6 Me naza: 4 Zils. | 0 | Dasl #39 5 Gn) 40m) Sy ely 210 3 6 OF |), 33 Ve aainess | 7 1 | 46 / 8 eee | | 88 0} 4 Oram 2, | 0 2 rN | 10 Ores 7 | 24 ) Oe 17 | 9 0 1 11 1.47 0 1 12 vt 142 | 10 Or t2 13 1 26 fener la ie | 160) 14 9 | 43 | 12 Br | 15 6 | 39 | 13 ih 1) i OK G | Seam pie Dil AD) | 17 Ase AA | 15 Ors wowl 18 lees Orr. 2 oO) 18 | 16 O27 | ie O35 —————— TUG EEE an meee ars one 66 | 570 | 10.3 | 20° | 473 || 4.0 1 Ssh Al Le | (> al 5 | 51 2 Tl) 49: 2 ees 3 17, | 635 Vass Des? B 4 0 23 | 4 0 "| Sem evSe |) 250s) 0 g 6 5 6 Oy) as 5 On 2c 6 0 | 35 atalian nes Meer one hes 33 | 149 eee 8 | 209 3.6 134 A. FRANKLIN SHULL TABLE 28 Showing number of male- and female-producers in the progeny of four sister indi- viduals of Hydatina senta, one line being bred in spring water, the other three respectively in solutions of ammonium chloride, ammonium carbonate, and ammonium nitrate. AMMONIUM SALTS SPRING WATER | - — —- — - CHLORID CARBONATE NITRATE NO. OF | NO. OF NO. OF | NO. OF NO. OF NO. OF NO. OF | NO. OF a? tots) fou fe} (9) | a? 22 oo? ee) 30 19 es | ea ie 10 28* 2 17* 8 OG vias g* 11 1* 0 9* ial 4* 2 11 8 ie 0 3° live Os 1 | 41% HO Clause) | Ad 14 | Oe Gy + ines 22 29 | 3 29 292 6 2 39 35 19 | 0 38 0 6 8 25 22 12° | 1 8 0 13 8 35 11 iy | Liga aes 10 17 1 13 25 19 | eye | 34 2 4 3 42 19 26 8 44 4 35 11 292 20 35 0 33 0 35 el 7 10 25 0 18 0 35 12 15 15 21 1 18 6 32 0 16 7 16 13 16 0 19 2 al 3l 10 15 232 | 296 60 360 80 | 290 76 | 288 Per cent of ee ee 14.2 21.6 20.8 *Remainder of family not recorded. Experiment XXVIII. Ammonium hydroxid. On July 6, 1910, a female from the same pure line as those of the preceding experi- ment was placed in a solution of ammonium hydroxid, made by diluting strong NH,OH to 5000 times its volume with Great Bear spring water. Its progeny for thirteen generations were bred in the same solution. The strength of the original hydroxid was not known. ‘Table 29 shows this line, and a control in Great Bear spring water. LIFE CYCLE OF HYDATINA SENTA 135 The ammonium hydroxid, like the salts, reduced the proportion of male-producers to less than half that of the line in spring water. Here again it seemed unnecessary to repeat the experiment. Influence of beef extract on the percentage of male-producers The experiments with beef extract were designed chiefly to test the question whether extractives, which are present in feces, influence the proportion of male-producers. Liebig’s extract was used, because of the absence from it of certain classes of substances which are present in other brands. TABLE 29 Showing number of male- and female-producers in the progeny of two sister individuals of Hydatina senta, one line being bred in dilute ammonium hydroxid, the other in spring water. Sprine WATER } AMMONIUM HyprRoxIp | PER | No. oF | PER ene ce Heras poe” | Rae Meanll) oo" | Ee ina 4 27 1 Ol 34. | ve 8 23 2 0 2 | oe 0 29 3 0 zi ae 0 12 | 134 0 27 en 2 11 5 7 28 Gericke ae 4 28 6 12 14 | We 14 21 | (oon tod | Giewatetey, 15 21 | Siar 4 22 eee eee 14 25 9 | 1 20 2 30: | 10 1 18 9 31 | 0 ge 10: 3 40 12 6 34 | Zi coils hae 0 1 11) eee LGnuh eee 2| 13 3 50 0 24 | 12= gre ee 4 24 | 1S tee | 0 18 | | A Se eee | 22 i | | | || Wotaters : es Sate 409 22.8 | | 34 326 | 9.4 | | I 136 A. FRANKLIN SHULL TABLE 30 Showing number of male- and female-producers in the progeny of two sister indi- viduals of Hydatina senta, one line being bred in spring water, the other in a solu- tion of beef extract. A and B are separate experiments. Sprinc WaTER | Berer Extract EXPERIMENT | | || NO.OF | | PER NO. OF PER | oko | ee | no, or | eae | ee eu seat ed aoe oe ( 1 7 | AG 1 oe aT 2 | 25 2 1A" pele | Paie 49 Be hdl aie a \iupess Salleee Ae |) Baas | | 5 ilis39 5 1) |) 45a) | 38 Osi 6 ai) 29 ee 2p 5 9 | 13 lin 4 Lees 8 Su asi 0 5 9 17 \ 930 | 5 Ait 29 10 ty oF | 0 31 iBT Si ioe 6 0) 4°20 a 12 0. || 29 0 14 ‘s 13 Oulae [ee Daley AO } Ou ent iy es | 3 | 36 | Tiel 2 | ee) 6 | {36 | | 0 54 | ee | Oo es | | | | tOrs)> 28 | 11 0: 1-20 | OQ) ees | 12 [> al 31 [ lay shal | | aaelate Potal ee ee 124 | 358 25.7 | | 27 | 630 4.1 dy) |) XO esa 1 Guilecse 2 OM) | 0 | 54 Sp ail qa! 17) || ieee Oo 23 B @ | 2074 | Oe ts 4 oO ) “19m 3. Neo iC 2a0 5 P| 0) 25 | & | agen | Pati fits "Stal 5) 0 a en | 18 | 139 | 114 | We ech RES LIFE CYCLE OF HYDATINA SENTA BIZ Experiment XXVIII. Two sister females were isolated June 23, 1910, from one of the other experiments being performed at that time. One was placed in a solution of Liebig’s beef extract, the other in spring water. The beef extract was dissolved in spring water, and a 1 per cent solution kept in stock. This stock solution was boiled once or twice a day to prevent the growth of bacteria, and in the intervals was set in the cool water of a spring. Loss by evaporation was restored with distilled water. The stock solution was diluted with Great Bear spring water when ready to be used. In the first experiment, A, table 30, a 0.03 per cent solu- tion was used for the first two generations, a 0.04 per cent solu- tion thereafter. In B, a female was removed from the line in beef extract in A to spring water, so that here also the strength of the beef extract is 0.04 per cent, the line in the extract being the last part of the corresponding line in A. In both experiments there are considerably fewer male-pro- ducers in the beef extract than in spring water. Experiment X XIX. The preceding experiment was repeated, but this time two strengths of the extract were used, one a 0.04 per cent, the other a 0.05 per cent solution. Both lines are repre- sented in table 31. The results of these repetitions agree closely with the first experiment, even showing a smaller percentage of male-producers in the stronger solution of the extract. There can scarcely be any doubt that beef extract tends to reduce the pro- portion of male-producers, and it seems quite possible that these could even be wholly excluded by sufficiently strong solutions of the extract. | Influence of creatin on the percentage of male-producers The positive results from the beef extract in the experiments of the preceding section suggested, among other things, that extractives may alter the proportion of male-producers. One of the commoner extractives, creatin, was selected to put this matter to the test. The creatin used in these experiments was not pure, but contained an admixture of creatinin. 138 A. FRANKLIN SHULL TABLE 31 Showing number of male- and female-producers in the progeny of three sister indi- viduals of Hydatina senta, one line being bred in spring water, the other two in solutions of beef extract of different strengths. SPRING WATER | 0.04 Per Cent Beer Extract | 0.05 Por Cent Breer Extract { | NO. OF | | NO. OF | = | NO. OF | GENEEA- | ied Solon | SENS | Ress Sieve GBNERA- Ago | nore ihe ees 8 | 23 1 ae 2s eee 0 | 320 Do 0 29 Pedi” 0 23)! 32 Oy is 2) a BNA a OMe COR ao a 1540) 98 0 9 EY oy 2 ll | mee 7% all 0 4 Beene. 5.2 4 28 | 4s ao |e: 0 11 Ber crs 33k 14 21 | 40 15 tot pale rer 15 OTe es 0 26 5 i Morn sem te OL ce eee 14 |) 925 aes ORE eto" || te TOmr 319 Aa UF alee Lligees: | «8%. lee 34 Mes i 8 4 34 | Opie 8 Did scoembens ee ec bet 1 So lead 0 30 LO fas 16) |; Bl9 pal rst0 0 209 1 iio 37 eee Oops ee | | Onn) 24 | | THI lees | | | 0 | 33 Total........| 87 | 290 | | 6 | | 275 eee igee: | || | Per cent of | | | Gui s ase ab | 23.0 | 2.1 | 0.6 Experiment XXX. From three sister females, three lines were reared, starting on July 18, 1910. One line was reared in a 0.02 per cent solution of the crude creatin in Great Bear spring water, one in a 0.025 per cent solution, the third in spring water alone. ‘Table 32 shows the three pure lines in detail. Experiment XXXI. This is a repetition of Experiment XXX, but with weaker solutions of creatin in both parts, a 0.01 per cent and a 0.015 per cent solution being used. In both of these experi- ments some of the eggs laid in the creatin solution did not hatch in the usual short time required in spring water. In such cases the creatin was diluted after the death of the parent, and many of the LIFE CYCLE OF HYDATINA SENTA 139 TABLE 32 . Showing the number of male- and female-producers in the progeny of three sister individuals of Hydatina senta, one line being bred in spring water, the other two an creatin solutions of different strengths. SPRING WaTER | 0.02 Per Cent CREATIN | 0.025 Per Cent CREATIN rar ae | =a NO. OF NO. OF | || wNo.oFr | TREES NO. OF | TOO. GENERA: | Berio a ron, | GuNEE A a Doi 1 1} | | i: ga ean 14 25 me KO | a ea 2 30 2 ii, 9 Lil oll ee Gr 3s PO ey 3 ie is. | Gul er Cite tees ay ea! 40 4 8 278 Wend Ouse 22 | 4 15 lewat ne eS opt iy 0 17 Say eres | 16 19 5 0: 1 ae 5 0 16 0 24 0) Lata SG Ooh ty AY eee hace Oh | 6 i 24 | 1a} 33 Ruwais ame le 10 18 O), jeaiene Seam mee a7 jo esx (ae eaten 22 11 7 0 39 8 0 36 Tele Nee U.S 4 36 0 27 9 1 |) 36 CO Nee ae 9 a 0) |) eae eto Quite We OE so 1 Fe ot | Ov see 1 Gis LOW at) ste 6 36 OF 4) 0, Aes 0 15* iT eee eae 7 14* | <0 20 12 1 16* | 10 1 28* | | 0 18 | | be tt O; slave ual | | Total... 101 | 375 10 | 405 4 | 354 Per cent of NOs ak a .2 | | 2.4 1.1 *Remainder of family not recorded. eggs then hatched. Table 33 presents the details of the experi- ments. In both of these tables the same result appears, that is, a lower percentage of male-producers in the creatin solution than in spring water, and a lower percentage in the stronger solution than in the weaker. The differences are as great as were those of the beef extract, and are too marked to leave any doubt regarding the influence of creatin. 140 A. FRANKLIN SHULL EXPERIMENTS WITH EXTERNAL FACTORS Those who have hitherto worked on the life-cycle of Hydatina have inclined strongly to one or the other of two extreme positions. On the one hand are those who hold that the proportion of male- producers at any time is a function of the external conditions then obtaining; on the other hand are those who believe that that proportion is determined by internal factors, uninfluenced by TABLE 33 Showing the number of male- and female-producers in the progeny of three sister individuals of Hydatina senta, one line being reared in spring water, the other two in solutions of crude creatin of different strengths. faa SprING WATER 0.01 Per CENT CREATIN \| 0.015 PER CENT CREATIN | = GENER oe of 5 oO GENERA | ber om “5 a | GENERA ae Q | oom ieee a 4 24 a) Co ala ae eat 3 1 Oe 4 Oks eee 0 18 \ Oil 225 On| = «728 ee 22 iat 2 2 46 Oyj duets oa Ca Aj) 36 3 Buf], 39 3° AVG) i) sag Geen ee 9 31 4 0 43 oO | 48 eee ey 5 0 25 4 0 28 hs nee Gelso Gi) oth aOn oD 5 0 36 See ate Wo aes 0 15 6 0 | ~30 OE cl a, 0 | eM Uh 0 30F 7 o | 16* We sli ee te el | | Totals 2aeraeos: | 198). 11 286 | 2 207 Per cent of | \ CO aie | Pal | 3.7 | 0.9 *Remainder of family not recorded. external agents. Clear expression is given to the latter view by Punnett (’06), who concluded that the only differences to be found in the proportion of male-producers were the differences in distinct pure lines, and that those differences depended on the character of the gametes uniting in the resting egg from which each pure line sprang. In my earlier work (Shull, ’10b) it was shown that this conclusion of Punnett’s did not hold in certain LIFE CYCLE OF HYDATINA SENTA 141 cases there presented, where differences in the proportion of male-producers were obtained within the same pure line. That did not, however, exclude the possibility that such internal differences do exist. In the hope of finding pure lines showing different behavior with respect to the life cycle, search was made for rotifers in different localities near New York. But this species was nowhere found except at Grantwood, N. J. Eventually some specimens were obtained from Baltimore, Md. Experiment XXXII. A culture of Hydatina was obtained from Baltimore through the courtesy of Prof. H. 8. Jennings, and brought to New York, on March 23, 1910, by Prof. T. H. Morgan. The culture was labeled ‘‘Hydatina (Old Culture) Curtis Bay, March 6.”’ One of the females from this culture was isolated March 23, and from her sixty successive generations were bred. On the same day, a female was isolated from. the pure line used as control in Experiment XVIII, which was originally obtained from New York, as described in that experiment. From this female over fifty generations were reared. Both pure lines were bred in Great Bear spring water, and the same food was used every day for both. The conditions were kept as nearly alike as possible. It was believed that any internal difference between the two lines would be shown in so large a number of generations. In table 34 the two lines are compared in detail. In the three months and a half through which this experiment extended, the New York line produced only 11.1 per cent of male-producers, the Baltimore line 18.5 per cent. Had such a difference been obtained in any of the experiments with external agents, it would have been taken as good evidence of a positive effect of the environment. It can scarcely be regarded as other than good evidence here. The difference is not due to some sudden increase in the number of male-producers in one line without a corresponding increase in the other line, for taken by and large the periods of many male-producers occur about the same iime in both lines. Nor is the difference in the two percentages due to the fact that the experiment was terminated at one time rather than at another; for the experiment could have been brought to a close at any time, even after a single generation, and the Balti- 142 A. FRANKLIN SHULL TABLE 34 Showing the number of male- and female-producers in the progeny of two females of Hydatina senta, not related to one another, when reared under the same conditions. One female was collected at New York, the other at Baltimore. New York Pure LINE BatutTiIMorRE Pures LINE NO. OF DATE OF | NO. OF DATE OF aie ee | emp ec nh ae |Mar.26| 0 32 1 | Mar. 25 8 22 Dae: | Pel) ee 40 2 26 1 49 Chae ee 29 7 40 3 28 18 37 Aaa ewer Loe 31 1 25 4 29 10 37 ee | Apr. 1 0 3 5 31 1 Al (Se Ae einer ie 3 0 19 6 ADEs s0L 0 16 eee 5 1 50 7 3 0 39 SN Ra) ch oan cule! 34 8 4 1 27 6 0 2 9 6 23 27 on 9 0 24 10 7 5 46 TCS ce feeiean til 0 iC) fee en 9 7 42 Gas ie sea 13 0 Pas | 12 11 1 34 Thee 15 0 2* 13 12 0 Q* TRS oka 16 0 EB) | 14 14 0 Q* 14er.. 18 0 Dee a5 15 0 2* pee 20 0 2a 16 17 0 2* 1635: 22 0 DEA eet 19 0 2* eA eae ae, ey 24 0 4* | 18 20 0 Q* 1S: 27 0 Cre 22 0 2* TQ aes 29 0 onal ae 20) 24 0 Q* | 29 1 DA ANE Eat 26 0 Q* May 1 0 26 22 27 0 13 De OE 2 0 7 23 29 4 39 Z 0 3 24 May 1 0 25 1 0 2 25 2 5 41 1 0 1 26 3 0 31 oF 3 0 14 7 5 4 44 3 0 31 28 7 3 46 7 OEE 6 1 34 29 9 5 37 08: brithv aa 9 0 39 30 10 23 24 eae Mp 11 1 8 31 12 18 25 age RON Si tie Sl 16 32 14 3 37 PRs: 15 8 30 33 16 6 41 Pfs 17 0 10 34 18 4 41 oR 19 1 27 35 19 a 26 29 ee 21 16 12 36 21 28 11 30. ees 23 0 31 37 23 13 24 LIFE CYCLE OF HYDATINA SENTA 143 TABLE 34—(ContTINUED) New York Pure LINE BALTIMORE PuRE LINE NO. OF DATE OF NO! OF | NOUOF NO.OF | DATE OF ROMO ENOM GT eS el ei Bee ea ee SiMe), SN49, 25 Geet 35: «|r Sseael | 28 10 BOM ae 95s | 26 1 38 39 | 26 6 35 Se ee 28 | 3 23 | 2400 27 1Saealay ot 3y8 Saas 30 | 0 2 41 29 29) | 7 | 31 | oF} 3 42 | 31 Pla ow 6 Bepty fee. <8) Jurie. “1 0 33* | 43 |June 1 mvs 36... 3 1 18% | 44500 4 Dilan 18* 37... 6 15 g* 45 | 6 Oo | 2* 385. 8 i a 1 | 46 8 | Ca eee : 12 100 Peta" )| (47 9) 9 30 39) | Ne: a NO. OF | NO. OF KO. cat |sNO. OF NO. OF BAe oF | NO. OF NO. OF Seen ACen: ly on | Coane | Dees Seanlneie t 1 0 9 1 2) 24. 1 Dp | nals: 0 2 2 15 | 37 2 4 | 39 1 2 3 LR, cA 3 0 | 25 0 | 26 4 4 | 47 4 5 | 41 3 Oa 5 2 | 45 5 a aes Ne LL, 2 Ome 6 2d aos 6 4 | 44 | OQ} 2 ri Io 5 7 3 | 46 Oe Tf 8 pe Re 4 Or) ala © “M31 | ras The’ Wwe 34 | | | i staan | een ee Het Aa et 86 | 305 | 16 239 Percent Ofc GP .1.. | 1.5 | | 21.9 6.2 her Seance 1 | 0 2 iy ol: ~ Same 1 4 | 39 PAL 2 2 16 30 2 0 | 25 f #0F%} 26 3 505.41 ts WP Mpa ai elO ah) 7 4 17sk $28 4 | (9) eu = J 0 Bla oie i2,.\ 42 Pichi Seall at Sper ates eee iG 10 | 43 Ge Nn : 46 ) | | ot | | | ess Or) 14) | | | | | 0% 1,31 | Uy 4 Ly) ee | ALOE RRS ees oo. oe ane ea l22 63 | 221 | 16 | 226 Romcontioneien tl. 16 | | 92.1 liu Pkt 146 A. FRANKLIN SHULL was In a medium which always contained traces of this mixture. Precautions were taken to obviate any error in this regard. In the first place, it seemed likely that the extreme dilution to which any substance peculiar to either environment was subjected would have prevented that substance from exerting any appreciable influence. It must be remembered that the Baltimore pure line was bred for eleven generations under the same treatment as the New York line, before the crosses were made. The first female, and the females of each generation thereafter, were isolated in certainly not more than four drops of water. This was at once diluted to forty or more, or to one-tenth of the original concen- tration. In eleven generations, the concentration of any substance originally present would be only 10! times the concentration of that substance at the outset. Unless the substance had enor- mous powers of propagation, it would be negligible. But to pre- clude any possibility of error, an amount of water approximately equal to that in which the males were transferred in making the crosses, but containing no rotifers, was taken from each line and placed in the dishes of the other. So that from that time on both of the pure line were being reared in the same mixed environment as were the offspring of their cross. The same precaution was taken in each crossing experiment. In both of these experiments (table 35 A and B) the pure line derived from the cross has a decidedly higher proportion of male- producers than did that part of either parent pure line which was bred at the same time. Moreover, it is higher than either of the parent lines produced as a whole, the entire series of genera- tions in the New York line having produced but 11.1 per cent, the Baltimore line 18.5 per cent. It is to be noted that the resting eggs in these crosses were kept at room temperature, whereas many eggs in nature are subjected to continued low temperature. While no experiment of as great duration as a winter season was attempted, some eggs were kept for a brief time, as described in the next experiment, in an ice chest. Experiment XXXIV. Two repetitions of the preceding experi- ment were made, except that the resting eggs obtained after cross- ing were kept for some days at a low temperature. ‘Two females LIFE CYCLE OF HYDATINA SENTA TABLE 36 147 Comparison of two distinct pure lines of Hydatina senta with another pure line derived from a cross between the first two, the winter eggs of the cross having been kept for twelve days at low temperature. A and B are separate experiments. New York Pore LINE | Pure Line From Cross BALTIMORE PurRE LINE EXPERIMENT | | i | | | NOaot | NOMOR NO.OF |) ho | No. oF| No. or || N°-°F | no. oF No. oF ee a ees | Oe ea ioe | ae 1 0 FAN Dri 4 1 5 | At 0 an) 2 Se) 22 2 Ouslanoil 0 zai 33 | 21 29 3 4 | 44 0 Hey oA 4 | 49 4 3 i 146 2 ON 14 5 >) Eas 5 5. | 87 0 31 6) oat, 6 D3, | 24 AY 3 eee 7 est) 7 | is: | 35 4 0 | 39) 8 3 | 50 / 8 Beall Sexe 5 1 8 9 9 | 40 9 6 | 41 BH Tistet 6) 10. | aS 10 4 | 4l ii She) _ 320i), 11 em 9 11 22) IG 8 Orie 10 12 |e One aes 12 28 | 1 9 1 27 | | | i) al 12 | | | | | | | lie Motall..: %.. + | 58 | 234 | | 120 | 382 | 121 | 404 | | | Eee Per cent of | | | eC 19.8 23.9 1? 13u0) 1 0 bf 1 LOO 8 i) 1 Beal 0 Swale 22 Oar i's <9 Ouest 0 Palle s3 CO) alee |e 4 | 4A hr) 1 | | On 23 4 Bh Ze Be: J Pear Owl 14° | 4 Gees 5 Behe oe ] Of Bile Mes 0455 So Az Gay 2oaneed 3 IU o4e i 6, areas vi 1S SADR 4 Cals 39 |. 2% ~ ee ees 5 1 8 [ Gat 16 | | Totalh..ais: 33 | 155 | 90 | 235 || 58 | 248 Per cent of | | | i.) At ee | 8716 || 18.9 148 A. FRANKLIN SHULL from the Baltimore pure line were mated, April 13, 1910, with males from the New York pure line, and resting eggs obtained April 14 and several following days. These eggs were put, with the females, into an ice-chest at 8° to 10° C. on April 14, and left until April 26, when they were again brought to room temperature. On May 1, two females had hatched, one from each parent. From these two females, the lines designated ‘‘Cross”’ in table 36 A and B respectively, were reared. The parts of the parent (New York and Baltimore) pure lines reared at the same time are given for comparison. Here again the pure line derived from the cross yields in each case more male-producers than do the parent lines, though the differences are small, especially in the first two experiments. The smallness of the difference is due, not to a lower proportion of male-producers derived from the cross than in Experiment XXXIII, but to a higher proportion in the parent lines than in the former experiment. The percentage of male-producers in the parent lines fluctuates considerably, and the delay in develop- ment due to low temperature caused the cross to be compared with the parent lines at a time of many male-producers in the latter. Obviously, an experiment of such extent is needed that the error due to these fluctuations may be reduced to a minimum. Such an experiment is the following one, in which the reciprocal cross is made. Experiment XXXV. On May 14, 1910, a female of the New pure line was mated with a male of the Baltimore line, and resting eggs obtained May 15 and following days. The eggs were kept at room temperature until May 24, when one of them yielded a female. From her, the line in table 37 designated “‘Cross”’ was bred. This line is compared with those parts of the New York and Baltimore lines that occur simultaneously with it. That large fluctuations in the proportion of male-producers in any line do not vitiate this experiment seems probable from the fact that the parts of the New York and Baltimore lines here given include approximately the same percentages of male-pro- ducers (10.8 and 15.9 respectively) as did those lines as a whole (11.1 and 18.5 respectively), as shown in table 34. If these figures LIFE CYCLE OF HYDATINA SENTA TABLE 37 149 Comparison of two distinct pure lines of Hydatina senta with another line derived from a cross between the first two, the cross being the reciprocal of that in tables 35 and 36. New York Pure Line Pures |.ine From Cross Motalieee sews Per cent of one) NO. OF | NO. OF KD, fot | NO. oF | NO. OF iy Se See hs | Sens Ho 238 t | d5ageeo Seales 2 Wane Call 25 0 2 3 | 13, aoa 0 3 | 4 |. 74 ieee Qi) 33* 5 30 || 19* fe t8* 6 2F ay 15 g* 7 0 Q* 1 1 8 8 ela 10 | 14 6. Clgeze 1 24 9 22) Naog 4 |) 20 10 ee) 0) 0 9 11 22 Niele OF otk 12 ib fay Ory) iz 13 25 | 19 0 1 14 19 | 26 0 3 15 | 209) 35 0 | 25 16 10: We25 Sealy 22 17 1b, 2h Ona 14 18 7 P16 0 1 19 Wee Ne ite 1 7 20 17) ell Gg al2 21 8.) ao Omelzee| a 22 { }45 | 0 6 23 317/429 Ogi atizigs| 24 9 13 0 2 | 25 8 i 0 | 14 | 26 17 | 30 0 1 27 re ei ely 324. | 0 1 0 6 | | 49 | 403 | 413 | 684 | | 10.8 37.6. || Ba.LtTIMORs PurRE LINE | | Nh OS NO. OF | NO. OF Bear tbl Se ieee 1 6 | 35 2 1S OD 3 | 29 i 4 | Dene sa 5 Pan 28 6 Tiga Pl Fol 7 0 | 2 8 O ze 9 91, $36 9 | 35 10 I, Wake re 3g 11 age ists 12 ee ee) 13 O: |, 38 14 B91 295 0 | 26 15 0 | 14 On eat 16 2 | 22 0 8 0 | 20 17 14 |) 98 18 O | 14 19 pila og 20 Os: 38 21 O. |. 18 22 0 | 36 124 | 652 15.9 * Remainder of family not recorded. 150 A. FRANKLIN ‘SHULL are trustworthy, as I am convinced they are, we have here the remarkable result that the pure line from the cross yields not only a greater percentage of male-producers than does either parent line alone, but decidedly greater than the percentages of both parent lines combined. This result is so remarkable that it became of interest to breed an individual of the pure line from the cross back to a member of one of the parent lines. This was done in the following experiment. Haperiment XXXVI. Several females of the pure line derived from the cross between a New York female and a Baltimore male, in the last experiment, were mated with males of the New York line on June 13 and 14. Of the winter eggs laid by these females June 14 and following days, one hatched June 22. The female from this egg gave rise to the line shown in the middle column of table 38. By an extension of meaning, I shall speak of the whole line derived from the cross between the New York female and Baltimore male as F,, and of the whole line derived from the cross between an F female and a New York male as F:. With F. are compared those parts of the parent (F; and New York) lines which occurred simultaneously with it. A comparison of table 37 with table 38 is most interesting. In the former, where two rotifers in no way related to one another were crossed, the F; gave rise to a line having a much higher pro- portion of male-producers than either parent line; in the latter table, where two related rotifers were crossed, the pure line result- ing from the F, has a proportion of male-producers intermediate between those of its two parent lines. It may also be remarked that in the former experiment F, contained larger families and pro- duced more generations in a given time than did either parent line; whereas, in the latter experiment, the size of family in F: and the number of generations in a given time is intermediate between those of the two parent lines. If this is not an isolated case, it is important. LIFE CYCLE OF HYDATINA SENTA til TABLE 38 Comparison of New York line of Hydatina senta and of the line (F,) derived from a cross between the New York and Baltimore lines, with another line (F2) derived from a cross between the F; and New York lines. New York Pure Line F2 | F, NO. OF NO. OF I NO. OF lee | GENERA- | "oo | cal os Biss aor caine ee | De OF jy ae eee Qt 1 OP) a5 1 15 | 21 DAL ANGIE. ; 0 1 2 3 4 | 2 7H 16 1 i 3 Lose saul 3 Se aT 6 | 12 4 19 | 13 4 Od ES Bee tn eN Aa Oye) al? 5 4 | 20 5 Re eae FMC eee et: 0 6 6 Sse 6 Pes Gusseeeetey al. PO pe ales 0 | 13 | % le Siec 19-99 ew 2 7, 10 48 8 pas 7 cores CO O14 8 OF 19 9 8 | 3 BA eae gd Toe 0 1 9 O 1-208 | 10 171) 30 leas aS Ih 32 10 20 iNse la 11 Aen OF Pe ote Ou el 11 On Waoeal 12 8 | 428 0| 6 12 ipa fe S| 13 Q | 29 herons) mal 0 | 14 i fe | oe0 6 13 Over Ge| 15 Zane 120 1 0 lies 16 Aa 28 0 4 14 0 4 | 17 14 | 21 a0 1 15 O% 12) 18 lars) 2d AES eel Testor 16 tae | LOR BIN PEO | 225 i ae ee eecore |e 17 10a) pee] | le 1 | 0 Be | Ori "s3 | | 0 4 | DDS esis, ck 0 4 | Do Oe oe 0 12 | Gaeien| | TEEPE eee. ot 0 2a 0 4 otal ceey | 203: | 88 | 363 | 188485 Per cent of | | et ae eC) Te | 13:7 27.9 152 A. FRANKLIN SHULL DISCUSSION Those who have studied parthenogenesis and sexual repro- duction in Hydatina have drawn very different conclusions regard- ing the causes of the transition from one mode of reproduction to the other. Some have been impressed with the influence of external conditions in causing or preventing the inauguration of the sexual mode, others have held that internal factors are the chief if not the sole agents in this transition. Among the former were Maupas (’91) and Nussbaum (’97), to whom, tem- perature and starvation, respectively, seemed sufficient to account for all the phenomena they observed. Punnett (’06), on the other hand, discarded both these external agents, and suggested that the factor which determined the proportion of male-producers (sexual females) is wholly internal, and is fixed at the time of fer- tilization. Whitney (’07), in his earlier paper, remained nearly neutral, merely stating that he found no evidence of external in- fluence. He likewise found no evidence for the one internal agent, zygotic constitution, proposed by Punnett. But in a later paper (Whitney, 10) he implicitly ranges himself on the side of those who hold external factors accountable for all variations in the percentage of sexual forms. In my own article (Shull, ’10b) all the evidence presented was in favor of external agents, though the possibility of internal factors was admitted. The evidence presented in this and the earlier paper can hardly fail, I believe, to convince one that both external and internal agents are involved in the production of the life cycle. It has been shown that starvation, of itself, is probably not one of the external agents that have an influence on the proportion of male- producers; but any attempt to bring about starvation by experi- ment may make it seem to have an influence, because food can not be introduced in diminished amount without introducing diminished quantities of other things. We have found that differ- ences in temperature may have an influence, as shown in this paper, but it is practically certain that this influence is an indirect one. If the effect of temperature were direct, it would be expected that its influence upon the proportion of male-producers would be LIFE CYCLE OF HYDATINA SENTA 153 exerted always in the same direction. A comparison of tables 18 and 19 with table 20 shows that this is not the case. A lower temperature may result in either more or fewer male-producers. The results of the several experiments are not contradictory if we assume that the influence of temperature is indirect. Let us suppose that all the conditions, whether external or internal, with the exception of temperature, which exist at a given time, tend to produce at a given temperature either a higher or a lower proportion of male-producers than prevailed previous to that time. If at a lower temperature the response of the rotifers to other conditions is less than at higher temperatures, all the results here obtained find their explanation. In tables 18 and 19 we may suppose that conditions at room temperature tended to pro- duce fewer male-producers; in the ice-chest, the rotifers did not respond to these conditions to the extent that they did at room temperature, and the result was more male-producers at the lower temperature. In table 20, it seems that a set of conditions was present at the end of January which tended to produce more male- producers, and that the low temperature of the ice-chest caused the rotifers to respond to these conditions to a smaller degree, the result being fewer male-producers at the lower temperature. This view encounters no difficulty in the fact that no difference in the the proportion of male-producers was obtained at a temperature of 20° and 24.°5 C., respectively; for if the temperature is suffici- ently high that the rotifers may respond to other conditions to the greatest degree of which they are capable, it may make little difference which of several moderately high temperatures prevails. In offering this explanation I have assumed that the response of the rotifers to both external and internal conditions may be modified by temperature. It is obviously possible to assume that the response to external conditions alone is so modified. In that case, the results shown in table 20 demand a set of external con- ditions which tended to increase the proportion of male-producers above that usually obtained from the same line in spring water. It is possible that such external agents exist. Whitney (10) believes that there are chemical substances having such an effect. 154 A. FRANKLIN SHULL So far, however, the chemical agents of which we have definite evidence, if added to spring water, all decrease, instead of increase, the proportion of male-producers. I have therefore preferred to assume, until more is known of the external agents, that the re- sponse of the rotifers to internal conditions may also be modified by temperature. More effective than temperature in modifiying the proportion of male-producers, are certain chemical substances, as a glance at tables 27 to 33 will show. Creatin has a remarkable effect in reducing the proportion of male-producers. Beef extract has an equal effect, but it is a mixture of many substances. Ammon- ium hydroxid and three ammonium salts, the chlorid, the car- bonate, and the nitrate have a distinct effect, but not as marked as creatin or beef extract. Urea may almost certainly be put in the same class. All these substances tend to reduce the propor- tion of male-producers. Unlike temperature differences, the effect produced by these substances was in every case of the same sign. It is to be noted, however, that all of the experiments with a given substance were performed with a single pure line, whereas those with temperature included experiments with two distinct lines. This may or may not make a difference in the result. To the above substances which have an undoubted influence may be added perhaps the degree of alkalinity. In several cases a greater alkalinity seemed to produce fewer male-producers, but the effects were slight and the results were not uniform. Whitney (’10) in a recent communication is in substantial agreement with the author, in that he finds chemical substances responsible for considerable effects upon the life cycle. He has experimented with the manure solution used by me, and from these experiments, together with certain observations, he con- cludes that certain substances in the manure solution affect the proportion of male-producers. But in the details of the conclu- sion we differ. In my two former papers (Shull, ’10a and ’10b) I had expressed the conviction that certain substances present in the manure solution prevented the male-producers from appear- ing. Whitney believes that a certain substance may be present which causes male-producers to appear, and that it is only when LIFE CYCLE OF HYDATINA SENTA 155 this substance is absent that female-producers alone occur. Inas- much as I used old food cultures (manure solutions) without caus- ing any epidemic of male-producers, Whitney holds that the sub- stance which does cause male-producers to appear is a transitory product of decomposition of the manure, which is therefore pres- ent only in new solutions, not in old ones He states his point clearly (Whitney, 710, p. 348): ‘‘I would maintain that there seems to be a definite but transitory chemical substance produced in appreciable quantities in the decomposition processes in newly made horse manure cultures that can so act upon the partheno- genetic females as to cause them to produce sexual daughter females. When this substance is absent, no sexual females are ever produced, but only parthenogenetic females are produced * * * ” Tn these words Whitney clearly implies the belief that no internal agent in Hydatina ever tends to produce sexual females (male-producers) and that therefore whenever male-pro- ducers appear they are a manifestation of some external agent. I had assumed, on the contrary, that internal agents probably tended to cause some male-producers to appear, without the direct aid of any particular substances in the medium, and that the pres- ence of certain substances prevented them. In further support of the view that some substance causing male-producers to appear is present in new cultures but not in old ones, Whitney cites his own earlier findings (Whitney, ’07) that the male-producers occurred predominantly in the earlier parts of the family. Since the food culture was new in the first part of the family and old in the latter part, one might expect that some chemical difference between old and new food cultures caused the male-producers to appear early in the family. That this evidence can not stand seems probable from data presented in the second of my papers (Shull, ’10b), where it is shown, from a large number of families, that male-producers do not occur more abundantly in the first half of the family than in the last half. Whitney has not found any specific substances which cause male-producers to appear. Search for them may well be success- ful and should certainly be made. But it is now certain that the absence of the substance which, he supposes, causes male-producers 156 A. FRANKLIN SHULL to appear is not the only means by which they may be prevented from appearing. The presence of creatin, the presence of beef extract, the presence of certain ammonium compounds and of urea, all tend to prevent the occurrence of male-producers. These facts should not blind us, however, to the possibility, which Whit- ney points out, that other substances may cause male-producers to appear. As to the internal factors, which Whitney is inclined to discard in accounting for all male-producers, I have shown elsewhere that these internal agents exist; and I believe that we may attribute to internal agents, alone or in combination with external agents, some of the phenomena which have been held to indicate the presence of external agents alone. Certain points of theoretical interest may be mentioned in connection with chemical substances. Inasmuch as the question was raised whether the action of temperature in altering the pro- portion of male-producers is direct, and was answered in the negative, it may also be asked whether that of chemical substances is direct. The chief evidence for supposing the influence of tem- perature to be indirect was the fact that it sometimes increased, sometimes diminished, the proportion of male-producers. Among chemical substances, on the other hand, with the exception of the degree of alkalinity, which produced differences too small to be of much value, the effect of each chemical was in every case tried of the same sign. This of itself would lead us to believe that the action of these substances is direct. But this is not necessary. The substances may, for example, induce a physiological state which is directly responsible for determining the mode of repro- duction. It may also be pointed out here that nothing in the evidence yet obtained shows whether these substances actually decide how events shall occur in a given cell, or whether, events having happened differently in two classes of cells, the substances in the medium merely decide which class may most rapidly and success- fully develop. The conclusion that certain external agents may modify the ratio of sexual to parthenogenetic females has, I believe, been LIFE CYCLE OF HYDATINA SENTA 157 completely established. In like manner, it can no longer be doubted that, as intimated above, internal factors may exist which modify thatratio. This is of particular importance because of the tendency which has existed to attribute all the phenomena either to external factors alone or to internal factors alone. Little can yet be said regarding the nature of the internal differences. Punnett suggested that the ratio of male-producers in a pure line depends upon the character of the zygote from which the pure line springs, and the nature of the zygote in turn depends of course upon the nature of the gametes. Were it not for the possi- bility of modifying that ratio by external conditions, much in the experiments described in this paper might seem to support Pun- nett’s view. When two unrelated individuals, coming from two pure lines which behaved differently with respect to the propor- tion of male-producers, were crossed, the zygote gave rise to a pure line which, when reared under the same conditions as the parent lines, yielded a higher proportion of male-producers than either parent line. The same result was obtained in every experi- ment of this kind. But when two rather closely related individ- uals, one from the F; line just mentioned, the other from one of the original parent lines (Experiment XXXVI), were mated, the zygote gave rise to a pure line having a proportion of male-pro- ducers intermediate between those of its two parent lines. If it had been found impossible to alter these results by employing different external agents in the different pure lines, we might per- haps be justified in saying that zygotic constitution is alone re- sponsible for the ratio of male-producers. It was possible, how- ever, to reverse the results. The F; pure line which was yielding more male-producers than either of its parent lines, was made, when reared in beef extract, to yield fewer male-producers than its parent lines. Other things being equal, some internal factor, perhaps zygotic constitution, causes different pure lines to yield different proportions of male-producers; but Punnett’s assumption that zygotic constitution determines that proportion regardless of external conditions is not justified. There are no pure ‘‘strains”’ producing 40 per cent of male-producers or 2 per cent. A line producing 40 per cent can be made to yield 20 per cent as long as 158 A. FRANKLIN SHULL the external conditions are properly selected; and any line with a not too high proportion of male-producers can be made to yield no male-producers. There is probably no such thing as a line which, under all circumstances, yields no male-producers. But even if we recognize that the internal factor is only one of several which together determine the proportion of male-producers, the internal agent may still not be zygotic constitution. It is quite possible that something, perhaps environment, may permanently modify the internal nature of a pure line, as Woltereck (09) believes may occur in the daphnians. Such a modified internal nature could not be called zygotic constitution. Whether such a modification can occur in Hydatina is unknown, but it is concelv- able; and so long as it is possible, we must not cling too tena- ciously to zygotic constitution as the sole agent in producing the internal differences described. It would be of great interest to know the process by which the internal factor, whatever it is, affects the life cycle. Does the cycle depend, internally, upon the quantity of something intro- duced by the gametes, or does it depend on segregable genes that combine and recombine in Mendelian fashion, or does it follow some rule not analogous to anything known in other animals? Furthermore, do any of these agents, external or internal, deter- mine whether the cytological changes accompanying a change of the mode of reproduction shall occur in a given cell, or do they merely decide which of two already differentiated classes of cells shall go on and develop? These questions must for the present remain unanswered. There is no evidence on which to base an answer to the second question; while if we attempt to answer the first, all we can say is that if the internal agents work either quan- titatively or in a Mendelian manner, the process is a complicated one. Results like those obtained from the crossing experiments with Hydatina have not been reported, so far as I know, from any other animal. OD a) = (oy) — sH OF 19 19 29 19 29 19 19 19 A) AN “Ul “Ul L061 L061 L061 ‘8 O€ OT L06T 8 OE OT LO6T ‘8 O€ OT LO6T 8 0 OT LO6T "8 GEOL LO6T ‘8 OF OT LO6T "8 Ge OL LO6T "B GE" OT LO6T ‘8 CY OT LO6T "8 Ge OT L06T "8 GE OT LO6T "8 GP OT LO6T ‘08 ‘9% ‘61 ‘ST vat ‘OT mal ‘00d ‘00d ‘09d ‘00d ‘00d 00d ‘00d ‘00d ‘00d ‘00d Deval ‘09d ‘0d ‘00d ‘00d =) a =) =) i=) + ;UOTJOBOI OT} JO UOTJOOATC 8 L “IVOGIAIGNI FHL dO HARWON “CWO 9970) 7462) aanjoaup 07 (QT “Ow “70TT) 4DSaDI DYWN'T fo suoyonas ponpurpur fo fisnmuny * WTAVL THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 193 tion of the larva was noted and recorded. Records in total dark- ness were taken in the same manner with the aperture for light entirely closed. Table 5 is a summary of all records for both species. TABLE 5 Reactiveness to directive light through the general range of intensity for migrated larvae of Lucilia caesar (Lots no. 6 [A] and no. 25 [B]), and of Calliphora vomitoria (Lot no. 24.)* REACTIONS SOURCE OF LIGHT INTENSITY IN C. M. i, aaeTee Guvomitorix + | = + 0 = 0 Diffuse daylight...... | ? CA) OF 250m 0 0 50 0 ANTEC Ned NRA ee eet oe 800. (A) 0] 50 0 0 47 3 Tinie armcla lito: niGee eens ene 0.56 (A)1} 44 5 3 44 3 linea Gepic nities 0.1764 (A) 5 es? 8 8 32 10 mesamde lights... 5.2 «,- 0.0342 (A) 6} 35 8 Da aa eats Iincamic elitohiiee seers 0.00705 sym) | S83 | ks 7 | 24 19 iaeamnic allie hie ee eee 0.00176 (B) 2} 28 20 8 iL? 25 Ime aracls lienite sete ee 0.00063 (B)5| 14 28 9 | 11 30 lim@zinel, Ibid. 5 oe koa lee 0.00007 CA) 1G ae 46 5 4 41 or —_ = ie Total darkness ((A)2| 2 46 * The reactions (+—0 = positive, negative, and indifferent respectively) are based on the movements of ten larvae given five trials each with an exposure of thirty seconds. Between trials—taking larva no. 1 first, then no. 2, etc., through the series—each individual was kept separate in a closed receptacle. An inspection of table 5 shows that in Lucilia caesar the lowest directive intensity is 0.00176 C.M. and for Calliphora vomitoria, 0.00705 C.M. This difference in sensitiveness was quite evident in all experiments in which both species were involved, L. caesar responding more readily and being more active than C. vomi- toria. Below the respective minimum directive intensities, light has, however, a dynamic effect until an intensity of 0.00007 C.M. is reached, when there is neither a directive nor a dynamic effect. The larva under such conditions remained perfectly quiet, as though in the dark. This is illustrated by fig. 24, which shows the tracings made by larvae in total darkness. There is a certain 194 WILLIAM BRODBECK HERMS amount of stimulation due to rolling the larva into place; under such conditions it makes many randon movements, is clearly un- directed, and soon comes to rest. The large number of indifferent reactions recorded for the lowest intensity and total darkness is based on the behavior just described; whereas the reactions recorded in the same column for higher intensities may also be based on movement to right and left, neither positive nor negative (commonly designated as zero movements). . 5. Light graded in intensity Two methods of light grading were employed. The first was by means of the light grader and was of non-directive nature v.e., vertical light directed on a horizontal stage on which the animal moved. A field of light was thus secured ranging from a maximum of 70.69 C.M. to 0 within a given distance. The sec- ond method, illustrated by fig. 11, gave a field of graded light directive in its nature, but gradually increasing in intensity over a given area in the direction of the rays. Such a field was produced from the vertical filaments of a 66 cp. incandescent lamp (L) by placing an opaque screen (C) in front of it in such a manner that the light from the entire length of the filament could reach the horizontal stage at a point (B), one metre distant from the upper- most portion of the filament. On both sides of this point (B) the light gradually diminished in intensity; toward the filament it diminished because less and lessof the filament wasinrange of the stage, and away from the filaments it diminished because of the radiation of the light. The behavior of the larvae in reference to the two methods will be discussed separately: first, behavior under non-directive graded light, and secondly, behavior toward directive graded light in the above sense. A. Non-directive graded light. A field of light of this character was produced ‘by means of the light grader already mentioned. Triangular diaphragms of various altitudes were used, the extreme lengths producing fields of light 2 em. broad and respectively 10 cm. and 2 em. long. The intensity of the light in the strongest THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 195 region was 70.69 C.M. and it gradually diminished to nothing at the other extreme. Of the two extreme lengths mentioned, the longer resulted in a gradation of 7 C.M. per centimetre and the shorter in a gradation of 35 C.M. per centimetre. Since the sarcophagid fly-larvae are negative to ight, one would expect them to turn to the darker portion on entering such a field of graded light. This would also be expected when one considers the results obtained by means of light on two sides, as illustrated by figs. 3 and 4. The courses of the larvae always lay more or less transverse to the rays, the greater deflection being toward the light of lower intensity regardless of the size of the luminous field. When the larvae were placed midway between two balanced lights of like luminous area (likewise when of unlike area, but like intensity), their course lay transversely to the rays, as illustrated by fig. 5. These results are in accord with the statements made by Loeb (’05): “If there are two sources of light of different inten- sities, the animal is oriented by the stronger of the two lights. If their intensities be equal, the animal is oriented in such a way as to have symmetrical points of its body struck by the rays at the same angle.” If, now, the negative fly larva is in a field of graded light, one would expect it in creeping to take a course toward the darker side of the field until stimulation (light from above)became equal on both sides, and decreasing in intensity. Clearly the opposite direction would be out of the question, since that involves a grad- ual increase in intensity, though there would be a chance for equal bilateral stimulation. Since locomotion is involved and conse- quently directive stimulation in order to bring the larva in right relation to the field of light, it was necessary to start the animal off in the proper direction by means of a light from behind the larva. which could be controlled. This was first accomplished by means of a7 ep. incandescent light. Thus the larva was properly oriented and would continue traveling in the same direction for some time after the light was turned off. The more frequent result on reach- ing the graded field was that the larva passed into this area of light, exhibiting the usual random movements, but the after effects of the directive light, though the light was turned off ten 196 WILLIAM BRODBECK HERMS EXPLANATION OF FIGURES 3 TO 10 3 Calliphora vomitoria. Course of a larva with light of differing intensity from opposite sides. 4 lucilia caesar. Course of a larva with light from two directions, and differing in intensity. 5 Calliphora vomitoria. Course of a larva with balanced light on opposite sides. 6 Calliphora vomitoria. Behavior of larvae in non-directive graded light, under a directive intensity of about 50 C.M. 7 Calliphora vomitoria. Behavior of larvae to graded non-directive light, after having previously been in the dark. Note the strong deflection of the path due to the after effects. 8 Lucilia caesar. Behavior of larvae to graded light while under the influence of a weak directive stimulus (about 0.5 C.M.) 9 Lucilia caesar. Movements of the larvae when forced into the negative end of a field of light grading at the rate of 7 C.M. per cm. There was a con- tinuous directive intensity of about 0.5 C. M. 10 Lucilia caesar. Movements of a larva when forced into the negative end of a field of light grading at the rate of 35 C. M. per cm. There was a continuous directive intensity of about 0.5 C.M. THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 197 —_— 2640CM 159 CM. < —— 733 0.M —_. 733 CM. 198 WILLIAM BRODBECK HERMS to twenty seconds before, were sufficient to keep the larva mov- ing in the general direction taken at first (fig. 7a). Less fre- quently the larva withdrew after a few random movements and then took a course along the border of the field either to the right or to the left (figs. 6 right hand tracing and 7b), or in a very few cases turned directly back on its own course. Apparently the ‘‘starting off”? was brought about by means of an intensity too high in proportion to the non-directive light. Therefore in all later experiments a directive light of about 0.5 C.M. was employed. Under such conditions the larvae very sel- dom went into the field of light: but, once in the field, they passed through in the manner described, again with no constant relation to intensity. It was then decided not to turn off the directive light, but keep it on the larva continuously. The result of these experi- ments is shown in fig. 8, which represents the usual reaction either to right or left. The larva on arriving at the edge of the field of higher intensity withdrew and then moved somewhat transversely to the directive rays which however, soon caused it to take up its usual longitudinal orientation, as a result of which if once more entered the more intense field, but again withdrew. This behavior continued until the region of higher intensity was passed where- upon the course finally followed the rays of the directive light. Over 150 recorded experiments, as illustrated by the figures were made for fields varying from 10 cm. to 2 em. in length. An equal number of unrecorded experiments (7.e. unrecorded by larval traces) were made in which the light passing through the plate glass stage was reflected out of the light grader by means of the mirror always in place. There is clearly no constant relation between the movements of the animals and the graded field. It was further found that the larvae could be forced entirely through the graded field from the dark (0) end to the brightly illuminated end (70.69 C.M.) when an intensity of 50 C.M. or over was used to direct them. This was not possible when the directive light had an intensity of only 0.56 C.M. Under this in- tensity the larvae would, however, pass farther into the darker end of a field grading at the rate of 7 C.M. per centimetre, than into one grading at the rate of 35 C.M. per centimetre. Though THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 199 the relation was not constant the usual distance was about five times as far in the former field (fig. 9 and 10). Mast (’07) found that Volvox, which is positively phototatic, was deflected toward the more strongly illuminated side in graded light and concludes (p. 141) that ‘‘The direction of motion in Volvox exposed to light is consequently regulated by the intensity of the light on opposite sides of the colonies regardless of the direc- tion of the ray.’”’ This conclusion, as Mast shows, is in direct opposition to the statement made by Loeb (05) and already referred to. From the experiments already enumerated and others to fol- low (B), it becomes quite evident that the results obtained with sarcophagid fly-larvae are quite in accord with Loeb’s (’05) con- clusions, and that the statement made by Mast (’07, p. 136) is quite apropos at this juncture. ‘‘Let it be clearly understood that in the criticism of Loeb’s conclusions, I do not wish to inti- mate, that because the reactions of Volvox or any other organism do not take place in accord with those conclusions, they neces- sarily cannot hold for the organisms Loeb worked with.’’®> Mast’s further statement on the same page in reference to that investi- gator’s results is, however, equally applicable, viz. ‘‘I do, however, wish to state and emphasize that in my opinion his experimental results as quoted above, do not warrant his conclusions, even for the animals worked on, much less for all organisms which orient to light.” B. Dhrective graded light. A field of light, directive with reference to the larvae, graded to higher intensities in the direction of the ray was produced in the manner already explained. The field (AB, fig. 11) was 36 em. in length and 25 em. in width, pro- viding ample room for long journeys under these conditions. The grading was from the point A, with an intensity of less than 1 C.M., over a distance of 36 cm. to a point B, with an intensity of 66 C.M. The larvae were started about 2 cm. beyond the point A, where orientation away from the light took place. Locomo- 5Loeb’s conclusions, were based on results obtained from experiments on blow-fly larvae among other species (Loeb, ’90, pp. 70-71). 200 WILLIAM BRODBECK HERMS tion under such circumstances brought the larvae into successively higher intensities, and in consequence numerous trial movements were produced and creeping ahead was accomplished very slowly. As the larva advanced into regions of still higher intensities, its trial movements were continued, though reduced in number, but the rate of locomotion was increased. A very pronounced change in behavior was evident when the point B was passed and the path lay in light decreasing in intensity. The rateof movement Fig. 11 Outline of apparatus to produce a field of light increasing in intensity in the direction of the rays. L, a 66 cp. incandescent lamp elevated on a stand (S) ; C,an opaque screen; DD, an opaque screen with an oblong aperture about the size of the incandescent bulb, to eliminate side light; AB, a field of light produced by the lamp (L), gradually increasing in intensity from A to B. increased perceptibly, trial movements were visibly eliminated, and the course was more nearly a straight line. The rate of move- ment for a distance of 10 em. on the brighter side of the point B was greater by 10 per cent than for a like distance on the other side of this point. Thisaverage is based on continuous movements of ten larvae each given a single trial after 12 to 14 hours of rest. In the field AB there was a very marked tendency on the part of the larvae to take a diagonal course to the right or the left. Out of 50 trials on 10 larvae, 50 per cent of the paths were diagonal, THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 201 whereas under usual conditions with like intensity such diagonal courses were reduced to about 15 per cent in the same number of trials. Out of 50 trials, 4 per cent (two trials by the same larva) resulted in a return to the point A, the larva refusing to go into regions of higher intensities. This larva in its remaining trials (three in number) took a sharply diagonal course. Under the heading of ‘‘ Accuracy of Orientation,’’ Walter (’07, pp. 79-80) notes that the negative planarians subjected to direc- tive light showed a strong tendency to take a path in a diagonal direction, and calls attention to the similar case found by Smith (02, p. 469) for the earthworm. Walter believes this to be due in the case of planarians, to imperfect orientation resulting from the crescentic pigment shields of the eye, which would permit a diag- onal path to the right or left to a certain degree without allowing light to stimulate the retina. In the eyeless earthworm, though Walter does not suggest this, the diagonal path may have been due also to imperfect orientation. Furthermore, the arrange- ment of the sense organs on the segments, as shown by Harper (05), would certainly permit a more or less diagonal course, 7.e., a turning from side to side to a certain degree as the worm crawls would be possible without subjecting the sense organs to stimu- lation from light. A very much more perfect orientation is possible on the part of the fly-larva because of the localized condition of the photore- ceptive function as discussed on page 205, ete. In these organisms the receptive surface is restricted to the extreme anterior pole, and as the animal travels away from the light, this part of its body lies in its own shadow, so that the creature is continuously oriented within a narrow range of shadow. 6. Intensity and rate of movement While experimenting with various intensities of light, it became apparent that the larvae crawled more rapidly as the directive light became more intense. The question naturally arose,—What is the relation between the intensity of the light and the rate of movement. This matter was tested in the following manner, 202 WILLIAM BRODBECK HERMS The larvae were started in directive light from a drop of tap water (slightly colored with Methylene blue), which afforded the necessary moisture for crawling upon a sheet of paper. The indi- vidual was given two to three centimetres in which to gain proper orientation. This brought its extreme posterior end on the start- ing line, from which the larva was timed by means of a stop-watch over a distance of 10 cm. The course of these animals when once properly oriented is nearly straight, and the time is based on con- tinuous movement; larvae which paused in the course were removed and not used again till after they had rested. This measure was very seldom necessary. The light intensity was cal- culated for the middle point of the course, since the results in rate will be more nearly in accord with the givenintensity as an approx- imate average. The tabulated results are based on the movements of ten larvae, each given a single trial (except as above mentioned), with a rest of from one half to one hour before exposure to the next intensity. This method was pursued in order to guard against excessive mechanical stimulation or exhaustion. No heat screen was used, since no difference was found in average between the rates of larvae in high intensities with and without a receptacle of water interposed to absorb the heat. TABLE 6 The relation between the intensity of directive light and the rate of movement of sarco- phagid larvae, based on the time in seconds required for migrated individuals to travel ten centimetres. The intensity of the light is calculated for the middle point in the course. | RATE FOR 10 cm. SOURCE OF LIGHT INTENSITY IN C. M. L. caesar C. vomitoria sec. sec. Incandescent........... 0.00176 30.10 Incandescent........... 0.00705 29.66 Incandescent...:....... 0.56 27.64 55.86 Incandescent........... 325.0 24.02 50.28 Incandescent........... 1057.0 21.34 3000 Ame lig hithenteerrt \ereonis 5000.0 18.86 29.16 THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 203 One must conclude from the summary of the experiments in table 4 that the rate of movement increases with the intensity. Davenport and Cannon {’97, p. 32) say for Daphnia, ‘‘Since there is no close relation between diminished intensity and the longer time required for migration, it seems more probable that this longer time is not the result of lower intensity, but that it is due to diminished precision of orientation showing itself in hesi- tating movements.” Although this statement was made for a positive organism, it might nevertheless be inferred that the same conclusions would hold for negative organisms as well. This is in part true of the sarcophagid fly-larvae. Certainly there are many more random movements in very low intensities, as illus- trated by fig. 16 (p. 219); these movements disappear largely if not entirely at high intensities, e.g. fig. 19 (p. 219). But it cannot be concluded that the increase in rate of movement in high intensities is due entirely to precision of orientation, since the light not only has a directive but also a dynamic effect. In table 5 it is shown that there is such a result even when the direc- tive element has ceased. By closely observing the larva as it travels in high intensities, e.g., 1000 C.M., it can readily be seen that the crawling movements are greatly accelerated. This was particularly noticeable when the larva was under the conditions shown in fig. 4. When the animal passed into the field of higher intensity (1056 C.M.) its longitudinal contractions and elonga- tions were perceptibly increased in rapidity after re-orientation, and the sidewise movements of the head were visibly less. Again, in a series of experiments discussed on page 199, in which the larvae were moving away from the source of light in a field gradually increasing in intensity, both phenomena were evident, viz., the sidewise movements decreased in number, while the rate increased. The sidewise or random movements were, however, greatly exaggerated under the conditions already pointed out. Yerkes (’00) in a further study of Daphnia also concludes with Davenport and Cannon that the increase in rate for these forms depends chiefly upon precision, but found evidence of a ‘‘ quicken- ing of the swimming movements.”’ 204 WILLIAM BRODBECK HERMS Davenport and Cannon found that the relation existing between the rate of movement (Daphnia) and the intensity of the light could be expressed thus: with one-fourth light, about 118 per cent of the time with full light. Yerkes expressed this in the following ratios: ratio of intensities 5.12: 1, and ratio of rates 1:1.25. By calculation such ratios may be roughly approximated in table 6, however, only with regard to the higher intensities. No like ratios could be established for the light of lower intensities, since under such conditions the course of the larvae is not continuous, but is greatly influenced by random movements. It must also be noted that the maximum rate for any single larva (1.64 second per cm.) was already attained in the 1057 C.M. intensity by one individual and not exceeded in 5000 C. M., though the average for this intensity is 1.88 sec. per em. (See also Nutting, ’08, in this connection.) It is apparent that the rates for the two species are quite differ- ent, but the ratios hold approximately for each. It was quite out of the question to secure a set of even approximately uniform rates in low intensities for C. vomitoria, because of the extreme individual variation, due to hesitation and wandering. 7. Sudden change in intensity One is naturally led to ask the question: What is the natur> of the response when a light is thrown suddenly from above upon the animal as it creeps in the dark or in anillumination of low intensity. If one considers for a moment what the result would be upon an animal having eyes, one might be led to expect a similar result under similar conditions when a fly-larvae is suddenly illuminated, viz. that it would pause momentarily, at least, and probably be thrown out of orientation. But it must be considered that in the sarcophagid larva we have to deal with an eyeless organ- ism. Various observers have found that certain organisms are thus affected to a greater or less degree. Yerkes (00, pp. 416-417) found that neither Daphnia nor Cypris responded by turning, but that both species quickened their movements as the result of sudden illumination from above. Jennings (’04, THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 205 pp. 49-50) describes what he calls a typical motor reaction for Euglena when, swimming toward the source of light, the illumina- tion is suddenly decreased. Mast (’06, p. 370) found that “‘Sten- tors which are oriented to a given light respond with the motor reaction to an increase in intensity of the light, they are for the time being thrown out of orientation.’’ It was also found by Walter (07, p. 63) that Planaria gonocephala ‘‘showed a decided response —either some change in course or a wigwag motion of the anterior end—more frequently when suddenly subjected to dark than to light.” To test the larvae of the sarcophagids to sudden changes of intensity, the light grader was employed so as to throw light from above upon the animals as they crawled on the glass stage at the focus of the lens. By means of a rectangular opening, instead of one triangular in outline, an ungraded field of 70 C.M. throughout was produced. The larvae were caused to crawl in the direction of a given area which could be suddenly illuminated by with- drawal of adiaphragm, when the individual had reached the proper position. The results were quite uniform; whether the larvae were crawl- ing in the dark, in 0.5 C.M., or in 50 C.M. directive light, all pro- duced random movements, and were more or less thrown out of orientation. The after-effects of the directive light on the larvae traveling in the dark, were sufficient to cause such individuals to retain the original general direction even after having been sud- denly illuminated. The accompanying figure (fig. 12) shows clearly the nature of the reaction. On the other hand by a com- parison of fig. 16 to 19 it is quite evident that sudden decrease in intensity, e.g., decrease from an intensity of 960 C.M. to total darkness, does not throw the larvae out of orientation, nor does it cause any apparent disturbance at the moment. §. Localization of the function It has already been pointed out by Loeb (’90, pp. 71-72) that only the raysof light striking the oral poleof the larva are effective in orientation. This conclusion, however, was reached by means 206 WILLIAM BRODBECK HERMS of rather crude methods: the larvae were placed on a board, which was thrust forward out of the shade in such a manner that the oral pole of the larva was subjected to sunlight. This caused the animal to withdraw its head and take up a position parallel with the rays. A similar experiment with reference to the aboral pole of the larva did not result in a like orientation. Sever- ance of the anterior segments also resulted in an inability to orient when subjected to light. Loeb rightly lays little stress on the results of this latter method. The experiments cited are characterized by the following statement made by Loeb earlier in the same paper (p. 21). “‘Die Thatsachen die ich nachzuweisen habe, sind von so einfacher Art, dass fast jedes technische Hiilfsmittel dabei entbehrt werden kann.”’ Later observations by many different investigators have proved that the reactions of the lower organisms to light are not of the simple nature inferred by Loeb. It will be observed that the position often occupied by the feed- ing larvae in reference to light must lead one to suspect that the posterior parts are not strongly sensitive, if at all. The head under such conditions (7.e., feeding) is buried in the tissues of the flesh, while all the rest of the body may be protruded in full daylight. Furthermore, when the larva travels away from a source of light, its aboral portions are fully exposed to the light, while the head is obviously kept in shadow as much as possible. The manner in which the larvae wave the head about in response to weak inten- sities leads one also to suspect that the rays falling on this part serve as a directive agency. A pencil of light may be employed with which to explore the entire body of the larva in order to ascertain the sensitiveregions or region. Even with a fine pencil of light there will be some diffu- sion, but by means of a pinhole aperture (fig. 2, J) diffusion can be reduced to a minimum as compared with the length of the larva. All parts of the length of the animal were carefully explored, but only one region was found where the light caused the larva to turn, and that was at the very tip of the oral end. By throwing the pencil on this region continuously, the larva could be forced to crawl in a circle as illustrated by fig. 13. Blackening this region THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 207 with a mixture of lard and lampblack, and then il‘uminating that portion, failed to produce any turning until the substance was rubbed off in crawling. On blackening one side of this region only and exposing the larva to ight from overhead there were produced the typical circus movements with the pigmented side toward the centre of the circle, as found by Holmes (01) for negative terres- trial amphipods. It should be said that each larva was first tested for the normal reaction before the pigment was applied. It was also possible by careful manipulation to snip off the seg- ment possessing the anterior hooks (the first segment), but the results were most unsatisfactory, since the larva is almost wholly dependent upon these hooks for locomotion, and consequently its reactions were questionable. Moreover, larvae thus operated on die in a few days. Another series of experiments was tried with the light pencil apparatus, which gave further evidence toward the restriction of photo-sensitiveness to a very limited region at the oral pole of the larva. The individuals were started from a drop of tapwater on the slate stage (fig. 2, 7) toward the light pencil (A). As soon as a fair start was made the light (L) was turned off. If the con- clusion already reached is to hold good the fly larva should be more or less sharply deflected to its left side on encountering the pencil of light. The moisture on the animal from the drop of water left a trail on the black stage so that its movements in the dark could be traced after again turning on the light. Ten migrated larvae of C. vomitoria were tried, each five times, and out of this total of fifty trials with the light striking the right side of the individual 80 per cent of the courses were deflected to the left. When the light pencil acted on the left side of the larva, again 80 per cent of the courses were deflected, but this time to the right. It is quite evident that this signifies a well balanced bilateral condition of the sense organs. The remaining 20 per cent of the trials represent courses taken straight through the light pencil without deflection; there were no deflections toward the source of light. If any deflection was to take place, it occurred each time when the head of the larva came into the light; as soon as the head passed through the illuminated THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 2 208 WILLIAM BRODBECK HERMS area into the darkness beyond, no further deflection took place. The light pencil at the point of experimentation was about 2 mm. in width, and individuals which had gained unusual headway might rush, so to speak, through the light because of its narrow proportions. The larvae usually paused on encountering the light and made trial movements in a very striking manner, occasionally 12 eats ss Fig. 12 Lucilia caesar. Course of a larva that was creeping in a directive in- tensity of about 0.5 C. M., when ungraded light of 70 C. M. was thrown upon it from above. Orientation is clearly influenced. Fig. 13 Course of a larva of Lucilia caesar when the light pencil is played continuously on its head. Fig. 14 Lucilia caesar. Typical course of the larvae when encountering the pencil of light. Fig. 15 Course of a larva of C. vomitoria showing the influence of the after- effects of directive light. The individual was started under a directive intensity of 3 C. M. and crept in total darkness from X to Y, when a new directive intensity of 0.56 C. M. was applied from in front. stretching the anterior segments so far that the darkness on the other side was reached, when the larva continued on its way. The larvae of Lucilia caesar were not givenasmany trials, but ten larvae, each given one trial for the right and one for the left side, were deflected sharply each time as is illustrated by fig.14. This again bears out the conclusions that this species is more sensi- tive to light than C. vomitoria. THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 209 9. The adult sarcophagid In an earlier paper on the sarcophagids, I (’07, p. 49) assumed that the eyes of these animals are of much importance in orienta- tion, because of their relatively large size. But attention was also called to the fact that the chemical sense is probably of more im- portance in detecting the presence of food, since it could hardly be assumed that the vision of these animals is so acute that so small an object as a fish could be seen at any great distance. On several occasions a dead fish wrapped tightly in folds of paper was carefully enclosed in a-tight box so that odors from the fish could not be detected. The package was then carried to a location far removed from the beach where no flies were to be seen and there opened up and the fish exposed. In ten minutes many sarcophagid flies were hovering about, and some eggs had already been de- posited. It can hardly be assumed that these flies found the fish through sharpness of vis‘on. It is the object of the following series of experiments to test the eyes of these flies for their image forming powers. Other insects have been worked on to ascertain their powers in this respect, among them the mourning cloak butterfly (Vanessa antiopa) by Parker (’03) and Cole (’07), which species will form a basis for comparison. A second question to be considered is one of distribution, alluded to in the Introduction, viz: Is there any relation between the pho- totropism of the two species and the fact that L. caesar is primarily a fly of the fields and C. vomitoria more or less a household form. An answer to both of these questions may be sought by the use of an apparatus described by Cole (07, pp. 840-346), but some- what modified to meet the present need. The method and apparatus may be briefly described as follows. Two lights of different areas were used, one, a single Nernst fila- ment on a 110-volt circuit placed back of a metal sheet in which a narrow slit was cut; the size of this slit could be regulated by means of a sliding shutter. The second light was produced by reflection upon a vertical plate of ground glass. The source of this reflected light was a two-filament Nernst lamp whose rays were thrown upon 210 WILLIAM BRODBECK HERMS a vertical white screen standing at an angle of 45° to the ground glass. Placing a photometer midway between the two areas the lights were balanced by manipulating the sliding shutter. The area of the smaller light after balancing was 7 sq. mm., while the area of the larger field (the illuminated ground glass) was 52,800 sq. mm., or a ratio of approximately 1:7500. The intensity of each source was 12 ep. The first set of experiments was tried on the migrated larvae. The individuals were placed midway between the twolights, and in all cases they took a straight path without turning toward either light. This behavior would be expected under light of equal intensities and at equal distances acting bilaterally upon these organisms. It must therefore be concluded that the size of the luminous area has no influence on the movements of the larvae. To test the adults a glass cylinder 20 cm. in diameter and 25 em. in height was placed between the two lights so that the axis of the cylinder coincided with the region of equal light intensity. A small glass vial with a rectangular stage of black cardboard around its neck served as receptacle for the individual flies as they were tried. After transferring the fly from its original receptacle into the vial, the latter was placed inside the glass cylinder, tilted so as to rest on the edge of the stage with the longitudinal axis of the vial on the line of equal light intensity. The cylinder was then covered with a sheet of black cardboard. The flies, which are negatively geotropic, naturally crawled up the vial and emerged with the: light from either side striking the two eyes equally. Under these conditions fairly accurate results might be expected. On protruding the head from the mouth of the vial the fly usually paused for a moment, then either crawled out upon the stage or flew immediately to one side or the other of the cylinder. Less often it crawled the entire distance. In transferring the individuals from one vial to another their negative geotropism and positive phototropism were made serviceable. A little practice and pre- caution were necessary to recapture the individuals after libera- tion in the cylinder without crushing or losing them. Ten specimens of each species were exposed first to the two areas separately and then to the two simultaneously. THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 211 By inspection of table 7 it may be seen that Lucilia caesar is much more strongly phototactic than Calliphora vomitoria, for under all conditions the former turned more frequently toward the single light or the larger area than the latter. It should also be added that Lucilia caesar is far more reponsive to light, as is evident from the following data. The average time that expired between the moment when the headof the fly was protruded from the mouth of the vial and the time of arrival at the side of the cylinder, based on 25 reactions of ten individuals to the larger area alone, was 8.1 seconds for L. caesar and 21.8 seconds for C. vomi- torla. TABLE 7 Summary of reactions of adult sarcophagids to opposing lights of the same intensity, but form sources whose areas were as 1 to 7500. Cc. VOMITORIA L. CAESAR Number Meee Response in percent of trials Response in percent | of trials Direction of reaction...) + — 0 + | — 0 | To areal (alone)...... | 62 226 50 100 0 (Oe | 25 To area 7500 (alone)...| 72 18 | 10 50a oa ees 0 | 25 To area 7500 (both | | | used simultaneously)| 62 36 2 100 \eeteon 288 | 3) | 76 In view of these facts an answer may be given to the second question proposed at the beginning of this section. The more frequent presence of C. vomitoria in houses and like situations is due chiefly to its relatively low degree of responsiveness to light, so that odors from darker places may attract it more readily. On the other hand L. caesar is very strongly phototactic and con- sequently would seek the open, and if by chance it should find: its way into darker places its responsiveness to large luminous areas would soon lead it tc escape. It may be assumed that the two species are equally chemotactic, which assumption is justified at least by observation. Since C. vomitoria is less strongly photo- tactic, individuals least so might easily be attracted into fairly dark places, and would not soon be compelled to leave because of their phototropism. 212 WILLIAM BRODBECK HERMS Hence it seems reasonable on the evidence at hand to conclude that because of their different degrees of phototropism C. vomi- toria is of more importance as a household scavenger (or pest, as the case may be), while L. caesar is distinctly a scavenger of the open fields, lake beaches and the like. A further inspection of table 7 leads one to conclude, that both species are about equally positive to the larger luminous area, which indicates (if we accept the test as conclusive) that the image forming power of the two species is about equal, with pone a a slight advantage in favor of L. caesar. It was shown that the eyeless larvae took a straight path be- tween the two lights without turning, while the adults, which pos- sess compound eyes, turned toward the larger luminous field more frequently by 26 to 41 percent (excess over smaller), which indi- cates discrimination. Since the intensity of the two lights was equal the discrimination must be due to the size of the luminous field and the consequent image formed in the eye. Therefore, these experiments afford a test of image forming powers. How do the results from the sarcophagid flies agree with those from Vanessa antiope? Cole (’07, pp. 380-382) found 87.2 per cent of the responses of Vanessa were toward the larger light, an excess of 75 per cent of the whole. Though the ratios of the two lights in Cole’s experiments (1:10000) were not the same as those in mine (1:7500), it seems quite likely that the eyes of the mourn- ing cloak butterfly are better adapted to the formation of images than the eyes of the sarcophagids. The flesh-flies from the nature of their habits are probably more dependent on odors and would necessarily not need image forming eyes of a very perfect character. Observations made by other investigators further justifiy the findings of Cole; e.g. Parker (’03, p. 461) calls attention to the manner in which Vanessa alights with spread wings and is thus found by other individuals of the same species, involving a well developed image-forming power. Also Latter (’04, p. 88) ‘‘once observed a Brimstone butterfly visiting flowers of the Dog violet scattered along a bank, and picking out these flowers to the exclu- sion of all others with great precision, not even approaching other blue flowers that were present.” THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 2s The fact that Vanessa antiope alights in sunny spots, as shown by Parker (’03, p. 461), is correlated with its reaction to luminous areas, and similar observations made on Lucilia caesar by the writer lead to a similar conclusion. It was frequently noted that when a dead fish was placed under a leafy tree so that a patch of sunlight fell upon it, flies would soon be hovering about the carcass. As the patch of sunlight left the fish, the flies also disappeared. This behavior was noted, and afterwards the fish was always placed in a patch of sunlight or in the open, and not in a position where shade and patches of bright ight intermingled. This con- dition does not hold true in large shady areas produced, for exam- ple, by a building or other large object where there is an absence of sunlit patches. The experiments with reference to image formation in the sar- cophagid flies are of further interest also because these observa- tions place at least this family of Diptera in line with other groups worked on by Cole (07), who was, however, unsuccessful with his experiments on the fruit fly, Drosophila ampelophila. IV. APPLICATION TO GENERAL THEORY OF ANIMAL BEHAVIOK During the progress of the experiments described and discussed in the preceding pages, it was a matter of concern to analyze the movements of the flies in order to ascertain their method of orien- tation and the factors involved. The matter for consideration in the following pages may be briefly stated in the form of three questions, viz., (A) How is the animal oriented by light? (B) How does the animal orient to light? (C) Why is there this be- havior toward light? The first question is concerned with exter- nal, the second and third with internal factors. A. How is the animal oriented by light? Sarcophagid fly larvae are stimulated to motion by light and that motion is away from the source of light. As early as 1853 the relative importance of intensity and direc- tion of light were made the object of some observation, at which time Cohn (’53) pointed out that Stephanosphaera collected in relatively darker situations and avoided bright light. Later this 214 WILLIAM BRODBECK HERMS author (Cohn, ’66, p. 164) advocated the view that the direction of the rays rather than intensity was the more important factor. “Weitere Versuche haben jedoch erwiesen, das nicht die Intensitat sondern die Richtung der Lichtstrahlen es ist, welche die Bewe- gungen der mikroskopischen Organismen beherrscht.”’ From that time on investigators have favored ore or the other of the two views. Davenport (’97, p. 211) designates the effects produced by the direction of the ray as phototactic and that pro- duced by the ‘‘ difference in illumination of parts of the organism”’ as photopathie. It is quite evident that both phototaxis and photopathy play an important réle in the movements of the flesh-fly larvae. The path of these individuals is largely pre-determined by the direction of the rays, its response being phototactic in that respect,while, on the other hand, the creeping animal keeps its head as far as possible in the shadow of its own body,—a photophathic response. This statement, based on the evidence already discussed, is in direct opposition to the conclusions of Holt and Lee (01, p. 462) who say: ‘‘Experimental study and a review of the literature on the subject have convinced us that the phenomena thus far reported do not demonstrate either that direction of ray and inten- sity of light operates separately, or that any distinction should be made between phototaxis and photopathy as independent forms of irritability,’ and further (p. 479) ‘“‘The direction of the rays has, in itself, no effect whatsoever, on the movements of the organism.’ The conclusions reached by Walter (07) on plana- rians is more or less in agreement with the conclusions of these authors. Walter states that, in general, intensity rather than direc- tion is ‘“‘the operative factor in light reactions,’ but he modifies this by the further statement that ‘‘At the same time there is much evidence that the intensity utilized by the organisms, is intimately associated with, and powerfully modified by the direc- tion of the light.’”’ These conclusions of Walter are also in accord with those of Loeb (’93, p. 101) on another species of planarian (Planaria torva), which is not phototactic, but reacts in a very striking manner to changes in light intensity (“unterschiedsemp- findlich”). On the larvae of Calliphora vomitoria, however, THE PHOTIC REACTIONS OF SARCOPHAGID FLIES DAS Loeb’s (90, pp. 70.71) conclusions are different, as the follow- ing quotation shows: ‘‘so konnte ich auch fiir die Muscidenlarven nachweisen, dass sie unter dem Einflusse der Richtung der Strah- len gezwungen waren, auch von Stellen geringer Lichtintensitit in solehe von hoéher Lichtintensitit zu gehen.” It is perfectly clear that sweeping statements with regard to the action of intensity or direction of the rays cannot be made. Whereas one species of organism is almost exclusively influenced by the intensity, as in planarians, a second group of organisms is chiefly influenced by the direction, as in the sarcophagid fly- larvae, and in both cases the two factors are more or less involved in each other. Furthermore, as has been pointed out by other workers (e.g. Walter, ’07, pp. 144-145), factors of a more or less disturbing or obscuring nature enter into behavior in general. Thus, there is an element entering into the behavior of the flies under consideration which has received little or no attention by former investigators either in fly-larvae, or in other lower organ- isms, namely the after-effects of light stimulation. For at least 15 to 20 seconds after the light has been turned off, the larva of a flesh-fly may continue creeping in a straight line without an in- crease in the number of random movements. This phenomenon is well illustrated by the following series of figures (figs. 16-23 inclusive). In all cases the path of the larva was in total darkness for a period of from 15 to 20 seconds preceding the change in direction due to the sudden illumination from in front. The after-effects are still more clearly demonstrated by figures 6-10 inclusive. The trails of the larvae in figs. 6 and 7 are sharply deflected and the succeeding paths are in a new direction and as straight as though the larvae were perfectly oriented to a contin- uous bilateral stimulus. In figs. 8-10 the movements of the ani- mals are again influenced by the after-effects. One would expect the path of the individual to make a sharp angle at the edge of the more intense field and to take up at once a course parallel to the rays of the directive light. Instead, the path is in its beginning diagonal, plainly influenced by the after-effects of the more in- tense light. 216 WILLIAM BRODBECK HERMS Clearly, this element must have a profound influence on the behavior of the individuals. Certainly, the seeming indifference to light on the part of the individual whose trail is reproduced in fig. 15, is nothing more than the result of the after-effects of previous stimultion, since the trail soon conforms to the new direction of the light rays. Using this individual as an example, it is to be noted that the larva was perfectly negatively photo- tactic, moving away from the light in the direction of the arrow. The directive light was turned off when the larva was at the point X, and thence to the point Y the larva continued on its way in total darkness undirected save for the after-effects; at Y the larva was suddenly illuminated by directive light from in front. Evi- dently regardless of the new stimulation it continued on its way (except for an increase of random movements) directly toward the light. At this juncture there is an apparent response, which, unless the history of the case were known, might be interpreted as positive phototaxis. The real conditions are obscured by the after-effects. Though the experimental evidence is not sufficiently complete to warrant a general statement, it appears from the observations made, that the after-effects are (within certain bounds) propor- tional to the intensity which produced them. This phenomenon is accordingly one which has an obscuring effect, comparable to that of mechanical stimulation referred to on p. 191, and noted in other organisms by several investigators, among them Towle (’00) for Cypridopsis, Holmes (’05b, p. 319) for Ranatra, and Walter (’07, p. 130) for planarians. B. How does the animal orient to light? The sarcophagid fly- larvae orient negatively to light and move away from the source, following very precisely the path of the rays. This behavior is also adhered to even when the course lies in a field of light increas- _ ing in intensity in the direction taken by the creeping larva, result- ing, however, in uncertainty of orientation and a consequent irregular path. Two distinct stages may be recognized in the process of photo- taxis after stimulation; first, orientation, which may be direct or THE PHOTIC REACTIONS OF SARCOPHAGID FLIES Zaled indirect; and, secondly, locomotion, a movement toward or away from the source of light depending on the existing relation between the organism and the stimulus. Many organisms are subject to stimulation on being illuminated, but fewer respond by orientation to the light. Of these (disregard- ing reactions to bright patches of light and intensity alone) the larger number by far move either toward or away from the source. The principal concern relates to the two stages in the phototactic process after stimulation and may be expressed by the question: How does the animal orient, and after it is oriented how does it continue on its relatively straight path toward or away from the light? Three theories based on reflex re-ponses of the organism have been advanced to explain the method of orientation. The oldest of these theories is the tropism theory of Verworn (95, pp. 419-446) and Loeb (’97, pp. 439-441), advanced by the latter as an appli- cation of Faraday’s conception of lines of force. It is defined by Loeb (06, p. 140) as follows, ‘‘the animal is turned automatically until symmetrical points of its surface are struck equally by the lines of force. As soon as this occurs the animals must keep this orientation, and therefore have no further choice in the direction of their motions.”’ The second theory is that of Jennings (the method by trial error) which is not entirely new, but in its present application is dis- tinctly in advance of previous views. The following extracts from the writings of Jennings (’04, p. 237) will serve to define the theory. “On receiving a stimulus that induces a motor reaction, they try going ahead in various directions. When the direction followed leads to a new stimulus, they try another, till one is found which does not lead to effective stimulation” (p. 252). ‘“‘This method involves many of the fundamental qualities which we findin the behavior of higher animals, yet with the simplest possible basis in ways of action: a great portion of the behavior consisting often of but one or two definite movements, movements that are stereo- typed when considered by themselves, but not stereotyped in their relation to the environment. This method leads upward, offering 218 WILLIAM BRODBECK HERMS at every point opportunity for development, and shows even in the unicellular organisms what must be considered the beginnings of intelligence and of many other qualities in higher animals.” The third theory (the method by random movements)is that of Holmes and is, as its author suggests (Holmes, ’05a, p. 106), ‘‘a form of the trial-and-error method minus the element of learning by experience.” it 1s also regarded as ‘‘more indirect.” Its definition (p. 102) is briefly as follows. ‘‘Of a number of ran- dom movements in all directions only those are ee up which bring the animal out of the undesirable situation.’ How well this latter method of orientation fits the case may be seen by an examination of figs. 16 to 25. Not less may be said of the second method, indeed, as has already been maintained by the writer (Herms ’07, pp. 80-81), there is so little difference be- tween the two methods in their application to fly-larvae, that they might be regarded as equally applicable were it not for the qualifying statement of Holmes ‘‘minus the element of learning by experience.”’ There seems little ground for Coubting that either the second or third methods find their application in the behavior of these organisms under the given intensities (figs. 16, 17, 20, 21, 22, and 25); but increasing the intensity lessens very decidedly the number of random movements, as illustrated by figs. 18, 19, and 23. This resultis in accordance with the statement of Loeb (’88, p. 3) that the orientation of the animal in the direction of the rays is more precise as the intensity increases. It was also found by Harper (05, p. 17) in the earthworm (Perichaeta bermudensis) that ‘‘random movements are a feature of less strong light, tend- ing to disappear with the increase of intensity, and are replaced by direct orientation in very strong light.” To test the influence of a range of intensities on the production of random movements, the larvae were started in a very low direc- tive intensity (0.5 to 1.0 C.M.), and after the individuals had oriented and were crawling away, the directive light was turned off leaving the larvae to creep in total darkness for 15 to 20 seconds, when a new directive light from in front of the animals was sud- denly turned on. It may be seen by the courses illustrated in figs. 16 to 23, that sudden darkness had no apparent effect on the Fig. 23 THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 219 16 17 18 19 22 23 e __. Vgeenth- ~H. | 25 OHCVOVOESE oaks . caesar. Random movements under an intensity of 0.007 C. M. . caesar. Random movements under 0.56 C. M. . caesar. Random movements under 325 C. M. . caesar. Random movements under 960 C. M. . vomitoria. Random movements under 0.56 C. M. vomitoria. Random movements under 64 C. M. . vomitoria. Random movements under 325 C. M. . vomitoria. Random movements under 960 C. M. Fig. 24 Ramdom movements of C. vomitoria in total darkness for one minute. The larva is undirected. Rest is the usual state under such conditions, but me- chanical stimulaton due to handling caused the larvae to be restless and to produce random movements. Fig. 25 C. vomitoria. Randon movements under 0.14 C. M. 220 WILLIAM BRODBECK HERMS larvae, quite the reverse from a sudden increase of i!lumination. For C. vomitoria, where the after-effects were so pronounced in experimenting with low intensities, no results were obtained to illustrate this principle in the same manner, but fig. 25 is substi- tuted and represents the matter quite as clearly. The courses seen in this series of figures (fig. 16 to 25) show without further ex- planation that the random or trial movements are characteristic for low intensities, and become fewer with the increase in inten- sity until finally the orientation is to all intents and purposes direct, 7.e., the animal turns directly away from light of high inten- sity. Thus there is almost a perfect gradation between the in- direct method of random movements and the direct tropism scheme of behavior with regard to orientation. Therefore it seems prefer- able in reference to the organisms in question to designate this as a combination method of orientation, uniting the two general schemes in one. The larger number of random movements pro- duced by C. vomitoria is again indicative of its lower degree of sensitiveness as compared with L. caesar. With the orientation of the animal to the source of light there is still to be considered the second step in the process of photo- taxis, 7.e., the movement away from the stimulus. This step is accomplished, as is evident from an examination of any of the figures, by the action of the light rays operating bilaterally on the organism, it must keep its general direction. Any deviation from this predetermined path is, however, corrected by trial movements in a more restricted sense. The circus movements produced by larvae when one side of the head is pigmented is further evi- dence that these organisms when once oriented follow the direct or tropism scheme of behavior. C. Why is there this behavior to light? It has been shown that the sarcophagid fly-larvae respond to light, orient negatively and creep away from the source of light, in short are negatively photo- tactic. What is the relation between this behavior of the larvae and the conditions under which they exist normally? It will be recalled that the eggsof the adult femalesare deposite | on dead animals found in light situations. The larvae while feed- ing are largely protected from light and birds by the carcass, THE PHOTIC REACTIONS OF SARCOPHAGID FLIES 220 but as the moisture is eliminated and the larvae become full grown they are left in a rather precarious position. The remnants of the fish or other dead body can then be easily dislodged by the winds or other mechanical means, and the larvae are then exposed to the ravages of birds, as has already been stated. The heat of the sun also would then result disastrously to them. The importance of moisture in the economy of the flesh-flies has been pointed out on several occasions (Herms, ’07, pp. 49, 67, and 75). It must be evident that the phototactic response of these larvae is highly adaptive to the situation. By this means the larvae find places of shelter from light, away from the desiccating influence of the sun, removed from preying birds and other disturbing elements. This response, coupled with their strong thigmotatic reaction (Herms, ’07, p. 82) as seen in burrowing, affords a natural and almost complete protection for the individual and consequently for the species. V. GENERAL SUMMARY 1. The egg-stage of Lucilia caesar and Calliphora vomitoria covers a period of from 8 to 48 hours; the feeding period from 2 to 7 days for the former species, and from 3 to 9 days for the latter; the prepupal period usually from 2 to 7 days (extreme 59 days); the pupal period usually from 8 to 17 days (extreme 34 days). The most variable in length is the prepupal period. Constancy in environmental conditions results in uniformity. The shortest time observed between emergence from the pupa and egg deposi- tion was 9 days for Lucilia caesar and 12 days. for Calliphora vomitoria. 2. The usual length of life of an imago flesh-fly is about 30 days, regardless of sex. The longest recorded life of a single adult individual was 63 days. 3. Pearce 273 2 The chief differences in the course and results of regulation of pieces 276 3 The course and results of regulation in relation to the size of the piece and the region of the body represented................... 284 4 Tabulation and graphic presentation of data.............. meee se 291 LUNES OW eh ae 9) URGES GLARS 2 foment A OM SR 2, Pe 300 ioauneyOresence Gla, SeCOndyZ00Id .. <)... <).as. veers eines + Sedieg os ore 2a 300 Pee Dhe CUuestOncor 0.2202) 0.0114 7 0.7309 0.0578 °¢ 76.85 | 83.53 November || 1910 | 0.3168 | 0.1464 | 0.0258 | 0.1206 | }— pees. | 0.3170 | 0.0949 0.0734 | 0.0175 | 0.2436 0.0791 | 76.85; 83.71 TABLE 7 I jay Wl peau a IV Vv Wier | Vil Vl WEIGHT OF TAILS IN GRAMS | PER CENT OF WATER Date TOTAL | DRY SUBSTANCE WATER oD REGEN- | aaa, ERATED pea) (oa | Beever | | November 97 eas ae 0.2903 | 0.1040 0.0165 0.0875 84.13 Marcu 1 0.2127 | 0.0680 | >} 0.2124) 0.0102 | + 0.5911) 0.0578 | > 73.57 |, 83.53 1910 ; 0.3005 | 0.0722 0.0102 0.0620 85.88 Average...... 0.2678 | 0.0814 | 0.0708) 0.0123 | 0.1970 | 0.0691 | 73.57 | 84.51 346 SERGIUS MORGULIS ee TABLE 8 ee Lie ape comer TN) Mex EMP cco VICES a0 VII | Wiericea OF TAILS IN GRAMS | PER CENT OF WATER DatE TOTAL | DRY SUBSTANCE | WATER jit: —_ | OLD REGEN- | : Oily z TAGE, ERATED De oie eh ct eer) ot )en Patt November | | | 30. 1909 to | | 0.2165 | 0.0730 0.0108 0.0622 | 85.21 Mare 5 0.2725 | 0.0832 | ¢ 0.1810) 0.0126 | ¢ 0.5040; 0.0706 | ¢ 73.69 84.85 1910 : 0.1960 | 0.0925 0.0146 | | 0.0779 | 84.22 ar — oe et =| Led oe Average.....| 0.2283 | 0.0829 | 0.0603 Ce | 0.1680) 0.0702 | 73.69 84.76 TABLE 9 I I | Meg eeive || y, VI vu VII WEIGHT OF TAILS IN GRAMS PERCENT OF WATER Marcu 1, 1910 i a — as 7 TO | | ‘ Apri. 13, 1910) TOTAL | mee eae a ce ip ERATED ee Regen- | Ola Regen- Old | Regen- cca TAIL | erated | erated erated | Average.....| 0.280 " 0.038 ‘0 0.0702 0702 0.0026 | 0.2099 i 0.0212 | 74.94] 89.08 water (average for four individuals), the tails which had been regenerating in their place for one month and had attained an average length of about 6 mm. contained 89.08 per cent water, or fully 14 per cent of water more than the old tails. From the other four tables (5 to 8) it will be learned further that three months after the operation, when the regenerated tails had already attained an average length of about 15 mm., the average per cent of water varied from 83.6 to 84.76 per cent. The old tails fall within two groups, those with about 73 per cent of water (73.57— 73.69) and those with about 76 per cent (76.2-76.85). The increase in the content of water is therefore different for the sepa- rate series, but if the average is taken for all four series (5 to 8), it will be found that the regenerated tails three months old con- tain7 per cent of water more than the original (amputated) tails (82.08 per cent and 75.08 per cent respectively). THE PHYSIOLOGY OF REGENERATION 347 We thus discover that also in the case of the salamander the per cent of water in the regenerating tissue increases rapidly at first (89.08 per cent), then decreases again, tending towards the normal (82.08 per cent). Although these observations are not enough to trace in fullness the changes in the water content of the re- generated tails of Diemyctylus, yet they suffice to substantiate the former results on Podarke as well as to throw light upon some points which remained obscure in those experiments. SUMMARY As the curve of formative growth and that of posterior regenera- tion are essentially alike in their important characteristics, it seemed desirable to investigate the question whether the factors in the two processes are also similar. Numerous experiments on both plants and animals have demonstrated the fact that in formative growth the per cent of water rises to a maximum during the period of rapid growth, and then falls as the organism ap- proaches the adult condition. With this in view, I have studied the water content at successive stages of regeneration in a poly- chaet—Podarke obscura. The result is practically the same as in formative growth; soon after an operation the water content rapidiy rises, reaching a maximum approximately between the first and second weeks; subsequently, it begins to decline. As it was found, furthermore, that the period of maximum water content and the period of maximum regenerative activity approximately coincide, as in formative growth, the similarity between growth and regen- eration was thereby shown to be still greater. Close analysis, however, revealed that, while from the point of view of the end result (7.e., the rise and fall of the curve of the per cent of water) growth and regeneration are alike, the two processes involve dis- similar factors. In formative growth the increase in size and in the per cent of water are brought about through imbibition of water from the surrounding medium; in regeneration this does not seem to be the case, as is shown by a comparison of the abso- lute quantities of water and of dry substance at various stages. The regenerating animals, whether fed or starved, lose in weight 348 SERGIUS MORGULIS a process which presents three definite phases of regulation of the water content in the organism. First comes a period of rapid loss in weight, when proportionally more dry substance than water is lost, the per cent of water, therefore, increasing. This is followed by a period of rather slow decrease in weight, when practically no water is lost, and when the regenerative activity and the water content both reach a maximum. Lastly there comes a period dur- ing which proportionally more water than dry substance is lost, the per cent of water thus declining. Wien, September 2, 1910. BIBLIOGRAPHY Brzoup, A. von’ 1857 Untersuchungen iiber die Vertheilung von Wasser, organ- ischer Materie und anorganischen Verbindungen im Thierreiche. Zeitschr. f. wiss. Zool., Bd. 8, 487-524. BraLaszewicz, K. 1908 Beitrige zur Kenntnis der Wachstumsvorginge bei Amphibienembryonen. Bull. Internat. Acad. Sei. de Cracovie, Cl. Sci. math. et nat., année 1908, October, pp. 783-835. Davenport, C. B. 1897 The role of water in growth. (Contribution from the Zool. Laboratory at Harvard College, no. 80.) Proceed. Boston Soc. Nat. Hist., vol. 28, no. 3, pp. 73-84. Dursin, Marron L. 1909 An analysis of the rate of regeneration throughout the regenerative process. Jour. Exp. Zodl., vol. 7, no, 3, pp. 397-420. Kraus, G. 1879 Ueber die Wasservertheilung in der Pflanze. I. Festschr. z. Feier des hundertjihrigen Bestehens d. Naturf. Gesell. in Halle, pp. 187-257. Loss, J. 1892 Untersuchungen zur physiologischen Morphologie der Thiere- II. Organbildung und Wachsthum. Wiirzburg, 82 pp., 2 Taf. Minot, C.8. 1907 The problem of age, growth and death. 3. The rate of growth. Pop. Sci. Monthly, vol. 71, pp. 193-216. Moraeutis, 8. 1909 Contributions to the physiology of regeneration. 1. Experi- ments on Podarke obscura. Jour. Exp. Zo6l., vol. 7, no. 4, pp. 595-642. Prerrer, W. 1903 The physiology of plants. A treatise upon the metabolism and sources of energy in plants. 2nded.,Vol.2. Translated by A. J. Ewart. Growth, reproduction and maintenance. Oxford, viii + 296 pp., 31 figs. Scuaper, A. 1902 Beitrige zur Analyse des thierischen Wachsthums. Eine kritische und experimentelle Studie. I. Theil: Quellen, Modusund Lokal- isation des Wachsthums. Arch. f. Entwickelungsm., Bd. 14, pp. 307-400, Taf. 15-25. EXPERIMENTAL METAPLASIA 1. THE FORMATION OF COLUMNAR CILIATED EPITHELIUM FROM FIBROBLASTS IN PECTEN G. HAROLD DREW Beit Memorial Research Fellow EIGHT FIGURES (THREE PLATES) CONTENTS [har aGhoVeAorn, Gavel TENA ie anes boo oOo BOMB BMmeue. cols on Saecmecbagpenpooour . 349 Description of tissues involved in the experiments....................-.--- 353 IN IPSTHAVGYEIG) 4 Gi ako dre hn Getehare & Blea aceue AUARS bite EERE ct. Ct a eee 356 Results of the implantation of pieces of the mature ovary into the adductor muscle, and the subsequent development of ciliated epithelium from the AOEOB Asis OrMed AtOUNG 16. c nics 2. <0. |: te meets eka ows s fe cines . 859 Experiments to determine more exactly the relation of the presence of ova- rian tissue to the formation of ciliated epithelium from fibroblasts........ 364 Summary of results of experimental work .........-..-.--.------2- sees 369 ID Reece Gi IERIE aoe cold obb One omenOeemdEUS hb conc. boodcouUroemeouodc 5 a) Wostrace ‘of ‘experiments performed: +’... .....ts-nea dete ted. ahs oe vie oe 5 Sic} LEGER He (ohdelo le a8 aks oe eae Putte RERUN coc cee = cnc MELAS DRIER ROE: 374 INTRODUCTION AND REVIEW The experiments described in this paper were performed on Pecten maximus and Pecten opercularis at the Plymouth Labor- atory of the Marine Biological Association of the United King- dom. In the course of some investigations as to the mode of formation of new fibrous tissue in Pecten, undertaken by Mr. W. De Morgan and myself (Drew and De Morgan’10) we transplanted various tis- sues, such as the digestive gland, gills, gonad, etc., into the mid- dle of the adductor muscle, and studied the process of fibrous tissue formation around the implanted mass. We also injected sterile Agar jelly made with sea water, and since then I have per- formed similar experiments with sterilised cotton wool, cork, elder THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 May, 1911 349 350 G. HAROLD DREW pith and other foreign bodies which are presumably unaffected by the body fluids of Pecten. In these experiments it was noticed that the reaction of the tissues to the implantation of portions of the ovary presented, in the later stages, marked differences from the reaction to any of the other tissues or foreign bodies employed, and it was with the object of explaining these differences that the work here described was undertaken. Briefly summarised, my results show that after implantation of a portion of the ripe ovary into the adductor muscle, a layer of fibroblasts is formed around it, and coincidently the ovarian tissue is invaded by phagocytes and degenerates. After the lapse of about six days no trace of organised ovarian tissue remains, but there is left a cyst surrounded by fibroblasts, and containing blood corpuscles and a quantity of small granules having the orange color of the yolk substance. After the lapse of about 20 days more, the innermost fibroblasts gradually change their shape, and form a layer resembling columnar epithelial cells, which later become ciliated. Eventually the whole cyst becomes lined with well defined ciliated epithelium, which persists at least for 120 days, which is the longest period I have yet succeeded in keep- ing the animals alive in the experimental tanks. I consider that there is some evidence to show that this change of the fibroblasts into ciliated epithelium is a reaction to the presence of some defi- nite chemical substance within the cyst. In the vertebrate many cases of the change of a tissue of one type into another type are known to occur, and to such changes the name Metaplasia has been given. According to Adami, (’08) they have this in common, that ‘‘ Epithelial (epiblastic and hypo- blastic) tissues can only be converted into other forms of epi- thelial tissue, and one form of mesoblastic into another form of mesoblastic.”’ Such examples as the conversion of cartilage into bone, or of connective tissue cells into fat, are cases of physiological meta- plasia of mesoblastic tissues, and it is to be noted that these changes occur in the adult and not only in the course of development. In- stances of pathological metaplasia are numerous. Thus among the epithelial tissues we have the change of the columnar ciliated EXPERIMENTAL METAPLASIA Sol epithelium of the larynx into a squamous type as the result of chronic irritation, the:conversion of the pavement epithelium of the bladder into the columnar type found in papillomatous over- growths, and a similar change in cases of ectopia vesicae where even glandular crypts of a very simple type may appear. Of special interest are the experiments of Wolff (93) on the eye of the larval newt and salamander. In these animals, if the lens be removed, it is regenerated from the iris, and it has also been shown that the retina itself can, under certain conditions, produce bodies resembling lenses. Here we have a case of meta- plasia between totally dissimilar tissues, differing greatly in their mode of formation, but which still are both of epiblastic origin. A somewhat similar case is that described by Saxer (’04) who found a tissue resembling columnar epithelium lining the cysts that are of relatively common occurrence in gliomata. Among the mesoblastic tissues there are many common in- stances of metaplasia. For example, the ossification of tendons and muscles, the formation of true bone in the lungs, in old pleural and pericardial adhesions, and in the fibroid valve of chronic endocarditis. Harvey (’07) has experimentally caused the forma- tion of bone in the walls of arteries of rabbits by injury, and a similar formation is often found in eases of arterio-sclerosis of long standing. Examples of the change of tissue derived from one germ layer into closely allied tissues derived from the same germ layer might be multiplied indefinitely, but it must be noted that there are many pathologists who deny this frequent occurrence of meta- plasia, and explain these changes in other ways. Foremost of these is Ribbert. He considers, for example, that the replace- ment of columnar by squamous epithelium is due to the over- growth of included islands of squamous cells, under altered con- ditions of environment, and he has other explanations for most of the cases here cited. His conclusions, as summarized by Adami (08) are to the effect that ‘‘only tissues that, while externally different, possess nevertheless the same histogenetic capacities, can undergo metaplasia, one into the other.’”’ On the other hand, Leo Loeb (’99) has recorded that in cases of epithelial regeneration ae G. HAROLD DREW in vertebrates, he has observed epithelial cells migrate into the underlying tissues, and take on the appearance of fibroblasts. Much of the work that has been done on regeneration is of interest in this respect. Braun (’03) has pointed out that in re- generation in tadpoles, epitheltum may give rise to nerve tissue. There isthe work of Barfurth(’91-’00) and Fraisse(’85)on regenera- tion of the tail in several Urodela, and in the tadpole of the frog, and of Towle on the limbsof Plethedon. These observers find that each tissue only reprodices tissue similar to itself, but their experi- ments were necessarily confined to types in which regeneration occurs, and dealt only with the phenomena consequent on amputa- tions. Much more work has been done on the regeneration of single tissues or organs, and in these cases it has been found that like always reproduces like, and that metaplasia does not occur. To turn now to invertebrates, we have the observations of Miss Reed, recorded by Morgan, (’04) on the regeneration of the claw of the crayfish and hermit crab, in which it is conclusively shown that the whole claw with its muscles, etc., is regenerated from the ectoderm, 7.e. metaplasia of ectodermal into mesodermal cells occurs. Again there are the experiments of Koeber (’00) on the regeneration of the pharynx of Allolobophora, in which he shows that though the epithelium lining the pharynx is developed from the ectoderm, yet in the course of regeneration it grows from the endoderm continuous with that lining the alimentary canal. Flexner (’98) has pointed out that in regenerating Planarians the surface epithelium may give rise to distinct nerve elements, and in this case, though both the surface epithelium and the nerve tissue are originally derived from the same germ layer, yet we have metaplasia occurring between tissues which are histologic- ally totally different, and indeed the process may in some ways be considered analogous to the regeneration of the lens from the iris in the eye of the larval newt and salamander described by Wolff (94). It thus appears that, whatever may be the case in vertebrates, in lower types of animals there is much evidence to show that me- taplasia may occur between cells derived from different germ layers, and between cells possessing widely different histological EXPERIMENTAL METAPLASIA oOo characters. With regard to the cytology of the changes involved in regeneration and metaplasia, K6lliker (’85) considered that re- generation of an organ or tissue cannot occur unless that organ or tissue contains cells of an embryonic character, or at least contains elements that are able to assume embryonic characters. The question has been more fully dealt with by Adami (’00), who comes to the conclusion that the fully differentiated cells of a tissue proper never arise from cells that are themselves fully differentiated, but during physiological regeneration arise from certain ‘mother- cells’ which are normally present in the tissues. He considers that ‘under abnormal conditions, the fully differentiated func- tioning cells of certain tissues are capable of proliferation and giv- ing rise to cells of like nature, but this is only after a preliminary reversion to a simpler, more embryonic type.” Tt will be shown in the present paper that under the experimen- tal conditions, the fibroblasts of Pecten revert to a somewhat un- differentiated embryonic type, and then become converted into columnar ciliated epithelium. DESCRIPTION OF TISSUES INVOLVED IN THE EXPERIMENTS An excellent and detailed account of the anatomy of Pecten maximus and Pecten opercularis is given by Dakin (’09) in his monograph on Pecten. The two species are very closely allied, and except in point of size, resemble each other both anatomi- cally and physiologically to a remarkable degree. The adductor muscle consists of two portions, bound together by the same sheath of connective tissue, but differing in structure. The larger, white and semi-transparent, consists of striated fibres. The fibres of the smaller, which is of an opaque and dead white appearance, and lies against the posterior surface of the larger mass, are non-striated. It was into the larger mass that all material in these experiments was introduced. Superficially the muscle is covered with a layer of columnar epithelium con- tinuous with that lining the mantle. There is a large blood supply to the muscle from the adductor artery (Dakin), and it contains numerous lacunar spaces. Scat- 304 G. HAROLD DREW tered through it are numerous strands of connective tissue. These contain fibroblasts with very elongated, deep staining nu- elei, and long fibrillar processes. The gonad consists of a semicrescentic mass attached at its base to the adductor muscle. When ripe the male proximal por- tion is creamy white in color, and the distal female part is of an orange or vermilion hue; the boundary between the male and female portions is sharply defined. In Pecten maximus the loop of the intestine reaches almost to the apex of the ovary, whilst in Pecten opecularis it does not ex- tend much beyond the testis. Microscopically the gonad consists of branched tubules, termin- ating in alveoli lined with germinal epithelium: when ripe the alveoli are crowded and distended with ova or sperm, and it is not always easy to trace the connecting tubules. These tu- bules join up to form two main ducts which are lined by columnar ciliated epithelial cells, the height of which is about twice the width while the cilia are about as long as the cells (Dakin). Traced towards the alveoli, the cells lining these ducts become shorter and lose their cilia, and in the smallest tubules are of a flattened almost squamous type, where finally they appear as if they were directly continuous with the germinal epithelium. The ripe ova ace of an orange or vermilion color, measuring about 50 uw in diameter, the nucleus is relatively large, the nucle- olus conspicuous, and the cytoplasm crowded with yolk granules. The spermatozoa are small and of the typical shape, with a long flagellum attached to the broad end. The blood of Pecten is a slightly cloudy, colorless fluid, it does not coagulate, but when shaken a number of small, white, floccular masses appear, which soon fall to the bottom of the tube leaving the supernatant fluid clear and transparent. These masses consist of blood corpuscles, agglutinated to form plas- modia. The corpuscles although varying in size, are only of one kind. They are amceboid bodies, which when expanded protrude a number of slender pseudopodia. When contracted, they are ovoid or spherical. There is a single compact nucleus, staining EXPERIMENTAL METAPLASIA 355 readily with methylene blue. The cytoplasm is finely granular, and stains with eosin, but there are no large eosinophil granules. According to Cuenot,(’91) they originate in a ‘glande lymphatique’ situated at the base of the gills. In a former paper (Drew, 710) I have shown in the case of Cardium norvegicum, that when the corpuscles come in contact with a rough foreign body, or with injured tissue, they possess the power of agglutinating and forming a compact plasmodial mass. In this way bleeding from a small wound is stopped. When the edges of a wound are covered with this mass of agglut- inated corpuscles, proptoplasmic strands are formed across the wound, connecting the plasmodia, these strands thicken and con- tract, and so approximate the edges of the wound. I have repeated these observations on Pecten, and find that the same phenomena occur, and that a similar plasmodial mass of agglutinated corpus- cles is rapidly formed around any tissue implanted into the adduc- tor muscle. That Lamellibranch blood corpuscles are capable of a phago- eytic action towards degenerated cells has been shown by De Bruyne (96) in the case of Mytilus edulis, Ostrea edulis, Unio pic- torum and Anodonta cygnea. Sir Ray Lankester (’86 and ’93) has shown that certain corpuscles of Ostrea edulis have a phago- eytic action on diatoms and minute green algae, and I have shown (Drew, ’10) that the corpuscles of Cardium norvegicum have a phagocytic action on bacteria, and are attracted towards extracts of dead tissues. It has also been shown that the cor- puscles of Pecten maximus exercise a similar phagocytic action on dead cells (Drew and De Morgan ’10). The formation of fibrous tissue around a foreign body implanted into the adductor muscle has been described in a former paper (Drew and De Morgan’10). The normal fibroblast is an elongated cell, with a spindle shaped nucleus and an indefinite amount of cytoplasm which appears to be drawn out and connected with the neighboring cells by fine fibrillar processes. ‘These cells are usu- ally connected with each other by slender strands of some col- laginous substance, which forms the groundwork of the fibrous tissue, and in the normal resting stage it is often impossible to dis- 356 G. HAROLD DREW tinguish between these collaginous strands and the fibrillar pro- cesses of the cytoplasm. When about to divide amitotically, the fibroblasts become shorter and thicker, the cytoplasm van- ishes, and an oval or round nucleus with a reticulated arrangement of the chromatin results; eventually this splits in two and the two halves separate. The process is shown in fig. 1. The details of fibrous tissue formation differ in the early stages according to the degree of irritative action of the foreign body, and the consequent amount of inflammation produced. If the inflam- mation is very slight, as was the case in most of the experiments about to be described, the implanted body is first surrounded by a thin layer of agglutinated blood corpuscles. This is followed by the rapid amitotic division of the fibroblasts in the neighborhood, they lose the typical spindle shape of their nuclei, and the new formed cells consist of rounded or oval nuclei with a scarcely per- ceptible amount of cytoplasm. These rounded cells migrate towards the implanted body, and arrange themselves in layers around it, the nuclei become elongated, and the proportion of cytoplasm increases. Finally a cyst wall of typical fibrous tis- sue is formed, surrounding and completely shutting off the im- planted body. METHODS Both Pecten maximus and Pecten opercularis can be obtained in large numbers by dredging in the neighborhood of Plymouth. It was found necessary to allow these animals to become accli- matised to living in the laboratory tanks before proceeding to the experimental work. When first placed in the tanks, the mortal- ity was heavy, often amounting to 30 per cent in the first three days, but after the lapse of about a week the survivors appeared to be fully acclimatised to the changed conditions, and often re- mained healthy for some months. Experiments on animals whose health was doubtful were of no value, both because the shock consequent on the injection of the foreign body frequently caused death, and also because the reaction of the tissues was not normal in unhealthy specimens. When a Pecten is healthy, it lies with the valves of the shell EXPERIMENTAL METAPLASIA 307 slightly apart, the tentacles are expanded, and it responds rap- idly to any stimulus by closing the shell; when held up in the air the water which drains away is clear and contains no slime. An unhealthy specimen lies with the valves of the shell wide open, there is little or no response to stimuli, and the valves only close under pressure. The tentacles are retracted, and the gonads, gills, and tissues generally look flabby and unhealthy. The water which flows out between the valves is slimy and viscid, and this is generally the first sign of deterioration. When making an implantation of the ovary, a healthy speci- men was chosen in which the gonad was of a full orange or ver- milion color, and obviously distended with ova. The valves of the shell were wedged apart with a cork, and the interior well washed with a brisk stream of sterile sea water from a wash bottle that had previously been steamed for some time in a ‘Koch.’ The adductor muscle was then severed with a scalpel, and one valve of the shell turned back. The extremity of the ovary was cut off, and while held by forceps, was very thoroughly washed with a stream of sterile sea water, then it was placed in a steril- ised Petri dish containing a little sterile sea water, and kept care- fully covered. All instruments were sterilised by boiling in dilute caustic soda solution, and were washed in sterile sea water to remove any trace of the caustic soda immediately before use. They were always re-sterilised between each experiment. When not in use it was found more convenient to keep all steel instruments in lime water instead of drying them, as they are particularly liable to rust after being exposed to the action of sea water. The transplanting needles, made of a platinum and iridium alloy, resemble large hypodermic needles. Two sizes were used, the larger for experiments on P. maximus, measured about 6 cm. in length, and 1 mm. in diameter, the smaller for experiments on P. opercularis was of the same length, but about half that diameter. Into the hollow needle a somewhat longer stilet fits closely, and works like a piston. Any material taken up in the point of the needle is sucked in by drawing the stilet back, and again ejected by pushing it forward. Small portions of the ovary 358 G. HAROLD DREW were cut off by fine scissors and drawn up into the needles in this way. In the earlier experiments portions of the ovary were injected into the muscle through a hole bored in the shell. The holes were drilled in the convex or right valve by an ordinary dentist’s drill, the head of which was prevented from penetrating too deep by a lapping of thread. The spot selected for drilling was sterilised with a saturated solution of corrosive sublimate, washed off with a solution of hydrogen peroxide (20 vols.) or distilled water, care being taken not to allow any of the sublimate to run between the valves. The transplanting needle was then introduced to the required depth, slightly withdrawn and its charge projected into the channel. The hole was then thoroughly dried, and stopped with sealing wax. In later experiments it was found that this proceeding, which occupies a good deal of time, was unnecessary, and the implantation was made directly into the muscle from the side. By this method there is a slightly greater risk of sepsis and the consequent death of the animal, but this is more than com- pensated for by the saving of time which it entails, and this is of importance when a large number of experiments have to be made. In some cases much larger pieces of the ovary were implanted by simply making a longitudinal slit in the muscle with a small scalptl, and inserting the ovarian tissue with fine pointed forceps. The wound thus made is closed by the contraction of the muscle, but the risk of sepsis when this methodisemployed is considerable, and only a small proportion of the animals survived the experi- ment long. During the experiments the animals were kept in the laboratory tanks, or in basins with a continuous flow of water. Exposure to too strong a light should be avoided, and if the animals are required to live long it seems to be of advantage to cover the basins with green glass, or to moderate the light in some other way. When required for examination, the animals were killed by wedg- ing the valves of the shell apart with a cork, aad then placing in dilute spirit. When dead, one valve of the shell was removed, and the adductor muscle carefully sliced with a razor until the orange color of the implanted ovarian tissue could be seen shining EXPERIMENTAL METAPLASIA 359 through the semi-transparent muscle. A small cube of the muscle containing the ovarian tissue in the middle was cut out and placed for about three hours in Zenker’s fluid, well washed, treated with dilute iodine, and finally embedded in paraffin. Serial sections were then cut, and stained in very dilute Delafield’s hamatoxy- lin. Other stains and fixatives were used for especial purposes, but the above procedure was found to be the most satisfactory as a routine method. It is noteworthy that even after the ovarian tissue has been im- planted into the muscle for as long as four months, the orange color is not lost or even diminished in intensity, so that the site of the implantation can always easily be distinguished. RESULTS OF THE IMPLANTATION OF PIECES OF THE MATURE OVARY INTO THE ADDUCTOR MUSCLE, AND THE SUBSEQUENT DEVELOPMENT OF CILIATED EPITHELIUM FROM THE FIBRO BLASTS FORMED AROUND IT The sequence of events after the implantation of pieces of the ripe gonad of one specimen of Pecten maximus into the adductor muscle of other animals of the same species is ideatical with that occurring when the same experiments are performed on Pecten opercularis. Pieces of the ripe ovary after ejection from the transplanting needle into the muscle, are roughly spherical in shape, and measure from 1 mm. to 0.5 mm. in diameter according to the size of the needle used. Very soon after implantation such a piece of ova- rian tissue becomes surrounded by a thin layer of agglutinated blood corpuscles, and the track of the needle is closed by a similar mass of blood corpuscles forming a plasmodial mass, which, by its contraction, draws together the tissues that have been displaced by the passage of the needle (Drew, 710). This condition can be seen In sections from animals that have been killed about one hour after the implantation has been made. If the operation has been conducted aseptically, the resulting inflammatory reaction is very slight. There appears to be a definite determination of the blood cells towards the ovarian tissue, but there is nothing approaching 360 G. HAROLD DREW the condition of venous engorgement and stasis that occurs when a septic tissue is implanted (Drew and De Morgan ’10). After a time fresh blood corpuscles penetrate the thin agglu- tinated layer, and start a phagocytic action on the ovarian tissue. Meanwhile the fibroblasts in the walls of the blood spaces, and in the intermuscular connective tissue in the neighborhood, undergo division. This division is amitotic, and commences about twelve hours after the implantation. Before division the fibroblasts lose their spindle shape and become oval: a split then appears at one end, and progresses in the plane of the long axis of the nucleus until two daughter nuclei are formed, attached to each other at one extremity, and inclined at an acute angle to one another. These gradually straighten out until they form an hour glass shaped mass of nuclear material. Finally the two nuclei are separated at the constriction, and two oval or circular cells are produced, having large nuclei with relatively very little cytoplasm, and bearing no resemblance to the spindle shape of a resting fibro- blast. There follows a migration of these cells with round and oval > nuclei towards the site of the implantation. They eniefly follow the course of the strands of fibrous tissue bounding the blood spaces, but many migrate in all directions between the muscular fibres. On reaching the layer of agglutinated corpuscles surrounding the implanted tissue, the fibroblasts arrange themselves in rows; and their nuclei elongate in such a direction that their long axes form arcs of a circle surrounding the implanted ovary. This surrounding layer presents a somewhat stratified appear- ance. At first it contains a number of blood-corpuscles, but these eventually are removed, probably by autolysis, leaving only the fibroblasts. In these experiments the layer of fibrous tissue formed in this way was always very slight, usually not more than two or three cells thick. If by any error sepsis occurred, it was followed by a violent inflammatory reaction, and if the animal survived, by subsequent great formation of fibrous tissue. Meanwhile the implanted ovarian tissue shows signs of degen- EXPERIMENTAL METAPLASIA 361 eration. The cells of the zerminal epithelium, the connective tissue cells, and the cells lining the oviduct, lose their normal appearance, the chromatin of the nuclei becomes aggregated into small darkly staining masses, and the outline of the cells becomes less distinct, the cilia of the oviducal epithelium soon vanish. Coincidently the whole mass of tissue 1s invaded by blood corpus- cles which exercise a phagocytic action and slowly remove the degenerated material. The mature, or nearly mature, ova show a much greater resistance to this degenerative process than any of the other cells. Thus, after the lapse of about three days from the implanta- tion, we have a mass of ovarian tissue which is invaded by phago- cytes and shows signs of degeneration, and is surrounded by a layer of fibroblasts forming a definite cyst wall. The fibroblasts are mostly oval in shape, with little or no perceptible amount of cytoplasm, and have not taken on the appearance they present in the resting state. From the fourth to the sixth day degeneration of the ovary con- tinues, and when cyst formation takes place, as in the cases here described, the degeneration is complete and every trace of organ- ised structure has vanished by the sixth day. When the degen- erative changes are complete, the site of the implanted ovarian tissue is occupied only by blood cells, and by a granular substance, which must either be formed during the process of degeneration, or be left after all the other substances composing the ovarian tissue have been rendered soluble and so have escaped from the cyst. If the cyst is cut open, and the contents examined under the microscope, it is seen that the granular matter is of an orange color, and that many of the blood corpuscles have ingested par- ticles of this substance. As the orange color of the cysts remains unchanged, and undiminished in intensity, even after fourmonths, it appears that this substance is unable to escape from the cyst through the surrounding layer of fibrous tissue, and the same must hold true for blood corpuscles within the cyst, which have ab- sorbed this substance by phagocytosis. It seems probable that such blood corpuscles, after ingesting granules of this-substance, 362 G. HAROLD DREW degenerate, and eventually become dissolved, leaving the gran- ules behind. Fig. 2 shows the condition at the fifth day. The degeneration of the ovarian tissue is practically complete, though traces of the ova still remain: this tissue 1s surrounded by layers of oval fibro- blasts, and the fibroblasts in the neighborhood are still dividing and migrating towards the cyst wall. Blood corpuscles are mak- ing their way into the cyst between the fibroblasts forming its wall. The subsequent changes occur more slowly. The fibroblasts forming the innermost layer of the cyst wall remain unchanged for some time, but those forming the outer layers regain the typi- cal elongated spindle shape, of the resting fibroblast. Then, after a period varying from eighteen to twenty-five days from the im- plantation, the fibroblasts in immediate proximity to the degen- erated ovarian tissue alter their appearance. The nuclei become rounder and the cytoplasm of each joins up with its neighbor, forming a faintly staining and somewhat indefinite continuous layer (fig. 3). Sections of a little later date show this layer more defined and also a change in the character of the nuclei. An ag- gregation of the chromatin resembling a nucleolus appears, and from this thin strands of chromatin radiate to the periphery. No cell walls are visible between the nuclei, which appear to have reverted to an embryonic type. Fig. 4 shows this condition, the contents of the cyst are surrounded by a continuous layer of nu- clei with definite nucleoli, and these nuclei are embedded in a mass of cytoplasm having no dividing cell walls. In the course of a few days these nuclei again alter in shape (fig. 5), they become smaller and more oval, and the nucleoli dis- appear. The surrounding cytoplasm becomes more definite, and stains deeper, and a distinct boundary or basement membrane appears between it and the layers of fibroblasts. Shortly after this, long slender cilia appear from the inner bor- der of this layer of cells (fig. 6). These cilia are very delicate and at first irregular in length, varying from a length about equiva- lent to the depth of the cells to.more than twice that amount. The date of the appearance of cilia varied from 21 to 32 days, and seems to have some relation to the amount of ovarian tissue EXPERIMENTAL METAPLASIA 363 implanted. Thus it certainly occurred earlier when large cysts, measuring about 4 mm. in diameter, were produced by cutting the muscle and inserting large pieces of the ovary, than when a transplanting needle was used. In the course of time, lateral walls dividing the cells appear, and these are usually clearly visible about 40 days after the im- plantation. Thus eventually there is formed a closed cyst, the wall of which is lined with typical columnar ciliated epithelium and surrounds a mass of orange-colored granular debris, in which are numerous blood corpuscles in varying stages of degeneration (figs. 7 and 8). This cyst remains unaltered for at least 120 days, which is the longest period during which I have succeeded in keep- ing the animals alive under experimental conditions. So far the sequence of events after implantation of pieces of the ovary producing subsequent cyst formation have been described, it will now be necessary to enter into the modifications of this process that occur when cyst formation does not take place. If extremely small pieces of the ovary are implanted, or if, as sometimes happened in the experiments, the ovarian tissue was distributed thinly all along the track of the needle, cyst formation around the whole implanted mass does not occur. In such cases there is at first a very slight formation of a surrounding layer of agglutinated blood corpuscles, but beyond this, if the experiment has been carried out aseptically, there is very little reaction of the tissues to the implantation in the first few days. The implaated tissue often appears quite normal for as long as a week, or eight days, but after this, degenerative changes set in, and by the thir- teenth day at latest, all trace of life in the cells of the oviduct, germinal epithelium, or ova has vanished. Meanwhile the de- generating tissue is invaded by blood corpuscles and fibroblasts. The latter tend especially to invade and travel along any remain- ing framework of connective tissue that may be left in the implanted mass, and thus to forma dense mass of new fibrous tissuein the placeof theold. From the examination of a large num- ber of sections of different stages of this process I am of the opin. ion that all implanted fibroblasts die, and that all the new fibrous tissue is derived from the cells of the host, though the matter would be extremely difficult to prove definitely. 364 G. HAROLD DREW As the actual disintegration of the ripe ova in the course of the degenerative changes occurs very slowly, the invasion of fibro- blasts causes the ova to become completely surrounded by new fibrous tissue. Thus are produced a number of minute cysts containing very few ova, or possibly only one. Subsequently these cysts become lined with columnar ciliated epithelium, de- rived from the innermost layer of fibroblasts forming the cyst wall, in the manner already described. It often happens, even when the whole implanted mass becomes encysted, that small aggregations of ovanear the outside form sep- arate cysts, and also develop a lining layer of ciliated epithelium. Similarly when small pieces of ovary are implanted, the fibro- blasts, by traveling along any connective tissue framework pres- ent in the ovary, may divide the whole cyst into several parti- tions separated by thin layers of fibrous tissue, which subsequently also form ciliated epithelium. EXPERIMENTS TO DETERMINE THE RELATION OF THE PRESENCE OF OVARIAN TISSUE TO THE FORMATION OF CILIATED EPITHELIUM FROM FIBROBLASTS An attempt was made to cause the formation of ciliated epi- thelium by the implantation of portions of the ovary that had been killed in various ways, but all these were unsuccessful. The following methods were tried: 1. Heat. Small portions of the ovary were boiled in sea water for various times ranging from 2 minutes to 15 seconds, and then implanted in the muscle in the usual way. A considerable inflam- matory reaction resulted, followed by extensive formation of fibrous tissue: a very large proportion of the animals died within a week of the implantations, and often the track of the needle did not heal up. In none of these experiments did the animals sur- vive for more than 15 days, and in all these there was an opening leading to the surface from the implanted tissue. Pieces of the ovary that had been heated for two minutes to 70°, 60,° 50,° and 45° C. before implantation gave similar results, but heating to 40° C. for two minutes in some cases did not affect EXPERIMENTAL METAPLASIA 365 the ovarian tissue, that is to say, cysts lined with ciliated epithe- lium were produced in a small proportion of these experiments. Theimplantation of pieces of ovary that have been heated to 30° C. produces the same results as the implantation of unheated por- tions. 2. Cold. Small pieces of the ovary were frozen (at a tempera- ture of —20° C.), and allowed to thaw slowly. The thawing process took about an hour, then after the lapse of 15 minutes the ovary was again frozen. This process was repeated four times, and then the pieces of ovary were implanted in the usual manner. The animals were killed after 35 days, and sections showed that a moderately intense inflammatory reaction had resulted; the implanted tissue was invaded and nearly completely replaced by blood cells, and was surrounded by a thick and compact layer of fibroblasts. No ciliated epithelium was present, nor did the inner layer of fibroblasts show any of the changes preliminary to its production. 3. Chemicals. Pieces of the ovary were treated for one hour with various protoplasmic poisons, then well washed in sterile sea water, and implanted. Solutions of the following substances were tried: 1 part corrosive sublimate in 2000 parts of sea water. 2 per cent phenol in sea water. 2 per cent of 30 per cent formalin in sea water. 1 part of 20 vols. hydrogen peroxide to 10 parts of sea water. 0.5 per cent of potassium cyanide in sea water. 20 per cent alcohol in sea water. 0.5 per cent of chloroform in sea water. In every case the animals died within ten daysof the experiment, showing signs of intense inflammation and often liquefaction of the tissues at the site of the implantation. 4. Degeneration in vitro. The ovary was thoroughly washed in sterile sea water, cut in small pieces, and each piece placed in a sterile test tube with a little sterile sea water. Similar pieces were placed in test tubes containing the blood of Pecten, collected under aseptic conditions. The ovarian tissue was allowed to degenerate in these tubes for three days, then cultures were made 366 G. HAROLD DREW with a loopful of the fluid from each tube on sloped fish broth peptone gelatin, made with sea water, and the pieces of the ovary were implanted into the muscle in the ordinary way. It was found, considering only those cases which the cultures showed to be sterile, that about the same proportion of the animals survived as in check experiments, but whether the degeneration had occurred in blood or in sea water, in no case was there any pro- duction of ciliated epithelium. Experiments in which the ova were removed from the ovary and then injected were unsuccessful. If the ova be shaken out into sterile sea water, centrifugalised, or allowed to settle, and then injected with a hypodermic syringe, it was found that a large proportion of the animals died within a week, and in those that survived it was impossible to find the ova on dissection. To ob- viate this difficulty the centrifugalised ova were placed in a ster- ilised solution of gelatin in sea water, at a temperature just above the point of solidification, this was allowed to cool, and portions of the jelly implanted into the muscle: unfortunately this always resulted in the rapid death of the animal, presumably because the gelatin has some toxic action. Agar jelly could not be used for this purpose because it solidifies at too high a temper- ature, and also is not dissolved by the body fluids of Pecten. A number of experiments were performed to prove that the development of the lining layer of ciliated epithelium of the cysts was not produced merely as a result of irritation, and as a reaction to the implantation of any foreign body. In a previous paper (Drew and De Morgan ’10) the result of the implantation of the tis- sue forming the gills and digestive gland of Pecten, and of sterile Agar jelly, has been studied. In addition to these, such substances as sterilised cotton wool, cork, elder pith, and small portions of sterilised silicious sponges were implanted to act as a source of irritation. In all these cases the formation of a cyst wall com- posed of fibrous tissue took place, but there was no development of ciliated epithelium, and the same holds good for cases where the organ of Bojanus of Pecten maximus was implanted into other animals of the same species. The transplantation of the ovarian tissue of animals of different EXPERIMENTAL METAPLASIA 367 species into Pecten maximus or Pecten opercularis in every case caused death in a very short time. Such experiments were tried with the ovary of Cardium edule, Cardium norvegicum, Glycim- eris glycimeris, and various species of Tapes and Venus. On the other hand, after the transplantation of the ovary of Pecten oper- cularis into the muscle of Pecten maximus and wce versa, the ani- mals survived indefinitely, but there was no development of cil- iated epithelium within the cysts formed round the ovarian tissue. The implantation of portions of the male gonad produced a violent inflammatory reaction, with subsequent very extensive formation of fibrous tissue for some distance around the site of implantation. No cyst formation took place, and no trace of the spermatozoa could be seen in sections made three days after the experiment. The implantation of large pieces of the testis (more than about 1 mm. in diameter) usually caused the death of the animal in three or four days. If small pieces of the ripe ovary be placed in sterile sea water containing a little sperm, then well washed and implanted, a sim- ilar inflammatory reaction and free formation of fibrous tissue results, only a small proportion of the animals survive, and in these no formation of ciliated epithelium occurred. Similarly, if some days after implantation of pieces of the ovary, a little sperm suspended in sterile sea water be injected with a hypodermic syringe into the exact site of the original implantation, inflam- mation aad free fibrous tissue formation is set up, and the ovarian tissue is either absorbed, or becomes surrounded by a large area of dense fibrous tissue, and again the formation of ciliated epi- thelium is prevented. With the object of eliminating the possibility of the formation of the ciliated epithelium lining the cysts from the epithelium covering the adductor muscle, which conceivably might have been invaginated by the introduction of the transplanting needle, a series of experiments was undertaken in which the implanta- tion was made through a hole bored in the shell, and afterwards closed with sealing wax in the manner previously described. In these experiments the ciliated epithelial lining of the cysts devel- oped in exactly the same way as when the implantation was made laterally through the muscle.. 368 G. HAROLD DREW Another possible explanation of the development of the ciliated epithelium would be to consider that it was derived from the cil- iated cells of the oviduct which ramifies through the ovary, though it would be difficult to understand how these cells could migrate to the walls of the cyst, and there form a continuous lining, while all the other parts of the ovarian tissue degenerate. To test this view two series of experiments were made. In one series only portions of the ovary taken from the extreme apex on the convex side were implanted: in this part of the ovary there is no oviduct, only the alveoli with the contained ova being present; the area thus free from the ciliated oviduct is small, but there is sufficient to make at least three implantations from each ovary with cer- tainty that no ciliated cells are being introduced. In the parallel series of experiments portions of the ovary containing as much of the oviduct as possible were implanted. In both cases similar cysts lined with ciliated epithelium were produced. Other ex- periments were made in which pieces of the oviduct, which in its main branches is easily seen from the surface, were dissected out as carefully as possible, shaken in sterile sea water to remove any adhering ova, and then implanted: in these cases complete ab- sorption and replacement by fibrous tissue often occurred, but in cases where cyst formation took place there was no formation of ciliated epithelium. Thus it is proved that the formation of this layer is independent of the presence of the ciliated cells of the oviduct. Another point investigated was the relation of the ripeness of the ovary to the formation of the ciliated epithelium. It was found that this reaction did not take place as a result of the im- plantation of the spent or immature ovary, but only occurred when an ovary which was obviously full of ova, and of a bright orange or vermilion color, was used for the experiment. After the animals had been kept in the experimental tanks of the Labor- atory for some time, the ovary often lost its bright color, and though full of ova became somewhat pale and unhealthy looking: experiments in which portions of the ovaries of such animals were implanted gave very uncertain results; sometimes the cysts were lined with ciliated epithelium, but more often this was absent, EXPERIMENTAL METAPLASIA 369 and the cyst wall consisted only of fibrous tissue. If a thoroughly ripe and healthy looking ovary be used, and sepsis does not occur, it can be said with certainty that all surviving over thirty days will develop ciliated epithelium lining the cysts. SUMMARY OF RESULTS OF EXPERIMENTAL WORK The implantation of small pieces of the ripe ovary of Pecten maximus or Pecten opercularis into the adductor muscle of an- other animal of the same species results at first in the formation of a closed cyst within the muscle, lined with layers of fibroblasts. Complete degeneration and disintegration of the ovarian tissue within the cyst occurs in a few days, and then the cyst contains only an orange colored granular substance, presumably derived from the yolk, and numbers of blood corpuscles. After the lapse of from 21 to 32 days, changes occur in the innermost layer of fib- roblasts lining the cyst, they revert to an embryonic type, and afterwards become converted into columnar ciliated epithelium, which forms a continuous layer lining the cyst. The changes resulting in this formation of ciliated epithelium from fibroblasts can be followed clearly step by step, and once formed, the ciliated cells persist unaltered for at least 120 days, which was the longest period for which the animals could be kept alive in the experi- mental tanks of the Laboratory. Experiments were performed showing that this change is not produced by the implantation of any of the other tissues of Pec- ten, by neutral foreign bodies which would merely act as a source of mechanical irritation, by the transplantation of the ripe ovari- an tissue of other Lamellibranchs, or by the transplantation of pieces of the ovary of Pecten opercularis into the adductor mus- cle of Pecten maximus and vice versa. Other experiments showed that the development of ciliated epithelium does not occur if pieces of the immature or spent ovary be implanted, and that it is prevented by treating the ripe ovary with a suspension of the sperm in sterile sea water before implantation. Also that it does not occur if the ovary be killed by physical or chemical agents before implantation. A series of THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 370 G. HAROLD DREW experiments were made to eliminate the possibility of the origin of the ciliated epithelium lining the cysts from the ciliated cells of the oviduct, which might be present in pieces of the ovary that were implanted, or from the layer of epithelial cells forming the outer coating of the adductor muscle, which might be carried inwards by the transplanting needle. It thus appears that the conversion of fibroblasts into ciliated epithelium is a specific reaction following the implantation of the ripe living ovary. These observations are the result of nearly a thousand experi- ments, of which the majority have been performed on Pecten opercularis. DISCUSSION OF RESULTS It appears that the conversion into ciliated epithelium of the inner layer of fibroblasts liming a cyst formed round a piece of the ovary, which has been implanted into the adductor muscle of Pecten, is a specific reaction that occurs only when the ripe liv- ing ovary of an animal of the same species is implanted. The reaction takes place long after all trace of orgaaised structure in the implanted tissue has disappeared, and it is difficult to con- ceive of its being due to any other cause than the presence of some definite chemical substance within the cyst, which is character- istic of, and specific for, each species. It cannot well be a fluid existing preformed in the ova, as in this case it would soon escape through the reticulate and easily permeable layer of fibroblasts first formed round the implanted mass, and for the same reason, considering the relatively great length of time necessary for the reaction to occur, it is not probable that it is a fluid formed at one definite stage in the process of degeneration. If a fluid at all, it seems on these grounds likely that this substance is slowly and continuously formed for a considerable period within the cyst. Examination of the contents of the cysts shows in all cases where the development of ciliated epithelium has occurred, that an or- ange granular substance, and blood corpuscles in various stages of EXPERIMENTAL METAPLASIA 371 degeneration, are present. These orange granules resemble in appearance the orange colored yolk substance of the ripe ova, and the amount of this granular substance within the cysts seems to be independent of the length of time during which the implanted tissue has been allowed to remain in the muscle. If implantation of pieces of the ovary of approximately equal size have been made, examination of the contents of a cyst after 6 days will show as much of this substance present as in a similar cyst after 120 days, hence it appears that this substance cannot escape through the cyst wall. When it is considered that the development of the ciliated epithelial lining only occurs as a reaction to the implan- tation of ripe ova, containing a plentiful supply of the orange colored yolk substance, there is at least a possibility that the or- ange substance within the cysts bears a close relation to the yolk substance, and that the development of ciliated epithelium from the fibroblasts lining the cyst is a specific reaction to its presence. Though based on no experimetal evidence, I would suggest as a possible explanation of the phenomena, that some substance is formed as a result of the ingestion of these orange granules by the blood corpuscles, and their subsequent degeneration within the cyst: that the granules themselves remain unchanged, and are again set free on the disintegration of the corpuscles, and that their action on the protoplasm of the corpuscles is merely catalytic. This substance, produced from the blood corpuscles, is probably a fluid, and would be slowly and continuously formed as long as blood corpuscles could pass through the walls of the cyst. The action of this substance on the fibroblasts forming the walls of the cyst is to delay their return to the spindle shape typical of the resting condition, and eventually to set up those changes in the mner layer of fibroblasts resulting in their conversation of cili- atedepithelium. Once this epitheliallayeris complete, the access of fresh blood corpuscles to the interior of the cyst would be hindered or prevented, and so the formation of this substance would tend to stop; at the same time the outer layers of fibroblasts would regain the resting state, and form a layer of typical fibrous tis- sue which would tend still further to prevent the ingress of fresh blood corpuscles. 372 G. HAROLD DREW An obvious objection to this theory is that the ciliated epithe- lium is not produced after the implantation of pieces of ovary that have been killed in any of the ways described, but it must be taken into consideration that in these cases the implantation of the dead ovarian tissue was always accompanied by a more or less intense inflammatory reaction, and so the experiments are scarcely com- parable to those in which portions of the living ovary were used. Also it is probable that the chemical composition of the ovary was altered by the methods of killing. If by future experiments, and more extended observations, it can be fully substantiated that similar cytological changes take place in other types, as a reaction to the presence of some definite chemical substance, the fact should have an important bearing on the chemical theories of development, and possibly on the origin of certain abnormal heterotopic growths. ABSTRACT OF EXPERIMENTS PERFORMED Total number of implantations, including animals that died before exami- MAtIOMMCADOUE), ¢ lcclsttee cus weston Fan ee ocak, hee ue eee eee ara sic ee 950 Number of animals examined in studying stages up to 20 days............... 215 Number of animals in which cysts lined with ciliated epithelium were pro- duced (not including those which died between 20 and 130 days, of which it is probable that most produced such cysts).............-.-+-----++.-- 68 Experiments on effect of heat on ovary before implantation .................. 42 Experiments on effect of cold on ovary before implantation..........-.....-. 18 Experiments on effect of chemicals on ovary before implantation. ............ 48 Experiments on effect of degeneration on ovary before implantation.......... 22 Experiments on implantation of centrifugalised ova...........-..-+.-++--+55 10 Experiments on implantation of ova in gelatin..............--..--0.05 +2000 10 Experiments on implantation of foreign bodies...............-+..--205+20055 33 Experiments on implantation of ovaries of other animals.............-.-..-: 46 Experiments on implantation of ovary of P. maximus to P. opercularis........ 12 Experiments on implantation of ovary of P. opercularis into P. maximus...... 12 Experiments on implantation of male gonad..............--....-2+eee sees 20 Experiments on implantation of ovary first treated with sperm..............- 14 Experiments on implantation of portions of ovary containing no oviduct..... 12 Experiments on implantation of portions of ovary containing oviduct........ 14 Experiments on implantation of unripe Ovary......-...--. +++ esse eee eee eee 20 EXPERIMENTAL METAPLASIA BS LITERATURE CITED Apamt, J. G. 1900 On growth and overgrowth and on the relationship between cell differentiation and proliferative capacity; its bearing upon regen- eration of tissues and the development of tumours. The Medical Chronicle. 1908 Principles of pathology, vol. 1. Philadelphia and New York. Barrurty, D. 1891-1900 Regeneration. Ergebnisse Anat. und Entwickl- Merkel und Bonnet. Braun, M. 1903-1904 Jahresber. der Anat. und Entwickl. Curnot, L. 1891 Etudes sur le sang et les Glandes Lymphatiques. Arch. de Zool. Expér. et Gén. Deuxieme serie, tome 9. Paris. Dakin, W. J. 1909 Pecten. Liverpool Marine Biological Committee mem- oirs, 17, London. DeBruyne, C. 1896 Contribution A 1’étude de la Phagocytose (1) Arch. de Biol., tome 14. Paris. Drew, G. H. 1910 Some points in the physiology of Lamellibranch blood-cor- puscles. Quart. Journ. Micro. Sci., vol. 54, part 4. Drew, G. H. anp De Moraan, W. 1910. The origin and formation of fibrous tissue produced as a reaction to injury in Pecten maximus. Quart. Journ. Micro. Sci., vol. 55, part 3. FLEXNER, S. 1898 The regeneration of the nervous system of Planaria torva and the anatomy of the nervous system of double-headed forms. Jour- nal of Morphology, vol. 14. Fraisse, P. 1885 Die Regeneration von Geweben und Organen bei den Wirbel- thieren, besonders bei Amphibien und Reptilien. Kassel und Berlin. Harvey, W. 1907 Journal of Medical Research. N. 8. vol. 12. K6iirker, A. 1885. Die Bedeutung der Zellenkerne fiir die Vorgainge der Vererbrung. Zeitschrift fiir wissenschaftliche Zoologie, vol. 42, p. 44. Kroeser, J. 1900 An experimental demonstration of the regeneration of the pharynx of Allolobophora from endoderm. Biol. Bulletin, vol. 2. LANKESTER, Str E. Ray 1886 On green oysters. Quart. Journ. Micro. Sci., vol. 26. 1893 Phagocytes of green oysters. Nature, vol. 48, p. 75. Lors, L. 1898 Some activities of the epithelium. Johns Hopkins Hosp. Bull. Vol. 9. 1899 An experiment-study of transformation of epithelium to con- nective tissue. Medicine. 374 G. HAROLD DREW Moraan, T. H. 1904 Germ layers and regeneration. Archiv fir Entwickl.- mech. der Organismen, vol. 18, no. 2. Rresert, H. 1904 Geschwulstlehre, Bonn. Saxpr, F. 1904 Ziegler’s Beitrage, vol. 38. Towts, E. W. 1891 On Muscle regeneration in the limbs of Plethodon. Biol. JByoull, INE Wourr, G. 1893. Entwickelungphysiologische. Studien 1. Die Regeneration der Urodelenlinse. Arch. fur Entwickl.-mech. der Organismen, vol. 1. 1894 Bemerkungen zum Darwinismus, mit einem experimentellen Beitrag zur Physiologie der Entwickelung. Biol. Centralb., vol. 14. PLATE 1 EXPLANATION OF FIGURES 1 Stages in division of a normal fibroblast. 1000. 2 Portion of a cyst wall after 5 days. Below is the degenerating ovarian tissue invaded by phagocytes, and this is divided from the muscle by several layers of fibroblasts, mostly in the active state. Above is a blood space bounded by fib- rous tissue, some of the fibroblasts of which are dividing. X 800. 3 Portion of a cyst wall after 20 days. The ovarian tissue has completely de- generated. The inner fibroblasts have changed the character of their nuclei and become connected by a continuous layer of cytoplasm. 800. REFERENCE LETTERS b.c. Blood corpuscles. div.fbl. Dividing fibroblasts. b.sin. Blood sinus. fbl. Normal resting fibroblasts. cil.ep. Ciliated epithelium. mig.fbl. Migrating fibroblasts. deg.ov. Degenerating ovarian tissue. msl.fbr. Muscle fibres. msl.nuc. Muscle nuclei. (N.B. The structure of the muscle tissue is merely indicated in the figures; details of striation, etc., are omitted.) EXPERIMENTAL METAPLASIA PLATE 1 G. HAROLD DREW msl. fbr. div. fbl. b. sin. msl. nue. b.c. mig. fbl. fbl. fbl. lyr. [os ©: deg. ov. msl. fbr. fbl. fbl. lyr. deg. ov. b.c. G. H. D. del. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 375 PLATE 2 EXPLANATION OF FIGURES 4 Portion of a cyst wall after 23 days. The nuclei of the changed inner layer of fibroblasts have developed definite nucleoli, the cytoplasm is more definite than in fig. 3, and the cells are suggestive of an embryonic type. X 800. 5 Portion of a cyst wall after 26 days. The layer of cells bounding the degen- erated ovarian tissue has become well defined, with a distinct basement membrane: the character of the nuclei has again altered, they are smaller and the nucleoli have disappeared. X 800. 6 Portion of a cyst wall after 30 days, showing development of long rather ir- regular cilia. The fibroblasts of the outer layers of the cyst are assuming the rest- ing stage. X 800. 7 Portion of a cyst wall after 98 days. The formation of ciliated epithelium is complete and dividing walls have appeared between the cells. The nuclei are smaller than in the stage represented in fig. 6. and the cilia are somewhat shorter and more regular. X 800. 376 EXPERIMENTAL METAPLASIA PLATE 2 G. HAROLD DREW r BS go @ eS — msl. fbr. BE BESeEeee~ ,,,, BROLDECBODIS | ae ite a we : 4 co @ Ta ‘ — msl. fbr as EES ———— — : ——— aD —— SE 4 BD EE ED ay A LP DE Elle: Ss Scis5 CED 6 deg. ov. msl. fbr. msl. nue. fbl. lyr. cil. ep. deg. ov. b.e. msl. fbr. msl. nue. fbl. lyr. —— cil. ep. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 PLATE 3 EXPLANATION OF FIGURES 8 Complete cyst lined with ciliated epithelium, of which fig. 7 represents a portion. The bands of fibrous tissue on each side of the cyst are formed in the track of the transplanting needle. X 400. 378 EXPERIMENTAL METAPLASIA PLATE 3 G. HAROLD DREW --— deg. ov. Ki Ss ies 2 ra a oa ‘ msl. fbr. — — b.c cil. ep. a4 fbl G. H. D. de THE EFFECTS OF SEMI-SPAYING AND OF SEMI- CASER ATION, ON THE SEX OGRATIO -OF » THE ALBINO RAT)MUS NORVEGICUS ALBINUS.) HELEN DEAN KING The Wistar Institute of Anatomy and Biology The widely accepted view of a hundred years ago that the sex of an individual depends entirely upon which of the ovaries sup- plied the egg is generally credited to Hippocrates (460-377 B. C.). In spite of a considerable amount of adverse evidence, this theory was revived by von Seligson in 1895, and very recently it has been advocated by Dawson (’09) and by Calhoun (10). The first two of these recent advocates of the theory are physicians, and much of the evidence that they offer in support of their views is derived from clinical cases that have come under their own obser- vation. Calhoun’s conclusions are the results of an investigation of stock breeding on a western ranch. Medical literature contains descriptions of many cases of one- sided ovariotomy which show that eggs capable of developing into individuals of either sex are produced in each ovary. Authen- tic records indicate that in man, as well as in cattle, the removal of one testicle does not lead to the production of offspring of one sex only. Evidence of this kind, however, is either ignored entirely by von Seligson, Dawson and Calhoun, or its authentic- ity 1s questioned. Among the first to make an experimental investigation of the cause of sex in mammals was Henke (1786). This investi- gator operated upon pigs, dogs and rabbits, removing an ovary or a testicle from each of the individuals used in the experiments. The results reported as having been obtained when these animals 381 382 HELEN DEAN KING were mated are very remarkable. In every instance a litter was composed of males when the left ovary and the left testicle of the parents were lacking, and entirely of females when the oper- ation had removed the gonad from the right side of each parent. In one of his experiments Henke mated a bitch that had been sprayed on the right side with a dog that had been castrated on the left side, but no litter was produced. From the results said to have been obtained in these experiments Henke concludes that in all mammals each ovary and each testicle has its own kind of ‘germ.’ Eggs from the left ovary can only be fertilized with ‘samen’ from the left testicle, the resultant individual always being a female; conversely, male eggs from the right ovary can only be fertilized with ‘samen’ from the right testicle. Most modern zoélogists would not consider these conclusions warranted, since Henke made but a small number of experiments and appar- ently had no controls of any kind. Ignoring the manner in which Henke carried out his experi- ments, von Seligson (95a, ’95b) uses the results to support his own theory, which is that of Henke expressed in more modern terms. Von Seligson himself operated upon four female rabbits, removing the left ovary from two of them and the right ovary from the remaining two: each of these rabbits was subsequently mated twice with normal males. Von Seligson states that the two females that were spayed on the left side produced males only, and that the other two rabbits had litters containing only females. The results of these experiments can hardly be con- sidered to afford conclusive evidence in support of von Seligson’s claims, since all details are wanting regarding the manner in which the experiments were conducted. Von Seligson does not mention what precautions, if any, were taken to safeguard the experiments; and in no case, apparently, did he make an autopsy to ascertain whether or not the operation had been successful. The publication of von Seligson’s theory caused a considerable amount of discussion among physicians, particularly in Germany, and a number of papers soon appeared in various medical journ- als giving birth records, after one-sided ovariotomy, which were not explicable according to the theory of Henke and von Selig- SEX RATIO OF THE ALBINO RAT 383 son. Many of the writers of these papers stated their belief in the theory that sex is determined in the ovary, but very few of them put any faith whatever in von Seligson’s contention that male eggs are segregated in the right ovary and that female eggs are produced only in the left ovary. Goenner (’96) repeated von Seligson’s experiments on rabbits, and also extended them. He removed one testicle from each of four males and one ovary from each of five females, all of the ani- mals being about six months old when the operation was per- formed. From the various matings in which animals were paired that lacked the gonad on the same side of the body Goenner obtained only three litters, each of which contained both males and females. Four of the 16° young contained in these litters died before their sex was ascertained ; of the remaining 12 animals, four were females and eight were males. These results indicate that von Seligson did not properly distinguish the sexes of the individuals in the various litters that he examined; but they do not give convincing evidence against his theory, since unfortun- ately Goenner does not state whether he killed the females and ascertained if the gonads had been entirely removed by the oper- ation. Dawson’s revival of the right and left ovary hypothesis for man has one important modification: the spermatozoan is not con- sidered to have any influence whatever in determining sex. According to Dawson’s theory, therefore, spermatozoa from either testicle are able to fertilize eggs from either ovary. Cal- houn is of the opinion that the spermatozoan may possibly have something to do with sex, and she suggests that this matter be investigated experimentally. Evidently this writer is unac- quainted with much of the literature dealing with the question of sex-determination in the higher forms. In order to test the truth of Dawson’s hypothesis, Doncaster and Marshall (710) made a small series of experiments last spring on the albino rat. One female was spayed on the-right side, and -a second one on the left. As soon as these rats had recovered from the effects of the operation, they were mated with normal males. The female that was spayed on the right side gave birth 384 HELEN DEAN KING to a litter containing seven young. The sex of only five of these individuals was ascertained; four of them were females, and one a male. The female lacking the left ovary produced a litter of five young, of which three were males and two were females. The breeding females were killed soon after the birth of their litters and dissected. Each was found to lack the ovary and part of the fallopian tube on the side of the body that had been oper- ated upon. These experiments are not open to criticism on account of the manner in which they were carried out, and the results show very conclusively that, in the albino rat, eggs cap- able of developing into individuals of either sex can come from either ovary. Doncaster and Marshall rightly argue that because Dawson’s theory is not valid for the rat is not a proof that it is also invalid for man; but they believe, as do probably most investigators, that ‘‘definite proof for another mammal detracts from its probability.”” Various cases in which one-sided ovariotomy in woman has been necessitated by disease have fur- nished evidence against the theories of von Seligson, of Dawson and of Calhoun that is fully as convincing as is that which the experiments of Doncaster and Marshall give for the albino rat. A number of investigators, among whom may be mentioned Rauber (’00), Beard (’02), Schultze (03) and Russo (’09), main- tain that sex is determined in the ovary, although they do not believe that the male-producing eggs are segregated in the right ovary and that the female-producing eggs are all contained in the left ovary. This theory has a considerable amount of evi- dence in its favor, and if it be true, it is evident, as Schultze (’03) has stated, that ‘“‘in der Ovogenese ist die Lésung der Geschlechts- bildung enthalten.”’ No advocate of this theory has ventured a suggestion as to the relative distribution of the male-producing and of the female-producing eggs in each ovary, and there is the possibility that eggs of one kind may be produced in much greater numbers in one ovary than in the other. On the current hypoth- esis that spermatozoa are dimorphic and that the male determines sex, the possibility also exists that many more spermatozoa of one kind are produced in one testicle than in the other. If there is a constant difference in the relative distribution of the various SEX RATIO OF THE ALBINO RAT 385 kinds of germ cells in the gonads, this difference should be shown by a distinct alteration of the normal sex ratio among the young produced by mating animals from which one of the gonads had been removed. To test this point a series of experiments on the albino rat (Mus norvegicus, albinus) was started in the fall of 1909. The results obtained in these experiments are given in the present paper. The rats used in this series of experiments were operated upon by Dr. J. M. Stotsenberg of The Wistar Institute, to whom I am greatly indebted for this assistance. In all cases the operation was performed on the rats when they were 16—20 days old, the ovary or the testicle being removed while the animal was under the influence of ether. Full details regarding the manner in which the operations were made will be given in a forthcoming paper by Dr. Stotsenberg. About half an hour after the opera- tion the young rats were returned to their nest, and they remained with their mother until they were one month old when they were fully able to care for themselves. The sexes were separated when the animals were two months old; and the rats were mated for the first time when they were about four months old. Each pair of breeding animals, earmarked for identification, occupied one of the standard cages used for the rat colony of The Wistar Institute. It was not possible, therefore, for the experiments to be invalidated by promiscuous breeding. The sex of a newborn rat cannot be ascertained with any degree of certainty unless the animal is killed and dissected. When rats are 14-16 days old, however, the sexes are easily distinguished, as Dr. Stotsenberg has discovered, since the mammae in the females are clearly visible at this time. After this period the hair covers the entire body. and it becomes very difficult to dis- tinguish the sexes in the living young until they are several weeks old. Cuénot (’99) ascertained the sex of 255 young albino rats belonging to 30 different litters. He found a slightly greater number of males than of females; the sex ratio being 105.64 males to 100 females. Records that I have made of the sex of 452 young albino rats, belonging to 80 litters, give a sex ratio of 107.33 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4. 386 HELEN DEAN KING males to 100 females. Apparently, therefore, in the albino rat, as in man and various other mammals, there is normally a nearly equal proportion of the sexes among the young, although in all species there seems to be a slight excess of males. THE EFFECTS OF SEMI-SPAYING ON THE SEX RATIO OF THE ALBINO RAT Six females, belonging to two litters born in October, 1909, were operated upon when they were 16 days old. From three of these females the right ovary was removed, and from the remain- ing three the left ovary was taken. Two of these females, one spayed on the right side and the other spayed on the left side, never had a litter although they were paired with normal males for five months: both of these rats died of pneumonia when they were about nine months old. When dissected each female was found to have but one ovary which appeared normal in every respect. The only reason that can be suggested for the failure of these rats to breed is that they had been attacked by pneu- monia when they were immature and therefore were never in a physical condition to bear young. There is evidence that rats may suffer from pneumonia, in an incipient form, for a consider- able length of time with no manifestations of the disease other than a loss of weight and a failure to breed; and it is only when this disease has nearly run its course that the characteristic difficulty in breathing, which indicates the formation of pus nodules in the lung tissue, becomes at all noticeable. Table 1 gives a summary of the number of young produced by the four semi-spayed rats when they had been mated with normal males. The letter R or L after the number given the rat indi- cates that the right or the left ovary had been removed. Each litter of every female contained young of both sexes; and although there were more females than males in the total number of individuals that were produced, the excess of females was too small to be considered as significant. The two females spayed on the right side had a total of five litters which contained 22 individuals; nine of these were males and thirteen were females. SEX RATIO OF THE ALBINO RAT 387 The five litters produced by the two rats that were spayed on the left side contained 25 young, of which thirteen were males and twelve were females. These results show that the sex ratio is not affected in the slightest degree by the removal of the right or of the left ovary from the breeding females, and that each ovary pro- duces, in approximately equal numbers, eggs that are capable of developing into males and eggs that can develop into females. Henke and von Seligson maintain that it is not possible for a male to be produced when the right gonads are lacking in the breeding pair, or for a female to develop when the left gonads have been removed. They also state that it is impossible to fertilize the eggs of an ovary with spermatozoa from the testicle on the Table 1 NUMBER | NUMBER | AVERAGE NUMBER | NUMBER A aes SRaNG | ba Saag | Be G2) eee 3 12 4.0 4 8 PE (GR eee ee 2 10 5.0 5 5 3 (Gh). a 16 me 10 6 4h (Ui) 2, 9 4.5 3 6 10 47 Ai 22 25 opposite side of the body. To test the truth of these hypotheses for the albino rat the following series of experiments was made: 1. Female no. 2, which had been spayed on the right side, was mated with a male from which the right testicle had been removed. A litter containing five young was obtained; three of these indi- viduals were males and two of them were females. 2. Female no. 3, spayed on the left side, was mated with a male castrated on the left side. There was one male and one female in the resultant litter. 3. Female no. 1, lacking the right ovary, was mated with a male that had Boeri castrated on the left side. This female had a litter containing five young, of which three were males and two were females. 388 HELEN DEAN KING 4. Female no. 4, spayed on the left side, was mated with a male which lacked the right testicle. The litter produced con- tained six young; two of which were males and four were females. These results show conclusively that eggs from either ovary of the albino rat can be fertilized with spermatozoa from either testicle. They also prove that males can be produced when the right gonads are lacking in the breeding animals and that females can develop when the left gonads of the breeding animals have been removed. Table 2 gives a summary of the distribution of the sexes in all of the young produced by the four semi-spayed females. Table 2 NUMBER | NUMBER | AVERAGE NUMBER | NUMBER panera | wee | rn eae eae ILC 8) nae | 4 17 all Ul 10 oh OR Woe cae | 3 15 5.0 8 7 3 (be: 4 18 4.5 iit of ANE) ee 3 15 5.0 5 10 | The most striking fact brought out in the above table is that the litters average but 4.64 young each; a result which can doubt- less be justly attributed to the removal of one of the ovaries from each of the breeding females. It is very probable that in the rat ovulation takes place in both ovaries at the same time, and that the litter of a normal female contains young that have developed from eggs derived from each ovary. Presumably, therefore, the removal of one ovary would cause a decrease in the size of the litter by lessening the number of eggs that might have been fer- tilized at the same time. The average number of individuals in the 30 litters of albino rats examined by Cuénot was 8.03; while the 80 litters I have obtained contained an average of only 5.6 young. My records, however, are made up in great part from litters produced in SEX RATIO OF THE ALBINO RAT 389 inbreeding experiments, and it is well known that inbreeding causes a marked decrease in the number of offspring. A normal sister of two of the semi-spayed rats was mated four times to a normal male. She had a total of 24 young, of which fourteen were males and ten were females. The average number of indi- viduals to a litter in this instance was six. One of the females operated upon by Doncaster and Marshall gave birth to a litter of seven young; but none of the semi-spayed rats used in these experiments ever had a litter containing more than six individuals. It seems probable, therefore, from the data shown in table 2, that semi-spaying causes a decrease in the average size of the litters, although it has no appreciable effect on the sex ratio, and apparently does not decrease the number of litters a female can produce. Two of the semi-spayed females used in these investigations died from pneumonia; the other two were etherized when it became evident that they were out of condition and would not breed again. An autopsy was made in each instance, and in no case was there any ovarian tissue on the side of the body that had been operated upon. The remaining ovary appeard normal in every female, and there was no marked increase in its size to compensate for the loss of the other ovary, as Doncaster and Mar- shall found to be the case in each of the two rats upon which they had operated. As these investigators operated upon adult rats and killed them about two months after the operation, it seems probable that the noticeable increase in the size of the re- maining ovary must have been due to some pathological condi- tion, and not to a normal ‘compensatory hypertrophy.’ There is a considerable variation in the size of the ovaries of the same rat, as well as in those of different rats; and it is not improbable that the same ovary varies in size at different times. A series of careful measurements would have to be made of a number of ovaries, removed from females at different times in the month and at different seasons of the year, in order to obtain proper standards by which to measure any apparent deviation from the normal size. 390 HELEN DEAN KING THE EFFECTS OF SEMI-CASTRATION ON THE SEX RATIO OF THE ALBINO RAT In February 1910, one testicle was removed from each of six males, belonging to two different litters. One male, castrated on the right side died before reaching maturity, so that two males castrated on the right side and three males castrated on the lett side were available for the purposes of these experiments. Except in the cases already noted, these males were mated with normal females. The number of offspring produced, together with the distribution of the sexes in the various litters, are shown intable 3. In this table the letters R and L refer to a castration on the right or on the left side respectively. Table 3 MALB NUMBER NUMBFR AVERAGE NUMBER 5 OF OF YOUNG MALES FEMALES Ry OH EON LITTERS YOUNG PER LITTER i053 ena 4 GF 4.1 10 7 DyORN bPiode 4 18 4.2 8 10 Su Gly 2 14 7.0 9 | 5 Alen: 4 21 5.2 9 | 12 Bri). 3 18 4.3 6 7 17 83 4.88 | p 42 AI Practically equal proportions of the sexes were obtained as a result of this series of experiments. The two males that were cas- trated on the right side had a total of 35 offspring, of which 18 were males and 17 were females; the sexes were equally divided among the 48 offspring of the three males from which the left testicle had been removed. These results show that the sex ratio was not affected by the removal of the right or of the left testicle from the breeding males. In these experiments, as table 3 shows, the average number of young in a litter was 4.88, which is very little higher than that obtained in the former experiments (table 2). The records for the litters produced by the semi-castrated males, as shown in SEX RATIO OF THE ALBINO RAT 391 table 3, include the four litters obtained when these males were mated with semi-spayed females. If the records for these four litters are excluded, the remaining thirteen litters are found to contain a total of 65 individuals, which makes an average of five young to each litter. This is doubtless a low average for the litter of a normal albino rat; but it is very little lower than the average size of the litters produced in the stock colony of The Wistar Institute this past year. The small size of the litters obtained in these experiments was probably due to some ex- ternal factor, and not to the castration of the breeding males. The results obtained in these experiments indicate that, if there is a dimorphism of the spermatozoa which is associated with sex-determination, both female-producing and male-produc- ing spermatozoa are developed in approximately equal numbers in each testicle of every normal male. elts.ps049) 38 A. Agassiz (Bull. Mus. Comp. Zodlogy, 1892) records the production of “‘per- manent albinos”’ in Gasterosteus, as a result of keeping these fishes on a white bot- tom. The time required for such a change is not stated, however, and no evidence is offered to prove that the alteration really was permanent. 436 FRANCIS B. SUMNER opposite directions. The pupil of the eye is crescentic in form, owing to a semicircular projection from the dorsal side of the iris. This must largely eclipse the visual field overhead, though, as will be pointed out presently, the fish can see in this direction, even without any special movement of the eye. In most of these experiments, glass jars were used, whose bottom areas were not large in comparison with the size of the fish.*® Except in special cases, the walls of these jars were left un- painted. Since the fish commonly lay close to one or another side of the jar, and its head was never more than a few centimeters distant from one of the vertical walls, the animal’s visual field necessarily included much that lay outside of the jar alto- gether. The different portions of this ‘landscape’ must have varied greatly in their degree of illumination, from the bright side toward the window, to the comparatively dark side viewed in the opposite direction. That the fishes actually did, at times, give attention to things seen through the walls of the jar, was evi- dent from the fact that their eyes frequently followed my hand or other moving objects held in their vicinity. It was likewise found that the fishes could see objects directly overhead, since they sometimes rose with great celerity to take food which was dropped in from above, even in a larger tank with 25 or 380 em. of water, and certain more timid specimens became agitated when- ever I moved objects directly over them, although at a consider- able distance above the surface of the water. In the experiments thus far described, reference has been made to the bottom alone. In some cases the fish itself covered a fourth, or even more, of this bottom, while another large part of the animal’s visual field was necessarily occupied by the vertical walls of the jar, together with the lights and shades of the room *9 Most of the rectangular jars used had bottom dimensions of 15 by 20 em. (These are the external measurements. In reality, the movable plates were some- what smaller). Rectangular jars of a larger size were used, measuring 20 by 30 em. at the bottom, the artificial patterns being 17 X 27 em. The cylindrical jars employed had a diameter of 20 em., and a height of 12 em. The fishes ranged in length from 8.2 cm. to19cm. No.1hadalength of 11.4 cm., no. 4 a length of 11.2 cm., no. 1] a length of 8.9 em. ADJUSTMENT OF FLATFISHES 437 outside. Add to this the view overhead, which, as we have seen, was not wholly ignored, and it is truly a matter for surprise that the bottom was the surface chiefly concerned in evoking these pig- ment changes. To what degree, if any, surfaces lying in other planes might influence the fish was tested by special experiments. A fish (no. 8) which had first been photographed on a gravel bottom (fig. 8a) was used in a series of such tests. This specimen, whose length was 12.4 cm., was placed in a glass jar having a bot- tom diameter of 20cm. The bottom of the jar was painted black, but the walls were left transparent. Thus when this vessel was set into a larger jar and surrounded by gravel,*® the latter could be clearly seen by the fish. In this contrivance, the animal became much darker in the course of the next few days, the annuli being reduced to rings of white dots. These last remained conspicuously pale, however, so that the fish was far from being concealed on this bottom. (Fig. 8b represents the condition at the close of seven days.) The same specimen was now transferred to a contrivance exactly the reverse of the last, 2.e., one having the walls painted black, and the gravel visible through the bottom. A partial return to the gravel appearance was noted in the course of one day, and this increased during the next day or two, although the original appearance was not resumed in full, even after the lapse of ten days"! (fig. 8c). The fish was next transferred to a Jar having a white bottom, 20 cm. in diameter, and black walls. Within two days it was notice- ably lighter, and after three days ‘‘very much lighter and more homogeneous.” It is doubtful whether any further change oc- 40 Under water, of course, so that total reflection from the walls of the jar was avoided. “The fish, after seven days, had been changed to asmaller jar, having 9 diameter of only 16 cm., but no certain change was noted. Gravel was now (after ten days, ) placed in the bottom of the dish, so that the fish lay upon this directly, rather than upon a glass bottom. It is probable that some further change took place in the direction of adaptation to the gravel, for my notes state later that the fish now harmonized quite well. [ do not believe, however, that this higher degree of adap- tation had any relation to the tactile stimuli derived from direct contact with the gravel, but rather to an alteration of the visual stimuli. 438 FRANCIS B. SUMNER curred in the next six days, at the end of which it was photo- graphed, after being transferred to a Jar having a black bottom and white walls (fig. 8d). By this time, however, the fish appeared to have wholly lost its power of response, for it changed but slightly during a stay of six days in this jar, six days in a jar whose walls and bottom were both black, five days upon a coarse reddish gravel, and two days upon the dark, mixed sand.*” Similar experiments were tried with a number of specimens, but several of them became blind or otherwise diseased before decisive results were obtained. The most pronounced success was achieved in the case of fish no. 9. In fig. 9a, this fish is shown lying upon the bottom of dark mixed sand, on which it had been kept for two days.“ The tests to which it was further subjected were as follows. It was first put into a dish 16 cm. in diameter and 7 cm. deep, having the vertical walls painted black, and the bottom covered with fine gravel, cemented in place with plaster of Paris.44 The length of the fish was about 15 centimeters, and it must have covered from one-third to one-half of this bottom, while the head must at all times have nearly or quite touched the black vertical wall of the jar. In the course of a few hours, notwithstanding, the fish had changed somewhat in the direction of harmony with the gravel, while fig. 9b shows its condition on the following day. The appearance is certainly quite different from that manifested upon the sand, and is not ill-adapted to the new bottom, though it is recorded as ‘“‘somewhat darker in appearance than the gravel, and gravel pattern not particularly good.”’ Transfer to a larger jar (20 cm. diameter, 12 cm. high), also with black walls and gravel bottom, did not result in any appre- ciable change, but upon removal to a large open tank, having gravel of the same sort at the bottom, the fish resumed the maxi- mum degree of resemblance within a few hours. The animal was 42 Cf. p. 433, above. 43 It had previously been well adapted to the fine gravel. After transfer to sand, it changed considerably within an hour or less, and the maximum effect probably resulted within a day or less. ‘4 This was done in order to prevent the animal from covering itself with the gravel. ADJUSTMENT OF FLATFISHES 439 then returned to the larger jar used previously (20 cm. diameter, with black walls and gravel bottom), but the maximum gravel effect now persisted for two days, when the fish was again removed. Later, this same specimen was put into another jar (20 cm. diameter) having a black bottom and white walls. After five days (no observations being made in the meantime) the fish was of a “pale brown or buff, with pale spots still lighter’ (fig. 9c). My record says: ‘‘not only not black, but not a dark fish.”’ The animal was next transferred to a jar of the same size with bottom and walls both black. Within less than a day there was a very pronounced change: “‘ Fish so dark and so devoid of conspicu- ous markings as to be pretty well concealed in jar. Not noticed at first” (fig. 9d). The transfer was again made to the white- bottomed iar, and back again to the all-black one, with similar results in each case. From the records of the two foregoing specimens, as well as of many others, it is quite plain that the bottom, even when it is of very limited area, and largely concealed by the fish itself, may exert a predominant influence in determining the appearance assumed by the latter. On the other hand, it seems plain from the later behavior of the second of these specimens that the vertical wall may also exert an important influence upon the animal. A comparison of fig. 9¢ and fig. 9d will sufficiently illustrate this point for the present. This entire question was much more search- ingly tested with Lophopsetta maculata (see below). In order to determine whether a surface directly overhead would have any effect upon the color-pattern of Rhomboidichthys specimens were placed in the large tank (p. 427), lighted from be- low by means of a mirror. A plate of opaque white glass, of the same size as the bottom of the tank, was covered with small, irregular blotches of black paint. Four corks, 35 mm. long, were fastened to the painted surface of this plate, one near each corner, and served as legs when the contrivance was placed at the bottom of the tank. In this position, the spotted side faced downward, at a distance of about 3 cm. above the eyes of the fishes. The three specimens used in this experiment had all been unmis- takably influenced by this spotted plate when this was placed 440 FRANCIS B. SUMNER beneath them, assuming a much blotched appearance resembling that which was commonly shown upon a bottom of fine gravel. Upon the removal of the plate from beneath them, they had re- turned to a nearly unspotted condition. The spotted plate, now mounted on corks, as above described, was next inserted above the fishes (under the surface of the water, of course). The plate, in its present position, was brightly lighted by the mirror below. That the fishes could see this spotted surface cannot be doubted. Nevertheless, not one’ of the specimens showed any appreciable influence, even after several days.** Return of the spotted plate to the bottom of the tank, beneath the fishes, resulted in each case in a resumption of the blotched condition within a few hours at the most. With two specimens an attempt was made to force the animal to look directly upward. The eyes, or rather the eye-stalks, were tied together by means of threads stitched through the skin. This drastic treatment resulted in the blindness of the fishes, and no significant results were obtained. Relation between the degree of illumination and the character of the response The foregoing experiments were conducted in a laboratory room of medium size, lighted from one side only. Different Jars were exposed to very different amounts of light, and the degree of illu- mination for all of them varied greatly, of course, with the weather and with the time of day. Nevertheless, no undoubted relation was discovered between the intensity of the light and the rapidity or extent of the adaptive changes. In a few instances, it is true, certain of the pigment patterns were found to be much less con- spicuous on dark, cloudy days. It has been pointed out, however, that the completeness of these adaptations varied at different times in the same individual, even when no external cause was dis- coverable. And even if some specimens were actually affected at times by the intensity of the illumination, this was certainly not 45 The fishes were observed through the walls of the tank, by raising the dark curtains; also by removing the spotted plate. ADJUSTMENT OF FLATFISHES 441 the rule. The dark tone assumed by the fish upon the dark sand did not give way to a lighter shade when the animal was brought into direct sunlight; while fishes on a white bottom acquired the maximum degree of pallor, even though this white bottom was heavily shaded. This state of affairs has already been pointed out by other observ- ers,“° and indeed it would seem to be a necessary one in order that the color should be adaptive or cryptic. For it is obvious that the fish itself is shaded or lighted equally with the surface on which it lies, so that the relation between the two remains unaffected by the degree of illumination. A rather curious corollary may be drawn from this last principle. Suppose that we have two aquaria side by side, one with its inner surfaces painted white, the other painted a perfectly neutral gray. Suppose, now, that the white aquarium is heavily shaded, so as to admit comparatively little light, while the gray aquarium is well illuminated. Under these conditions, the fishes in the white tank should, according to hypothesis, assume the maximum degree of pallor, while those in the gray tank should continue to display some of their dark pigment, in amount depending upon the shade of gray employed.47 Now experiment shows that this is precisely what happens. The fishes in the shaded white tank blanch to their fullest extent, while those in the gray tank become gray. It is obvious, however, that the dimly illuminated white bottom of the one tank may be actually darker than the brightly illuminated gray bottom of the other, in the sense that the former may reflect an absolutely smaller amount of light to the observer than the latter. The theoretical bearings of these facts will be discussed in a later section. At present, I shall confine myself to an account of cer- tain experiments upon Rhomboidichthys. Two glass jars, 20 em. in diameter and 12 cm. deep, were used. The bottom of one of these was painted gray of ashade nearly match- 6 Most clearly by Keeble and Gamble for schizopod and decapod crustacea (Phil. Trans. Roy. Soc., Series B, vol. 196, 1904, pp. 353 et seq.) ; likewise by Bauer for isopods (Centralblatt fiir Physiologie, 1906, p. 459). 47 It is needless to point out that gray isnot acolor. I pure, it reflects white light, though of reduced intensity. 442 FRANCIS B. SUMNER ing that produced on a color-wheel by combining two parts of black and one part of white.‘® The gray was not, it is true, perfectly neutral, being somewhat ‘cold,’ 7.e., tinged with blue. That this fact played no part in the results seems likely from my later experi- ments (p. 461). The side of the jar toward the window was.left transparent, the opposite side being covered with white cardboard to increase, by reflection, the illumination of the bottom. In addition to this, a reflecting screen of white cloth, inclined at a suitable angle, was poised above. The white-bottomed jar had walls which also were painted white, but the light was largely cut off from its interior by a cylinder of sheet iron, painted black, which encircled the jar and projected upward for a distance of 12 cm. above its top. That the bottom of the gray tank was actually far lighter than that of the white tank, 7.e., reflected far more light, is readily seen from fig. 11/—12b (plate 13), which reproduces a photograph taken in the laboratory with the jars arranged as nearly as possible in the same manner as during the test. Several experiments were made with these jars, with more or less instructive results. Only one of these tests, however, was so clear cut and decisive that it deserves to be described in detail. Specimen no. 11, which had previously been kept for a long period upon white and other pale backgrounds, but was in the present case taken from the black magnetite sand (after twelve days) was placed in the gray-bottomed jar. It became appreciably paler within an hour, much paler within a day, and, after two days ‘probably no darker than the gray bottom.’ Specimen no. 12 which had likewise been on various backgrounds, but came, in this instance, directly from gray, was placed in the (shaded) white jar. This specimen grew noticeably paler in the course of a day, and the pallor increased for a day or two more. (The color- book was here referred to for comparison.) At the end of four days, the two specimens were examined under identical conditions of illumination, when it was found that no. 11 (on the gray bottom) was decidedly darker than no. 12 (on 48 Contrary to what one might suppose, such a gray is far from being dark. ADJUSTMENT OF FLATFISHES 443 the white bottom). Photographs were now made, in which the two fishes were taken together, first on the gray bottom (fig. 11g- 12c, plate 13), then on the white bottom. Both of these showed very plainly the difference of shade between the two fishes. The specimens were then transposed, no. 11 being placed upon the white bottom, no. 12 being placed upon the gray bottom. After two days, no. 11 appeared to be paler than 12, and after four days it was certainly so. They were compared, as before, and again photographed together, first upon the gray bottom (fig. 11h-12d), then upon the white bottom. The relative shade of the two fishes had obviously been reversed. The two animals were once more transposed, and with similar results, though no photographs were taken. Is the behavior of the fish influenced by the degree of its adaptation to the background ? As has already been pointed out, most specimens covered them- selves more or less with the gravel or sand in which they lay. In some cases, the marginal fins only were concealed; but in a few instances the entire body was buried, only the eyes protruding. So far as my observations go, the fish was just as likely to cover itself with the bottom material when its color and pattern were highly adapted to this as it was when they were glaringly ill- adapted. When specimen no. 10 was taken from a pale back- ground and placed in black sand, it buried itself with extreme rapidity, and remained completely concealed. In this instance the fish was at the outset utterly out of harmony with the bottom. It was noted, however, that this tendency to hasty concealment beneath the sand was just as marked after the fish had assumed a shade not far different from that of the latter. In contrast to the last example, specimen no. 11, though even more conspicuously out of harmony with its background, did not, at first, make any endeavor to bury itself when placed on the magnetite sand. In order to test the question whether the fish, when offered the choice, tended to select a background in harmony with its own 444 FRANCIS B. SUMNER shade, a plate of glass was employed as a bottom, having an area of 17 x 27 cm., and divided transversely into a black and a white half. Fishes nos. 11 and 12 were used in these experiments. Both were healthy and active. The former was, at the time, adapted to a very pale bottom, the latter to the dark sand. The fishes, one at a time, were placed upon this background, in the neighborhood of the division between the white and black halves, and in such a position that they could plainly see both. This experiment was repeated a number of times with each fish, the latter being left in some cases for more than an hour upon this bottom. No preference was shown for one surface more than the other. The fish commonly remained very near to where it was placed, whether or not it was adapted to the surface which immedi- ately surrounded it, and in no instance crossed over into the oppo- site side. On the contrary, it happened that in more than one case, the dark fish moved further back into the white area, and vice versa. Each of these fishes was then placed upon bottoms of dark and of white sand. No. 11 showed no disposition to burrow in either. No. 12 covered itself very little with either sand, and with one no more than with the other. Such experiments are of course not entirely conclusive, but, when taken in connection with other observations, they at least render it improbable that the fish exercises much selection in respect to the shade of its background. The behavior of this animal is thus not at all in accord with that of the decapod crus- tacean Hippolyte varians, as described by Gamble and Keeble. The réle of sight in these reactions Various previous observers®® have recorded that blind fishes failed to undergo adaptive color changes, and it has been pointed out that both specimens which have become blind through natural causes, 49 Quarterly Journal of Microscopical Science, 1900, p. 601. (See especially plates 32 and 33.) 50 H.g. Pouchet, Mayerhofer and Secérov, in works already cited. ADJUSTMENT OF FLATFISHES 445 and those which have been deprived of their sight experimentally, cease to adjust themselves to the shade of their background. Certain of my own fishes which failed to respond adaptively to changes of bottom were found to have become diseased in one or both eyes, although, as has been stated, some of the most refrac- tory individuals had not lost their sight. One of the fishes, both of whose eyes were affected, acquired a peculiar appearance which was not noted in any normal specimen. It assumed a rich brown color, with specks of orange, the whole effect being much more decorative than the somber hues ordinarily displayed by this species. Another specimen, one of whose eyes had already become blind, took on almost precisely the same appearance after I had cut the optic nerve of the sound eye. In both cases the fishes remained conspicuously out of harmony with the gravel or sand on which they were kept. My most complete experiments in blinding fishes were later made upon Lophopsetta, but a few which were made with Rhom- boidichthys deserve recording. It was at first my endeavor to cover the eyes with some opaque coating, without thereby causing any injury.*! This proving impracticable, I next tried the effect of searing the corneas with a red-hot platinum wire. The results, with specimens no. 10 and 11, are detailed in the next paragraph. The effect of cutting the optic nerve has already been described for one specimen”. I will add that this fish was subsequently kept for 22 days upon a white marble bottom. A slight paling was noted at first, and this, as subsequent experiments showed, might have occurred equally well on any bottom. Thereafter, no change was noted, and the fish remained fairly dark as long as kept under observation. One result of blinding, manifested in two of the foregoing speci- mens, is of peculiar interest. Specimen no. 10, which had been 51 Mixtures of lampblack with certain fatty substances were tried, but it was found that these would not adhere for more than a very few minutes. 52 Aside from the resulting loss of sight, it is probable that there is a distinct shock effect from the cutting of the optic nerves. Thus specimen no 10 (see below), when in a pale condition after the searing of the corneas, turned dark immediately, upon the cutting of the optic nerves, and this dark condition presisted until the death of the fish four days later. 446 FRANCIS B. SUMNER kept most of the time during a period of two months upon white and pale gray bottoms, was transferred to the black magnetite sand for a period of six days. At the end of this time the fish was nearly or quite as dark (fig. 10d) as are most of these fishes when adapted to a very dark bottom. The animal was then blinded by searing the corneas with ared-hot platinum wire. The effect was a conspicuous paling of the body (fig. 10e), which became evident in a short time and persisted for some days, after which the darker condition began to return. Substantially the same results were obtained from specimen no. 11, which had been kept for an even larger proportion of the preceding two months upon pale bottoms, and had been only 3 days upon the black sand at the time of blinding. Within a few hours, the fish returned completely, or nearly so, to the extremely pale condition which it had acquired upon its earlier backgrounds, and remained in this condition for a day, after which the observa- tions were brought to an end. These results are readily intelligible on the assumption that the pale condition had, to a certain extent, become fixed in the nervous mechanism during the long sojourn of the fish upon such bottoms. The transfer to a dark bottom resulted, without much delay, in the acquirement of a darker appearance, but this condi- tion was not, at first, a stable one, and its maintenance seemed to depend upon the continuance of visual stimuli. There are, it is true, certain facts which seem irreconcilable with this hypoth- esis, as we shall see later. But the foregoing experiments have been repeated upon two other species, and the facts themselves are beyond question. Moreover, fishes which had undergone no extended sojourn upon a pale bottom did not (with a single excep- tion) become pale when blinded. Changes having no relations to visual stimuli As stated in an earlier paragraph, decided changes occur in the disposition of the skin pigment, which have no relation to the background or, indeed, to any visual stimulus whatever. Cer- tain very conspicuous phenomena of this sort were observed ADJUSTMENT OF FLATFISHES 447 during the course of the present experiments. The fishescommonly assumed a very different appearance when disturbed or when swim- ming ‘voluntarily’ in the aquarium from that displayed when at rest.*3 Such changes generally followed certain laws, though there were abundant exceptions to these. 1. A fish of pale or medium shade generally became darker*! when disturbed, and at such times dark spots or blotches commonly came into view.®» In a few specimens highly colored red specks appearedatsuchtimes. Theresting condition was resumed in afew minutes or seconds after the fish settled upon the bottom. These phenomena were manifested even by those blinded specimens which had secondarily become pale. 2. In certain cases very dark fishes, which had recently been considerably paler, assumed a lighter hue when caused to swim around. These changes were so inconspicuous that I was no* at first certain of their reality, but their ocurrence was confirmed by observations upon at least three fishes, after transfer to the magne- tite sand. 3. When the fish was in the highly contrasted condition, with conspicuous white and black areas, this appearance commonly diminished, or even wholly disappeared, when the animal was dis- turbed. Its skin then assumed a medium shade, and the markings became inconspicuous. The same monotone was commonly assumed when these fishes swam about without known external stimulus. Indeed, in the case of certain specimens, I found it a very easy matter to discern the fish’s ‘intention’ to begin swim- ming by the disappearance of the spots and the assumption of this monotone. Thus fig. 4g was taken upon such an occasion. Upon settling down upon the bottom, the skin pattern gradually came into view, and generally attained its maximum distinctness within comparatively few seconds. The effect of these latter changes 53 Pouchet, Van Rynberk, Townsend (op. cit.), and others, have called attention to pronouced color differences, in some species, between the resting condition and conditions of activity or excitement. 54 This darkening, under the influence of disturbance, is the only change of this sort recorded by Van Rynberk, who believes it to be of constant occurrence. 5>Pouchet (op. cit., p. 76) records the appearance of such spots in the turbot. 448 FRANCIS B. SUMNER upon the observer was much like that which one experiences in watching the development of a photographic plate. Indeed it not infrequently happened, in those cases in which the maximum adaptive effect was not displayed by the fish at the time of its being disturbed, that this maximum effect appeared for a brief period after the animal settled down, only to diminish again after a few moments. 3. EXPERIMENTS UPON RHOMBUS Although Rhombus maximus was the species which first arrested my attention in the show aquarium by its extraordinary adaptation to the gravel bottom, no striking results were obtained in the laboratory from the single specimen which I used. More- over, the species was too large for convenient manipulation. Two specimens of Rhombus laevis were, however, used with some interesting results. Both of the specimens showed a high degree of adaptation to the fine gravel, used in the foregoing experiments, and one of them (the other was not tested) likewise acquired a high degree of harmony with the dark sand. Both specimens became much paler when placed upon the white marble bottom of a large aquarium, though neither attained such an extreme condition of pallor as did Rhomboidichthys. One of the two, at the end of a stay of forty-six days, ‘“‘harmonized pretty well with the now much stained marble bottom,’’ though the maximum degree of adaptation had probably been brought about long before this. Even after this extended sojourn upon the marble, however, I note of this specimen, after transfer to gravel, that ‘‘within a short time, certainly in less than an hour, the spots had come distinctly into view, and on the same afternoon the fish harmonized pretty well with the gravel.” After a short sojourn on the gravel, the corneas of this fish were rendered opaque by the application of silver nitrate, the animal being then returned to the same bottom. After the lapse of a day, the fish was very much paler than before the operation, and not far different from the condition when on marble. After two days, however, the gravel condition (7.e. the darker, spotted ADJUSTMENT OF FLATFISHES 449 condition) had, to a considerable extent, reappeared. Subsequent experiments with this fish led to the suspicion that enough light still passed through the corneas to influence the changes. After complete extirpation of the eyes, the fish, which was finally re- turned to the marble-bottomed tank, remained “rusty brown, with inconspicuous markings.”’ 4. LOPHOPSETTA MACULATA Since certain important points were left in an unsatisfactory state by the experiments at Naples, this line of work was resumed during the following summer in the laboratory of the Bureau of Fisheries at Woods Hole. The fish which was chiefly employed in these later experiments was the common ‘window-pane’ or ‘sand-dab,’ Lophopsetta maculata (Mitchill), another member of the turbot group. Lophopsetta proved to be a far less favorable object for studies of this sort, since, on a white surface, it never attained such an extreme degree of pallor as did Rhomboidich- thys, and its capacity for displaying adaptive skin patterns, though far from wanting, was much more restricted.® The experiments with this species were therefore concerned chiefly with the rela- tive influence of different portions of the visual field, the time of reaction, effect of blinding, etc. Especial attention was likewise given to the problem of how the fish is able to conform the shade of its skin to that of its background, irrespective of the degree of illumination. A few experiments with natural bottoms (gravel and sand) were also tried, but without any very striking results. In the large exhibition aquaria the adaptive reactions were, however, some- times rather impressive. Some specimens assumed a character- istic appearance upon gravel, which was decidedly different from that displayed upon sand, and the changes were sometimes fairly rapid. Upon the former material, spots, both light and dark, came into view rather conspicuously, while upon the latter, 58 Indeed, of the nine species of Pleuronectidae and Soleidae which have been observed by the writer, Rhomboidichthys podas appears to possess by far the highest capacity for adaptive changes of this sort. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 450 FRANCIS B. SUMNER a fairly homogeneous brown or buff tone was commonly assumed. The adaptations of Lophopsetta are not to be compared, however, with such appearances as those exhibited by Rhomboidichthys. The rich mosaic effect sometimes displayed by the Mediter- ranean species would seem to be structurally impossible in the American one. As a compensating advantage, from the experi- menter’s point of view, the latter may be obtained in far greater numbers. The experiments with this fish were nearly all performed in the cod-hatching boxes of the Woods Hole station. These are wooden boxes, with a bottom area of 30 x 70 centimeters, and containing water to a depth of 18 centimeters. They are built in rows of 12 each. In the course of the present investigations, they were painted variously, as will be described in connection with the separate experiments. No photographs of Lophopsetta were taken. Blotched surfaces A few experiments were tried to test the capacity of this species to adapt itself to a highly contrasted pattern. Four speci- mens were placed (two at a time) in one of the boxes just de- scribed, the walls and bottom of which had been painted white with small irregular daubs of black scattered throughout the entire surface. All of these fishes responded unmistakably to this stimulus, the ground-color becoming (or remaining) medium pale, while certain stellate or irregular black blotches came distinctly into view. The fishes thus acquired a piebald appearance, quite different from anything which was observed o. a background of uniform shade. Another interesting case was noted, in which these dark blotches appeared upon an extremely pale specimen which had been kept for three days in a box having white (unspotted) walls and bottom. It was found, in this instance, that patches of very dark vegetable debris had accumulated at the bottom of the box in the neighbor- hood of the fish’s head. Removal of this debris resulted in the dis- appearance of the spots, while a later accumulation led to their return. It must be remarked, however, that in the specimen under ADJUSTMENT OF FLATFISHES A451 consideration the dark blotches were vaguely visible much of the time, even when the fish was on a homogeneous ground. The pres- ence of dark spots in its neighborhood merely served to accentuate these. Direction of the stumulus A point which was tested much more thoroughly than at Naples was the relative influence of the bottom and of the vertical walls of the receptacle in determining the changes of shade on the part of the fish. The bottoms employed were, as just stated, 70 centi- meters long and 30 centimeters wide. The length of the fishes varied from 24 to 35 centimeters, and their area probably ranged from 200 to 400 square centimeters, or from 10 to 20 percent of the area of the bottom. The fishes lay, much of the time, near one or another wall of the box, and the larger specimens naturally could not at any time reach a position very far removed from the latter. Boxes were employed having 1, walls and bottomipainted black; 2, walls and bottom white; 3, walls black, bottom white; 4, walls white, bottom black. In addition to this, false walls of galvanized iron were made, which were painted white or black or both. These could be inserted into any of the boxes without disturbing the fishes. 1. In boxes of the first type, the fish became (or remained) as dark as it was capable of being. The shade varied, of course, with the specimen, but was usually a very dark brown, and fairly homogeneous, though certain small white spots sometimes showed distinctly. Usually the fishes were very inconspicuous in the black boxes. 2. Inthe white boxes, the fishes commonly attained a consider- able degree of pallor, assuming a shade which may perhaps best be characterized as buff, 7.e., a pale yellowish or brownish gray. In this condition, while of course far less conspicuous than previ- ously, they could not be regarded as very well concealed, at least from a nearby observer. The harmony with the pale bottom was furthered by the fact that, with the withdrawal from view of the dark pigment, not only the marginal fins (already partially transparent), but the adjacent portions of the body proper, be- 452 FRANCIS B. SUMNER came fairly translucent, so that the underlying surface was more or less visible through them. 3. In the black-walled, white-bottomed box, different fishes behaved differently, depending upon the individual peculiarities of the specimens, or upon their previous treatment. When placed here in the dark condition, most specimens remained fairly dark, even after a lapse of some days. But they were, notwithstanding, commonly affected somewhat by the white bottom, being notice- ably paler than those in an all-black box, and in some cases exhibit- ing a peculiar blotched or marbled appearance, as if attempting to adapt themselves to black and to white at the same time.*’ In one instance, a fish became nearly as pale as did the average specimen in an all-white box. Two fishes which had been placed in this box in the dark condi- tion, and which had remained dark for four days, were transferred to an all-white box. In the latter they attained nearly or quite the maximum degree of pallor within a single day. Upon being returned to the black-walled, white-bottomed box, they remained pale for two days, 7.e., as long as they were kept there. Two other specimens, which had remained dark for two days in this box, became pale in seven hours or less when the white mov- able wall was inserted. Upon removal of the latter, at the end of one day, however, the fishes promptly began to darken, and be- came nearly or quite as dark as before. Yet another two, which were put in when pale, remained so for two days. Thus, while there can be no doubt as to the influence either of the white bottom or of the black walls in these experiments, the relative importance of the horizontal and vertical surfaces seems to differ in different cases. The same is seen to be true of the next sort of box considered. 4. In the white-walled, black-bottomed box, dark fishes in every case remained fairly dark, though they were in some cases influenced by the white surfaces around them, for about half of 57 No such contrasts were here produced, however, as were shown upon the spotted bottom. (See above.) ADJUSTMENT OF FLATFISHES 453 the specimens took on a somewhat paler shade, looking asifa very thin ‘wash’ of white had been spread over their bodies.*? Pale fishes placed in this box either became nearly as dark as when kept in an all-black box (three specimens), or at least of a medium shade (one specimen). One of these specimens acquired the mottled appearance referred to above. 5. The movable walls, when painted uniformly, were used in order to change the outlook of the fish without otherwise disturb- ing it. The results obtained from their use were the same as those following the transfer of the animals from one tank to another. They need not detain us here. Highly interesting results were obtained, however, with a wall painted partly black and partly white, the line of division between the two areas being horizontal. In the first experiments the white and black portions were of equal extent, that is to say, the wall, which was 18 centimeters high, was divided into a white and a black half. Later, it was painted so that the white band occupied only a fourth of the height of the wall. The movable wall, thus painted, was used only in the white- bottomed, black-walled box. It served, therefore, to add a cer- tain amount of white to the vertical surfaces of the box. Some of the results from its use seem worth recording in detail. Two fishes (dealt with together) which had remained dark in this box, were found to become pale when an all-white movable wall was introduced, returning to the dark condition, however, when this was removed. The half-white, half-black wall was then in- serted, the white half being uppermost. No change occurred, even after 8 hours. Upon the reversal of the wall (white half now 58 Here, and inall similar cases, it was absolutely necessary to place fishes together in the same box before comparing them. The white walls, in the present case, reflected enough light upon the surface of the animal to give it a paler appearance than when in an all-black box. Accordingly, specimens from one of the latter were transferred to the present box, and a comparison made with the fishes which had been kept in this for some time. The reverse change was likewise made, both lots — being compared together in all-black tank. A mirror was also used in examining specimens kept in dark boxes, the fishes being illuminated by reflected light. Without such precautions, one may easily be led into error in judging of the less pronounced changes of shade. 454 FRANCIS B. SUMNER down) the fishes became decidedly pale within an hour. A second inversion of the wall resulted in the fishes becoming much darker (about medium shade) in 43 hours. It would seem, however, that even in this position the white surface did have some influence, since the fishes did not become very dark until the removal of the wall (leaving the vertical surfaces entirely black), when they became so within two hours. Two other specimens were, at the outset, subjected to the same tests as the preceding ones, and with substantially the same results. After several further changes of the visual stimulus, they were again subjected to the influence of the black-walled, white-bot- tomed tank, in which they now displayed a somewhat intermediate shade. The black-and-white metal wall was next put in (now one-quarter white), the white band being below. Aftertwenty minutes, one fish had acquired nearly the maximum degree of pallor, but the other had undergone no change. Two more fishes kept in this box for two days, remained dark, one being of about maximum darkness, the other somewhat paler. The black-and-white wall (one-quarter white) was now inserted, | the white band, as before, being below. After two days, both fishes had become pale, though not of maximum pallor. After removal of the wall, they remained in this condition for a day. Thus, it is interesting to note that when the bottom and the adjacent zone upon the vertical walls were white, even though this zone were no broader than 43 centimeters, the fishes reacted much (though not quite) the same is in an all-white tank» When, on the other hand, the bottom was white, and the adjacent zone upon the vertical walls was black, even the presence of an over- lying band of white, 9 centimeters wide, was not sufficient to call forth a truly pale condition in any of the four specimens thus tested. It would hence seem, at first glance, that quantitative relations alone could not determine which of these two components of the visual stimulus should prove effective. In endeavoring to decide this point, however, one must distinguish between the potential and the actual visual fields. What the fish, from a given position, ADJUSTMENT OF FLATFISHES 455 can see is not necessarily the same as what it commonly does see. It may well be that the animal’s attention is chiefly centered upon areas which do not rise much above a horizontal plane. I shall discuss this point more fully later. Rapidity of these changes The average time required by Lophopsetta to attain the highest degree of pallor, commencing with the dark state®* was probably less than two days, and the change was commonly noticeable within a single day. One particularly refractory specimen was kept for four days in a white box before any undoubted change occurred. The change, when it did come, was rather abrupt, though the highest degree of pallor was not attained until the lapse of 6 to 7 days. Specimens were found, on the other hand, which changed decidedly within a few hours, when placed for the first time in a white tank, and, in one case at least, the maximum degree of pallor was assumed in less than twenty-four hours. After the first experience of this sort, it happened, in many cases at least, that subsequent changes were undergone much more rapidly. Thus specimens which required several days for the first change to the pale shade often completed this change with- in a few hours, after one or more such transpositions. One dark fish, for example, when placed for the first time in an all-white box, showed little or no evidence of paling after one day, and did not blanch to the fullest extent until the lapse of about three days. After being returned to black, it was recorded, at the end of 19 hours, as being nearly or quite as dark as originally. When trans- ferred to the white box for a second time, the fish became decidedly paler in less than a minute, and within an hour was nearly or quite as pale as at any time previously. Whether or not a similar shortening of the reaction-time may be brought about in the case of the reverse change, 7.¢., from light to dark, was not fully determined. A change in this Freagiant is,in 59 Nearly all of the specimens, when first brought into the laboratory, were much nearer the darkest than the lightest condition. They were, too, frequently kept for some days before being used, in a large stock tank, painted black within. 456 FRANCIS B. SUMNER most cases, areturn to amore nearly normal state, and presup- poses a previous (commonly recent) change from an original dark condition. Condition in total darkness The fishes were examined at night, after three hours or more of darkness, by means of an electric flash-light. This was done on two different occasions, and with a considerable number of fishes in various conditions. With one or two possible exceptions, these fishes were of nearly or quite the same shade as when last observed in the daytime.*® Even specimens which had but recently as- sumed the pale condition were found to have retained this after the withdrawal of the visual stimulus. Certain observers have reported among fishes characteristic differences of color during ‘sleep’ or at least at night.“ I have found no evidence of such in the case of Lophopsetta. Experiments were tried in which fishes of different shades were shut up in a light-proof box. In the first of these, two specimens which had become very pale and two of maximum darkness were put into the box together. After five days the two dark ones were found to be dead. The other two, though much darker than when put in, still remained distinctly paler than those kept in neighbor- ing black boxes. They assumed the darkest condition after a few hours’ exposure to light in such a box. In the next experiment, one pale and one dark fish were kept in the light-proof box for a period of seven days. The pale specimen had previously passed 6 days in an all-white box. When the fishes were examined at the end of their stay in darkness, the dark speci- men was found to be as dark as before; the other, though now fairly dark, was distinctly paler than the former. It acquired the darkest shade, however, after a few hours’ exposure to light in a black box. . 60 In making such comparisons, I could only refer to my own notes describing the condition of these fishes in the daytime, and to my recollection of this. Differ- ences may have appeared which escaped me. 61 H. g. Verrill (American Journal of Science, 1897, p.135). Verrill’s observa- tions were made by dim gas-light, mainly between midnight and 2 a.m. ADJUSTMENT OF FLATFISHES 457 Thus it is plain that the shade assumed by the fish under the influence of visual stimuli tends to be retained for a considerable period after the latter are withdrawn.” Experiments with blinded fishes Any method of permanently destroying the sight of an animal must necessarily involve a considerable nervous shock, and it might be fairly objected, in the lack of further evidence on this point, that such results as are described below may be due, in part at least, to this shock, rather than to the loss of sight alone. Thus any mere failure to respond adaptively after the operation is not, in itself, a decisive proof that vision is a necessary element in the reaction. Such doubts are, to be sure, greatly weakened in the present instance by the fact that the blinding of one eye was found to have little or no effect upon most specimens. In order to meet fully this objection that we may have to reckon here with a ‘shock’ effect, I endeavored in the first place to use a bandage of black cloth, fastened over the eyes. It was necessary, however, to stitch this bandage to the margin of the head, and this, of course, involved an injury to the fish. Moreover, the fric- tion or pressure of the cloth soon damaged the eyes and led to blindness. Accordingly, I gave up all attempt to blind the animals without inflicting injury, and adopted the plan of cauterizing the surface of the eyes with silver nitrate. This resulted at once in an opacity of the cornea. After the lapse of a few days, the latter fell from the eye, exposing its interior to the surrounding water. Even in this condition, the'retina (or optic nerve) frequently remained for some days (7 or 8, in certain cases) decidedly sensitive to light, as was shown by reflecting daylight upon the head witha murror, or by the ing 62 For the pike, Mayerhofer (op. cit.) regards darkness as a “strong stimulus to an extreme contraction of the chromatophores,”’ since fishes which were thus kept became much paler after afew days. On the other hand, Secérov and some others report the acquirement of a deeper shade in total darkness. 68 The use of a coating of opaque material had been found to be impracticable with Rhomboidichthys. 458 FRANCIS B. SUMNER use of a flash-light at night. In many cases the eyes moved un- mistakably, or the fish even swam away, as a result of the stimulus. Altogether, 16 specimens were deprived of the sight of both eyes by cautery, while three others were blindfolded. Of this total, 8 fishes were in the dark condition at the time of the blinding; 9 were in the pale condition, and two others in an intermediate state. As regards results, the following general statements may be made: 1. Dark specimens, excepting those having the history speci- fied below (3), remained dark after the destruction of sight. 2. Pale specimens, after blinding, remained as pale as before for about a day, after which they gradually grew darker, and be- came indistinguishable from those which had been blinded when in the dark state. The duration of this peristence of the pale condition after blinding seemed to bear little relation to the length of the previous sojourn upon a pale background. Thus fishes which had been kept in white boxes for only two days before being blinded retained the pale condition about as long as specimens which had been thus kept for fourteen or seventeen days. 3. Specimens which had passed considerable periods (seventeen to twenty-five days) in a white or pale gray box, and then, before blinding, returned to black just long enough to cause them to resume the earlier dark shade (twenty-four hours, or less), became pale again within a few hours after blinding, and remained thus for about a day, after which they gradually became dark again. Three of the four specimens thus treated reverted, after blinding, to nearly or quite the maximum degree of pallor; the fourth became distinctly paler, though not so pale as it had been. The results of these experiments upon Lophopsetta are thus in complete agree- ment with those obtained from the use of Rhomboidichthys and Rhombus. On the other hand, with a single exception (see below), none of the ordinary dark specimens became paler as the immedi- ate result of blinding. 4. The shade assumed by the blinded specimens was not there- after influenced in any appreciable degree by the background. 64 One apparent exception is to be recorded among all the specimens used. This fish was of a fairly dark shade at the time of blinding. Some hours after transfer ADJUSTMENT OF FLATFISHES 459 Change from all-black to all-white boxes called forth no visible response. 5. Whatever the original shade of the fish, that which was finally assumed was, as already stated, a dark one. But the final condition was not, in the majority of cases, that of maximum dark- ness. It was frequently a shade distinctly paler than this, though in all cases one nearer to the darkest than the palest condition. Certain blinded specimens displayed a distinctly abnormal appear- ance which I never observed in an uniajured fish. On the other hand, some specimens remained very dark, and of normal appear- ance, to theend. For example, one fish (pale when blinded) was of about the maximum degree of darkness, even after forty-one days.® With six specimens, the sight of one eye only was destroyed. In three cases, this was the right eye, in three others the left. Since the two eyes are rather differently directed with reference to the bottom, I thought it worth while to look fora possible differ- ence in the effect of the two operations. Of these six specimens, four retained the power of adaptive change nearly or quite unim- paired. Indeed one of these, for rapidity and completeness of the adjustment, remained one of the most favorable specimens which I encountered. Of the two remaining fishes, one appeared to have very largely lost the power of change while in the other, this was considerably to a white receptacle, the animal was found to be very pale. It must be borne in mind, however, that the immediate result of cauterizing the eyes was not complete blindness, but that the corneas were merely rendered opaque. In this exceptional specimen the opacity might not have been complete. . & According to Pouchet, the shade assumed by a blinded turbot was always an intermediate one. Mayerhofer, experimenting upon pike, found that theimmediate effect of blinding was a paling of the fish, this being followed by the assumption of a more intensely colored condition than before the operation, accompanied by a disappearance of the dark bands. The further history of the specimen depended upon whether it was kept in the dark or in the light. If the former, the pigment tended to disappear. If the latter, the pigment cells not only persisted on the back and sides, but developed upon the (normally pale) ventral surface. This last phenomenon suggests the artificial production of pigment upon the lower side of flounders in Cunningham’s well-known experiments. A460 FRANCIS B. SUMNER impaired. In both of these cases, it was the right eye®* which had been destroyed, but I do not regard this fact as of any significance, since in the third specimen thus treated there was little or no impairment of the pigment reactions. Since a very decided inhibition of these reactions was also noticed in certain specimens which were injured in other ways, without being blinded (p. 465), it seems probable that the shock of injury and not the loss of the sight of one eye was responsible for such impairment of the chromatophore function as was ob- served in these last cases. This, of course, is reason for suspecting -a similar shock effect, perhaps an even greater one, in the case of those fishes both of whose eyes had been destroyed. Certain facts which I have recorded above may indeed be referred to this cause. But it must not be forgotten that we have, quite apart from these blinding experiments, conclusive evidence of the part played by sight in these reactions. Relation between the degree of illumination and the shade assumed In discussing the experiments upon Rhomboidichthys, I pointed out that the degree of illumination of the background had little or no effect upon the reaction which the fish underwent. As a special illustration of this principle, it was shown that a fish became paler upon a white bottom, even though this was heavily shaded, than upon a gray bottom exposed to a considerable measure of light. The latter surface, in my experiments with Rhomboid- ichthys, was shown photographically to be very much lighter than the former. Identical results were obtained with Lophopsetta. One of the boxes was painted gray of a shade close to no. 499 of Klincksieck 66 T.¢., the eye which belonged morphologically to the lower, unpigmented side of the body. Pouchet (op. cit., p. 88) had just the opposite experience, finding that one turbot whose left eye was destroyed failed to respond as well as previously, though no such impairment was observed in a specimen whose right eye was de- troyed. He suggested a possible physiological correlation between the left eye and the skin of this (the upper) side. ADJUSTMENT OF FLATFISHES 461 and Valette.*? The gray paint used was not perfectly neutral, it is true, being, when fresh, slightly tinted with blue. After exposure to sea-water, however, it changed somewhat, becoming ‘warm’ (i.e., slightly yellow) instead of ‘cold.’ It was probably in this condition during most of the period of the experiments.** The gray box was situated near the window and was well lighted throughout much of the day, though not exposed to direct sunlight. Another box was painted white within, but its interior was rather heavily shaded by a tent-shaped contrivance of gal- vanized iron. This was painted black within, and had a long cleft in the top, partly for the admission of light, partly to permit of observations. The cleft was largely closed by strips of wood, the amount and distribution of the light being thus controlled. No photographs were taken of the surfaces upon which the fishes lay, but I feel sure that the difference in the amount of light reflected from the two bottoms was as great, or greater, than in the experiment with Rhomboidichthys. Four fishes were used in the present experiment. Two speci- mens at once were kept in each of the two boxes. From time to time, those from one box were transferred to the other box, for brief periods, in order that all four might be compared directly under identical conditions of illumination and of background. Such direct comparisons were also made with other pale fishes kept in a neighboring white tank which was well lighted. At the close of the first phase of the experiment, the two speci- mens in the gray box were found to be decidedly darker than those in the shaded white box. More pigment was visible in the skins of the former than in those of the latter, and they were of a gray appearance, in contrast to the yellowish or slightly pinkish appearance of those in the white box. The latter, moreover, were 87 Prof. Yerkes, who kindly matched a sample of this paint upon a color wheel, reports that 75% of black and 25% of white gave the desired shade. 68 Tf anyone wishes to maintain that this slight element of color (aside from. shade) probably played some part in the results, I cannot absolutely refute him. I can only say that my experiments as a whole make this seem to me highly improb- able. 462 FRANCIS B. SUMNER found to show no appreciable differences of color or shade from specimens which were kept in a well lighted box, having a white interior. The two sets of fishes, which had now been in their respective boxes for six to nine days, were next transposed, 1.e., those from the gray box were transferred to the covered white one, and vice- versa. At the end of 5 days, the relations in respect to pigmenta- tion were found to be reversed, those which had formerly been darker now being paler and vice-versa. One of those which had previously been very pale was now (when on gray) recorded as “one of the best cases of resemblance in respect to general color tone which I have had.’’** Reference should be made here to one exceptional specimen which, when dark, was placed in the shaded white box for three days with little or no effect. , Upon being removed, to an wn- shaded white box, on the contrary, some change was noticeable during the same day, while on the next day the fish was very pale. In the case of this specimen, therefore, it would seem that the scant illumination of the interior of the former box had exerted an inhibitory influence.’° On the other hand, this result may have been due merely to my having dealt with a rather refractory specimen, which was on the point of changing at the time of re- moval from the shaded box, and would have done so if left there. How ts the fish able to adjust itself toa bottom of given shade, independently of the degree of illumination? As was pointed out earlier, (p. 441) it is plain that, in order that the change of shade on the part of fish should be adaptive, the latter would have to behave exactly as described. When, however, we begin to inquire as to the visual stimuli responsible 69 Indeed, a number of specimens, aside from those employed in the present experiment, harmonized quite strikingly with this and other gray bottoms which were used. This harmony was enhanced by the transparency of the fins and mar- ginal portions of the body, but was also due, in no small measure, to a disappear- ance of the yellow and brown tones and the assumption of a nearly pure gray. 70 Tt may well be that the degree of illumination at times affects the rate of ad- justment (Cf. Mayerhofer, op. cit., p. 554), but not its character. ewe ADJUSTMENT OF FLATFISHES 463 for the changes just recorded, the case becomes decidedly puzzling. For anyone with any knowledge of optics knows that gray—at least a perfectly neutral gray—is not acolor. Such a gray reflects all the components of white light in their normal proportions. It differs from white only in this, that it reflects a smaller fraction of the total quantity of light which falls upon its surface. Gray is thus relatively darker than white, but not always absolutely darker. When we ourselves judge of an object as being gray or white we make an allowance for the degree of illumination to which it is subjected, and this last is inferred from the totality of the visual field. But how about the fish? It is not in the position of an outside observer, with abundant standards of comparison at hand. This tank, with its painted surfaces, would seem to constitute for the time being its entire environment. How, then, if the walls of the shaded white box reflect absolutely less light to the animal’s eyes than do those of the brightly lighted gray box, does the crea- ture take on a lighter shade in the former than in the latter? So far as I can see, we are limited to two alternative explana- tions: either (1) the fish takes into account the degree of illumina- tion, just as we do, and makes due allowance for this in judging of the paleness or darkness of the background; or (2) it makes a direct visual comparison between its own surface and that of the background and endeavors to bring the former into harmony with the latter.7! In this second case, since the body of the fish itself is lighted or shaded to an equal extent with the background, it would have to become fully white in order to conform even to a dimly lighted white background. Let us take up the latter of the foregoing alternatives first. In order to test the question whether the fish compares its own appearance with that of its background, I have tried the expedient of concealing from the view of the animal its own skin color. For 71 Such hopelessly ‘anthropomorphic’ languagemay shock the sensibilities of the ultra-mechanistic reader. I therefore hasten to explain that no consciously rea- soned mental processes are here implied. The whole chain of events could doubt- less be stated in purely physiological terms, were we more familiar with the facts, but so, for that matter, might our own behavior. 464 FRANCIS B. SUMNER this purpose, I have employed two methods, that of staining the skin of the fish and that of covering it with a cloth, stitched along the margin of the body.” At first I made a full-length ‘swimming suit’ for the animal, but this did not seem necessary, since from the position of its eyes, only the anterior portion of the body can fall within its range of vision. Accordingly, a mask only was employed in my later experiments, apertures being made for the eyes. Now I have devoted considerable time and trouble to experi- ments of this sort. Eighteen fishes were provided with cloth masks or coverings for the body, and 8 others were stained in various ways.”* Fully satisfactory tests were, however, found to be difficult, if not, indeed, impracticable. It was, for example, very hard to cover the head completely with cloth, and at the same time permit of an unobstructed view for the eyes. In the earlier experiments, the body alone was covered, leaving the head, or most of it, exposed. The results from such are, I think, wholly in- conclusive. Stains, unless rubbed in with considerable force, were found to affect only the mucus covering the body, and to be removed with the discharge of this secretion. Moreover, all of the methods employed were open to one seri- ous objection: they injured and sooner or later killed the fish. Under such circumstances, it would be expected that disturbances of the normal reactions should occur, and such was indeed the case. A merely negative result in any instance, 7.e., the failure to respond to a given stimulus, cannot therefore be regarded as of great importance. We cannot, on the other hand, deny the sig- nificance of any positive results which were obtained, if the experi- ments were otherwise above criticism. Suppose, now, that the anterior parts of a dark flounder be covered with a white mask, and that the animal be placed in a white tank. The fish would see itself as white. According to the hypothesis we are testing, there would seem to be no reason for change. The converse experiment might be performed with a pale 72 This suggestion of covering the fish with a cloth I owe to Professor Parker. 78 Potassium permanganate and silver nitrate were the stains chiefly used. Both imparted a very dark shade to theskin. A white stain proved to be impracticable. ADJUSTMENT OF FLATFISHES 465 fish wearing a black mask and placed in a dark-walled tank. In this case, likewise, no change of pigmentation should occur, if a visual comparison between the animal’s skin and the surrounding bottom is a necessary element in the reaction. Yet such changes did occur in a considerable number of my experiments, and in several cases they occurred under such circumstances as to go far, I believe, toward refuting the hypothesis in question. In two such instances, where a white mask was employed, the fish (at first dark) did become pale upon a white bottom, and this reaction, in the case of each specimen, occurred again after a second trial. Once more, a pale fish, which had been kept for ten days on white, was stained (anterior parts only) with potassium permanga- nate. This produced a continuous dark brown mask, covering as much of the animal’s skin as it was enabled to see without bending the body. The fish, after return to the white box for a while, was later transferred to a black one. It became plainly darker in five hours and fairly dark ineight hours. Thenextdayitdied. Another specimen gave similar results, though not so well marked. It must be allowed that in none of these cases was the shade assumed as pale (or as dark), as in normal specimens, but this I believe was due to the inhibitory effect of such severe treatment. That the latter is the true explanation is rendered probable by the fact that reactions were quite as likely to be inhibited which con- formed to the requirements of the visual comparison hypothesis as were those which were contradictory to it. Thus one dark fish, which was covered with a dark mask and then transferred to white showed little or no change in the course of three days. Fairness compels the mention of a case in which the reaction (in this instance to a white bottom) was almost wholly inhibited for two days by the presence of a white mask, but occurred within the next few hours after removal of the latter. In this case, the stitches and marginal portions of the cloth were left in situ, and it cannot be said that the effects of injury had been lessened by removing the other parts of the mask. This result, which was obtained much less conclusively with two other specimens, may be held to support the view that what the animal sees of its body is a deter- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, no. 4 466 FRANCIS B. SUMNER mining factor in the reaction. I believe, however, that the inhibi- tory influence of the mask, in all these experiments, was due not only to the injury inflicted by needle and thread, but to the inter- ference of the cloth with the respiratory movements of the opercu- lum. This interference would of course cease with the removal of the overlying portion of the mask. One test of the hypothesisin question, which was made uninten- tionally with Rhomboidichthys at Naples, must be referred to at this point, for I regard it as of greater significance than all of these experiments with artificial masks and stains. Specimen no. 10, which had been kept for fifteen or sixteen days in a marble- bottomed tank, and had in consequence assumed a high degree of pallor, was transferred to the coarse dark sand used in so many of my experiments. The fish immediately buried itself with great rapidity, and remained so, with only its eyes protruding, during its entire sojourn upon this bottom. It is probable that it never emerged (in the datyime, at least) except when forced to do so by myself, and at such times it concealed itself with extreme rapid- ity. Nevertheless, after two days, this specimen was nearly as dark as the sand, and after five days it was described in my notes as harmonizing almost perfectly with this material. After another extended sojourn (twenty-six days) upon white and pale gray backgrounds, the fish was placed upon the jet-black magnetite sand. In this, it displayed the same tendency toward conceal- ment, remaining buried, except when forced to leave cover. Never- theless, the fish was quite plainly darker after the lapse of a single day, and of a very dark shade after the lapse of six days (fig. 10d), although my notes state that ‘‘when placed here, the fish seemed almost white in comparison with the sand.’”’ There surely had been little opportunity in this case for the fish to ob- serve the appearance of its own body. The foregoing experiments with these two species of fish, al- though not free from contradictions, certainly do not bear out the visual comparison hypothesis, but rather come very near to refut- ingitaltogether. Areally satisfactory test of the alternate hypoth- esis seems likewise very difficult in practice, and, although I have devoted much time to the matter, I have, at the present writing, ADJUSTMENT OF FLATFISHES 467 no experimental results of interest to offer. The essential require- ment for such a test would seem to be that the background to which the fish is to cause its own shade to conform shall be illumi- nated from a source of light independent of that which falls upon the animal’s eyes from overhead. Thus this background might be made to appear dark or the contrary, relatively to the light which seemed to illuminate it. A human observer, under similar cir- cumstances, would be deceived, and would misjudge the shade of the surface in question. Would not a fish do the same.” After considerable experimenting, I believe that I have devised an apparatus calculated to fulfil these conditions. This apparatus I will not describe here, since I am not prepared to report upon any results from its use. Owing to a lack of flounders of the right species, the experiments must be deferred until the coming sum- mer, when, I hope, it will be possible to settle the question at issue by a few decisive tests. SUMMARY AND CONCLUSIONS Flounders of several species were found to undergo marked changes in their color pattern or their general shade, when trans- ferred from one type of bottom to another. The range and char- acter of these pigment changes, and the nature of the stimuli which provoked them, were subjected to considerable experimental inquiry. The following are some of the principal facts which were revealed through these investigations: 1. Fishes became very pale (in one species extremely so) upon a white background; dark brown or nearly black upon a black one, and of various intermediate shades upon bottoms of gray, brown, ete. 2. The animals appeared to be limited in their capacity for adjustment almost wholly to black, white, brown and gray tones. Bright red or yellow backgrounds, for example, failed to call forth adaptive responses, at least during periods quite sufficient 7™4 Again the spectre of ‘anthropomorphism’ may seem to rear its head. But no. An optical illusion does not presuppose intelligent judgment (or misjudg- ment), any more than does an ordinary normal perception. 468 FRANCIS B. SUMNER for the other changes which are here described. In other words, the skin pigments which were displayed seemed to be restricted to components of the more habitual backgrounds encountered by such fishes. 3. Upon a homogeneous ground, the pigment of the skin was commonly much more uniformly distributed than upon a back- ground having a diversified appearance. 4. Upon a mixed background, such as was afforded by one of the ordinary sands or gravels of its customary habitat, the fish took on a definite color pattern, which varied with the texture of the material, and was oftentimes in striking harmony with the latter. 5. Artificial backgrounds, containing variously distributed areas of pure black and white, called forth far more contrast in the skin patterns than did the less contrasted tones of the sand and gravel. 6. The principal markings constituting these various skin patterns were found to be permanent, in the sense that they always reappeared in the same positions, and even when the animal adapted itself to a homogeneous background, the outlines of most of these spots were still distinguishable. In the case of Rhom- boidichthys podas, the arrangement of these spots was, in its essentials, constant for all members of the species. Regarding the other fishes used, I cannot speak with the same certainty. 7. The patterns assumed were consequently limited, in great degree, by fixed morphological conditions. Thus squares, cross- bands, circles, etc., were never copied in any true sense, by the fishes. 8. Within the limits thus imposed, the capacity of one of these species (Rhomboidichthys podas) to adapt itself in respect to the distribution of its skin pigments was often remarkable. For ex- ample, experiments with painted squares and circles of black and_ white showed that the resulting skin patterns depended not only upon the relative amounts of black and white in the background, but upon the degree of subdivision of the areas of the latter. As an example of this last point, when the background was divided into areas 2 millimeters square, a finer grained appearance was produced in the fish than when 1-centimeter squares were used. ADJUSTMENT OF FLATFISHES 469 9. Thus, while the adaptation was most complete upon such backgrounds as formed a part of the natural habitat of the species, it was plainly not restricted to these cases, and the pigment was at times disposed in ways which, it seems likely, were quite foreign to the previous experience either of the individual or the race. For example, the extremely pale, and perhaps also the very darkest conditions; likewise the vividly contrasted black-and- white condition, without intermediate shades, which was assumed by certain specimens upon some of the artificial backgrounds. Accordingly, the notion that the fish is limited to a few stereo- typed responses, representing the most familiar types of habitat, must be rejected at once. 10. Fishes of the same species differed greatly in their indi- vidual powers of adaptation, and some seemingly normal specimens possessed this power in a very limited degree. Again the same fish acquired with practice (if this word may be allowed) the power of changing much more rapidly than before. The time required for a radical change of shade or of pattern ranged from a fraction of a minute to several days. 11. In the case of Rhomboidichthys, the underlying surface (more strictly, that part of the bottom immediately surrounding the fish) appeared to be the one chiefly effective in calling forth these changes. The influence of the vertical walls of the vessel commonly seemed to be a subordinate one, even in cases where the fish was so large that it covered a considerable fraction of the bottom, and was obliged to lie constantly with its eyes close to one or another side of the jar. Fairly conclusive evidence was offered, however, of the influence of the vertical walls of the latter, even upon this species. What the fish saw directly overhead seemed, on the contrary, to exert a negligible influence upon the color pattern. 12. With the sand-dab, much clearer evidence was obtained of the influence of the vertical walls of the receptacle. These, at times, appeared to have an effect as great as, if not, indeed, greater than, that exerted by the bottom. It must be noted here that this difference between the two species is perhaps to be attributed to the differing positions of their eyes. Those of Rhomboidich- A70 FRANCIS B. SUMNER thys are situated at the ends of movable stalks, so that this fish must be able to obtain a much nearer view of the bottom than is possible for Lophopsetta. 13. Within very wide limits, the degree of illumination of the background was found to have little or no effect upon the shade assumed by the fish. As a special example of this principle, fishes in a white receptacle, even when the latter was heavily shaded, became paler than fishes in a gray receptacle, even when this was exposed to bright light. In such cases, the dimly lighted white bottom of the one tank was actually darker than the brightly lighted gray bottom of the other, in the sense that the former reflected an absolutely smaller amount of light to the observer— whether human or piscine—than the latter. A rather curious problem was raised by a consideration of these facts, which was dealt with at some length and was made the object of special tests. 14. A specimen of Rhomboidichthys which was transferred while extremely pale to black sand, acquired a very dark shade, even though the fish remained persistently buried in this material, with only its eyes protruding. Again, specimens of Lophopsetta having their skin deeply stained, or wearing masks of cloth, were found, in some cases, to undergo pronounced adaptive changes, despite the fact that their body surface was disguised in this way. It is thus rendered highly improbable that any direct visual com- parison on the part of the fish between its own body surface and the surrounding background is an essential factor in the pro-. duction of these changes. 15. Fishes (Rhomboidichthys), when given the choice of two backgrounds, displayed no preference for the one which conformed more nearly to their own shade at the time. Likewise, specimens which were glaringly out of harmony with a given shade of sand appeared no more likely to conceal themselves beneath its sur- face than when their skin color was adjusted very closely to this. 16. When examined at night, after several hours of complete darkness, the fishes (Lophopsetta) were found to be in the same condition of pigmentation as when previously observed by day- light. Pale specimens, which were kept 5 to 7 days in a black- painted, light-proof box, became considerably darker during this ADJUSTMENT OF FLATFISHES 471 period, though remaining distinctly paler than dark control speci- mens with which they were compared. They acquired the same shade as the latter, however, after a few hours’ exposure to light in the same box. 17. Experiments with fishes which had been deprived of their sight confirmed the findings of earlier investigators that the unim- paired functioning of at least one eye is necessary for the adjust- ment of the animal to its background. If blinded when in the dark condition, the fishes ordinarily remained dark, though they did not always permanently retain the darkest shade which is displayed by a normal specimen. If blinded when pale, they re- mained pale for about a day, but reverted to a darker condition, representing more nearly the resting state of the chromatophores. An interesting special case was discussed of fishes which had been adapted for a considerable period to a pale background, and after- wards for a brief period to a dark background. These reverted to the pale condition after blinding, though this later gave place once more to the dark state. 18. Destruction of the sight of one eye (whether the left or the right) had little or no effect upon the chromatic reactions of the majority of specimens of Lophopsetta. 19. Tactile stimuli, if effective at all, certainly played a quite subordinate part in evoking color changes of an adaptive nature, for the fishes responded as promptly to patterns painted upon the under side of strips of glass as to bottoms of stones and gravel whose complexity could be discerned by touch as well as by sight. 20. Very decided changes in the markings, as well as the general color-tone of the body were at times called forth by tactile or other non-visual stimuli, and the fish, when swimming, commonly presented a decidedly different aspect from that shown in the rest- ing condition. But such changes as these belong to quite a differ- ent class from those which form the chief subject of the present paper. Certain of the facts above summarized, deserve further dis- cussion than was devoted to them in the body of the text. 472 FRANCIS B. SUMNER We have seen that the skin of some of these fishes commonly assumes a nearly homogeneous tone upon a bottom of uniform color and shade, while presenting a more or less pronounced pattern upon a bottom of diversified appearance. Abbot Thayer?® has pointed out that the breaking up of a uniform color tone by markings of any sort makes for concealment, and this is particu- larly true against a diversified background. This principle, without question, accounts for much of the effectiveness of the various patterns assumed by Rhomboidichthys and other floun- ders, and we must not be in too great haste to point out specific resemblances to particular backgrounds, merely because the fish ceases to be conspicuous upon these. I think, however, that a careful consideration of the experiments as a whole, and partic- ularly of the facts referred to in paragraph 8 of the summary, forces us to the belief that there may be very specific relations between the distribution of light and shade in the background and the pigment pattern assumed by the fish. Had we to do here merely with a general paling or darkening of the entire body surface, affecting spots and ground color to an equal extent, or even were there at the disposal of the fish one of two of these pigment patterns, corresponding to certain of the most frequent types of bottom, we might ascribe this power to a few comparatively simple reflexes. But we have seen that the responses are far from being as stereotyped as this. Certain areas become paler and others become darker, each more or less inde- pendently, and in varying degrees, depending upon the cireum- stances. At one time we have a large dark blotch covering a given portion of the surface; at another time, the pigment of this blotch has practically disappeared from view; at another yet this area has become broken up and diversified by the appearance of paler specks within it. Most of this change, too, is brought about by variations in the conspicuousness of groups of pigment cells — * Popular Science Monthly, December, 1909; also book by Gerald Thayer entitled ‘‘Concealing Coloration in the Animal Kingdom. A Summary of Abbot H. Thayer’s Discoveries,’’ N. Y., 1909. It is likely that few biologists can follow Mr. Thayer in the unbridled zeal with which he strives to universalize this and the other important principles of animal coloration which he has discovered. ADJUSTMENT OF FLATFISHES 473 which never wholly fade from view. The pale specks which serve to ‘“‘stipple”’ the dark blotches and give to them a fine-grained appearance (fig. 4a) may, in large part, be distinguished in the nearly solid blotches of the coarser pattern (fig. 4b).76 When we add to this complexity, the additional complexity due to differences of color proper (as distinguished from shade), it is difficult indeed to conceive of a nervous mechanism competent to bring about such changes. But conceivability is surely a poor criterion of possibility in biology, and we cannot see that a non- mechanical (7.e.,vitalistic) interpretation of these phenomena would help us in the least. For, on the sensory and motor sides, this baffling complexity of mechanism would have to be granted in any case, and the only thing which the vitalist could do would be to posit a non-mechanical coordinating agency, which adapted the means to the end. But this, as has so often been pointed out, is a merely formal solution of the difficulty, and one totally impotent as a principle of scientific explanation. That the stimuli which call forth these changes are visual rather than tactile has been shown, in my experiments, by the use of perfectly smooth glass plates, having the pattern painted upon the lower surface. That these stimuli are received through the eyes, rather than through the skin is, of course, not wholly proved by destroying the animal’s sight, since the objection may always be raised that we have to do with inhibition through shock. On the other hand, the recent experiments of Parker’? show pretty con- clusively that the skin of at least some marine fishes is insensitive to light, even when the latter is of very high intensity. Were it proved, however, that such a general sensitiveness to light and shade was highly developed in the skin, it is impossible to see how re- sponses to a pattern could be brought about through any organs except the eyes, for these alone are provided with the lenses neces- sary for the production of images. 78 As stated earlier (p. 416), the chromatophores themselves are probably dis- tributed with much greater uniformity than the complexity of pattern would at first lead us to suppose. The position of the spots—actual and potential—may be largely determined by the position of the nerve termini. 77 American Journal of Physiology, October, 1909. Fishes of nine species were used in Parker’s experiments. 474 FRANCIS B. SUMNER But aside from the evidence which they afford of the réle played by the eyes in these changes of color, the blinding experiments seem to show that vision is necessary in order that the pigment cells shall remain in a given state of tonus, exception being made to the case of those fishes which are blinded in a uniformly dark state, representing most nearly the resting condition of the chromato- tophores. Continued adaptation to a iess usual background, €.g., a very pale one, may result in the new condition becoming more or less fixed. The latter may persist for a time after loss of sight, but the more habitual state of tonus finally reasserts itself. The cases mentioned at the close of section 17 of the summary might be explained by supposing that the pale condition had become in considerable measure fixed, so as to reappear after the stimuli responsible for the secondary dark condition had been withdrawn by destruction of the sight. The ultimate return to the dark state would be intelligible here as in the case of fishes which are blinded when pale. But if the foregoing interpretation is correct, it is hard to understand why any unusual state of the chromatophores which has but recently been acquired should not give place to the more habitual condition as soon as the light of day is withdrawn (e.g., at night). But this was found not to be the case. Here, as so often happens, the simple and obvious explanation does not seem to contain the whole truth. Evidence has been offered which seems to show conclusively that the plane in which a given surface lies with relation to the fish determines in some cases, whether or not it shall be effective in calling forth a given change. It was not made certain, however, that even in such cases, the matter was not decided by purely quantitative relations within the visual field. For, as was pointed out, we must distinguish between the potential and the actual visual fields. That the horizontal surface lying immedi- ately about the fish is the one which is generally most potent in determining the reactions of Rhomboidichthys, might be due entirely to the fact that the animal’s gaze is commonly turned in this direction. In experiments upon Lophopsetta, we found (p. 454) that when the bottom, plus the wpper half of the vertical walls, were white, while the lower half of these walls was black, ADJUSTMENT OF FLATFISHES 475 the dark fishes which were used did not turn pale. On the other hand, the white bottom, plus the white lower half, or even lower fourth were found to call forth this change. These facts, which at first thought would seem to indicate the operation of some other factor than the relative amounts of black and white, do not in themselves force us to such a conclusion. If we assume that the animal’s field of attention (due to the position of the eyes or other- wise) extends but little above the horizontal plane, the facts may, indeed, be explained on a purely quantitative basis. But this is probably not the whole truth. For it seems to follow from the considerations offered below that differences in the direction of different portions of the visual field probably condition the reac- tions of the fish in another important respect. This problem is more clearly allied than might at first be supposed to another one which has received considerable attention in the present paper. I refer to the modus operandi of the stimuli which lead the fish to become very pale on a white surface and ‘gray upon a gray surface, irrespective of the degree of illumina- tion.’”* As I have already pointed out (probably quite needlessly ), a surface of pure gray reflects white light, with no alteration except a diminution of its intensity. A human observer distinguishes a given object as gray, rather than white, only by reference to the degree of illumination to which it is subjected, and this last he infers from the appearance of the remainder of the visual field. Suppose, for example, that our visual field should for the moment consist of a single uniform surface, of which we had no prior knowledge, illuminated by a light of unknown intensity. Under such circumstances, we should be at a loss to say whether the sur- face was gray or white.7’> Once we have an idea of the degree of illumination, however, and we make the necessary correction for this, as, for example, when we view a piece of white paper in the twilight, we commonly pronounce it to be white, despite the abso- 78 The reader, if interested in this part of the discussion, is advised to refer directly to the treatment of this question in the body of the text, particularly pp. 440- 443 and 460-467. It would be impossible, without much undesirable repetition, for me to restate the entire problem here. 79 Colors, of course, would still be distinguished. 476 FRANCIS B. SUMNER lutely small amount of light reflected from it. In the same way, the interiors of the white vessels used in my experiments seemed to the outside observer*° to be white, and those of the gray vessels to be gray, despite the fact that the latter actually appeared far lighter in a photograph. Are the perceptions of the fish similarly determined? How can such a thing be possible, in cases where the uniform walls of the receptacle constitute practically the entire visual field of the ani- mal? Without any outside standard of comparison, how can a heavily shaded surface of white appear paler than a brightly lighted surface of gray ? We have seen that one simple solution of this difficulty would be to assume that the fish makes a direct visual comparison be- tween its own body surface and the bottom on which it lies. If the former is adjusted to the latter, the absolute degree of illumi- nation which is common to the two is a matter of no possible consequence, for the object of this adjustment is the concealment of the animal. This hypothesis was, however, rejected, in view of pretty conclusive experimental ce" ‘ence. Furthermore, it does not accord well with the fact (p. 443) that the behavior of the fish does not seem to be influenced in other ways by the color- phase in which the animal happens to be. What, then, is the standard of comparison by which the fish (or its unconscious nervous mechanism, if the reader prefers) deter- mines the shade to which the skin is to conform? In other words, if, as has been demonstrated, the absolute amount of light reflected from the background is not the only factor in the effective stimulus, what other one is there? As just stated, the human observer would decide the point by reference to other ele- ments of the visual field. From the fish’s point of view, the only other element of the visual field, besides the bottom and walls of its tank, is the illuminated area overhead, representing the source of light—commonly sunlight reflected from objects outside the tank. May not, then, the ratio between the light reflected from the near- 80 At least they did soto me. It is quite possible that one who did not appreciate the density of the shadow might have judged otherwise. ADJUSTMENT OF FLATFISHES 477 by surfaces within the tank and the light which enters the latter from above be that factor of the total stimulus which renders pos- sible these accurate adjustments of the shade of the fish’s body to that of its background?*! I think that this is the true solution ot the problem, and I hope, with apparatus already constructed, to put the question to experimental test, as soon as material is available. That such a relation between the light intensities of two por- tions of the visual field may form an integral part of the immediate perception,without the necessity of rational mental processes, I think will be granted by all. Now naturally the fish knows noth- ing of the distinction between the source of the light and that part of the environment which is illuminated by it. There is for the animal but one continuous visual field, though this may not all be apprehended at once. The latter is constituted by various areas, differing in luminosity or in color. Those portions of this field which lie below, or but little above, a horizontal plane passing through the animal itself are the ones to which the appearance of the latter is adjusted. It is these which, as already seen, probably occupy the focus of attention most of the time. Those portions which lie more nearly overhead, and thus ordinarily beyond the focus of attention, must, however, serve in some way as a criterion by which the shade of the rest of the visual field isapprehended. With a given amount of light from outside the tank, a greater or a less amount of reflected light from the bottom would of course imply a lighter or a darker shade in the latter. On the other hand, with a given (absolute) amount of light reflected from the bottom, the occurrence of a low degree of illumination overhead would lead the animal to attribute a paler shade to this bottom (7.e., to see it as paler) than if the source of light were a brilliant one. 81 These words, and in fact my entire discussion of this problem, down to the end of the next paragraph, were written before I had any knowledge of the almost identical hypothesis which was put forward some years ago by Keeble and Gamble (Phil. Trans. Roy. Soc., Series B, vol. 196, 1904). Under these circumstances, such a verbal coincidence as isto be noted in comparing their statement with mine is rather surprising. On p. 358, these authors state: ““. . . on the white and black grounds the animal . . . appeals for pigment-guidance to the amount of light scattered or absorbed from the ground; or, as we put it previously, it is a : SeGirectose: reaction to the ratio— —— light.” reflected 478 FRANCIS B. SUMNER Experiment alone (see p. 467) can place beyond question the accuracy of this interpretation. What is needed especially is a satisfactory determination of just which elements of the visual field it is to which the animal conforms its own appearance, and which ones it is that serve as a criterion by which the shade of the background is apprehended. So far as I cansee, differences of direction from the animal’s eyes are the only ones which can be invoked in differentiating these two sets of stimuli, and Keeble and Gamble (whose treatment of this problem was unknown to me when the foregoing discussion was written*? incline to the same opinion. They hold (p. 354) that ‘“‘in some way, the eye differen- tiates between the direct and the irregularly scattered light,in other words, it displays a certain dorsi-ventrality.’”? Under ordi- nary conditions, the background is below and the source of light above. But the authors find that, if the conditions of illumination be artificially reversed, the ‘‘background”’ being above the animal and the light entering from below, the reaction to the former is the same as when it lies beneath them. Thus, they hold, ‘‘the dorsi- ventrality is probably not due to apermanent structural difference in the two sides of the eye.’ It is not clear, however, from their account of this experiment, that the conditions of illumination were not complicated by total reflection from the bottom of thejar. Unless the animals looked directly downward, or at least within a certain angle with the bottom, they would see, not a brightly lighted field below them, but the reflections of objects in the upper portions of the tank (pp. 427,428 of the present paper).** 82 See foot-note 81 88 T cannot feel quite sure that the experiment of Bauer (Centralblatt fiir Physi- ologie, 1906), in which he used electric lights, placed above and below the glass container, is not open to the same objection. From this experiment and others, Bauer concluded that the assumption of a dark shade by Idotea was determined by “‘Simultankontrast,’’ irrespective of the position of the contrasting portions of the visual field. This is certainly not true of fishes, as is shown by my ex- periments. (See particularly p. 451 et seq.) The experiments of Mayerhofer, likewise, (op. cit., pp. 553, 554) in which a mirror was placed below the glass container, inclined at an angle of 45°, appear to me to be inconclusive, owing to the same apparent technical defect; and it is significant in this connection to note that fishes lighted from below, in this way, assumed the same shade as those kept in total darkness. ADJUSTMENT OF FLATFISHES 479 A word in regard to the utility of this power of color change in the life of the organism. Despite the recent reaction against extravagant applications of the protective coloration principle, it is difficult to doubt, in the present instance, either that this faculty has some use, or that it has been developed in some way because of its use. The end to be attained seems to be concealment and nothing else. No appeal to thermal regulation,* to possible ‘“‘photoreceptive’ or ‘‘photosynthetic’ functions of the skin pigments, nor any other purely physiological explanation of the phenomena seems adequate. A complete explanation must regard ecological factors as well. Whether the utility of these changes to the fish consists primarily in their concealing the latter from its enemies or from its prey cannot, however, be stated with- out a greater familiarity with the bionomics of these species than the present writer possesses. I learn from several trustworthy observers that flounders of various kinds are preyed upon by sharks and other large fishes. The only information which I have relating directly to the enemies of any of the species which have been discussed in this paper, is the statement of Mr. Vinal Ed- wards that he has taken sand-dabs, along with other flounders, from the stomach of the cod. It is quite probable a prior that all of the species are similarly preyed upon. As regard the prey of these fishes, I can but offer my own obser- vation that specimens of Lophopsetta, when recently brought into the laboratory, frequently regurgitated the ‘sand-launce’ (Ammo- dytes americanus), sometimes in considerable numbers. It is not unlikely, therefore, that the cryptic coloration of flounders is of advantage in concealing them from smaller fishes* until the latter come within easy range. These few meagre statements of course illustrate the paucity of our direct evidence upon the whole question of the utility of cryp- tic coloration, and indicate the inferential nature of most of our conclusions in this field. February 23, 1911. 84 As suggested by Max Weber et al. (Van Rynberk, op. cit., p. 568 et seq.) 85 Invertebrate food may perhaps be left out of consideration here. EXPLANATION OF PLATES The photographs, with the exception of le (by Dr. Victor Bauer), were all taken by the author. The water-color sketches (plate 6) are the work of Mr. V. Serino, an artist in the employ of the Naples Station. The figures all relate to a single species, Rhomboidichthys podas (Delaroche), and all are reduced to approximately one half the natural size. Figures bearing the same number represent different views of the same fish. These are arranged with a view to easy comparison. Thus the order in which the views of a single specimen are presented does not necessarily bear any relation to the order in which the corresponding changes were undergone in the course of the experiments. For an account of the photographic methods employed see pp. 418-421 of the text. PLATE 1 EXPLANATION OF FIGURES Views of specimen no. 1: a, on a dark, mixed sand (after one day); 6, on fine gravel (after one day); c, on fine jet-black (magnetite) sand (after four days); d, on a very coarse reddish sand (after eight days); e, on a coarse gravel, devoid of sand (after two days). ADJUSTMENT OF FLATFISHES PLATE 1 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10,NO.4 481 PLATE 2 EXPLANATION OF FIGURES Views of specimen no. 1: f, on painted squares, 2 cm. sq. (after four days); g, do., 43 cm. sq. (after seven days); h, do., 1 em. sq. (after four days); 7, do., 2mm. sq. (after one day). 482 ADJUSTMENT OF FLATFISHES PLATE 2 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 483 PLATE 3 EXPLANATION OF FIGURES Views of specimen no. 1:j, after three days on background figured; k, after four- teen days on white marble bottom (the fish was in reality much paler than the pho- tograph would seem to indicate); J, after three days on the background figured; m, after six days on the background figured. 484 oO PLATE 3 ADJUSTMENT OF FLATFISHES SUMNER FRANCIS B. feoveee 00000 0 00 eee 6 @e08ee0e060828 8 OF @ 0 O %.% —,eeCevee of Cee eo; bs ist, @ @ e@: Beet ha be toe & @6@ . @ Be og ot ewer ®@@x Hee eso POC ee Ceo tecor ©©8 e808 08080008 O22 »© 000 ele oes cer. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No.4 485 PLATE 4 EXPLANATION OF FIGURES Views of specimen no. 2: a, on dark mixed sand, partly covered with this material (after two days); b, on white glass plate, shortly after transfer to this following sojourn on dark sand; c, on coarse gravel (after two days); d, on white glass plate, shortly after transfer to this, following sojourn on coarse gravel. (Compare this withb. Ind, the gravel pattern has persisted to some degree, despite an immediate partial disappearance of this). 486 ADJUSTMENT OF FLATFISHES PLATE 4 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 4) i PLATE 5 EXPLANATION OF FIGURES Views of specimen no. 2: e, upon fine gravel (after two or three days); f, upon dark, mixed sand (after two days). Views of specimen no. 3: a, after one day on present ground; b, taken on the day when first placed on this sand. (Note the inferior power of adaptation shown by this fish, as compared with the last. The harmony with the backgrounds in- creased little if any beyond the condition shown in the photographs). 488 5 PLATE HES STMENT OF FLATFIS + } ADJU SUMNER FRANCIS B. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 489 PLATE 6 EXPLANATION OF FIGURES Views of specimen no. 3, in the ‘sand’ and ‘gravel’ phases. Unfortunately, the capacity of this specimen for such adaptations proved to be comparatively small. 490 ADJUSTMENT OF FLATFISHES PLATE 6 FRANCIS B. SUMNER V. Serino, del. THE JOURNAL OF HXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 491 PLATH 7 EXPLANATION OF FIGURES Views of specimen no. 4: a, after six days on pattern shown; 6, after three days on pattern shown (compare minutely with last); c, after nine days on the dark sand; d, after three days on the pattern shown; e, after three days on the pattern shown. oLATE ADJUSTMENT OF FLATFISHES FRANCIS B. SUMNER wevwewevwsegeuwgegeuweweivw~ A ee lo ceeue © 06.6. eeee8e ee @ YH a 2. 2 a. «2 @& rs mK THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10,No.4 493 PLATE 8 EXPLANATION OF FIGURES Views of specimen no. 4: f, after seven days on present background (condition of rest); g, same, when preparing to swim; h, after one day on present background; 7, after three days on a background of gray, of a shade approximately matching that produced on a color-wheel by the use of two parts of black and one of white. (The harmony between the fish and the bottom was really much greater than would seem to be the case from this photograph, which has made the fish seem darker). Specimen no. 5: view inserted to show a particularly striking gravel pattern (taken after eight days). (There are in reality a few particles of gravel on the back of the fish). 494 ADJUSTMENT OF FLATFISHES PLATE 8 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No.4 495 PLATE'9 EXPLANATION OF FIGURES Views of specimen no. 6: a, upon dark, mixed sand (after one day); 6, upon the same sand, with the addition of white pebbles (after three days); c, on coarse, reddish sand (after six days). Specimen ar showing conspicuously mottled appearance, unusual in a speci- men just brought to the laboratory. ADJUSTMENT OF FLATFISHES PLATE 9 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 497 PLATE 10 EXPLANATION OF FIGURES Views of specimen no. 8: a, after four days on material shown; b, in jar having black bottom, and transparent walls, surrounded by gravel (after seven days) ; c, in jar having black walls and transparent bottom, with gravel underneath (after ten days); d, taken on a black bottom, immediately after transfer from a white- bottomed, black-walled jar, in which the fish had remained 9 days. 498 10 v) PLATE NT OF FLATFISHES u ADJUSTME SUMNER FRANCIS B. ae Se a eeshay THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 499 PLATE 11 EXPLANATION OF FIGURES Views of specimen no. 9: a, on dark, mixed sand (after two days); b, in jar having black walls and gravel bottom (after two days); c, in Jar having white walls and black bottom (after five days); d, in jar having black walls and black bottom (after one day); (Comparison between the last two shows plainly the effect of the white walls in the former case). 500 PLATE 11 OF FLATFISHES FRANCIS B. ADJUSTMENT ( SUMNEEF THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 501 PLATE 12 VIEWS OF SPECIMENS 10 AND I1 10 a After fourteen days on white marble bottom. 11 a After fifteen days on white marble bottom. (Both of these fishes appear much too dark). 11 b Showing possible effects of black specks in the white field. 10 b-llc Showing condition of former fish, after thirteen days on the dark sand as compared with the latter, which had just been transferred to this. 10 c-1l d Taken on gray bottom (gray of the same shade as in 47). 10 d_ After six days on jet-black (magnetite) sand, following long sojourn on white and gray bottoms. 10e After blinding, during recently acquired dark condition, following long sojourn on pale bottoms. The result is a return to the pale phase. 11 e After eleven days on jet-black sand 502 ADJUSTMENT OF FLATFISHES PLATE 12 FRANCIS B. SUMNER THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 503 PLATE 13 VIEWS OF SPECIMENS 11 AND 12 12 a Against background of gray (same gray as in 47), immediately after trans- fer from dark sand. 11 f-12 6 Showing conditions of illumination in the white and gray jars, used in the experiment described on pp. 440-443. The bottom of the white jar appears much darker than that of the gray Jar. 11 g-12 ec Showing condition of these two fishes (photographed together on gray bottom), after former had been kept four days on the (lighted) gray bottom and the latter four days on the (shaded) white bottom. 11 h-12d Appearance of the same two fishes, four days after the reversal of the above conditions (11 being in white jar, 12 in gray). (The reader must not be mis- led by the altered positions of the two fishes in the later picture. The identity of each specimen is revealed by its size, no. 12 being the larger). 504 ADJUSTMENT OF FLATFISHE: PLATE 13 FRANCIS B. SUMNER | } nee ato Retr: THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 50 STUDIES ON THE PERMEABILITY OF CELLS EDMUND NEWTON HARVEY From the Zoological Laboratory, Columbia University THREE FIGURES CONTENTS irarters Cy CHAT Cts] Tabs ee essere epee Sore tn: SOA ec sis: sce a cr Pe ata en 508 Ul, LEI Aor | aed PS eo are eg oo eo ae OED MIN Airic etc icee ian arn Sc kre Eon Bam Us Jeune OnresCOncermMmespenmedpilitve +1... + seeeeene eee aie ei 509 Be IMietlavorcks @ie esis foramen ovlbhive a ceecbecgoecdcscupaedo spc enanmessaoe 512 MHeMMeMetratt omy Olam erdyESue ns. cael s ciceaeuneraehe Seam el neha cht nine ie 514 ie VMiechanismiotstaminesplanticellss-2..5-- neater eer ae eels 514 ay (ONAGIEUONAY/ EFI ORIG OO) AVN SIS Sn eee EP ROE 5 copra ae crib ao Bro dian o lowour oats 514 Loy MT Kova less ae 5 Powe ee ee he etn A Ens I. ales oasa yO aerate aes 515 G "Wineovey Ort machvoenordsh joy bolls Sauecs bese oneno css dcucnoudagoooue une 516 Gee NCtOUNOL tHe ACIG\iS ON the Gy€.. 2... ..25 o4 sae ponent doe ees sees « 516 @ SVBTENGIES TOE J OS ee eve i eS 517 Pelvelanion: to.Overton’s lipoid theory .:.222saeqsescdss- ses delays os See 519 Se wechanismion staining animal cells....05: ..daats.s< co: cones wee te eee es 522 Sm Le RT AREAS TCUNTRUIP Oy 5. PEC pees | aA a cs RR ROR ie Beton Coat ena he os 522 (3), INU RIINS ERRCRS ete a fale neo RP ere dee Nac ce se ey Rae eee 524 Whegnenctranioneotmallkaliesver eo. oc)... os. be ene a eae Nee oats arched 525 ee ViethodeandemnrevasOUSULESULGS:. ... s.i1s osteo iaeuee eirereresbais eng trie sccnie gen 525 2. Objections to the use of an indicator within a cell.................... 527 See peTMenmts waitin plamb.Gells: .......<+/.05aeeec ei eee seen ys nee sew atens O80 Ce Shon ClyaCiscsOClategealkKallGs:,,..4> a2: saree ccece ites © ama c aac ae 530 Dra Wiedklysdissociatedealialies....... «22 sfaestmsemse oes etek yates seek - 532 c Permeability of cells exhibiting protoplasmic rotation............ 536 d The concentration of alkali which stops rotation................... 537 e The effect of added substances on the penetration of NaOH and KOH 538 AP XpPeLIMentsmwitieanumal «cells: . + 12. csi aie ince ee altel: 541 CEPT GATING CHUTES ae teme re io ohn Sa caltd cc ne eyes Gh Sens 541 Die eri ere oc ieee re pee le 2. crt aos eee Stes JASN oc ag eee ee 547 JD WSCCUNSSL VOLES Ws 9 Re one Sa CEP 0E coc bt ee 551 SUN CATTA A OM-TRESIOU NBS en Gb. a ecg oe eee RRR TE Ore re SCL Oe or Rien epee eer 554 FSi inereerssy Lip geed pao es MME PRI es. 3 5, Sasha gs MO NSN dd tees han pe ewakaaded are 556 508 EDMUND NEWTON HARVEY INTRODUCTION 1. Historical Within the past fifty years a great deal of scientific research has been directed toward advancing our knowledge of the man- ner in which substances may pass into living cells and of the classes of substances which may or may not enter. Intimately connected with the question of the permeability of cells are the phenomena arising from the existence of an osmotic pressure without and within the cell, and the rigidity or turgor of plant parts directly dependent on the latter. This relation is most evident when we recall that the existence of a continual turgor of plant structures depends on the possession, by the individual cells, of a surface membrane which prevents the diffusion out- ward of most of the substances dissolved in their sap vacuoles. Questions concerning osmotic pressure must therefore always go hand in hand with questions concerning the permeability of the membrane whose presence is one condition for the exist- ence of that pressure. Historically the two subjects have de- veloped simultaneously. It is hardly within the scope of this paper to give a detailed account of the history of this complicated subject, so numerous are the papers dealing with its different phases. The funda- mental facts were outlined by three observers during the latter half of the last century. Their influence has been so great that I shall mention briefly the contribution of each. Nageli (55) first investigated the osmotic properties of the cell and made clear the cause of turgor and of plasmolysis. The word plasmolysis was introduced later by DeVries. It is to Pfeffer (77, ’86, 90) and DeVries, (’71, ’77, ’84, ’85) however, that we owe our present conception of the important réle played by diffusion and osmotic pressure. Both of these authors in- vestigated the magnitude of the pressures existing in plant cells and the properties of the cell membranes. Pfeffer has especially emphasized the conditions under which accumulation of sub- stances takes place; DeVries the importance of the vacuolar THE PERMEABILITY OF CELLS 509 membrane, designated by him the tonoplast. The generaliza- tions made by these three botanists, in which the discovery of semipermeable precipitation membranes by M. Traube (’67) has played a most important part, have been extended and con- firmed by a host of recent experimenters, Overton, Hoeber, Nathanson, Ruhland, Hamburger, R. Lillie, Koeppe, Gryns, Hedin, Asher, J. Loeb, and many others. Although most studies on permeability have been carried out on plant cells, the same essential relations are exhibited by animal cells. 2. Theories concerning permeability The more recent studies, especially those of Overton (’95, ’97, 99, ’00), have been concerned with the classes of substances which may or may not pass the plasma membrane. This at once raises two important questions. 1. How does a substance enter? 2. What is the nature of the cell surface or plasma mem- brane? . In answer to each of these questions several theories have been advanced. Let us consider first the nature of the plasma mem- brane. Quincke (’88), in order to account for the power of movement of amoeboid cells as well as certain peculiar osmotic properties, assumed that a thin film of oil was present at the surface. Over- ton has explained the very rapid entrance of ether and fat- soluble substances by assuming that the plasma membrane is composed largely of lipoids like lecithin. This view was later modified to explain the entrance of substances insoluble in lipoids by regarding the cell surface as a mosaic of proteid plus choles- terin (Nathanson, ’04, a) or proteid plus lecithin. On the other hand Pfeffer has always insisted that the membrane is chiefly proteid, while Robertson (’08) considers it a form of modified protem comparable to that which remains about droplets of chloroform shaken up with protein solutions and then washed repeatedly in water. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 510 EDMUND NEWTON HARVEY Three general theories, based on Van’t Hoff’s view of the driv- ing power in osmosis, have been advanced to account for the passage of substances through membranes :—the filter theory, the solution theory, and the chemical combination theory. The filter theory regards a membrane as a molecular sieve. Whether it is permeable to a given substance will depend on the relative sizes of the molecules of the substance and the interstices of the membrane. A study of artificial precipitation membranes has afforded considerable evidence against such a simple explana- tion. According to the second theory a substance must dissolve in the membrane in order to pass through, and according to the last theory the substance must combine with the membrane before it may pass. J. Traube, (’04, 08, ’09, 710) on the other hand, regards the driving force in osmosis, as an ‘Oberflichendruck,’ later called ‘Haftdruck,’ measured by the tendency of the substance to in- crease or decrease the surface tension of the solvent. ‘The mem- brane separating two phases is not the important thing but the difference in surface tensions, the phase of lowest surface tension tending to pass through the membrane into that of greater sur- face tension. Traube admits that the Haftdruck of the mem- brane may also be a determining factor (710, p. 533). At present there are two general views as to the classes of sub- stances which may diffuse into cells. These are based largely on conceptions regarding the nature of the cell membrane and the physical or chemical process by which a substance may pass it. The results obtained by different methods of investigation and their interpretation have been in many cases conflicting. Inasmuch as my studies on the permeability of cells show in the clearest manner the existence of two distinct classes of alkalies with respect to their ability to enter, a brief statement of the opposed views may not be out of place. Overton and Hoeber classify substances into lipoid-soluble and lipoid-insoluble. The former are found to enter cells very rapidly, the latter not at all when tested by the plasmolytic. method (p. 512). Yet the lipoid-insoluble substances are just those which we know by analysis and microchemical tests to THE PERMEABILITY OF CELLS ali occur in cells (inorganic salts, sugars, ete.). Further, the en- trance of KNO; can be demonstrated by the diphenylamine reaction, while the cell remains plasmolysed. Consequently Overton and Hoeber assumed that cells have a physical and a physiological permeability. The lipoid soluble substances enter in a purely physical manner, by simple diffusion, involving solu- tion in the lipoids of which the plasma membrane is assumed to be composed. The lipoid-insoluble substances enter in some other way, but not by diffusion. As evidence of this Overton calls attention to the fact that the neutral salts, sugars, etc., are just the substances which are known to pass into cells or through cells from regions of lower to regions of higher concentration without chemical change. Some other factor than diffusion must be involved.t. Hoeber (’09) now admits that the lipoid theory does not hold for dyes and that it must undergo more or less re- modelling (p. 78). On the other hand, Overton’s opponents draw no distinction between lipoid-soluble and lipoid-insoluble substances, but re- gard the entrance of any solute as a process of simple diffusion, (without considering how the substance passes the membrane), the only difference lying in the relative rates of diffusion. The presence of salts in cells in different proportions from the medium is explained as due to combination with proteids in the cell. Indeed, Moore and Roaf (’07) have gene so far as to deny any importance to the existence of asurface membrane in regulating the entrance of salts into red blood corpuscles, but regard the difference in salt content between cell and medium as entirely due to formation of salt proteid compounds. A great deal of confusion has arisen from the fact that a cell may change in permeability from time to time. Differences are 1It would seem that we must draw the distinction between the accumulation of salts in solution in vacuoles and the existence of salts (as determined by chem- ical analysis) in the protoplasm of cells without such structures. The former is a phenomenon comparable to the passage of NaCl through loops of intestine from dilute to more concentrated solution. Two bounding surfaces are involved. The latter is possibly explainable by the formation of ion-proteid compounds within the cell as developed by Moore and Roaf (Biochemical Journal, Vol. 3, p. 55, 1907) to account for the high K content of the red blood corpuscle. 512 EDMUND NEWTON HARVEY to be noted especially between rest and activity, due to stimula- tion. A further source of error results from the fact that the very substance whose penetration we are studying may change the permeability of the cell in the concentrations used. This naturally leads to a brief consideration of the methods of test- ing permeability used by various authors. They may be con- veniently classified as follows: 3. Methods of testing permeability 1. Plasmolytic or osmometric (depending on changes in volume and turgor of the cell). a. Direct observation of plasmolysis (DeVries, Overton). b. Indirect, by noting cessation of movement in motile bac- teria (Wladimiroff, Zeit. f. physik. Chem. 7 p. 527, 1891). c. Indirect, by weighing (Overton, Pfliiger’s Archiv. 92, p. 115, 1902, Loeb, Pfliiger’s Archiv. 69, p. 1, 1897 and E. Cooke, Jour- nal of Physiology, 23, p. 137, 1898). d. Indirect, by noting liberation of haemoglobin in isotonic permeating solutions (Gryns, Pfliiger’s Archiv. 63, p. 86, 1896). e. Indirect, by determining change in volume of centrifuged corpuscles (Koeppe, Gryns, Hedin). f. Indirect,by determining the concentration in sea water and the concentration, pure, capable of causing artificial partheno- genesis (Loeb. Univ. of Calif. Pub. 3, p. 81, 1908) 2. Observational a. Directly, as the entrance of dyes (Pfeffer, Overton, Hoeber, Ruhland). b. Introduction of an indicator (as neutral red) and’ subse- quent change. c. By some change produced in asubstance (as tannin) already present in the cell (Overton, Zeit. Physik. Chem. 22, p. 189, 1897). d. By microchemical tests (as the determination of KNO; by diphenylamin; Molish). THE PERMEABILITY OF CELLS 513 3. Analytical (chemical or otherwise) a. By analysis of cells themselves. b. By analysis of medium (chemically, or by determining freezing point or electrical conductivity) after diffusion into the cell has taken place. c. By analysis of the medium after diffusion from the cell has taken place. 4, Conductivity [of blood corpuscles (Stewart, Tang] and Burgarszky, and Roth) or of eggs (McClendon)]. 5. Alteration of function (on the assumption that to affect a cell the substance must enter). a. Test by toxicity. b. Test by narcosis (Overton). c. Test by change in manner of response to stimuli (Loeb, Dynamics of Living Matter, p. 842, New York, 1906). d. Test of effectiveness in causing artificial parthenogenesis (Loeb, J., Chemische Konstitution und physiologische Wirksam- keit von Sauren. Biochemische Zeitschrift, 15, p. 254, 1909). No one of the above methods can be claimed as universally better than any of the others. Recent researches have exposed sources of error in the application of the plasmolytic method (Osterhaut, 08). Mass analyses of cells give us no hint as to the location of the substance in the cell or the state in which it is present, in solution or in combination. The evidence (pp. 543-546) in the case of the inorganic hydrox- ides shows that these alkalies may produce functional changes and death of Paramoecium without entering in sufficient quantity to affect granules stained in neutral red within the cell, and its effect must be on the membrane. MHoeber regards the surface of the cell as the point of attack of the strongly dissociated sub- stances in general. Only after the cell surface has been funda- mentally modified does the reagent pass in. Whenever applicable the observational methods are most useful in studying problems in permeability, for they not only answer the general question concerning penetration of the substance in question, but also enable us to locate the reagent in ‘the cell and 514 EDMUND NEWTON HARVEY to determine changes in function associated with the entrance of a given quantity. Most of the present experiments were performed in the Zodlog- ical Laboratory of Columbia University. The study of marine eggs was made possible by a visit to the Tortugas Laboratory of the Carnegie Institution and the Marine Biological Laboratory at Woods Hole. I wish to express my indebtedness to Dr. Mayer for the many special opportunities for research offered me at the former station and to Dr. Morgan for permission to use a Colum- bia table at Woods Hole as well as for many helpful suggestions. THE PENETRATION OF ANILINE DYES 1. Mechanism of staining plant cells In studying permeability for alkalies, to be discussed below, penetration was indicated by the change in color of neutral red in which the cells had been stained. In order to determine under what conditions neutral red exists in the cell and the nature of the dye compounds which the alkali must decompose on enter- ing, I have conducted a few experiments on certain dyes with the above points in mind. The most interesting fact obtained is that basic dyes as a rule cannot enter cells in the presence of a trace of acid in the medium whereas certain acid dyes do not enter cells in neutral or weakly alkaline solution but readily stain and kill the cell in weakly acid solution. We should natur- ally suppose the explanation of the above results to be in the effect of the acid or alkali on the dissociation of the dye molecules. a. Overton’s hypothesis:—Overton had at one time supposed that only the free dye base of basic dyes might enter cells. Since basic dyes are combination of weak bases with strong acids we should expect them to be hydrolytically dissociated in water, thus: RC] + H,O = ROH + HCl Only the ROH and not the RCI might enter. Overton (’00) later abandoned this idea since he was unable to show that the dye acetates entered the cell more readily than the dye chlorides. THE PERMEABILITY OF CELLS IRS The combination of a weak base and a weak acid undergoes a greater hydrolytic splitting, more of the free dye base is produced, and the cell should stain more rapidly in the acetate. But such was not found to be the case. The addition of a slight amount of acid will prevent the hy- drolytic dissociation and this test must be a surer one than Overton’s. I have consequently come to the conclusion that Overton’s first hypothesis is the correct one. The detailed evi- dence for this is given below. The dyes used are mostly the same that Overton experimented with belonging to the triphenyl- methane and chinonimid groups. b. Hlodea:—Pfeffer (’86) first studied the absorption of aniline stains and gave us a clear account of the mechanism of accumu- lation. The dyes collect as granular colored tannin precipitates in Spirogyra. The case of Elodea is different. Neutral red is not typically precipitated, but collects in solution of a red color.? It likewise collects as a red solution in the vacuole from alkaline tap water of a pale yellow color. This suggested that the sap vacuole is slightly acid and in the acid condition neutral red cannot pass out. Consequently it is slowly accumulated. (Har- vey 710). I was thus led to try staining in weakly acid solution. While it is true that neutral red does not enter from the HCl or CH;COOH acid condition, the acid present in Elodea is prob- ably a very complex organic one. I have been unable to detect any red coloration by crushing the leaves on blue litmus paper. The following experiment shows that the dye will not pass into the cell in the acid condition. Elodea leaves were placed in each of the following solutions: | A. 50 cc. tap water + 1 drop 0.1 per cent neutral red. B. 50 cc. 0.001 n HCL + 1 drop 0.1 per cent neutral red. C. 50 cc. 0.001 n NaOH + 1 drop 0.1 per cent neutral red. After six hours the leaf cells in A and C were found to have accumulated large quantities of the dye while the cells of B con- tain no dye at all. Leaves in B are not injured for protoplasmic 2 In some cells red globules may be seen; In others red needle crystals. In alkaline condition neutral red is yellow; in neutral and acid condition, red. 516 EDMUND NEWTON HARVEY rotation goes on and when placed in A they were able to collect the dyestuff from the surrounding solution. The stain may be ‘adsorbed’ by the cellulose cell walls from acid solutions just as it is adsorbed by glass only when in the acid condition. But this phenomenon has nothing to do with the question of the per- meability of the cells for the dye, although on casual observation the appearance of dye accumulation may be given. Elodea cells fail also to stain in 2355 acetic acid, yet accumulate the dye if transferred to a neutral solution.’ c. Theory of indicators applied: Indicators as neutral red or litmus are very weak bases or acids. According to Ostwald, the color change is due to the transformation of the acid or base into a salt which is very highly ionized. The ions give the color to the solution and their color is different from that of the undis- sociated molecule of the free color base or acid, only slightly ionized in solution. Neutral red has the following structure: N (GH): Ne= Cae. | »S CesHe (CHs) NH, | HCL vor RCl a A small amount of acid converts all of the neutral red into dis- sociated R+ and Cl—. A small amount of alkali forms ROH, undissociated, and it is the undissociated color base which may enter cells. In the neutral condition a small proportion of free base ROH is present, due to hydrolytic dissociation. Conse- quently cells may be stained in neutral solution. d. Action of the acidis on the dye: That the effect of the acid is on the dye and not on the plasma membrane of the cells, de- creasing its permeability, is made probable by the following facts. 1. Certain acid dyes (eosin) enter only in the presence of dilute acid and fail to enter in alkaline solution. 2. The presence of dilute HCl does not prevent the toxic effect of heavy metal salts like CuCh. ? The observations refer only to the normal rotating cells and not to certain large cells filled with a mass of white granular matter, which stains in acid solu- tion. THE PERMEABILITY OF CELLS old 3. The relations of basic dyes are exactly comparable to those found by Overton (’97) for certain alkaloids which are weak bases, and do not enter cells in acid solution. In the acid an alkaloid salt is of course formed. I have observed that caffein, on the contrary, enters equally well in acid, neutral, and alkaline solution. It was compared with strychnin in similar percentage concentrations: Spirogyra cells were placed in the following solutions: A. 0.01 per cent strychnin sulphate in .%, NaOH tap water. 0.01 per cent strychnin sulphate in -¥, HCl tap water. 0.01 per cent strychnin sulphate in tap water. 0.0125 per cent caffein in ~¥, NaOH tap water. 0.0125 per cent caffein in -¥, HCI tap water. 0.0125 per cent caffein in tap water. 7ey NaOH tap water. 7eo HCl tap water. HO ato bo In A the strychnin passes into the cells and forms a granular reticulum and within twenty minutes the cells have lost their turgor and secrete a sticky substance. In C strychnin also enters but less rapidly and death results after about one hour. In B no strychnin precipitate is formed and the cells are normal after one hour. In D, E, and F, an equal amount of caffein precipitate is formed after 45 minutes in each case. The cells in G and H are quite unaffected by the acid or alkali after one hour. ‘ e. Spirogyra: Exactly the same permeability relations hold for Spirogyra, sea urchin and starfish eggs, and Paramoecium. The mechanism of accumulation is different in each case. In the study of dyes we have always to consider two points—in what condition the dye enters and by what means it is made visible within. There must always be an accumulation of some kind for otherwise the color would not be apparent in so small a layer. I have also studied the entrance of several others dyes in the acid and alkaline condition into Spirogyra and the same general law appears to hold. The results are best given in the form of a table which does not pretend to be exhaustive, but simply to 518 EDMUND NEWTON HARVEY show that the conditions determining absorption of neutral red are equally true of other classes of dye stuffs (table 1). Besides Pfeffer’s original monograph, the most important com- parative studies of permeability for the aniline dyes have been made by Overton, Ruhland, and Hoeber and Robertson. Overton (’00) studied the permeability of both plant and animal cells in neutral solution for many different dyes and the solubility of the same dyes in olive oil and mixtures of lipoids. He came to the conclusion that the lipoid-soluble dyes enter liv- ing cells and the lipoid-insoluble do not. It has since been found that there are certain exceptions to Overton’s conclusion. Ruhland (’08) pointed out that there are some dyes which are lipoid soluble and fail to enter living cells, others are lipoid in- soluble yet enter readily and still others which may enter, yet show no relation between rate of entrance and solubility in lipoids. Hoeber’s (’09) recent study of the same question gave a similar result to that of Ruhland with respect to certain dyes. He con- cludes that the facts correspond: better with the ‘Satz’ that basic dye stuffs are intra vitam stains and acid dye stuffs are not. Robertson (’08) has attacked Overton’s original position by a study of the partition coefficient of various analine dyes in ethyl- acetate, ethylbenzoate, triacetin and triolein. He came to the conclusion that the solution of an acid dye in fatty substances is increased by the addition of acid, the basic by the addition of an alkali. In other words the free color acids or bases are more _ soluble in lipoids than their salts. Robertson also studied the stainability of fat cells, connective tisue cells, and red blood corpuscles (fixed on a slide) in acid and alkaline solution. But the HCland NaOH used were so strong (;%;) that they must have killed the cells and no conclusions as to the permeability of living cells can be drawn from his experiments. I have observed also the stainability of the yolk platelets of the frog’s egg in dilute solution of analine dyes in the acid and alkaline condition. 'These bodies are likewise more readily stainable by the free color acids and bases than their salts. I will discuss this matter later. THE PERMEABILITY OF CELLS 519 The acid solutions contained ,%, HCl; the alkaline ,2, NaOH in tap water; neutral solutions were of glass distilled water. The acid had no effect on the Spirogyra during the short time of the experiment, under four hours. The dyes (all prepared by Gribler and Co.) were of such concentration as to give a very light colored solution in a layer 3 em. thick. Many dyes cannot be satisfactorily studied because they become colorless very rapidly in alkaline solution and less rapidly in neutral solution. The letter B in the next table placed after the dye signifies that it is basic; A signifies an acid dye. In the last column is given the stainable power of the yolk platelets of the frog’s egg in the same dilute dye solutions used in the study of Spirogyra. The platelets were obtained from the ovarian eggs of Rana catesbiana. They are regarded by McClendon (710) as a lecithalbumin ‘“ecomposed of 6 per cent lecithin and 94 per cent batrachiolin, a nucleo-albumin containing 1.2 per cent phosphorus, 1.3 per cent of sulphur, and 15 per cent of nitrogen.” The entrance of all the basic dyes studied is indicated in Spirogyra by precipitation with tannic acid as fine colored gran- ules, Just as is neutral red. On the contrary the penetration of the acid dyes is accompanied by a combination of the dye with the cell proteids, nucleus and pyrenoids appearing colored first, then cytoplasm and chlorophyll bands. The spiral bands are often distorted, the nucleus swollen and turgor is invariably lost. This is true of all the acid dyes given in the following table and is indi- cated by the word stained. As mentioned above the appearance of cell staining is often given by a union of the dye with the cellu- lose wall but this is readily detected by high magnification. It is obvious that only the presence of colored granules in the sap vacuole or the staining of the protoplasm should be regarded as criteria of permeability. 2. Relation to Overton’s lipoid theory It will be seen from table 1 that it is the free color bases or acids which enter cells and not their salts and this is the sameresult which Robertson obtained in studying the solubility of dyes in 520 EDMUND NEWTON HARVEY TABLE 1 . pre ye a ~ DYE | REACTION COLOR SPIROGYRA | acid pink colorless Neutral red (B)........ 4 | neutral pink red granules || alkaline yellow red granules | acid blue colorless Methylene blue (B) 4| neutral blue | blue granules | alkaline blue blue granules | } ( acid red | colorless Saffranin (B)......:.-.. | neutral red red granules | alkaline red red granules | acid violet | colorless Methy] violet (B)....... neutral violet | violet granules || alkaline violet | violet granules acid orange yellow colorless Bismark browne) neutral cree yellow nae gran- (| alkaline lemon yellow red brown gran- ules {| acid blue colorless Wpastoyaibn (ey. dhe bane neutral blue blue granules alkaline light purple blue | blue granules acid orange yellow colorless Chrysoidin(B)i....- neutral orange yellow brown granules | alkaline lemon yellow brown granules acid blue colorless Tolyindin blue (B) neutral blue blue granules alkaline blue blue granules acid vermillion stained red osinr (CAs)s abo ce teic mals. neutral vermillion colorless || alkaline vermillion colorless acid pink stained pink Bordeaux red (A)....... 4| neutral pink colorless {| alkaline pink colorless (| acid. blue violet stained blue neutral blue violet (fades | unstained Satireviolet (A)............5 4 slowly) alkaline blue violet (fades | unstained [ slowly) acid light yellow stained light Avarantia (A) -eeeeen ee yellow neutral yellow unstained || alkaline yellow unstained YOLK PLATELETS colorless colorless colorless colorless very faint blue blue colorless very faint red red colorless faint violet violet colorless faint yellow faint yellow colorless faint blue blue colorless faint yellow faint yellow colorless very faint blue blue red red faint pink red red colorless violet violet colorless yellow yellow faint yellow THE PERMEABILITY OF CELLS Spall fatty substances and fat solvents. It must not be forgotten that, as Mathews (’98) showed, the basic stains yield colored precipi- tates with proteids only in alkaline solution, the acid only in acid solutions. The same was found to be true of the staining of coag- ulated proteids as egg albumen. We should expect therefore that the lecithalbumen platelets of the frog’s egg would show the same staining relations that Mathews found for coagulated albu- men, even from very dilute solutions. It is probable that the acid or alkali affects the lecithalbumen as well as the dye. We might draw the conclusion from this that a dye only enters a cell when it combines with the surface membrane.‘ Yet I have never noticed that the plasma membrane of any cell becomes stained in dilute solutions of basic dyes. The most conspicuous fact connected with the staining of plant cells is that the stain passes through the cell protoplasm without affecting it in the least and collects in the vacuole. My studies on dyes have not been extensive enough to warrant generalizations as to the classes of dyestuffs for which cells are permeable nor as to the nature of the cell surface. It appears to be true—as a general rule, to which there are exceptions—that the substances (including alkaloids, alkalies, dyes, anaesthetics, etc.) more soluble in fat solvents or fatty substances than in water, penetrate cells with practically no resistance, while those compounds insoluble in ether and fats meet a marked resistance at the cell boundary as Overton has postulated. But whether we are to conclude from this that the boundary is lipoid in nature is quite another question. The evidence on this point is far from conclusive. Indeed, Traube has shown that the lipoid soluble substances, the easily permeating substances, are those having the greatest tendency to lower the surface tension of water in air, and, according to his theory of osmosis to pass into the phase of greater surface tension (into the cell). No lipoid membrane separating the two phases is required. 4 Mathews (’10) has recently concluded that the dyes penetrate by ‘combination with substances in the peripheral layer such as lecithin and the electro-negative proteins, soaps and possible other substances.’’ (p. 218). He regards the taking up of basic dyes by lipoid solvents, which act as weak acids, as a chemical com- bination. pape EDMUND NEWTON HARVEY 3. Mechanism of staining animal cells Animal cells show the same relations toward neutral red as do plant cells: Paramoecia were placed in the following solutions: A. 10 cc. tap water + 0.1 ec. X, HCl (,4,,) + 1 drop 0.02 % neutral red. B. 10 cc. tap water + 0.1 cc. X, NaOH (,4;) + 1 drop 0.02 % neutral red. Cao ec. tap water + 1 drop 0.02 % neutral red. After 30 minutes the individuals in A are unstained while those in B and C are very deeply stained. In one hour the Paramoecia in A show a faint pink color in some of their vacuoles, and later they become noticeably stained. This is probably due to the fact that the animals are constantly forming new food vacuoles. A small amount of neutral red enters with the fluid of the vacuole. The acid passing in at the same time is neutralized and the dye may then pass the wall of the vacuole and stain certain granular , bodies in the protoplasm. Paramoecia stain after some time in acid solutions not because the dye may pass the surface mem- brane but because it is engulfed along with the food of the organ- ism. The food eaten may itself be stained. If paramoecia stained in neutral red are centrifuged in an elec- trical centrifuge for one and one half hours it is easy to dif- ferentiate the bodies with which the dye unites. Six more or less distinct zones may be distinguished. These very soon mix again due to the constant rotation of the protoplasm. ‘Their relative volumes are indicated in fig. 1. Only two substances in Paramoecium are found stained (1) the food and granules in some of the vacuoles; (2) the minute granules which often form a ring about the food vacuoles. Macro and micronucleus, trichocysts, cilia and the clear fluid portion of the protoplasm of the living organisms are quite unstained. The staining of marine eggs is essentially similar to the stain- ing of Paramoecium. Owing to the presence of bicarbonates and phosphates in sea water considerably more acid must be added, than is necessary to change the color of neutral red from yellow to red, before all the dye is actually in the acid condition and consequently, before the dye will fail to enter the eggs. About 0.5 cc. X, HCl to 100 ec. sea water is sufficient to bring about THE PERMEABILITY OF CELLS 523 Fig. 1 Diagram of the areas which may be distinguished in a centrifuged Para- moecium, (electric centrifuge 1.5 hours at a radius of 6 em.) stained in neutral red. O, oil (?) globules; V, food vacuoles, the contained matter often red stained ; S, granules stained in neutral red; N, macronucleus; C, crystals. If the organism is uninjured, there is a very rapid redistribution of substances in the protoplasmic circulation. Figs. 2and3 Diagrams of the distribution of substances in centrifuged Chae- topterus (2) and Arbacia (3) eggs stained in neutral red. Elongation in the axis of the force has taken place. O, O1, Os, oil globules; in Chaetopterus of two sizes and of two densities O; and O:; N, nucleus; C, clear area; in Chaetopterus containing a few scattered yolk and minute red stained granules; in Arbacia containing at the surface a few pigment bodies (chro matophores) and numerous minute stained granules, quite unmoved by the centrifuge. At the time of fer- tilization these disappear, apparently going to form the substance which passes out of the egg and hardens to a fertilization membrane. Y, yolk; in Chaetopterus appearing pink from numerous minute red stained granules most of which col- lect in a mass (S) just under the oil. P, the pigment granules, appearing dark red from absorption of the neutral red dye. 524 EDMUND NEWTON HARVEY the color change of neutral red. The yellow color returns to some extent on standing. Both Arbacia and Asterias eggs stain in 100 cc. sea water + 2 cc. X, HCl but fail to stain if 3 ce. & HCl is added, even after one hour. They are quite transparent and uncoagulated. About half the egg of Asterias coagulate in 100 ec. sea water + 5 ec. % HCl after one hour’s time and those which are opaque and coagulated become faint pink in color. Apparently the dye is adsorbed by the proteid coagulum. The mechanism of absorption of neutral red is practically the same asin Paramoecium. In all the eggs thus far studied (Cum- ingia, Arbacia, Asterias, Toxopneustes, Hipponoé, Holothuria and Chaetopterus) it combines with very definite granules which often differ in color, always in specific gravity and generally in size, from other granules in the egg. In markedly pigmented eggs like Arbacia or Cumingia, it is the pigment granules which become stained, but in the eggs of Holothuria, the large yolk granules are orange and the dye absorbing granules are small and colorless. The latter are much the heaviest granules present and pass to the outer pole of the egg when centrifuged. That this is not due to an increase in weight from taking up of the dye may be shown by first centrifuging the eggs and then staining them. Exactly the same areas stain as if the experiment had been re- versed, the eggs first stained and then centrifuged. The two statements made above are true of all the eggs studied except Chaetopterus. The stainable granules of Chaetopterus are specifically different from other granules in the egg, but they are not the heaviest. The difference is best made clear by refer- ence to figs. 2 and 3. The red area is found to be just under the oil and to consist of globules of varying size which have apparently been formed by fusion of very minute red granules. These may be easily seen over the clear area and scattered throughout the yolk mass, giving a pinkish tinge to that region. A similar aggregation of minute granules to form larger granules occurs in Toxopneustes also. Both fertilized and unfertilized eggs at first stain ‘diffusely,’ 7.e., the dye is localized in very minute granules, visible under high magnification. More and more of THE PERMEABILITY OF CELLS 525 these minute granules take the dye and at the same time fuse together to form clearly defined red bodies much like the chroma- phores of Arbacia eggs. The final stage is more rapidly attained in eggs with fertilization or artificial membranes. Inasmuch as the conditions under which the dye unites with the granules in the two types of eggs, fertilized and unfertilized, may be different, we cannot at present draw conclusions as to differences in per- meability to dyes in the two types of eggs. In brief the mechanism of staining Paramoecium or marine eggs is as follows: The dye enters the egg as the weak base, yel- low in color, and combines with a specific insoluble substance present, in the form of granules. The combination resulting, like- wise insoluble and comparable to a salt, is red in color just as are the water soluble salts of neutral red. The exact chemical nature of the granules with which neutral red combines is unknown. THE PENETRATION OF ALKALIES 1. Method and previous results Of the many methods which may be used in studying the pene- tration of various substances, the color change of an indicator within the cell is the simplest and most delicate for the detection of acids and alkalies. Many plant cells contain natural pig- ments which may serve as indicators. Both Pfeffer and DeVries made use of such pigments in their studies on permeability. DeVries (’71, p. 24) noted that the red sap of beet cells becomes brown to yellow brown in dilute NH,OH and the red color comes back again on washing in pure water. Pfeffer (77, p. 140) showed that the red sap of Pulmonaria petals and of the stamen hairs of Tradescantia becomes first blue then greenish in diluteammonia. KOHand K,CO,act like ammonia (p. 141). Pfeffer regards the dead and the living plasma tobe similarly easily permeable for dilute alkalies as well as acids. This is the view stated in botanical text-books at the present day and the subject is dismissed with few words and without further THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, No. 4 526 EDMUND NEWTON HARVEY comment. Evidence will be given in the body of this paper to show that the generally accepted view is only in part the truth, so far at least as the penetration of alkalies is concerned. Animal cells suitable for experimental studies contain no pig- ment exhibiting a marked color change in alkalies. We may overcome the diffiulty by introducing a dye which does so change. Neutral red is excellent for the purpose. In solution, it turns from red to yellow in an H ion concentration of 110 tor ilome and is perfectly harmless for the cells providing they arenot allowed to take up over a certain maximal amount. For the sake of com- parison neutral red was employed in plant cells as well. I have endeavored to answer for the alkalies the following general questions, many of which have already been settled for other classes of substances by previous workers in the field of cell permeability. 1. Do plant and animal cells exhibit essentially similar per- meability relations for alkalies? 2. Do living and dead cells exhibit similar permeability rela- tions for alkalies? 3. Are there distinct classes of alkalies in regard to permea- bility? 4. Are the effects of different alkalies reversible or irreversible after an equal amount (OH ion concentration) has entered the cell? 5. May the composition of the medium affect permeability without irreversibly injuring the cells themselves? 6. Are there any relations between functional changes and permeability for alkalies? 7. May alkalies produce marked functional changes without entering the cell? Many of these questions can be decided only with an especially favorable type of cell and the evidence one way or the other has therefore been given in describing the permeability phenomena of that particular cell. The connection between the penetrating power and the physical properties of the various alkalies are taken up in the discussion. I have taken the greatest precaution to THE PERMEABILITY OF CELLS 527 exclude sources of error and make sure that the permeability of ling cells is in reality the problem studied, a point not always carefully guarded against. For this reason the observations and experiments which follow are recorded in considerable detail. 2. Objections to the use of an indicator within the cell Despite the fact that mdicators offer the most delicate means of detecting alkalies (and neutral red is exceedingly sensitive in this respect, reacting to Na,HPQO,, there are four possible com- plications arising from its use in the cell, all of which would tend to make the amount of alkali entering, appear less than that which actually entered. Or, to put it in another way, more alkali would enter than we should calculate, judging from the concentration at which the color change takes place in pure water. 1. In determining alkalinity in a test tube we have only a very weak solution of the indicator; in the leaf cells of Elodea, neutral red is in concentrated solution (otherwise it would be quite invisible in so narrow a space) and also in combination with some complex organic acid. Enough alkali must enter to convert it all into the free base (ROH) before the color change occurs. 2. In Spirogyra neutral red is accumulated as a tannin com- pound which becomes straw color in the presence of alkali and red again on adding acid and begins to dissolve. This compound as well as the combination formed by neutral red with the granu- ules of animal cells require a greater concentration of alkali or a longer time, to turn them yellow than does the pure dye in dis- tilled water. By treating cells with chloroform or by rupturing their surface, as described under each organism investigated, it is possible to exclude the complication due to the above mentioned conditions. The concentration of alkali may be found which brings about a color change instantaneously. 3. The most serious objection to the use of neutral redasan indicator within the cell is, as I have determined experimentally, that in the presence of proteids like egg albumen, the dye (in solution) is unaffected by a certain amount of added alkali which 528 EDMUND NEWTON HARVEY apparently enters into combination with the albumen. The following experiment illustrates this: A stock solution of unknown strength in 0.1 N NaCl was made from Merck’s powdered egg albumen. When filtered it was opalescent. To 5-cc. was added one drop of 0.05 per cent neutral red and the solution titrated with 55 KOH. 0.6 cc. was added before the original pink color began to turn and 0.9 ec. so KOH before it was yellow. Phenolpthalein was then added and the solution found to be neutral. In all 1.6 cc. was added before the phenol- pthalein began to turn pink and 2.0 ec. before it became dis- tinetly pink. 0.6 ee. so NH,OH was also required before the neutral red began to fade and 1.1 cc. before a distinct yellow appeared. The solution was still neutral to phenolpthalein and 3.00 cc. were re- quired before a light pink appeared. The effect of chloroform on the power of neutralization was also determined. The solutions compared were: A 65cce. albumen + 10 cc. water + 1 drop 0.1 per cent neutral red. B_ 5cc. albumen + 10 cc. chloroform-saturated water + 1 drop 0.1 per cent neutral red. Exactly the same amounts of 5» KOH and s> NH,OH were required to induce the color change in A asin B. Chloroform has no effect on the combining power. Yet one drop (0.05 ec.) of s» KOH or NH,OH is more than sufficient to render 0.1 N NaCl solution alkaline to both neutral red and phenolpthalein. Any difference in permeability between KOH and NH,OH— and there is a very great difference—cannot therefore be attrib- uted to a difference in combining power of K and NH, The use of chloroform in killing cells must affect the surface layer of the cell in some way, and not the combining power of the cell pro- teids within. 4. It is quite possible that the cell (especially plant cells) may actively secrete an acid which neutralizes the alkali as it enters. But when we consider that the mass of the material studied rel- ative to that of the alkaline solution is very small and that the THE PERMEABILITY OF CELLS 529 KOH, let us say, would continue to diffuse in so long as neutralized at a practically constant rate, it would require an enormous pro- duction of acid on the part of the cell to take care of the KOH entering. For this reason alone it seems safer to consider that the inorganic alkalies do not enter (without affecting the surface membrane) rather than that they are neutralized as they enter. There is, indeed, some evidence that an acid is secreted into the vacuole of Elodea cells. If red stained leaves are placed in ¥) NH,OH until color change occurs and then are immediately trans- ferred to pure water, the cells are uninjured and the red color returns. It is often a distinctly brighter red than before. I have never noticed that Elodea cells placed in solutions of inorganic alkalies became a brighter red before the color change finally occurred. Again red stained leaves treated for one minute with chloro- form water appear a slightly brighter red than thecontrol. The chloroform apparently induces the formation of an acid which collects in the vacuole. Yet such leaves placed in 45 NH,OH are decolorized slightly more rapidly than normal leaves and in 7} KOH, 30-40 times more rapidly. The experiment shows the negligible effect of the acid in neutralizing entering alkali. The above considerations make the calculation of the strength of any alkali entering a cell very difficult. We can state this much, however, we can give the concentration of alkali in terms of the color change of the neutral red combination in each particu- lar cell, and the relation of this color change to structural or functional changes brought about by the presence of the alkali. We must assume that when red becomes yellow the OH ion con- centration of the various alkalies is equivalent. It must be borne in mind that the indicator in both Spirogyra and Elodea is in the cell vacuole and that the alkali to reach it must pass through the cell (plasma) membrane, the protoplasm, and the vacuolar membrane. On the contrary, the indicator in animal cells is in the protoplasm. 530 EDMUND NEWTON HARVEY 3. Hxpervments with plant cells a. Strongly dissociated alkalies: The leaf of Elodea, except at the midrib, is only two cells thick, a layer of small cells con- stituting the lower surface, a layer of much larger cells above. There is great variation in different leaves in the time for the color change to take place, as well as individual differences among the separate cells. A consequence of the latter fact is the decoloriza- tion (in K, Na, or N(C.H;),OH ) of the leaf in patches, groups of red cells becoming yellow before others. The phenomenon is not marked in Ca, Sr, and Ba. Spirogyra exhibits the same variability as Elodea. A comparison between the inorganic hydroxides was made with # alkali in tightly corked bottles, to prevent absorption of CO:. The water was redistilled from glass and was non-toxic to Spirogyra, Elodea and Paramoecium. Paramoecium is especially sensitive to commercial distilled water and may be used as an indicator of the purity of a water. Elodea showed rotation in commercial distilled water after 24 hours. Nevertheless the rate of entrance of NaOH is more rapid when dissolved in commercial distilled water than in pure redistilled water and more rapid in pure redistilled water than in tap water. The following table constructed from many experiments gives the relative rate of penetration of N(C2H;),OH, Na, K, Ca, Sr, and Ba hydroxides’. The actual times varied somewhat in indi- vidual experiments, but the relation order of penetration was Penetration of rm alkali into Elodea leaves. MINUTES | MINUTES INS OHS ia heer eee ate Bectna ae ee 25 He NIC CHIBI) (OLS IIMB lipis oye octo se crooace 30 KOHE Ae ope eee ee ee ere 99) SA MNUEIGCOREIME .,... . ; oto. Gea oe ee 0.5 CaCO agen ccs cae 23 Methydmanmiine. een heen 1 Srl@iED esis “fee een ccritcremoes 15 | Minine tbs liam ine setce lary piste 2 BalOM) rss saacteoet emcee eae 15 5] am indebted to the Chemistry Department of Columbia University for methyl, dimethyl, trimethyl, ethyl, propyl, and isopropyl amines. Tetraethyl- ammonium hydroxide was obtained from Merck and Co. The inorganic hydrox- ides were Eimer and Amend’s C. P. with the exception of Ba(OH)s, which was manufactured by Kahlbaum. THE PERMEABILITY OF CELLS 531 fairly constant. In all the experiments on alkalies, N means normal, equivalent to corresponding normal solutions of HCL. The rate of entrance when the cells have been killed in various ways is shown in tables 2 and 3. TABLE 2 Times for different concentrations of NaOH to decclorize the neutral red tannin compound within Spirogyra threads. In column A are figures for the living plant; in B for threads killed by boiling water and stained in neutral red; in C for threads killed by ~ HCL (8 mins.) and washed free of acid; in D killed by CHCls water (1 min.), and in E the threads were killed by a saturated solution of HgCle (3 mins.) and washed in water. The NaOH was dissolved in tap water and the solutions tested in corked bottles. CONCEN- TRATION OF A B C D E NaOH ee 0.5 minutes | instantly instantly instantly ah ate EY 2.5 minutes instantly instantly instantly ite Bisse. 9 minutes instantly instantly instantly orcs 40 minutes instantly instantly < 0.5 minutes < 0.5 minutes see Be 70 minutes instantly instantly < 0.5 minutes < 0.5 minutes _ Shed 1.5 hours instantly instantly < 0.5 minutes < 0.5 minutes 70 rae 3 hours instantly | instantly < 0.5 minutes < 0.5 minutes a Ae 2 hours instantly instantly < 0.5 minutes < 0.5 minutes isa 5 i 2 <18 hours instantly | 0.5 minute 1.0 minute 2 minutes 500 Nie AA | >18 hours instantly 1 minute 3 minutes 10 minutes en etn ote >18 hours 1 minute | 2 minutes 8 minutes cee Wey ica 2 minutes | 18 minutes TABLE 3 Penetration of different concentrations of NaOH into Elodea leaves. The NaOH was dissolved in tap water. | CONCENTRATION | LEAVES KILLED BY BOIL- LEAVES KILLED OF NOLS, LENSE Be ORN Ao ne ING WATER, AND DEEPLY NaOH STAINED IN NEUTRAL RED a SET cee 2 minutes instantly instantly a ond 8 aac eens } 9 minutes 1 minute | instantly | a PEs Eel Ave, sus | 45 minutes 2 minutes instantly Ted tale DlagHeune | 3 hours 5 minutes instantly | a Srey ee kek sla | unaffected after 18 hours 8 minutes 2 minutes It is found that all alkalies may enter with practically no resistance providing the cell surface has been destroyed in some way; it is immaterial how. Thus in sy NaOH Spirogyra threads retain their red color about 3.5 hours, but if treated with CHCl, Dae EDMUND NEWTON HARVEY saturated water and then placed in sy NaOH the red precipitate becomes yellow immediately. On adding dilute HCl the thread again becomes red and the precipitate begins to dissolve. As shown above, chloroform has no effect on the alkali com- bining power of egg albumen and presumably none on cell pro- teids. It must be that the inability of the alkali to enter is due rather to the inability to pass the surface layer of the Spirogyra cell than to a neutralization by proteid or acid after passing this layer. The color change in the tannin precipitate takes place practi- cally instantaneously in s¢) NaOH (or KOH, Ca(OH),, Ba- (OH), or Sr(OH).), if we cut the cell transversely so as to allow ready mixture of solution and precipitate. In weaker concen- trations the color change also occurs but it takes a much longer time. Thus, dead Spirogyra filamants (red stained) become decolorized in less than 15 hours in s¢30 NaOH, while the control in water remained red. Every cell which I have tested (Cabomba rosafolia, containing a natural red pigment, Elodea Canadensis, Spirogyra, Para- moecium, Vorticella and various marine eggs) has proved to be resistant to the entrance of the inorganic hydroxides, a condition which is lost on death (by chloroform, HCl, heat coagulation, drying, etc.) This post-mortem increase of permeability has ° been so often emphasized by many writers for very diverse sub- stances that it hardly requires special confirmation for the alkalies, except as showing the degree of impermeability which the normal living cell possesses. b. Weakly dissociated alkalies: —Exactly opposite results are obtained when ammonia and its primary, secondary and tertiary alkyl substitution products? are studied, instead of the inorganic hydroxides. All these substances pass into the cell with very little if any resistance. Elodea is more suited to experimentation than Spirogyra’ because the color change is more marked (tables 4 and 5). 6 If a red stained filament of Spirogyra is placed N, NH,OH it becomes dark green in one minute. On washing in water the red color soon returns. In weaker solutions of ammonia the formation of the NH:—tannate compound prevents a sharp color change in the neutral red-tannate compound. THE PERMEABILITY OF CELLS oao TABLE 4 Effect of NHsOH on red Elodea leaves. Glass distilled water used. Solutions in corked bottles CONCENTRA- TION OF NHsOH N N N_ N N_ AND N N | B ee! : Normal leaf.. instantly 1 minute 1 minute 13 minute 3 minutes6 minutes) Not entire- Unaffected ly yellow after 1 after 30 hour. minutes Slightly affected after 18 hours Chloroform treated leaf. instantly instantly) minute 1 minute 3 minutes6 minutes Dye diffuses out of leaf In Spirogyra, entrance of ammonia may be indicated also by the precipitation of tannin, and this method has been used by Overton (’97) who found that ammonia, the primary and secondary amines penetrate readily, the tertiary amines and quaternary ammonium bases do not, behaving in this respect like inor- ganic hydroxides. So far as I have observed trimethyl amine enters cells readily but not so quickly as the methyl or dimethyl] derivitives of ammonia. » If an Elodea leaf has remained in a solution of # NaOH (or K, Ca, Sr, Ba, N(C2H:;)s hydroxides of any concentration) until the color change to yellow has occurred, and then is immediately placed in pure water it is found that the leaf has been killed and the red color never returns.?. The capacity of again accumulat- ing dye is likewise lost. The entrance of a sufficient quantity of inorganic alkali to affect the neutral red produces irreversible changes in the cells themselves. But if a similar Elodea leaf is placed in an 4 NH.OH solution . the color change occurs almost instantly (about one minute). Furthermore, on transferring to pure water, the red color in the cells and the protoplasmic rotation characteristic of the plant returns. Evidently death of the leaf does not necessarily ensue from the ’ Except on immediately adding an acid. This shows that the dye is actually changed to yellow and not reduced to a colorless substance. The actual yellow color may be obscured to some extent by the green chlorophyll present. 534 EDMUND NEWTON HARVEY entrance of a concentration of OH ions sufficient to affect the neutral red combination. In the case of the inorganic alkalies and N(C.H;),OH death is to be referred to two possible causes. First, and of greatest importance, the alkali only enters after the cell surface has been destroyed. This is best illustrated by comparison of the mode of entrance of NH,OH and NaOH into Paramoecium (p. 546) and is strong evidence in support of Hoeber’s (06, pp. 260, 266-267) theory that the strong electrolytes, in general, produce their effects by a change in the colloids of the cell surface and not of the cell interior. Second, the combination of the strong alkalies with the cell surface proteids may be irreversible, whereas the combination. of the weak ammonia is easily reversible. The cell surface of the living as compared with the dead (by chloroform treatment) cells offers a highly resistant barrier to the entrance of the strong alkalies but both living and dead cells are almost equally permeable for the weak alkalies (see table 4). This suggests, but does not prove, that if small quantities of NaOH could enter without affecting the membrane, the cell would be as unharmed as in NH,OH. Ammonia has likewise a toxic effect but it is only manifest after a longer exposure, and is quite independent of the entrance of ammonia into the cell. Red Elodea leaves recover if placed in fresh water immediately after decolorization in 7 NH,OH. If left for five minutes the dye becomes red again but the cells eventually die. If left over 30 minutes even the red color fails to return. The leaf is of course killed. The inorganic hydroxides (and N(C.H;),OH) only enter the cell after they have affected the normal impermeability—in other words after they have rendered its surface permeable to them- selves. It seems best, then, to speak of a resistance of the cells for the strong and a permeability of the cell for the weak alkalies. Reversibility of the neutral red color change is quite indepen- dent of the death of the cell. The red returns after decoloriza- tion by the amines although they produce fatal after-effects. It likewise returns in cells first decolorized in ammonia and then killed with chloroform water, and more rapidly than in the THE PERMEABILITY OF CELLS AD control. Chloroform water induces the formation of an acid in the vacuole and the production of acid in solution of weak alkalies is probably one factor in the return of the red color. Failure of Elodea leaves to become red when once turned yellow by KOH is not due to the longer time required by the KOH to bring about the color change (the neutral red diffusing out slowly in the interval), because the leaves become yellow within one minute, if placed in 7 KOH yet in pure water the red does not return. We must seek an explanation of the reversibility of the NH,OH change, the irreversibility of the KOH change in the different degrees of hydrolytic splitting of the respective NH; and K salts of the acid with which the neutral red combines. If R is the acid radical within the plant and D the neutral red radicle, the reactions may be represented thus: RCOOH + DOH = RCOOD + H.O. RCOOD + NaOH = RCOONa + DOH RCOOD + NH:OH = RCOONH, + DOH The RCOONH, salt of a weak base and a weak acid undergoes _agreater hydrolytic splitting than the RCOONa combination, the NH.OH formed diffuses rapidly away and recombination of RCOOH and DOH again take place. The difference in resistance of the plasma membrane to NH,OH and NaOH is strikingly shown in the following experiment. If we remove Elodea leaves, after decolorization in NH,OH to chloroform water (one minute) and then place them in 75 NaOH there is never a return of the red color. Chloroform destroys the plasma membrane and the KOH may penetrate rapidly. On the other hand, if the leaf decolorized in 7) NH,OH is placed in 50 NaOH, without previous chloroform treatment, its cells become red again just as they would in pure water. The conditions for demonstrating any entrance of NaOH are here most favorable, the neutral red is already in the alkaline condition, the proteids have taken up the amount of NH,OH necessary before and alka- line reaction may be indicated, yet the NH,OH diffuses rapidly 536 EDMUND NEWTON HARVEY outward while the NaOH cannot pass in to maintain the dye vn the yellow condition. Eventually of course the leaf in 55 KOH be- comes yellow just as does the control. The amines show a behavior similar to NH,OH but, with the exception of trimethyl amine, are considerably more toxic and produce after effects which lead to the death of the cell. The entrance of just enough to affect the neutral red is typically fatal (table 5). TABLE 5 Effect of NHsOH and amines on red Elodea leaves. Concentration, a in glass distilled water. The red color disappears (decolorized) in less than 2 minutes in all solutions. The leaves are then trans- ferred to (A) tap water; (B) reo NaOH. Columns A and B give the results, respectively. A B INAISECONS bats Bobagnaest eet rere Cells become red in 15-30 min- | Cells become red again in 15-30 utes and still alive after 18 hrs. | mins. and only turn yellow | when control turns yellow NH;CH30H.................-....| Cells become red in 15-30 min- | Cells remain colorless | utes but dye diffuse out. | Colorless after 18 hours. NIEe(@H3) sO Estee se eee | Cells become red*in 15-30 min- | Cells remain colorless utes but dye diffuse out. Colorless after 18 hours. | INPSNCCABIY OSL A Are rae greater | Same as NHsOH | Same as NHsOH INES (@SEp) Oe neree nae Same as NHsCH;0H | Same as NH3CH3;30H INGEls (Csi; CEs) Olen. cee Same as NH;CHs0H | Same as NH3;CH;0H (normal propyl amine) | NH3(CHa)2eCHOH (isopropyl- Same as NH;CH;0H | Same as NH3CH3;30H AMMAN) oe carne Se Si ee eee | Controle sends n ere ere Seen Red and normal after 18 hours | Red > 2 hours Leaves treated with CHCl,, after decolorization in any of the above solutions, and: placed in x. NaOH never become red again; if placed in tap water all tend to become red but the red dye eventually dif- fuses out of the cells because the cell surface has been affected by the chloroform. ce. Permeability of cells exhibiting protoplasmic rotation. There appears to be no marked difference in the resistance of ‘rotating’ and quiescent cells to the penetration of NaOH or KOH. Ina number of experiments in which individual cells were watched rotating cells became yellow before non-rotating or vice versa. Comparisons of whole leaves are not of much value because of their great variability in resistance to NaOH. * THE PERMEABILITY OF CELLS 537 Judging from Hérmann’s (’98) researches and the comparison he has drawn between the rotating plant protoplast and muscular contraction a difference in permeability was to be looked for. The cessation of streaming induced by thermal, mechanical, chemical and electrical means strongly suggests that a shock- stoppage is comparable to muscle contraction and depends on a similar conditioning change. Even the details connected with elec- trical stimulation run parallel. According to Hérmann the pass- ing of a constant current through a rotating Nitella cell causes a cessation of movement at the cathode on the make and at the anode on the break. While the current is passing the streaming is slower at the cathode. A wave of shock stoppage may be pro- pagated from one region of a leaf to another and when tested electrically the stopped regions are found to be negative to rotat- ing ones just as the region of a muscle in contraction is negative to an uncontracted area. We might therefore consider that protoplasmic streaming is in some way connected with a high degree of electrical polariza- tion, a low surface tension and a surface membrane relatively impermeable to soluble substances, states typical of the resting condition of many types of cells. Each of these three conditions undergoes a change in the opposite direction, on ‘stimulation’ of the cell. As stated before I have been unable to detect any constant differences in permeability (or resistance) of rotating and non- rotating cells to KOH or NaOH. It is quite possible that several factors each of which alone may be sufficient to prevent rotation, are involved. d. The concentration of alkali which stops rotation:—In NH,OH (+) to so) rotation ceases just at the point where the bright pink sap begins to turn dull pink before finally becoming yellow. In NaOH the rotation ceases, begins again and finally ceases per- manently loag before the initial dull pink of the color change appears. The description of the two experiments in which the changes in individual cells was observed is as follows: 538 EDMUND NEWTON HARVEY Ammonia: Red stained Elodea leaves are placed in 4% NH,OH. Cells beconie yellow in less than one minute. Rotation ceases at the time the color change begins (# min.). After 1 min. the alkali is re- placed by water. The bright red color begins to return (20-25 mins.) and the cells are as red as they were originally in 1 hour. In 1 hour, 10 mins. jerky rotation begins and in 2 hours the original rapid ro- tation may be observed. Sodium hydrate: Red stained Elodea leaves are placed in gy NaOH under the microscope. Rotation ceases in about one minute, begins again slowly in 5-10 minutes, stops again after 15 minutes longer, and the red sap only begins to turn dull pink to yellow after 45 minutes to 1 hour. If the alkali is replaced by water, the sap never becomes red again and rotation never returns. e. The effect of added substances on the penetration of NaOH and KOH. As indicated in table 4 even chloroform treatment hardly increases the rate with which NH,OH may enter Elodea cells. Ammonia enters living cells as rapidly as dead ones. NaOH enters dead cells nearly as rapidly as ammonia, but living cells offer a high resistance to its passage. This resistance may be decreased by the addition of chloroform to the medium in amounts too small to have any irreversible effects in the absence of NaOH. On comparing the time for NaOH or KOH to decolorize red stained Elodea leaves in 7) solution alone and in gy solution plus dilute chloroform, alcohol, urea, glycerine, and various salts it is found that all have the effect of shortening the time which it takes for the NaOH to enter. The analysis of the experiment is somewhat complex. The effect of the added substance may be on the plasma membrane or on the alkali (affecting its dissocia- tion or combining to form more toxic compounds); or the alkali may allow the more ready entrance of the added substance with consequent rapiP destructive action and death of the cell which leads also to easy penetration of alkali. In other words, the action of two substances together may be additive. On aecount of the complexity and difficulty of interpreting results I discontinued further experimentation along this line. A few experiments are given. The effect of dilute chloroform solution must be attributed to decrease in resistance of the cell THE PERMEABILITY OF CELLS 539 surface. Such a concentration would have no effect on the alkali or vice versa. If the urea glycerine or salts change the condition of the plasma membrane it is surprising how little effect this has on the proto- plasmic streaming, which continues for many hours in solutions of these substances. If leaves are selected from the same or neighboring whorls on the same plant concordant results may be obtained. But dif- ferent plants and young and old leaves exhibit the greatest varia- tions in resistance to NaOH, as separate experiments will show. The solutions were contained in tightly corked glass vials to prevent absorption of CO.. Two to four leaves were tested in each experiment. Experiment 1. A. a> NaOH, } saturated with CHCl; tap water—decolorized in 13 minutes. B. + ) NaOH in tap water—decolorized in 90 minutes. Rota- tion ceases in leaves in this solution in < 15 minutes, but if re- moved to tap water (after 15 minutes) begins again in 16-20 minutes. C. i saturated CHCl; in tap water—Rotation ceases, but if removed to tap water (after 15 minutes,) begins again in < 15 minutes. One-sixth saturated chloroform increases the permeability of Elodea cells to urea. Leaves removed from solution C after ten minutes plasmolyse much less readily than control leaves of the same plant in 5 urea. Experiment 2. A. zy NaOH + 0.75 m C,H;OH in tap water —decolorized in ten minutes. B. zo NaOH + 0.37 m C.H;OH in tap water—decolorized in 19 minutes. C. «go NaOH in tap water—decolorized in 19 minutes. D. 0.75 m C.H;OH in tap water—rotation momentarily ac- celerated, then slowed and continued slow for > 1 hour. E. 0.87 m C,H;OH in tap water—rotation hardly affected, slightly accelerated if anything. Experiment 8. A. zo NaOH + 0.075 m NaCl in distilled water—decolorized in 4 minutes. 540 EDMUND NEWTON HARVEY B. <5 NaOH in distilled water—decolorized in 28 minutes. C. 0.075 m NaCl in distilled water—rotation unaffected after 24 hours. Experiment 4. In this experiment the effect of salts, and the penetration of NaOH into red stained Elodea and also Spirogyra were studied. The salt solutions are all in distilled water. SPIROGYRA | ELODEA min. | min i tap water NAO: v.47 War. eee ce eee Pe slo | 30 | 45 u distilled watersNaOH 2s 24.) Ake ee ce: | 10 | 35 SEINE) O) a iy = 2S Ea Ni) C] ee ees eee. «or 4 | 8 §, NaOH + fy (100 NaCl + 1 CaCh)....., ee 6 3 | 9 i) NaOH + ¥; (100 CaCl + 2.2 KCI). Pee, ety 5 NOt ae ee NaCl ae 2.2KCl1 + f 6 CaCl, ) Ae 2 | 6 Experiment 8. | SPIROGYRA ELODEA min. min. ay taDiwater iGO 2, 4.5ck sank eke nt Pea oc) 30 35 Ae distilledawater KOH.) fies: «acco o4 10 30 #, KOH + ™ glycerine in distilled water.. 2 See ell 10 19 KOH =- = Es glycerine in distilled water................ i 14 x KOE => urea andistilled water): 4... esses 5 19 , KOH i x trea. in. distilled waters... 5a | 5 14 Both the chloroform and alcohol in sufficient concentration allow the more ready entrance of NaOH (experiments 1 and 2). One-sixth saturated chloroform also retards plasmolysis by 7 urea. Such a retardation must be due to the fact that urea can enter chloroform cells more readily than normal cells. Urea penetrates normal cells slowly. A most striking effect is exerted by the neutral salts on the penetration of NaOH (experiments 3 and 4), especially with Spirogyra. A concentration which may enter in distilled water only after 10 minutes, passes the cell membrane instantly in +> NaCl. The effect on Elodea is similar. Addition of CaCl, prevents the ready permeability to NaOH®. In Spirogyra the 8 No precipitate of CaCO; is formed. THE PERMEABILITY OF CELLS 541 action is not marked but it is constant. It suggests that the effect of the pure NaCl is on the membrane, not on the NaOH. Lillie (11) has held, and especially emphasizes this in a recent paper, that the action of a pure isotonic solution is to increase the permeability of the cells exposed to the action, and antitoxic cations as Ca, prevent such an increase to a certain extent. 4. Experiments with animal cells a. Paramoecium: Although several observers have investigated the toxicity of and physiological effect of the inorganic alkalies, no study of their power of penetrating animal cells has as yet been made. It is generally assumed in consequence of marked functional alterations produced that the cell is readily permeable for them. On the contrary the permeability relations have turned out to be exactly similar to those of plants. The same two classes of alkalies may be recognized, the weak (NH,OH and amines) and the strong (inorganic hydroxides and N(C.H;).- OH), the former meeting a very slight resistance, if any, the latter a marked resistance. Neutral red was again made use of as an indicator. The Para- moecia were stained in a watch glass by adding just enough of the dye so that it is practically all taken up by them from solution. No abnormalities or functional changes appeared. If an exces- sive amount of neutral red is added the organisms cytolyse in a manner typical of NH,OH (see p. 543). The fact that Paramoecium has a mouth through which alkali may enter introduces no error into the experiments, for the mouth is not open but the point at which vacuoles form is protected by a surface film. Its osmotic properties are unknown but most probably are essentially similar to those existing over the rest of the cell. The course of an experiment is often very short and the alkali is dissolved in distilled water in which there is no food to be eaten. The change undergone by Paramoecium in alkalies is very similar to that which occurs when in the presence of a great many other toxic substances (alcohol, chloroform, chloretone, nicotin THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 542 EDMUND NEWTON HARVEY and other alkaloids, KCN and lack of oxygen (Budgett ’98) and has been designated as cytolysis. An excellent account of the process is given by Wulzen (’09). The essential effect is a change in shape, with the protrusion of clear drops (vesicles) from the surface and the separation of a more or less well defined membrane. On account of the change in shape (shortening and widening) it is difficult to say how much swelling accompanies cytolysis. The exact changes vary somewhat according to the alkali and the strain of Paramoecium used, but the sequence is fairly con- stant, as follows:— Motor reflex or avoiding reaction. Change in shape. Swim backward (not always observed). Color change appears (with NH,OH). Swim slowly in circles without making headway. Clear drops appear at surface. Swimming ceases. Drops fuse to a membrane.° Surface bursts. Color change appears (with NaOH). At a certain point the power of swimming is lost and at another point the red is changed to yellow and diffuses out of the cell. The relative times for these two events to take place is given in table 6. One drop of stained Paramoecia was mixed with 10 cc. of the alkaline solution in Syracuse watch glasses. A glass plate 2 ‘Membrane’ is used in rather a broad sense. The type of membrane depends on the Paramoecium and on the alkaliused. In Ca(OH)» the clear drops extruded rarely fuse and no definite membrane forms. In Ba(OH)2 or NaOH an irregular membrane may form but I have never observed cilia beating on it. In NH,OH a very definite membrane is lifted off, which must be the original surface film of the animal for the cilia may be seen beating on it. The original surface of Paramoecium within the membrane lifted off (in NaOH) is often perfectly clear and distinct and trichocysts may be seen beneath it. Lifting off of the whole ectoplasm plus pellicle by the accumulation of liquid beneath, occurs also in NaOH. Where this does not take place we must regard the membrane formed as either the pellicle or a haptogen film on the surface of the clear drops. The fact that this film is impermeable for NaOH points to the former alternative. THE PERMEABILITY OF CELLS 543 excluded dust and prevented evaporation. The water was redis- tilled in glass from K:Mn-sO; and NaOH and was non-toxic. The first third of the distillate was rejected. Two different cultures of Paramoecia were used and they showed characteristic differences, both as regards resistance to the toxic effect of the alkali and rate of penetration of the alkali. Different species of Protozoa show likewise quite different degrees of resistance. An Oxytricha, Chilomonas and a Colpidium in- troduced along with Paramoecium into certain of the alkaline solutions appeared quite unaffected while the Paramoecia them- selves were killed in a short time. The individuals used in experiments, the results of which are given in table 6, were large and the amines and ammonias pene- trated much less readily than in the second culture. The com- parative differences between the alkalies are constant however. In every instance the last seven substances enter Paramoecium readily and change the red dye to yellow; the first seven only enter long after all motion has ceased, and the organism is very much swollen and dead. If it burst or is crushed so that the surface is ruptured the alkali enters at once and turns the neutral red to yellow. Or if the animal’s surface is changed by CHCl; water or chloretone the alkali is found to enter immediately. A detailed comparison of the effects produced by NH,OH and NaOH on stained Paramoecium will serve to make clear the differences between the two groups of alkalies, as regards diffusi- bility through cell membranes. The second culture of smaller individuals was used in this comparison. troow NHs,OH—Paramoecia placed in this solution at first give the avoiding reaction. The movement is immediately slowed and the animals revolve slowly on their long axis first forward a short distance then backward. Change in shape begins immediately, the twist of the hind end becoming less marked. The change in color of neutral red also begins immediately; the red gradually fades and the animals are color- less in two to three minutes. Some individuals showed clear drops (vesicles) along the sides of the body in about four minutes. These individuals ceased movement in five minutes and disintegrated. The remainder (about half) simply swell often with protrusion of the NEWTON HARVEY EDMUND ah a Ker) oUBS 3UTBS oules 9UTeS JULES OSP0S ‘pojotduoo useq pry 9 eqQv} Jo SolTRy]R tayo OY} YIM syuottdxe oy} loqyye poureyqo ATUO sv YIM FFOrCHZD)N YILA pojse} oom vlooourvIeg JO UTRIYS PIT Vor a1uvs SinoT FZ 1o}fe SULAT] JUTeS Sures JUS oUuLvsS OFZOT as Jes dures OUTS sanoq #Z Joyye por puv SULATT ysour 4nq pBep Moy B isumoy g I9}Je por pure [BULIOU sinoy fZ 10yye yeurzou |isunoy 9 40zyR oules pod pus [eultou SInoY #Z% 10}; [eur -1ou :sinoy g Ia.ye pet pue yeuiou oules oures 0996 N 9 WIEVL | sanoy g 194y8 peep TV ‘sanoy Z 104;8 peep pue UaT]OMs 4sour sumoy #Z 10}JB -SUIATT pue sinog g Jeqye pel puv SULAT] Moz e@ tsmoy Z Ulygra peop Auswur imo0y | [ UL Moys AzaA o9n -UIUT [ UTTIIA [JoMs | } SIMOY FZ Iojje SULA] JSOUL {prep Moy B sInoyg g dojye per pues SUIIMAODOI {ANOY T uIqAIM pools :ajn -ULUL JT UIT FIM [Jes simoy FZ 10} JB [BuIIOU sinoy g Jojje pot ‘[eurlou sumoy FZ J9}j"8 [BUlIOU sinoy 9 19}}e poi pus [eurtou samoy $ Joye [eur0U soynurur g sojynUulUl gz | soynurm g | soynurur eg | soynulUI cg SaynurUr (Z Seynurur (2 SOyNUIUL YE SO]NUIUL YG soJNULUL CF 0821 iy) sojynurur g-) SoJNULUT G SOjNUIU ()T soynUlU F -9AOUT | saseeo b oLO'(“H70)N | queu | | J | osueyo Io[oo “(HO)eE SasBeo | JuUsTTXSAOUL S9SB00 “(HO)IS | JusTTAAOUL asueyo IO]O9 SaSBO0 quaure. our *HO)®) asueyo Iojoo sasBoo JusuUIdAOUL HOM osuByo ooo | sasBoo juoueAOUI HO®N NOILOTOS 40 NOITLVYLINADNOD 545 OF CELLS THE PERMEABILITY 9uivs aults OoUIes OUILES ouIBs | ourles eules smoy FZ 1018 [Bul -10U ‘sInoy 9 JoqjB pel pue [euriou sInoy FZ 1o}fe [BUL -10U ‘SsInoy 9 19}jB perl pus [vurli0U soy FZ Jojye [Bul -10U ‘Sdnoq g 199} pel pues j[eu0u SINOY FZ 10}JB [BUI -10U ‘sinoy g 109}e pol puv j[eurou SsInoy $Z% Ja}jB [Bur -10U ‘sInoy 9g 1a}jJe pel pues y[euiiou SINOY FZ JojyR [vUr -10u ‘sinoy g 103} pet pue yeuioU SIMOY FZ 19}}B [BUI -10U ‘SInoy 9 10}}B pel pue [eui0”u SatLoy FZ Lo} SUTAT] :noy I Je}jB ssep10joo pues ua[OMs A[}YSTIS sunoy $o 1oqye = BUTATT inoy T J9jjv ssey -10]00 puR UWaT]OMs sanoq $e 40 doqye 2 SUTATT NOY [ 10}fB sso] -I0[00 puG Uos][[OAS SINOY FZ 19}}G SULATT | fanoy T -I0]09 puB Ud][OA\s I0}}B SSo] SINOY FZ | dajye SuULATT ‘anoqg | J J0}}8 Ssop1o0foo | pue ueTfoms A]ITSTIS | SINOY FZ IoIV SUTAT | ammoy JT JejjR ssoey -10]09 pae uUs[[OMS OUles sayNUyUr g soqnurul pe sopnUrULr PT soinulUl ye SayNUIUT ()T soynulM og So]NUIU ZT S98 TNULUT (Ee SoyNULUL GT SoJNUTUL ZT soyNUlUL OF sunoy #Z Joye peus0U SInNOyY g 19jJB Ssoy | -10[00 pue [RUIIOU sa}NUyUl (g | SoJNUIUI F soynulu } soynuUlUl F sey NUIUI g soqynuyur g SOJNUTUT g soqynulta ¢ SOJNUTUL § sojnurur ¢ soqnurud J so}NuUrU g SoyNUIUL ZT sojynurur 9 soyNUTUr YG | | So{NurUr ¢*] aynuyUr | OYNULUT T SoyNUTUT Z soynurul Z SoyNULUL G’T SoyNUIUL CT OPOUTUL T OyNUTUL | So} NuTur fos | soynurUr Z saynurur ¢ SoynuUrUr G ~- 9uUTUIe sasRed ake -[AdoidosT queur | -aAout | | las aByo Ro) Co) sastveo _ quoeur | -9AOur ’ aurmepAd -O1d [GUIION 5 osunyo 1O] OD) || Oren sostaa | Oe sere qyueul -aAOUL osuvyo | r0]09 | SasBad queul -aAOUl ~(*HO)HN esuvyo | ro]o9 | a ae -(°HO)"HN quaut -dA0UL asuByo Ro) (oy) HO sasBaa (HO)*HN queul -d9AOUL J | | | | | aah ael) 1009 | Lo * sasveo HOTHN {UdUIdAOUL 546 EDMUND NEWTON HARVEY ectoplasm bearing cilia, but swim about normally although slowly for over fifteen minutes. A much more rapid rate of swimming is regained soon after the slowing, which takes place when first subjected to the action of the solution. say NaOH—Paramoecia placed in this solution swim rapidly forward but in less than one quarter minute they stop short and give the avoid- ingreaction. Many stop suddenly and swim rapidly backward. Change in shape begins immediately and in three minutes they are much shorter and broader and move slowly forward, circling about their long axis. In four minutes clear drops (vesicles) and also clear protrusions appear on the surface. The protrusions may contain many red stained granules. After eight minutes some individuals have burst and the neutral red, which up to this time has been red, immediately turns yellow. Most of the Paramoecia burst before twelve minutes but a few are still mov- ing very slowly and appear as red as when first placed in the solution. Essentially, the series of changes undergone in the two solu- tions is the same. The NH,OH meets no resistance at the sur- face and the red color may be seen to change gradually to yellow, which diffuses out leaving the organisms colorless, from the moment they are placed in the solution. The change is comglete before droplets appear at the surface or movement ceases. NaOH does not enter until after movement has ceased and the organism is enormously swollen and has lost all semblance to its original shape. Red granules may be present directly under the surface yet they remain red. Once the NaOH does begin to enter it does so rapidly and what is left of the Paramoecia becomes first yellow then colorless, in less than two minutes from the time the alkali begins to pass In. We must draw the conclusion, that the NaOH produces the changes in behavior, the vesicle formation, the cessation of move- ment and the final death of the animal, all by an effect on its sur- face membrane. So long as NaOH alone was studied I could never be sure that a small amount of NaOH, too small to affect the red stained granules, did not enter and was not responsible for the observed changes. But the comparison with NH,OH shows how low the OH ion concentration that is required to de- compose the red granules really is provided the alkali may enter THE PERMEABILITY OF CELLS 5047 freely. Ammonia, likewise, must affect the membrane in time since it produces change in behavior, vesicle formation, cessa- tion of movement and finally death, but the changes produced bear no relation to the time of entrance. Even in very dilute solutions NH,OH and the amines are able to ‘decolorize’ stained Paramoecia but it takes a longer time. Yet in equivalent molecular concentrations of KOH, NaOH, etc., the animals sometimes retain their red color for 24 hours. Generally they are found to be colorless in that time. The de- colorization is not due to the slow entrance of alkali because the same red individuals placed in distilled water are found to be colorless after 24 hours, although otherwise unchanged. A com- parison after a shorter time, six hours, must be made. The results are given in table 6. NH,OH enters the cell readily and sets free a small amount of the neutral red base from its granule combination. The freed base diffuses out into the medium and more NH,OH enters. Thus the process is repeated uatil the organism is quite colorless. That the same decolorization does not take place in KOH must be due to the fact that the KOH does not enter freely. In very weak concentrations (;:%1,) NH,OH fails to affect the red color of stained Paramoecia at all. This concentration is presumably below the limit necessary to free the neutral red from its combination with the granules. b. Marine eggs: In the following experiments the eggs of both Toxopneustes and Hipponoé were used. The sea water at Boca Grande,"! where the experiments were performed is markedly alkaline to neutral red and faintly so to phenolpthalein. If Toxopneustes eggs, unfertilized, are placed in 100 cc. sea water + 1.2 cc. X, NaOH sufficient alkali does not enter them to turn the neutral red yellow for over three hours. If chloroform is added to the sea water, the eggs almost instantly turn yellow. Chloroform likewise causes the eggs to swell (cytolysis), an effect prevented in plant cells by the presence of a cellulose wall, and the penetration of the alkali might be connected with the swell- 11 About twelve miles west of Key West and sixty miles east of Tortugas. DAS EDMUND NEWTON HARVEY ing. The following experiment in which cane sugar is added to the sea water shows that in the presence of chloroform the alkali may enter the eggs before swelling of the egg has begun. Sugar prevents rapid pushing out of the artificial fertilization membrane which is relatively impermeable to it. A. 10 ce. (50 ec. sea water + 10 cc. 2 m cane sugar) + 0.15 ec. X NaOH saturated with cholorform. Neutral red stained eggs are turned yellow in 3-4 minutes. After about 5 minutes swelling is noticeable. If an acid is added the eggs are turned pink again. B. Control (10 ee. (50 ce. sea water + 10 cc. 2 m cane sugar) BV koe. See O) ale Red stained eggs remain red for over three hours, in the mean- time undergoing irregular division and fragmentation. Even very small fragmented spheres retain their red granules intact, providing their surface is likewise intact. Just as in Paramoecium, observation of the manner in which the color change occurs points to the view that the alkali only enters after it has destroyed the surface. In immature Penta- ceros eggs the red staining graaules are present at the periphery separated from the alkaline solution only by the surface film of the egg. Yet they remain red for 15 minutes in ,¥, NaOH in 0.6 n NaCl. Once the NaOH begins to enter the color change is very rapid and the egg swells simultaneously. The same is true of Toxooneustes where the red granules are uniformly distributed. There is never a gradual entrance of alkali from the moment the eggs are placed in the alkaline solu- tion but after a certain interval the NaOH passes the surface and then it may be seen to move rapidly within the egg. Toxopneustes eggs even undergo irregmar division in hyperal- kaline sea water (100 ce. sea water + 1.3 ec. 4, NaOH) without the entrance of enough alkali to change the red to yellow. I have not experimented with NH,OH but it is probable, judging from my Paramoecium experiments, that this alkali would pass into the eggs freely and induce the color change before division, cytolysis, or any injury to the egg takes place. If such were the case it would show that the action of NaOH as a parthenogenetic THE PERMEABILITY OF CELLS 545 agent must be on the egg suriace alone and not the catalytic acceleration of any reactions within the egg through an excess of OH ions. A resistance to the entrance of NaOH is likewise shown by the eggs of Holothuria Floridana, Hipponoé esculenta, Pentaceros, reticulatus, and Asterias vulgaris. A comparison of the entrance of alkali into fertilized and un- fertilized eggs has shown that the fertilized eggs of Toxopneustes are much more readily entered by NaOH just after fertilization and again about the time of first cleavage. Only one experiment was performed toward the end of the breeding season and the eggs of the female used cleaved in the control in many instances somewhat irregularly. A stock solution of ,¥, NaOH in 0.5 m NaCl was made up. The unfertilized and fertilized eggs at intervals after fertilization were compared with each other as to entrance of NaOH, in separate watch glasses over a white back- ground. It is thus easy to see when all the eggs have been en- tered by the alkali, and their original red color changes to yellow. The following table shows the result. seevaenesrmecizinros | 10 AUR emamzety | Vantaa auEe ornare min. min. min. 2, 13 19 5 14 21 10 19 19 20 20 22 30 21 21 45 ie 20 55 21 21 65 20 20 The eggs begin to cleave about 45 minutes after fertilization. A somewhat similar result is obtained by comparing mature Asterias eggs fertilized and unfertilized, as well as eggs treated with acetic acid. The latter form artificial membranes. The results are shown in the following experiment. 550 EDMUND NEWTON HARVEY s = , NaOH in Lo | 0.6 m NaCl. SIRE AU 1 0 5 See Mes Ls} pee soe min. | min Fertilized eggs (4 minutes after fertilization)......... 5 | 15 Fertilized eggs (20 minutes after fertilization).......... 6 | 20 Wntertilizediescs (Control) eau eere ee eee. 11 30 Unfertilized eggs (5 minutes after acetic treatment)... 6 20 Contrary to the results obtained in the Toxopneustes experi- ment it will be noted that the starfish eggs did not regain their original resistance to NaOH a short time after fertilization. While it is of course true that in time NaOH may enter sea urchin eggs as well as Paramoecium or plant cells, and in this ‘sense they are difficultly permeable, it seems better, in consider- ing the inorganic alkalies and N(C2H;),OH, to speak of a re- sistance of the cell against their entrance rather than a permea- bility of the cell for alkalies as I have done in a preliminary re- port (10). The change undergone at the time of fertilization results in a surface less resistant to the penetration of alkali. At the same time the decrease in resistance for alkali is presum- ably connected with an increases in permeability for other sub- stances notably the salts of sea water, as indicated by the experi- ments of McClendon (711) and Lyon (’10). In working with Elodea (p. 539) I was able to show that small concentrations of chloroform which inhibited the protoplasmic rotation, but without any irreversible changes, increased the rate with which NaOH entered the cells. Exactly the same fact may be shown for the sea urchin’s egg as the following experiment indicates. Unfertilized Hipponoé eggs stained in neutral red were placed in these solutions. A. Ny; NaOH, + saturated with chloroform, in 3 m NaCl. B. 3; NaOH in 3 m NaCl. . C. 4 saturated chloroform in ? m NaCl. D. 4%; NaOH, 1 saturated with ether, in 3 m NaCl. E. + saturated solution of ether in 3 m NaCl. In solution A the alkali has turned the eggs yellow in 10 minutes, in D in 6 minutes and in Bin 20 minutes. Eggs in the chloroform THE PERMEABILITY OF CELLS 551 control, C, were uncytolyzed in one hour and about one-half of them cytolyzed in the course of two hours. Eggs of the ether control, E, were unaffected in 30 minutes and one-half of them eytolyzed in 45 minutes. DISCUSSION In an extensive paper, Barratt (’04) has studied the action of both acids and alkalies on Paramoecium; my results on alkalies are in fair quantitative agreement with his. Barratt came to the conclusion that neither the alkali nor acids produced their effect by a catalytic splitting of any substances in the organism but by a combination of the acid and alkali with the protoplasm. It was proved that the concentration of acids and alkali decreases in solutions in which a large number of Paramoecia had been placed. Three methods of determining this were used, viz.: (1) the use of an indicator, (2) measuring the electrical conductivity, (3) (05) determining the E. M. F. by means of hydrogen elec- trodes. Only a relatively small amount of acid and alkali com- bines. One hundred parts of living Paramoecium take up 0.25 parts of HCL and 1.5 parts of NaOH. I fully agree with Barratt that the toxicity of the alkalies bears no relation to the OH ion concentration. The order of toxicity for Paramoecium is N(C.H;),OH (?) found that, within certain limits, a rise of 10° of temperature caused a doubling of the rate of contrac- tion, while the results of Khainsky,?° 1910, showed that in Par- amaecium caudatum the vacuole contracted 2.86 times per min- ute at 16° C., 6 times per minute at 23° to 25° C., and 10 times per minute at 33° to 34° C., under the conditions of the experiments. It is clear then that excretion products in the case of many organisms have a profound effect on cell division and growth, and it is also clear that under favorable conditions of food and tem- perature the Infusoria excrete considerable amounts of carbon dioxide, together with various other end-products of metabolism, which may reasonably be expected to be evident through bio- logical as well as chemical tests. The ordinary ‘hay infusion’ teeming with animal and plant life is a microcosm in which every organism may and probably does in some degree affect the well-being of every other organ- ism present. Besides the obvious influence exerted by animals in feeding on other forms and by green plants through photo- synthetic processes, one would expect the effects of organisms on their environment by the elimination of products of their metab- olism, or excretion products, to be one of the most important. The interdependence of the organisms of a hay infusion is so com- plex that, taken as a whole, it is almost beyond the possibility of analysis, and accordingly the logical method of approach to the subject is to study the interaction of isolated organisms and small groups of organisms on themselves and on each other. The present paper presents the results which have been obtained from the study of the effects on the rate of reproduction of Para- maecium of: 1. Different volumes of culture medium; 2. Changing the culture medium at twenty-four hour and at forty-eight hour intervals; 3. Culture medium in which rich growths of paramaecia have occurred. 2° Kanitz, Biol. Zentralblatt, Bd. 27, 1907. 26 Khainsky, Archiv. f. Protistenkunde, Bd. 22, I, p. 1, 1910. EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM — 9563 EXPERIMENTS The organisms used in this work were from my pedigree cul- tures of Paramaecium aurelia and Paramaecium caudatum. ‘The experiments were begun on July 24, 1910, when the P. aurelia culture was at the 1903d generation and the P. caudatum was at the 113th generation, and were concluded on October 4, 1910, when the P. aurelia culture was at the 2070th generation and the P. caudatum culture was at the 288th generation. Emphasis is placed on the fact that the animals which formed the subjects for the experiments had been under daily observation for over forty months in the case of aurelia (which was the main culture employed), and over three months in the case of caudatum (which was used in certain experiments for com- parison). Consequently their rate of reproduction, and the exact conditions to which they had been subjected for nearly three and a half years, were known in the case of the main culture. Further, since the pedigree cultures were each originally started with a single individual, all the P. aurelia used in this work were ‘sister cells,’ and all the P. caudatum used were ‘sister cells.’ Therefore all the experiments were performed on the ‘same pro- toplasm’ of the respective species. Fig. 1 shows graphically the average daily rate of division of the four lines of the P. aurelia culture again averaged for each month of its existence to the time it was employed for this work.?7 1. The effect of different volumes of culture medium on the rate of reproduction of Paramaecvum A series of four experiments, two of sixteeen days duration and two of twenty days duration were made with P. aurelia. In all the work it was found that sixteen to twenty days was the most suitable length of time for the experiments, because those of less than sixteen days appeared too short to give conclusive results, and in those which were extended beyond twenty days, the ani- 27 For details of these cultures see Woodruff, Biol. Bull., 16, 4, 1909; Archiv f. Protistenkunde, Bd. 21, 3, 1911; and Jour. Morph., vol. 22, 2, 1911. LORANDE LOSS WOODRUFF 564 ‘94Bp OF BIN}[ND OY} JO OII[ OY} JO yYZUOW YORE TOF pasv1sAvy ulese ‘oANg[N9 OY} JO SOUL] ANOJ oY} JO UOISIATP Jo JVI A[LVp osdVIOAV oY} JUOSOAdoI So}VUIpPLO OYJ, “UOTYBIOUSS YIOOOS 94} 48 “OIG ‘TZ Joquieydag 07 “OST “T Avy uO 4xe4s WOT; ‘T aIN|[ND “vIjedne wuNTOevUTVIRG Jo AIO4SIY ayo[dUIOD =| “SIT 0002 O06 O08! = 00L! 9091 ler 008! 0b! O0e! 002! OO! 00! bOI 006 008 O0L 009 005 O0b 2061 008 002 0 “161 “das Wt Tf ‘Wt ACH Ob Le i) Ig ig S¢ 02 Gl a fl Ol l i] l EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 565 mals on the various amounts of medium were about thirty genera- tions apart, so that it might be suggested that this offered an ob- jection. The volumes of medium selected were two, five, twenty, and forty drops. The same pipet was used in measuring the liquid in all the work, and so practical uniformity was attained. Infusions of hay were used as a culture medium and the organisms were isolated on slides of different capacities, depending on the amount of liquid employed. ‘The slides were kept in moist chambers to prevent evaporation. The daily rate of division of the organisms in two, five, twenty, and forty drops of culture medium changed every twenty-four hours showed that, for example, those in five drops divided 2.4 per cent more rapidly than those in two drops, those in twenty drops divided 6.4 per cent more rapidly than those in two drops, and those in forty drops divided 7.4 per cent more rapidly than those in two drops (fig. 4, part A). The details of each of the four ex- periments (A, B, C, D) are evident in fig. 2 which shows the rate of division of each of the lines on the different amounts of medium averaged for each four days of the experiment. Experiments B and C are the most important ones because these comprised cultures on all the volumes of medium. A was carried to test the general method to be used, and D was carried to check up certain data. It is believed that the experiments are sufficiently comprehen- sive clearly to establish the fact that the rate of reproduction of specimens from pure lines of paramaecia, when bred under identi- cal conditions of temperature and culture medium, is influenced by the volume of the culture medium (within the limits tested vn the experiments), and that the greater the volume the more rapid is the rate of division. It being clear that in an increased volume of culture medium there is an increased division rate, the next point of importance is to determine to what factor or factors this is due. It is evident that it may be brought about by variations in 1, temperature; 2, pressure; 3, surface of medium exposed to atmosphere; 4, food supply; 5, excretion products of bacteria; or 6, excretion products of paramaecia. LORANDE LOSS WOODRUFF 566 -= sdoip Aqaoy ‘:---= sdoip Ajuomy ‘ =sdoip oAy ‘----=sdolp 0M} Ul UOISTAIpP Jo oyey ‘spotsod Avp aNOF IOF pesv1oAV ULVSE ‘TUNIPSU JO SOUUNJOA 9ATJOOdSaI VY} Ul SUISTURB.LO JO SOUT] INOF OY} JO WOTSTATP Jo 94¥a ATIBp ISVIBAB OY} JUesotdad soyvUIpIO VY, “WoT}oONporder jo oy¥vA Itay} UO ‘sInoy “nof-Azwanj AJOAO posuByo ‘WNIPe 941N}[ND JO SBUM[OA JUdJoyIp Jo yooyo oy} ouruttajep 04 (q ‘O “gq ‘Y) S}UIUTIOdxX9 INOF JO Solos OY} UL VI[BING UNIIOVWIBILY JO UOISTAIP JO 04¥1 VY} JO plooay = Z “BI i ©) : i Vv EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 567 The experiments were conducted simultaneously and in thesame place for all the volumes of medium, and therefore the temperature was the same. It is believed that the exceedingly slight pressure variations in the different volumes of water was without appreciable effect. There is certainly no evidence extant that free-living protozoa are sensitive to such exceedingly small changes, and therefore this factor will be dismissed as not entering into the effects noted. The question of increased surface exposure to the atmosphere in the larger volumes of media employed facilitating the exchange of gases, as oxygen and carbon dioxide, is possibly one of more importance, and accordingly care was taken to use receptacles of different capacities and shapes for the different volumes in an attempt to equalize the proportion of surface to volume of the different amounts of medium. Obviously it is practically impos- sible to make them actually equal, but certainly the precautions taken were sufficient to render this factor negligible. The food supply is obviously a factor of great importance. The culture medium consisted of an infusion of hay which was raised to the boiling point to eliminate the possibility of contam- inating the culture with ‘wild’ paramaecia, and was used after it had cooled. No precaution was taken to make the infusions exactly the same each time, but the same infusion was used for all cultures at the same time. This was made up fresh every forty-eight hours, beginning with the first day of each experiment, thus, in those experiments in which the medium was changed every twenty-four hours, the organisms were transferred to some of the medium still remaining in the stoppered flask from the day before. This flask was shaken before it was used, and there is every reason to believe that the organisms in each amount of medium received exactly the same food. Slight variations in the bacterial flora were avoided, it is believed, by the fact that at the beginning of each experiment all the paramaecia came from the same environment, and consequently the bacteria transferred with them when they were isolated would presumably be the same in each case, and if they were not the same, or if the new in- fections from the air, which must have occurred in the course of LORANDE LOSS WOODRUFF 568 -— -— + = sdoip Aj10] ‘---- = sdoap Aquemy ‘—— = sdoap ody ‘---- = sdoup 0M} Ul UOISTATp Jo oyvy ‘spotted Avp MOj LOf posvloav UILSB ‘UINTPOUT JO SOUINIOA DATJDOdSoL BY} Ul SUISTUBSLO JO SOUT[ INOJ oY} JO UOISTATp Fo oyea ATLVp OSBIDAV OY} JUOSIdad SoJVUIPAO OY, “Uoryonpoddead Jo ova troyy uo ‘sanoy 7Yyb2a-Ajsof AtoAe posuryo ‘UINIpetT o1N}]Nd JO SoUM[OA JUOLOFIp Jo JooHe oy} ourutlojop 04 (‘g ‘DH “gq ‘V) S}uoUL -110dxX9 INOJ JO Salsas oY} UL VI[VING UINIOIVWIBIG JO UOISTAIP JO 031 OY} JO plOooY | “SITY i) ©) gd Vv EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 569 the experiment, tended to render the media of the lines different, this variation was eliminated by cross infections of all the lines of all the cultures daily at the time of isolation. It is believed then, that every practicable precaution was taken to ensure as near absolutely the same food conditions as it is feasible to obtain, and consequently that the results have not been influenced by varia- tions in nutrition. It is not apparent that the consistent variations in the division rate in the different volumes of medium can be the result of the products of metabolism of bacteria, in view of the great care taken to maintain identical flora in all the preparations. It is possible, however, that slight irregularities in the curve may be explained on this basis and this point will be mentioned later. The remaining factor to be considered is the possible effect of the excretion products of the paramaecia themselves. That the infusoria in their ceaseless activity are continually voiding ap- preciable amounts of carbon dioxide and other products of me- tabolism is evident from the work of previous investigators. The question then is—is the amount excreted sufficient to affect the division rate appreciably under the conditions of the experiments? Taking the average rate of division during the experiments as one and one-half divisions per day, the average number of or- ganisms in a preparation during the first twenty-four hours would be one and one-third—one during the first sixteen hours, and two during the following eight hours. However improbable it may seem that this number of individuals can excrete sufficient toxic substances to have an appreciable effect on the division rate when diluted, for example, by twenty drops or by forty drops,?8 I believe the experiments outlined make this highly probable, and point to the conclusion that the variations in the daily division rate in the different volumes of water is due to the excretion products of the paramaecia themselnes. 28 With the division rate increasingly more rapid with increase of volume, there would actually bemore excretion products in the larger volumes than in the smaller. This however can be disregarded. 570 LORANDE LOSS WOODRUFF Fig. 4 Summary of the results of the experiments plotted in figs. 2 and3. A shows the per cent gained in division rate by Paramaecium aurelia in five, twenty, and forty drops of medium changed at fwenty-four-hour intervals, over the divi- sion rate of those in two drops changed also daily. B shows the per cent gained in the division rate by P. aurelia in five, twenty, and forty drops of medium changed at forty-eight-hour intervals over the division rate of those in two drops changed also on alternate days. EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM o71 2. LE ffect of changing the culture medium at twenty-four and forty- eight hour intervals, in each of the several volumes, on the rate of reproduction of Paramaecium If the conclusion reached from the previously outlined experi- ments is true, the effects of the excretion products should manifest themselves more clearly in cultures in which the organism remained in the medium a longer period, than in those in which the organ- ism remained in the medium for a shorter period of time. To test this a second series of experiments was carried out simultaneously with those already described, and in this second series the animals remained in the same medium for forty-eight hours instead of twenty-four hours. There were, then, the following series of cultures involved in this experiment: Ad 2 =P. aurelia in 2 drops of medium changed daily. Ad 5 =P. aurelia in 5 drops of medium changed daily. Ad20 =P. aurelia in 20 drops of medium changed daily. Ad40 =P. aurelia in 40 drops of medium changed daily. Cd 5 =P. caudatum in 5 drops of medium changed daily. Aa 2 =P. aureliain 2 drops of medium changed on alternate days. Aa 5 =P. aureliain 5 drops of medium changed on alternate days. Aa20 =P. aurelia in 20 drops of medium changed on alternate days. Aa40 =P. aurelia in 40 drops of medium changed on alternate days. Ca 5 =P. caudatum in 5 drops of medium changed on alternate days. Each of these cultures comprised four separate lines, e.g., Ad2—1, Ad2-2, Ad2-3, and Ad?-4, and the number of divisions of each of these four lines was recorded daily, at which time a single organism was isolated in fresh culture medium. Conse- quently the rate of division of Ad? is the average rate of division of the four lines comprising it. The culture in which the medium was changed at forty-eight- hour intervals showed that the organisms in a volume of five drops divided 5.3 per cent more rapidly than those in two drops; those in twenty drops divided 9.3 per cent more rapidly than those in two drops, and those in forty drops divided 9.25 per cent more rapidly than those in two drops (fig. 4, B). The details of each of the four experiments are graphically shown in fig. 3, Hi2 LORANDE LOSS WOODRUFF which gives the rate of division of each of the lines in the dif- ferent volumes of medium averaged for each four days of the experiment. This series of experiments covered seventy-two days and was run simultaneously and coextensively with those already outlined with which it is compared, and all the conditions to which each was subjected were identical, the only factor requiring special mention being the food content of the culture medium. It is stated in the description of the cultures of the Ad series of experi- ments that the culture medium was made up on alternate days— so that the organisms in this series were continued for two days on infusion that was made up at the same time, but which was supplied fresh (from the stock flask) at the end of the first twenty-four hours. Thus the only difference in the treatment of series Aa and Ad was that the change to a second supply of the culture medium was not made at the end of twenty-four hours. Therefore the only difference in the environment of series Aa aod Ad, at the beginning of the second twenty-four hours, was that Ad was put in a fresh portion of the medium in which it had been for the past day, and consequently a medium not contaminated by its own excretion products, whereas Aa was continued for the next twenty-four hours in the same portion of medium in which it had been living for the previous day. It is believed that cross infection rendered the bacterial flora of the infusion in the supply flask and that on the Aa culture slides essentially the same and observation also showed that the supply of bacteria on the cul- ture slides was ample. The results, then, of this series of cultures in which the organisms were isolated every forty-eight hours, confirms the general result de- rived from the series isolated every twenty-four hours, 1.e., that an increased volume of medium is conducive to more rapid muutvpuca- tion,2® and further it clearly shows that the gain in rate of division 29 Attention is called to the fact that Aa-20 gained 0.05 per cent more over Aa-2 than did Aa-40 and therefore is an exception. Cf. fig. 4, B. It is possible that another factor enters, or at least becomes perceptible after twenty-four hours in a volume as large as forty drops. The bacteria in this quantity of culture fluid may EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 5 ~J CO Fig.5 Record of the experiments on the effects of changing the culture medium at twenty-four-hour intervals and at forty-eight-hour intervals, when Paramae- cium aurelia is bred intwo dropsof hay infusion. The ordinates represent the aver- age daily rate of division of the four lines of organisms, again averaged for four day periods. Medium changed at twenty-four-hour intervals= ; changed at forty-eight-hour intervals = - - - -. A B C Fig. 6 Record of experiments on the effect of changing the culture medium at twenty-four-hour intervals and at forty-eight-hour intervals, when Paramaecium aurelia is bred in five drops of hay infusion. The ordinates represent the average daily rate of division of the four lines of organisms, again averaged for four day periods. Medium changed at twenty-four-hour intervals = ; changed at forty-eight-hour intervals = -- --. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 10, NO. 4 574 LORANDE LOSS WOODRUFF of Aa-5, Aa-20 and Aa-40 over Aa-2 is in every case greater than the gain of Ad-5, Ad-20 and Ad-40 over Ad-2, (cf. fig. 4). Again, from a consideration of the data of comparable cultures changed daily and that of cultures of equal volumes of media changed on alternate days, it is found that the gain of the series changed daily over those changed at fory-eight hour intervals is over eight per cent in the case of two drops and slightly over six per cent in the case of five, twenty, and forty drops. Consequently, as one would expect, changing the medium on alternate days has most influence in the smallest volume of medium (for details ef. figs. 5, 6, 7 and 8). The results of some of the experiments performed, for control and comparison, on the P. caudatum culture are plotted in fig 9, and a glance at this will show that they are perfectly consistent with those derived from the work on P. aurelia. A further check on the work is also brought out in the first series of experiments (A) plotted in fig. 9. At the end of the fifth period of this ex- periment, another culture was isolated, line by line, from Ca which was designated Cad, and thereafter in this the medium was changed daily. The result, as shown in the diagram, was that the organisms within the following eight days attained the same division rate as that of the culture Cd, thus clearly indicating that the variation in the division rate between Cd and Ca was effected by the duration in time to which they were subjected to the culture medium. Further, as a control to test the accuracy of the general method employed in all the work, a duplicate control culture, designated Ad-5dup, was carried and the number of divisions in this culture at the conclusion of the work was exactly the same as Ad-5. Of course it was an ‘accident’ that there should have been no varia- tion during the long series of experiments, but this indicates that such error as exists in the method used is very slight, and develop so fast that they exhaust their own food and produce excretion products in sufficient amount to be detrimental to the paramaecia, whereas in the smaller volumes of medium the animals keep the bacteria reduced so that they do not ex- haust their food and so continue to multiply, providing food for the paramaecia, but not sufficient excretion products to have a perceptible effect. EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 575 Fig. 7 Record of experiments on the effect of changing the culture medium at twenty-four-hour intervals and at forty-eight-hour intervals, when Paramaecium aurelia is bred in twenty drops of hay infusion. The ordinates represent the aver- age daily rate of division of the four lines of organisms, again averaged for four day periods. Medium changed at twenty-four-hour intervals= forty-eight-hour intervals = ----. ; changed at A B C Fig.8 Record of experiments on the effects of changing the culture medium at twenty-four-hour intervals and at forty-eight- hour intervals, when Paramaecium aurelia is bred in forty drops of hay infusion. The ordinates represent the average daily rate of division of the four lines of organisms, again averaged for four day periods. Medium changed at twenty-four-hour intervals; = ——; changed at forty-eight-hour intervals = ----. LORANDE LOSS WOODRUFF 576 "----= S[VAJo}UL INOY-JYSIa-AyAOJ Ye pasuByo SS puvw —— = S[¥A10jUI ANOY-ANoj-AYZUIMy Jv pasuvyo unIpeyy ‘sported Avp anoj IO} pasviaae UIBSB ‘SUISTUBSIO JO SOUT] INO} oY} JO UOISTAIP 9431 A[Iep ssBIOAG ay} JUOSeIdaI SoywUIPIO OY, “WOISNsuUt Avy jo sdoip aay ul poiq st uNnyepneo WNOsvUIBIB UsYM ‘STBAIOJUL NOY-JYSle-AJ1Oj YB PUB S[BAIO}UL INOY-INOJ-£}UIM} YB UINIPUL 91N}[Nd sy} SULSuUBYO Jo JooHe oy} UO syusuMtedxe Jo ploovoy 6 “SI Q ©) g Vv EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM 577 that the differences in the division rate in the experiments are far beyond the limits of error. It is clear then that all the data derived from the experiments outlined point to the conclusion that paramaecia excrete substan- ces which are toxic to themselves and that these substances are more effective, as one would expect, when the organisms are confined in limited volumes of culture medium. The logical method of pro- cedure is to determine the influence of media known to be con- taminated with the excretion products of large numbers of par- amaecia. 3. The effect of media in which rich growths of paramaecia have occurred on the rate of reproduction of Paramaecium As a culture medium for this experiment an infusion of chopped hay was made and boiled. After a thorough stirring, equal vol- umes were poured into sterile flasks of a capacity of 250 cc. One of these flasks was then seeded with five cc. of infusion containing twenty-five P. aurelia from the ‘stock’ left over from the pedi- gree culture, and the other flask was seeded with five cc. of the same infusion, from which the paramaecia had been removed. The flasks were kept plugged with cotton. There were then two flasks containing precisely the same culture material and bacterial flora, and the one differed from the other only in the presence of paramaecia. After these infusions had stood for ten days there was a heavy growth of paramaecia in one and none in the other, and the media were ready for the experiments. From the four lines of the pedigree culture of P. aurelia two separate cultures were isolated (designated A+P and A —P respectively), one of which was bred on hay infusion from the paramaecia-free flask (F —P), and the other on material from the flask inoculated with paramaecia (/+P). It was necessary, of course, to remove the paramaecia from the culture medium before it was used in the experiments, and this was done daily by filtering it through filter paper before it was used, and then examining it carefully under the microscope, and picking out with a pipet the few animals which had not been retained by the filter paper. As a precau- 578 LORANDE LOSS WOODRUFF tion, the infusion from the flask minus the paramaecia was simi- larly filtered so that there could be no possibility of error through the filtration process affecting the bacterial content or contami- nating the infusion. Further, since in the culture medium f’ +P the paramaecia had undoubtedly decreased the number of bac- teria present through feeding on them, the fluid, after the ani- mals were strained out, was inoculated with bacteria from the medium / —P, and as an added precaution /—P was inoculated with bacteria from +P. Thus, while the bacteria were reduced in +P through being eaten by the animals, this medium un- doubtedly contained more food for bacteria, and therefore, when inoculated, should develop bacteria more rapidly than the * —P culture medium, and therefore there would be at least as much food for the organisms on which the experiments were made in F+P asin F—P. It is believed then that, when the media were employed in the experiments, they were practically identical except that one con- tained the products of metabolism of a heavy growth of paramae- cia while the other was absolutely free from such contamination. The results of this experiment are shown graphically in fig. 10. A shows the results of a preliminary observation which includes the daily isolations for four days. B illustrates the results from another and longer experiment made by the reisolation of both series from the main pedigree cultures A. C gives clearly the same general results which were obtained when the whole experi- ment was repeated, this time with new flasks of culture medium, etc. As a further check on the results, fig. 10, B shows that, when after eight days subjection to the / +P medium, another culture was isolated line by line from it and put on the / —P medium, the division rate approached the rate of that continuously on the fF —P medium. And again when still another culture was iso- lated from the culture recently transferred from the F —P medium, and put back on the # +P medium, the rate of division of this new culture approached that of the series which had been continually on the #+P medium. A short series of experiments for comparison was made with 579 EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM eee eee =(d+) sjonpoad worjotoxe YIM wNIpeut oanyjno ‘— — — — = (q—) wnWerurieg jo sjonpoid worjotoxe oy} WOT} 9oIf WINIP -OUL 91N}[NO UI STUSIUBSIO JO WOISTAIP JO 9}¥Y ‘sporsod Avp INOF OF posvs9Av ULBSB ‘SUISTUBS.IO JO SOUT] INOF 9Y} JO WOTS -IAIp JO o3vd ATIvp osBI19Ae ayy quoseider so}VUIpLO OY, “BIpedne ‘gq jo uoronpordod Jo o}vt oy UO “BITAINe WNosBUIe -I¥q JO YMOIS Yort B poy1oddns svy YOryA “UIMIpeut v JO JooHe oY} OUTULIeJop 0} S}USUMTIgdxe oY} Jo pLod9Y OT ‘SI Ol 0) qd V 580 LORANDE LOSS WOODRUFF the same two culture media on the pedigree culture of P. caud- atum, and this gave the same general result. In this experiment the animals on the F+P medium died out (fig. 11). It is obvious then from these data that culture media in which Fig. 11. Record of an experiment to determine the effect of a medium, which has supported a rich growth of Paramaecium aurelia, on the rate of reproduction of P. caudatum. The ordinatesrepresent the average daily rate of division of the four lines of organisms, again averaged for four day periods. Rate of division of organisms in culture medium free from the excretion products of P. aurelia (—P) = --- , culture medium with excretion products (+P) =---- paramaecia have been living has a decidedly depressing effect on the rate of reproduction of paramaecia of the same pure pedigree stock as the contaminating animals, as well as on the rate of reproduc- tion of another species, P. caudatum. EFFECT OF EXCRETION PRODUCTS OF PARAMAECIUM OD81 CONCLUSIONS In this paper are presented the initial experiments of a series which is planned to elucidate, if possible, some of the complex factors at work in a ‘hay infusion,’ for example, such as those which determine the interdependence of the organisms, their sequence, time of appearance and disappearance, etc. The data outlined were derived from the study of the following points: (1) Theeffect of different volumesof culture medium on therate of reproduction of Paramaecium; (2), The effect of changing the culture medium daily and on alternate days on the rate of repro- duction of Paramaecium; and (3), Theeffect of culture medium, in which many paramaecia have been living, on the rate of reproduc- tion of Paramaecium. It is believed that the results obtained justify the following conclusions: 1. The rate of reproduction of Paramaecium aurelia and Para- maecium caudatum is influenced by the volume of the culture medium, within the limits tested, and the greater the volume the more rapid is the rate of division. 2. Paramaecia excrete substances which are toxic to themselves when present in their environment, and these substances are more effective when the organisms are confined in limited vol- umes of culture fluid. 3. The excretion products of paramaecia play an appreciable part in determining the period of maximum numbers, rate of decline, etc., of this animal in ‘hay infusions.’ ‘ bil é, vf 7 : ‘J i = j rt i» ‘ i¢ a . ) . 1¢'h ° ye 4e 7 2 ] - i } -” > = Ps ‘ ae f , ie ST Beh. , i: 5 me Aly ; “a ye! at Vit a . P , vy / aa ne , pdgas'y Tah ; : , Pie ehh | P yn i, or if s SY: ' ry bee ne i mi pin ae ger a spy Brits! | oe i Mal | OPAL Bo a O60 biphd wih , LI as wilh as i a ey 5 ye : fh) va PATE . ’ *£SeS= 2 sa ate i ie Soe ee ghee ie Caos _-F - a ; ud P94 pe Boe a etches beat “yf Tet oat ee, Hane