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Wee oe eee we ewe ates 265-7 we re et et hii ieee poss $i 9 0528 ee o-Ps thek abe ppreees tel “ae pores or he f +h hens at re% lets! rie 1 i titit! - ant AM 14 1 [ts35 eieseirsetetrerars F. ot SEN MAL,” - ee en etek K H Le fe De San Beet af eg Dh ee ates VP ery THE JOURNAL EXPERIMENTAL ZOOLOGY EDITED By WiuuiaM E. Castle Jacques LorB Harvard University The Rockefeller Institute Epwin G. CoNnKLIN Epmunp B. WILSON Princeton University Columbia University CuarRLES B. DAVENPORT Tuomas H. Morcan Carnegie Institution Columbia University HERBERT S. JENNINGS GrorcEe H. PARKER Johns Hopkins University Harvard University Frank R. LILuiz RAYMOND PEARL University of Chicago Johns Hopkins University CHARLES R. STocKARD Cornell University Medical College and Ross G. Harrison, Yale University Managing Editor VOLUME 36 JULY—NOVEMBER, 1922 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. . : 5s oh) #) f wy c.meyee pe . ; y } iy ’ ; ; ; w£; x 7? Aw iL | #/9as rite k \ > ‘\y are ue Fecniv een a t= ¥ 7 > , of yey ° > * a i Foe i “a% “ i gew ey ' : rae a dedieg: i olf , i“ TAL ; vty? ih ; ve “ ‘. ‘ ; Fa i ‘ines Here * me a a : le 3 a WV pat * ie f ad iu awlavl | Le pero : | he | i . ~ OSV We Ok OS niall) hi aa CONTENTS No. 1. JULY J. Witxt1amM BucHanan. The control of head formation in Planaria by means of anestheties.. Three figures =... 2. <> scerteivesis oes saps Mela geet ul J. M. D. Otmstep. The role of the nervous system in the regeneration of polyclad, Lurbellaria, Nine figuress. Sic vcldados Saag ee glace eis b+ yell 49 J. M, D. Oumstep. The role of the nervous system in the locomotion of certain marine polyclads. 2... 0.5.04 seme o.- + dese ates ee een e ee 57 LEONELL C. Strona. A genetic analysis of the factors underlying suscepti- bility to transplantable tumors. Thirty-three figures.................. 67 No. 2. AUGUST Rourn L. Purures. The growth of Paramecium in infusions of known bac- terial content. One figure. .... 2... ...0...%: asaete Soot he OS Cate eee 135 Cart G. Hartman AND WittiAM F. Hamintron. A case of true hermaphrodit- ism in the fowl, with remarks upon secondary sex characters. Two PREG MEM RULED) 5 Soa a son csc oats Gscretel oraah@iousia os Bihar ce eects aad etaiaie vee 185 G. H. Parker. The leaping of the stromb (Strombus gigas Linn.). Two BURRITO ee eR ats) es is: Salar a, uatro) oa thea pM eh eb soeea a arene Reo Ee Naaeaan 205 No. 3. OCTOBER Epearpo Baxpr. Studi sulla fisiologia del sistema nervoso negli insetti. II. Ricerche sui movimenti di maneggio provocati nei coleotteri. Quar- SOS a. « x Me oe odin 8 Renata WSU RNR eins wie area aera 8 211 F. B. Sumner anp H. H. Couiins. Further studies of color mutations in mice of the genus Peromyscus. Two plates (nine figures)............. 289 G. H. Parker. The crawling of young loggerhead turtles toward the sea.. 323 Wiui1am M. Goupsmitx. The process of ingestion in the ciliate, frontonia. Three plates (twenty-five Hpures). 3.0.0 seas acide dines da este meiows «ss 333 DonneELL Brooxs Younc. A contribution to the morphology and physi- ology of the genus Uronychia. Three text figures and three plates DEMIR UNC UGE G ERORITOR IN (ict < cihccsc isto a 2 s/stoie sieioiotetn ys «nies «cir muainns wi Srcatao > 353 iv CONTENTS No. 4. NOVEMBER W. W. Swinaie. Experiments on the metamorphosis of neotenous amphib- ians. Two text figures and two plates (eight figures)................. 397 H. P. Kserscoow AGERSBoRG. Some observations on qualitative chemical and physical stimulations in nudibranchiate mollusks with special reference to the réle of the ‘rhinophores.’ Two figures................ 423 Dwicut BR. Mrnnicn. A quantitative study of tarsal sensitivity to solu- tions of saccharose, in the red admiral butterfly. Pyrameis atalanta ESTSTIN 70) ONE OUEG S70. 8. cio okpevghinsss. dye cgnie, Ax.» ei cle Soothe ate Bene a ete er ee 445 Steran Kopré. Mutual relationship in the development of the brain and eyes: of Lepidoptera. One plate (six figures).....2... 0255... ¢0000nees 459 Steran Kopré. Physiological self-differentiation of the wing-germs grafted on:caterpillars: of the- opposite sexi... 2...) See. Patients uke eee eee 469 EE. J. Lunp. Experimental control of organic polarity by the electric cur- rent. II. The normal electrical polarity of Obelia. A proof of its Gxtahences- lwo lures. 5200 sens PAR 2, Balls SUR ae, Cope meee ee 477 PROMPT PUBLICATION The Author can greatly assist the Publishers of this Journal in attaining prompt publication of his paper by following these four suggestions: 1. Abstract. Send with the manuscript an Abstract containing not more than 250 words, in the precise form of The Bibliographic Service Card, so that the paper when accepted can be scheduled for a definite issue as soon as received by the Publisher from the Editor. 2. Manuscript. Send the Manuscript to the Editor prepared as described in the Notice to Contributors, to conform to the style of the Journal (see third page of cover). 3. Illustrations. Send the Illustrations in complete and fin- ished form for engraving, drawings and photographs being pro- tected from bending or breaking when shipped by mail or express. 4. Proofs. Send the Publisher early notice of any change in your address, to obviate delay. Carefully correct and mail proofs to the Editor as soon as possible after their arrival. By assuming and meeting these responsibilities, the author avoids loss of time, correspondence that may be required to get the Abstract, Manuscript and Illustrations in proper form, and does all in his power to obtain prompt publication. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 1 JULY, 1922 Resumen por el autor, James W. Buchanan. La regulacién de la formacién de la cabeza en Planaria por medio de los anestésicos. Mediante experimentos en masa el autor demuestra que la frecuencia de la cabeza en pedazos de Planaria puede regularse sometiendo dichos pedazos, durante cortos periodos después de cortarlos, a la accién de concentraciones apropiadas de cloretona, cloroformo, hidrato de cloral, éter y alcohol etilico; en tales concentraciones de estos agentes el aumento en la asimilacién de oxigeno después de la secci6n no se lleva a cabo. Las pruebas acumuladas demuestran que los factores que regulan la for- macion de la cabeza no son especificos y presta apoyo a las conclusiones de Child respecto a la naturaleza de estos factores, esto es, que la formacién de la cabeza esta determinada por las actividades relativas de dos factores antagénicos: 1) La ten- dencia de las células en la vecindad de la superficie anterior del corte a desdiferenciarse y a desarrollarse en la cabeza de un nuevo individuo; 2) La tendencia del conjunto del pedazo, con exclusion de las células destinadas al desarrollo de la nueva cabeza, a mantener la diferenciacién del antiguo individuo, ejerciendo un cierto grado de regulacién sobre estas células en la proximidad de la superficie anterior del corte, y tendiendo a impedir la formacién de una nueva cabeza. Los hechos encon- trados por el autor indican que los anestésicos alteran la fre- cuencia de la cabeza: 1) Por inhibicién directa de los procesos del desarrollo de las células destinadas a la formacién de una nueva cabeza, produciendo disminuciones en la frecuencia de la cabeza; 2) Mediante inhibicion del aumento de la actividad metabélica del conjunto del pedazo a raiz de la seccién; en pedazos de ciertas regiones este efecto supera al efecto directo de las células destinadas a la formacién de la nueva cabeza sobre los procesos del desarrollo, y en tales piezas la frecuencia de la formacién de la cabeza aumenta. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THH BIBLIOGRAPHIC SHRVICE, MAY 1 THE CONTROL OF HEAD FORMATION IN PLANARIA BY MEANS OF ANESTHETICS J. WILLIAM BUCHANAN Hull Zoological Laboratory, University of Chicago THREE FIGURES Regeneration in Planaria has attracted the attention of in- vestigators for more than a century, at least since 1791 (Ran- dolph, 795). Record of mass experiments is absent from the literature, however, until the closing years of the nineteenth century (Morgan, ’98). The present paper presents the results of an attempt to analyze with the aid of certain narcotics the conditions controlling the completeness of regeneration, par- ticularly as regards the head, in pieces of Planaria. The data consist of mass experiments which have involved the cutting of more than forty thousand worms and the handling of more than one hundred and twenty thousand pieces. THE PROBLEM Cross-sections from the body of Planaria do not always reconstitute new individuals with anterior ends like those in nature. Abnormalities have been frequently produced experi- mentally and described in many species and their occurrence explained by diverse theories. In Planaria dorotocephala Child (11 a) has for convenience distinguished five classes of anterior ends in regenerated pieces, each class continuous into the next. Figure 1 shows the external appearance of the five types: nor- mal (A), in which the head is the usual form of those in nature; teratophthalmic (B, C, D), in which the shape of the head is normal, but the eyes show some degree of abnormality ranging from slight reductions in size and approximation to the median line, to a single median eye; teratomorphic (#), in which the shape of the head is abnormal, reduced in size, and the cephalic 1 os 2 J. WILLIAM BUCHANAN lobes are approximated to the median line and the eye is single and median; anophthalmic (/), in which there is more or less anterior regeneration, but no eye; headless (@), in which there is no appreciable anterior regeneration, merely a healed wound. These external characteristics are indices of the degree of devel- opment of the cephalic ganglia (Child and McKie, ’11). The term ‘head frequency’ has been applied to the frequency with which heads of these types appear in a given number of pieces. An examination by Child of the nature of regeneration and head frequency in pieces from different regions of the body of several species of Planaria, particularly Planaria dorotocephala, has led him to the conclusion that the conditions controlling A B C D E G Fig.1 A,Normalform. B,C, D, three types of teratophthalmic forms. H, teratomorphic form. F, anophthalmic form. G, acephalic, or headless form. F the degree of regeneration are primarily physiological and quantitative, not morphological and specific. He supports this conclusion with data showing that controlled changes in the physiological conditions of the animals by feeding, starvation, temperature, mechanical stimulation, and the action of certain chemicals bring about controlled changes in head frequency, either increases or decreases, in pieces from any region of the body. Furthermore, it is evident from the data that changes in the physiological conditions in the animals which bring about changes in head frequency are quantitative changes, and not specific. For example, the rate of oxidative reactions, whether deter- mined by differences in age, or nutrition, or by chemical agents CONTROL OF HEAD FORMATION IN PLANARIA 3 such as KNC, is a fundamental factor in determining head fre- quency (Child, ’11 a, 719 a, ’20a). By investigation of the regeneration of pieces of the same length from different regions of the body of animals of the same size, Child showed that there is a definite gradation in head frequency in such pieces, decreasing from the head posteriorly to the region at which fission usually occurs, indicating there the posterior limit of the first zooid. Then it rises abruptly to decrease again toward the end of the second zooid (Child, ’11 a, 711 b). These two facts, that conditions controlling regeneration are physiological and quantitative and that there is an anteropos- terior gradation in head frequency in each zooid, when jointly considered lead to the inference that there must be some sort of quantitative physiological gradient along the axis of the animal. By means of certain poisons in solutions strong enough to kill slowly, Child (11 b) was able to demonstrate a well- marked anteroposterior gradient in survival time. The reagent most extensively used for this purpose was KNC, which is known to interfere with the oxidative reactions. In appropriate solu- tions of this reagent, as well as others, the anterior end of the animal dies first, indicating that there the rate of oxidative reac- tions is most rapid, since the protoplasm is most susceptible there! The death process then progresses posteriorly in each zooid, indicating the decreasing rate of metabolism from ante- rior to posterior (Child, ’11 b, 13 a, ’13 b). Child’s first conclusion, based on his results on regeneration and this differential susceptibility to poisons, that the gradient made evident by the data is a rough index of the relative rate 1 The differential susceptibility to external agents of regions of different rates of metabolism has been itself the subject of extended investigation and discussion (Child, ’13 a ’14b, ’15 a, ’20b, and other papers; Bellamy, 719; Hyman, 719 a, 21a). The ee ie been that regions of high rate of metabolism are more affected by certain ranges of concentration or intensity of action of external agents than regions of lower rate. To certain ranges of lower concentrations or lower intensities of action, however, the regions of higher rate of metabolism are better able to acclimate or recover from the effects of the agent or condition and consequently show eventually less effect of such treatment than regions of lower rate. 4 J. WILLIAM BUCHANAN of the metabolic processes, particularly the oxidative, in differ- ent regions of the animal, has received ample proof. This proof is afforded indirectly by subsequent work showing that the prog- ress of death due to lack of oxygen follows in general the same course as in KNC (Child, ’19 b); furthermore, as far as the work has been carried out, the rate of carbon-dioxide production measured both by indicator and the Tashiro methods furnishes direct proof that confirms the conclusions drawn from the sus- ceptibility data (Child, ’11 b, ’15; Tashiro, 717). And last, but perhaps more important, Doctor Hyman has shown that the relative rate of oxygen consumption of different regions agrees in every case with the susceptibility data (Hyman, ’21 b). The value of the susceptibility methods as an indicator of meta- bolic conditions has thus been sustained both by direct and in- direct proof. To recapitulate, the existence of an anteroposterior gradient in rate of metabolic processes, particularly those in- volved in oxidative reactions, in Planaria dorotocephala may be considered to have been adequately demonstrated. This gradient is known to involve the following physiological processes: the rate of oxygen consumption, the rate of carbon-dioxide produc- tion, the rate of death in several types of poisons, and the rate of death due to lack of oxygen. The nature of this gradient and its existence in other forms and role in morphogenesis in general have been discussed in a number of papers by Child, Hyman, Bellamy, and others. J. M. D. OLMSTED BIBLIOGRAPHY Cuitp, C. M. 1904 Studies on regulation. IV. Some experimental modifi- cations of form-regulation in Leptoplana. Jour. Exp. Zodl., vol. 1, pp. 95-133. Heatu, H., anp McGrecor, E. A. 1912 New polyclads from Monterey Bay, California. Proc. Acad. Nat. Sci. Phila., Sept., 1912, pp. 455-488. Luoyp, D. J. 1914 The influence of the position of the cut upon regeneration in Gunda ulvae. Proc. Roy. Soc. Lond., Series B, vol. 87, pp. 355-366. Morean, T. H. 1904 The control of heteromorphosis in Planaria maculata. Arch. f. Entw. Mech., Bd. 17, S. 683-694. Otmstep, J. M. D. 1918 The regeneration of triangular pieces of Planaria maculata. A studyin polarity. Jour. Exp. Zodél., vol. 25, pp. 157-176. tn . ; : . JC Al ae ‘7 = ea enw ELE ty Ce eal cj LF Le eid i! ‘ aH ~~ wel ty boll 7 an a 7 = + » rn Fine Pu rs r PAP AtD Sb dimmu! al! He wefan wexate ob aherty iii? iy orheegy Ss a H a i Teh ee P eee | Sith ree ae. Herat ape yatta ten» bp yi ae) eee Pe ey ee eee . A iia io) he ok r } t ed its ‘Fu me pry’ Le ne Ash)! ae bys) 771i eZ Resumen por el autor, J. M. D. Olmsted. El papel del sistema nervioso en la locomocién de ciertos policladidos marinos. Los policladidos Planocera californica, Phylloplana littoricola y Leptoplana saxicola exhiben cuantro tipos posibles de loco- mocién. Para los movimientos de natacién es necesario que los ganglios cefdlicos estén intactos. La destruccién parcial o la pérdida del cerebro impide el uso de este método de locomoci6n. La accion ciliar no esté regulada por el sistema nervioso y prac- ticamente no juega papel alguno en la locomocién. La loco- mocioén atdxica es un fendmeno puramente local, pero esta regulado por el sistema nervioso, puesto que puede abolirse por la cloretona. Euryleptotes cavicola se mueve por este tipo de locomocién solamente. La locomocién retrégrada detaxica esta regulada por los ganglios cefdlicos, cada ganglio regulando la progresion de la olas musculares de su mismo lado. Los cordones nerviosos sirven como conductores de los impulsos para la for- macion de las ondas de su mismo lado. Cuando.se corta un nervio las ondas desaparecen en el mismo lado de la operacién al nivel de la seccién. La locomocién de los policladidos es comparable en este respecto a la de los moluscos. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MAY 22 THE ROLE OF THE NERVOUS SYSTEM IN THE LOCOMOTION OF CERTAIN MARINE POLYCLADS J. M. D. OLMSTED Hopkins Marine Station of Stanford University and University of Toronto During experiments on the regeneration of polyclad turbel- laria of Monterey Bay, California, it was:noticed that there were pronounced changes in locomotion following certain operations which involved the central nervous system. Three species were studied with reference to the control of locomotion by the nervous system, Planocera californica, Phyllo- plana littoricola, and Leptoplana saxicola, with a few observa- tions on Euryleptotes cavicola. The methods of locomotion are the same in the first three species, the chief differences lying in the rate of progression—L. saxicola being the fastest moving and P. californica the slowest—and also in the relative frequency of each method in the different species. A general account of the locomotion of polyclads is given by Lang (’84) in his monograph on the polyclads from the Bay of Naples. A more detailed account of the locomotion of Lepto- plana tremellaria, also from the Bay of Naples, is given by Child (04), and of Leptoplana lactoalba var. tincta from Bermuda by Crozier (718). Child states that there are two chief methods of movement in L. tremellaria, ‘swimming and creeping.’ ‘The for- mer is an ‘undulating movement’ of the margins of the body, the latter involves ‘both muscular and ciliary activity.’ ‘‘When the animal is moving quietly—the cilia afford the chief motive power, although the slight muscular movements of the margin of the body are almost constant, portions being lifted from the substratum, brought forward, and again attached.’ But when strongly stimulated, ‘movements occur in rapid alternation on the two 57 58 LOCOMOTION OF POLYCLADS sides of the body and the similarity between this mode of pro- gression and the use of legs cannot escape the observer.” This is a type of locomotion common to gastropods and is called retrograde detaxic (Olmsted, 717). This type of locomotion was observed by Crozier to occur in his Bermudian species. The methods of locomotion described by these authors are seen in the species of polyclads from Monterey Bay, but they are in reality four distinct methods of progression. In these species the swimming movement involves the whole body, not merely the lateral margins as in Leptoplana tremel- laria (Child, ’04). P. californica resorts to this method more frequently than the other species. E. cavicola was never seen to release itself from the substrate, and therefore never swam freely. While creeping about a dish, P. californica will suddenly release its anterior end, raise it above the substrate, and initiate a series of waves which, from their resemblance to gastropod loco- motion, may be termed retrograde monotaxic (Olmsted,’17). The resulting movement is like that of a rug being rapidly shaken. The waves appear at the rate of two a second, and pass posteriorly at such a rate that two or two and a half waves are present at a given moment. An individual seldom swam more than 2 cm. in this fashion before resuming its creeping. The other two species seldom employed this method of locomotion except when falling through the water after creeping along the under side of the surface film, or when somewhat roughly dislodged from the substrate during active creeping. Swimming was never initiated from rest, but only occurred if the animal were already in motion. In this type of locomotion either the dorsal or ventral surface might be uppermost, the dorsal more often in P. littoricola. When transverse cuts were made at any level posterior to the cephalic ganglia of these polyclads, this swimming movement could no longer be elicited from brainless posterior pieces, but the anterior pieces containing the brain, no matter how short, exhibited a few swimming movements when falling from the surface. Likewise after splitting the head longitudinally so that the two halves of the brain were separated, an animal could not be made to swim, even if one cephalic ganglion remained in each J. M. D. OLMSTED 59 piece. The brain must therefore be intact for this movement to take place. The wave of muscular contraction in this method of locomotion involves the coordinated movements of both sides of the body, for the wave extends across the entire width of the animal. One might imagine that each cerebral ganglion could control the movements on its own side, and in those individuals with the brain split in halves each side might act independently. But this proved not to be the case. It was necessary that the entire brain be undisturbed. Again, it would seem reasonable that the impulse to start off this method of locomotion might originate from some stimulus at the anterior end of the worm, since preliminary movements are made by this portion before releasing the rest of the body from the substrate in preparation for swimming. But if the anterior ends were removed by a transverse cut immediately in front of the cephalic ganglia, it was found that the anterior brainless pieces were unable to swim, while the posterior pieces retained this power perfectly. The impulse to initiate the wave seems therefore not to occur as a stimulus from the anterior part of the body, but from within the brain itself. When the cephalic ganglia were removed by cutting out a small dise of tissue in the vicinity of the eyes, and the hole had filled in by regeneration, it was found that the brain could not be regenerated. Such brainless worms were unable to swim although no part was lacking except the brain. Ciliary movement does not play a prominent part in the locomotion of these four species of polyclads. When they are apparently at rest and making absolutely no progress at all, the cilia can be seen still beating. When the worms are placed in chloretone, the cilia ¢ontinue to beat, and if a worm becomes detached from the substrate to which it often adheres even when anesthetized, it will be carried along at a very slow rate by the cilia. The speed is the same whether the worm is on its dorsal or ventral surface, and is slower than any method of locomotion by muscular action. I am convinced that the cilia never function as the sole organs of locomotion under normal conditions. They may aid muscular locomotion, but do not act 60 LOCOMOTION OF POLYCLADS alone. Miss Stringer (’17) has made this same criticism of the accepted view of the means of locomotion in the triclad planarians and Crozier (18) claims that in the Leptoplana ‘tincta’ “when muscular waves are absent, no creeping progression can be de- tected.” Pieces of any size and from any portion of the body of the Monterey polyclads show this same ciliary movement in chloretone. This ciliary action is therefore not dependent on the central nervous system. The slow creeping movement is accomplished by means of the constant slight muscular contractions to which Child (’04) refers. There are no definite waves, but the entire ventral surface appears to be thrown into irregular ripples. This can be especially well observed when the polyclad is creeping under the surface film of the water. E. cavicola is especially favorable for observation, since the animal is a broad oval, some 3 cm. in length by 2 in width. The animal lays down a track of mucus as it proceeds. The midventral region is depressed and fairly quiet, as if this portion were holding on by suction, while the margins are espe- cially active. The rippling motion is due to momentary local release of a portion of the ventral surface from its point of at- tachment and a shifting of this area by muscular contraction. These worms are able to go both forwards and backwards and to turn to one side. The movement is slow and often irregular. Pieces formed by longitudinal cuts always moved in circles to- ward the injured side (cf. Child). With P. californica and E. cavicola the rate of progress averaged some 0.5 mm. per second. The other species moved slightly faster, but so seldom in a straight line that accurate measurements were not obtained. Worms with the cephalic ganglia removed and pieces from any region anterior or posterior to the brain exhibited this ataxic (Olmsted, ’17) type of locomotion. The rippling muscular movement persisted for some fifteen minutes in a saturated solution of chloretone in sea- water, but finally ceased if the animal were undisturbed. The slow creeping movement is therefore under control of that part of the nervous system in the immediate locality of each muscular contraction and is a local phenomenon. J. M. D. OLMSTED 61 Of the fourth type of movement Child makes the following remark, ‘The animals appear almost as if walking forward.” This ditaxic retrograde locomotion is brought about by the re- leasing of a small portion of the lateral margin at the anterior end of the worm, the pulling of this bit forward 2 or 8 mm. by con- traction of the longitudinal muscles, and its reattachment at a point anterior to its former position. The contraction once started proceeds in a wave down the entire margin to the posterior end. These waves alternate on each side of the body, so that the worm appears to stride along like a biped. This agrees in every respect with the process in gastropods. From records of observa- tions on one large specimen of P. californica, the rate of slow ditaxic locomotion is 0.21 em. per second, and the worm takes 0.33 step per second. Other records on the same individual gave 0.36 cm. and 0.4 step per second. When disturbed it could pro- ceed at the rate of 0.389 em. per second in 0.66 step. The rate of locomotion may therefore be varied both by an increase in the number of steps and the length of each step as well. For P. littoricola the ordinary movement was more rapid, 7.e., 0.4 em. per second in 0.11 step. When disturbed, there was very little increase—0.43 cm. in 0.13 step. This type of locomotion was not observed in E. cavicola. To determine whether the wave was initiated in the margin of the head, this portion was cut away. It was then found that waves started just posterior to the cut, but their rate was prac- tically normal. In another experiment a semicircular cut in front of the brain of L. saxicola severed the connection of the nerves of the anterior margin with the brain. This worm ex- hibited the walking movement perfectly, the waves starting on a level with the ends of the cut. The cut healed over in three days, and by the fourth day locomotion was perfectly normal, the waves starting at the anterolateral margin of the head. Headless specimens and those with the cephalic ganglia removed were unable to employ this type of locomotion, but anterior pieces from worms transected just behind the brain nearly always use this as their sole means of locomotion. THE JOURNAL OF EXPERIMENTAL ZOSLOGY, VOL. 36, NO. 1 62 LOCOMOTION OF POLYCLADS Splitting the head through the brain usually resulted in loss of this ditaxic type of movement. In two cases, however, parts of the cerebral ganglia remained intact. In one of these the cut removed a portion of the right half of the brain. For a week after the operation this worm performed the walking movement with its left side perfectly and moved in circles to the right. Later, an occasional wave on the right alternated with those on the left. This imperfect coordination continued even after a month, so that the animal was never able to move in a straight line. In the other case the brain was evenly divided. Each piece regenerated all its parts except the missing half of the brain. The smaller piece which contained the left half of the brain moved in circles to the right by means of a single wave which took its origin at the usual point and passed down the left margin. There was never any indication of a wave in the new tissue on the right side during the month it was under ob- servation. In the larger piece in which the left half of the brain was missing, waves passed down the right side quite normally with only an occasional alternate wave down the left side, so that this piece moved in circles to the left. By the end of a week, however, the waves on the left side made their appearance some- what more frequently and alternated some two or three times with waves on the right side. These waves never appeared in the new tissue, they began posterior to the cut. The worm remained in this condition for more than a month, still circling to the left and lacking perfect coordination between the right and left halves of its body. These experiments indicate that each cerebral ganglion con- trols the ‘stepping movement’ on its own side, each half being independent of the other. But for perfectly coordinated move- ments involving both sides of the body, the connection between the halves of the brain must be intact. In view of the fact that a few coordinated movements were possible in the two cases just described, where a portion or all of the brain had been removed from one side of the body, it is evident that a wave may appear on a side lacking the brain. But it must be remembered that there was at least one-half the brain present in the body, that the J. M. D. OLMSTED 63 wave appeared only in the old tissue, never in the new, and that it appeared infrequently and only after several waves had passed down the uninjured side. I should explain the appearance of the wave on the side lacking its half of the brain on the ground that through the physiological principles of ‘facilitation’ and ‘summation,’ the movement of a wave down the uninjured side was able to serve as a stimulus, which, upon being transmitted through the connecting commissure immediately behind the brain to the other nerve cord, was sufficient to initiate an occa- sional wave. (Cf. the diagram of the ventral nervous system of L. saxicola given by Heath and McGregor, 712, fig. 21.) If the stepping movement in ditaxic locomotion is controlled by the cephalic ganglia, the question arises as to the function of the longitudinal nerve cords. Various operations were tried in order to answer this question. It was found necessary to make observations fairly soon after cutting the nerve cords, for within three or four days a wound extending almost the entire width of the body would be joined together and perfect coordination re- stored. For example, in a specimen of L. saxicola a horizontal slit was made posterior to the brain, severing both nerve cords. The part anterior to the wound at once moved rapidly by the ditaxic method of locomotion and also attempted to swim freely in the water. The posterior part moved slowly by the ataxic method. The consequence was that the pulling of the anterior end caused the wound to gape in a wide circle. When the worm came to rest, the edges of the wound closed. Two days later the wound was healed and on the next day perfectly coordinated al- ternate waves passed down the entire length of the worm in a normal manner. The nerve cords serve as conductors of impulses which cause progression of the waves in ditaxic locomotion. This is nicely shown by cutting a portion from the side of a worm without in- juring the nerve cord. A wave starting at the head end of the body stops when it reaches the upper edge of the wound, for the muscles which would carry the wave on have been cut away. But the wave again makes its appearance at the proper time at the lower margin of the wound, and then continues on to the 64 LOCOMOTION OF POLYCLADS posterior end of the worm. ‘The impulse therefore was carried on through the nerve cord, though there was no visible effect accompanying its passage. But if the wound were near enough to the midline to have cut into the nerve cord, the wave on that side ceased entirely at the level of the wound. According to the diagram of the nervous system of L. saxicola of Heath and McGregor, there is only one cross connective between the two nerve cords, and that is situated immediately posterior to the brain. If the wound is made anterior to this connective an oc- casional wave will pass down the injured side as explained above, but if the wound is posterior to this connective there can be no conduction of the impulse to form a muscular wave. Local mus- cular contraction can still take place, however. In one case a long strip was cut from one side of the body of a worm, leaving a narrow bridge of tissue at the anterior end to attach it to the body. The ditaxic waves stopped when they reached the bridge of tissue, and reappeared at the posterior edge of the wound. No wave appeared in the strip itself. One could cause the strip to contract by pricking any portion of it, so that the local muscular response remained. Within two days the strip was attached to the body except at the extreme posterior end, but no waves ap- peared in it when the worm was ‘walking.’ On the third day the wound was entirely healed and waves passed two-thirds of the way down the strip. On the following day the animal moved normally. The union of cut ends of the nerve cord or of severed peripheral branches with the brain or nerve cords occurred in a remarkably short time, since perfect coordination was established in a very few days. But if apiece of the nerve cord were actually removed, there was lack of coordination for several weeks, be- cause of the relatively slow regeneration of lateral parts including the nerve cords. Nerve cords do regenerate, while the brain will not. The locomotion of these polyclad worms is comparable in every respect with that of gastropod mollusks. The types of move- ment are the same. Retrograde monotaxic, retrograde alternate ditaxic, and ataxic methods are clearly distinguishable. The part played by the nervous system is similar in each group J. M. D. OLMSTED 65 (ef. section of ‘Physiologie des Nervensystems,” by 8. Baglioni in Winterstein’s ‘‘Handbuch der vergleichenden Physiologie’). Slugs are able to move by means of waves on the foot when the connection with the brain is severed. Waves can also appear on isolated pieces of the foot. ‘This is correlated with the presence of an extensive nerve net in the foot with many cross connectives. Ataxic locomotion in the polyclads is likewise carried on in the absence of the brain and in pieces from any part of the body. Their nerve net must be responsible for this movement, since the movement is under the control of the nervous system in the im- mediate vicinity of the contracting muscles. In snails, such as Helix pomatia, the impulse which causes the normal peristaltic wave arises in the pedal ganglion, and is transmitted by the nerve cords of the foot, each of which serves a definite area. In a simi- lar way both monotaxic and ditaxic locomotion of the polyclads are controlled by the cephalic ganglia, and the nerve cords trans- mit the impulses to a definite area. This is another instance of evolution along the same lines in two quite different groups. SUMMARY The polyclads, Planocera californica, Phylloplana littoricola, and Leptoplana saxicola, exhibit four possible types of locomotion. For the swimming movement it is necessary that the cephalic ganglia be intact. Injury to or loss of the brain prevents the use of this method. Ciliary action is not under the control of the nervous system and plays practically no part in locomotion. Ataxic locomotion is a purely local phenomenon, but controlled by the nervous system since it is abolished by chloretone. Eury- leptotes cavicola moves by this type of locomotion alone. Ditaxic retrograde locomotion is under the control of the cephalic ganglia, each ganglion controlling the progression of mus- cular waves on its own side. The nerve cords serve as conductors for the impulses to wave formation each on its own side. Cutting a nerve causes the waves to disappear on that side at the level of the cut. The locomotion of polyclads is comparable in these respects with that of mollusks. 66 LOCOMOTION OF POLYCLADS BIBLIOGRAPHY Curtp, C. M. 1904 Studies on regulation. IV. Some experimental modifica- tions of form-regulation in Leptoplana. Jour. Exp. Zodl., vol. 1, pp. 95-133. Crozier, W. J. 1918 On the method of progression in polyeclads. Proc. Nat. Acad. Sci., vol. 4, pp. 879-881. Heatu, H., anp McGregor, E. A. 1912 New polyclads from Monterey Bay, California. Proc. Acad. Nat. Sci. Phila., Sept., 1912, pp. 455-488. Lane, A. 1884 Die Polycladen (Seeplanarien) des Golfes von Neapel. Leipzig. Oumstep, J. M. D. 1917 Notes on the locomotion of certain Bermudian mol- lusks. Jour. Exp. Zo6l., vol. 24, pp. 223-2386. Stringer, C. E. 1917 The means of locomotion in planarians. Proc. Nat. Acad. Scei., Dec., 1917, pp. 691-692. WINTERSTEIN, H. 1913 Handbuch der vergleichenden Physiologie, Bd. 4. Jena. Eines -) Resumen por el autor, Leonell C. Strong. Un andlisis genético de los factores responsables de la suscepti- bilidad 4 los tumores transplantables. La raza es el factor primario que determina si un individuo dado ha de presentar crecimiento progresivo o no ha de pre- sentarle, cuando alberga un tumor transplantable. La suscepti- bilidad y la ausencia de esta son manifestaciones de la consti- tucion genética del huésped. Varios factores fisiol6gicos secundarios, el mas importante de ellos la edad, funcionan en la determinacién del resultado de una reaccién determinada. Estos factores pueden denominarse contribuyentes o accesorios. El factor edad es una expresién del grado del proceso de la adquisi- cin de la especificidad de los tejidos, regulado hasta cierto punto por las génadas. La curva de susceptibilidad de la edad hacia los tumores transplantables para los individuos normales de una raza no susceptible presenta una notable semejanza con la curva de la actividad de las génadas. El factor sexo (que se encuentra en los ratones jOvenes) depende por lo menos de dos causas primarias: 1) del factor edad y 2) de la diferencia en actividad fisiolbgica de los sexos en diferentes periodos de la vida. La extirpacién de las génadas no cambia el tanto por ciento en masa de las reacciones para los individuos de una raza no susceptible. La gonadectomia produce en la variedad em- pleada un aumento significativo del tanto por ciento de las reacciones hacia ambos tumores, conforme sucede con ciertas caracteristicas morfol6gicas. Mediante extirpacién de las gonadas, la individualidad de los tejidos y el funcionamiento normal del factor edad pueden influirse. Los dos adenocarci- nomas de la glindula mamaria empleados en los experimentos han retenido una potencia de reaccién constante durante los experimentos. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MAY 1 A GENETIC ANALYSIS OF THE FACTORS UNDER- LYING SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS LEONELL C. STRONG Department of Zodlogy, Columbia University THIRTY-THREE FIGURES CONTENTS PArEnErOd GC GION. kyo cee eh kara Oe eS te iay'e eyatte er dfe yang toa ee Men Ue 68 1, Contributionsto the geneticsiof cancer. . os. 2.5 sci. lee => ae ae cin 68 EE ERROR es A REP cree ETE cys Meigs rs ete aslale SUNIL ec cat IRS 68 ba Moendeliancseenetations yop scot tala - dao -'d eepyneieo set: 69 Gr Mamba tion iypOvbesis; rene 2 fe. 74 se pees coc cee gerne ma seep arts 70 2. 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Apetatiscen tilrelitye (Csr) 6 io ci S405 betes thinks felel Aeteretebecore ates = 92 f. Age ati moculatiory, (GIRTAL) 3.5 1.3 opyaiesvofe oor age eonnt # chem yes wie Sanne 93 g. Age SUSCeptIDINy AGENT) os ce as ciy ga ole slain wn ie wine vc nle« 94 hy Composite age sUcepblOUluy sy. “hice. slew sonic cele awarntecian 94 67 68 LEONELL C. STRONG 4. Sexn(GQBrB) ZF ee es he oo aes Ohl Sells See ee eee 98 Rs BOx COTA) ncn s crtrars lercolelerns aie tereiane oie a. vce, evaiata tae 99 [Sex composite diprbvamd cst Aten emia enie ese ele tenia nee 100 Mee .GrONAASCCOMIY Saree sie ere hos Hila eres eS OLE TE ho a ree 102 n. Age susceptibility in normal and gonadectomized mice....... 109 o. influences ofoperation. 04.4 6. Cel Lhe Ako obh eee 110 oct Adaptation’ J. $6904.) 2" e.0 Rel PE et ee, ee ee 116 Eire General’ discussion 2): (0.5%. Seder ex Meme hol -ccucis| oka ee eae eee es ae 117 Le Weakite OL PIOVEG SLOCK Bae. varcceus, oe aut ade ove. otis Cenete ECT Rae 117 2. Fluctuations in growth energy or adaptation..................... 119 GS AG Speirs hore ie ene RA he eA i SP Ste ee Shad he ee GMOS OE ae SEP: 121 Sc) CI RL ALY SUPRA CEES Lome. ere ND aA As AO Pra Ea 123 HEP HOMAGE CEOMIY rrras cae ns cc ROkG wR Ot tite a ear ENG «ce OE LS ee eee 127 DVix(Conclistons kanes it. ce ee ace eiaie sate Cte eke aie eG ieteee aretha game 132 I. INTRODUCTION 1. Contributions to the genetics of cancer The first part of the following section reviews those genetic investigations that contribute to an explanation of the conclusions reached in this paper. Most of the work, unfortunately, has been published in periodicals not generally seen by medical investigators. Some of the work has been of such a nature that it has led only to skepticism. Three contributions to the genetics of cancer appear to be worthy of special significance. These relate to experiments on transplantable tumors, and involve— a. The recognition of the factor of race. b. The demonstration of mendelian segregation and recom- bination of factors that underlie susceptibility to transplantable tumors. c. The possibility that the causation of spontaneous neoplasms may be due to a process of mutation. a. Race. Up to within the last few years, most investigators in this field considered the phenomenon of race as a factor under- lying susceptibility to transplantable tumors to be of secondary importance. It has become evident, however, that market mice are not reliable for experimental work in cancer. Where the same tumor tissue is inoculated into mice that have been proved to be homogeneous in their genetic constitution, no rhythms of SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS _ 69 growth activity or fluctuations are observed. (See the work on the transplantable neoplasms of the Japanese waltzing mice discussed below.) The first work that showed the importance of race was that of Jensen. He discovered the interesting fact that if tumor tissue derived from a Berlin stock was inoculated into Berlin mice and into Hamburg mice there was a decided difference in the results. The percentage of susceptible individuals among the original stock was distinctly greater. He did not attempt any analysis of this difference. This work, however, attracted the attention of several students of genetics. Genetic research is primarily concerned in the recognition of races and variations within a species. The modern geneticist analyzes his variations by the method of hybridization. He must, however, be sure that the races used are homogeneous for all the factors that determine the differences encountered in the character under investigation. This fixation of characteristics is accomplished by inbreeding. b. Mendelian segregation. The next step was taken by Doc- tors Tyzzer and Little, working on two transplantable tumors that arose in a closely inbred strain of Japanese waltzing mice. The first (J. W. A.) was a carcinoma of the mammary gland that grew in 100 per cent of all mice (of that particular Japanese waltzing strain) inoculated. The first hybrid generation (Fi, produced by the hybridization of the two parent stocks) pro- duced sixty-two individuals, sixty-one of which grew the trans- planted tissue, even faster than animals of the original Japanese waltzing strain. No explanation of this apparent increased activity on the part of the tumor cell was offered. In the general discussion of this paper we shall consider this phenomenon and attempt a genetic explanation. In the second filial generation (F,, produced by mating the F, individuals inter se) only three out of the 183 mice obtained were found to be susceptible to the inoculated tissue. Tyzzer and Little applied the multiple- factor hypothesis to explain these results. This hypothesis postu- lates that in the production of certain characteristics several independent (or linked) mendelian factors may be necessary. 70 LEONELL C. STRONG Little and Tyzzer concluded that the simultaneous presence of from twelve to fourteen mendelian factors which were character- istic of the Japanese waltzing race was necessary for the pro- eressive growth of the transplanted tissue under consideration. They thus placed the inheritance of susceptibility to transplanted tumor tissue on mendelian grounds. Working with a sarcoma (J. W. B.), Doctors Tyzzer and Little were enabled to verify their first conclusion based on the car- cinoma, J. W. A. In this second case the results obtained were simpler and even more convincing. The parent stocks and their F, hybrids behaved as before. The F, generation, how- ever, gave twenty-three susceptible to sixty-six non-susceptible animals. By the use of back-crossing (produced by crossing the F, individuals to the non-susceptible race), Doctor Little (20) was able to analyze the mendelian factors more fully. The conclusions arrived at by this second analysis were: (1) That from 3 to 5 factors—probably four—are involved in deter- mining susceptibility to the mouse sarcoma J.W.B. (2) That for susceptibility the simultaneous presence of these factors is necessary. (3) That none of these factors is carried in the sex (X) chromosome. (4) That these factors Mendelize independently of one another. c. Mutation hypothesis. Theorizing from the Japanese waltz- ing mouse experiments, Doctor Tyzzer, in a general paper on “Tumor Immunity,” suggested that the cause of spontaneous neoplasms may be due to some sort of mutational process. The process of mutation is accepted by most modern geneticists as the cause underlying the production of diverse variations within a species. (In 1908, Williams made a statement to the effect that the cause underlying the tumor-cell formation may be analogous to the phenomenon known as mutation. He must receive the credit, therefore, of being the first to suggest such a possibility. ) SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS 71 2. Factors underlying susceptibility to transplantable tumor tissue a. Race as a factor has already received sufficient, preliminary consideration. b. Sex as a factor underlying susceptibility to inoculable tumor tissue is disputed. Certain investigators have been able to determine a significant difference between the sexes in their receptivity toward transplanted tumors. Others have been unable to determine any significant difference between the sexes. c. Age is a recognized factor underlying susceptibility to transplantable tumors as well as having some causative relation to the origin of spontaneous neoplasms. 1) A very young individ- ual from a susceptible race will sometimes fail to grow the transplanted tissue, although the same individual will do so if inoculated when it is one-half to three-fourths grown. At this age it is more susceptible than at any other period in its life- cycle. 2) Very young animals from a non-susceptible race will sometimes grow a transplanted tumor when inoculated, although no matured animal in that particular race grows the same tissue. d. Pregnancy. Several investigators have concluded that preg- nancy has some influence on the rate of growth of the neoplastic tissue. Leo Loeb has studied several reactions with transplantable tissues in relation to pregnancy. He determined that if carci- noma of the mammary gland be inoculated into pregnant females, the tissue would fail to grow, although no such behavior was encountered in the controls. Later he discovered the interesting fact that after autotransplantation, an adenofibroma of the mammary gland survived, but showed progressive growth only when the host became pregnant. Here we see that the factor of pregnancy can apparently differentiate between malignant and benign growths. According to Loeb, transplanted normal mammary-gland tissue behaves in the same way as adenofibroma tissue during pregnancy of the host. Further, he recognizes that the normal embryonal tissue reacts like carcinoma in the mouse, but evi- w2 LEONELL C. STRONG dently not in the rat. The result obtained with the transplanted normal tissue was to be expected—it is a matter of common knowledge that secretions from a corpus luteum can incite nor- mal mammary-tissue development. Loeb has concluded that— (a) there is a specific affinity of the transplanted tissue for a certain growth substance given off by the ovaries. This affinity is greatest in the case of normal mammary gland tissue and of adenofibroma of the mammary gland; less marked in the carcinoma of the mammary gland and lacking in the ordinary embryonal tissue. (b) A factor injurious to tissue growth operating in pregnancy. This may be either directly injurious substance or a shortage of ordinary foodstuffs due to the growth of the embryo. There are certain facts which suggest the first alternative rather than the latter. (c) Homoiotoxins seem to strengthen the second injurious factor, while their absence seems to favor the first aiding factor. (d) There seems to be variations in the strength of one or several of these variable factors in various species. 3. Prevalent conceptions concerning peculiarities and characteristics of the tumor cell (derived from investigations on trans- plantable tumors) a. Rhythms of growth. Of all the interesting peculiarities that the cancer cell is supposed to be endowed with, that of alternating periods of depression and growth is the most interest- ing. By plotting curves based upon the percentage indications! as ordinates and the interval elapsed before the tumor reached the inoculating point as abscissae, Bashford, Murray, and Bowen concluded that the transplanted tumor cell underwent cyclical changes of growth activity. It must be remembered, however, that these data have been collected from market mice. The objection has already been offered (Calkins) that if these were real rhythms of activity in the tumor cell, it could only be determined with accuracy by studying the rate of growth in one host only. The English investigators referred to maintain that the fluctuat- ing results obtained are due to the varying ability of the cell to adapt itself to the foreign-host tissue. This matter will 1 By percentage indications was meant the relative number of the mice inocu- lated that grew the tumor mass progressively. SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS 73 be taken up next under the consideration of ‘virulence or adaptation.’ Tyzzer and Little, on their work with the Japanese waltzing- mouse tumors (J. W. A. and J. W. B.), determined that every mouse of that strain inoculated grew the two tumors progressively and at a constant rate of growth (there was no significant differ- ence between the rates of growth of any two transplanted tumors in those series). Evidently, the results observed by Bashford, Murray and Bowen and Calkins may have been largely due to fluctuations in the genetic constitution of the ‘market mice.’ Where a definitely proved constant homogeneous race of mice is employed, no rhythms of growth activity are encountered throughout a period of over ten years. b. Virulence or adaptation. It has long been recognized that by continually inoculating tumor tissue into animals of a foreign strain, one can gradually increase the percentage of takes until the maximum is reached. The English observers were the first to determine that this percentage of positive tumor takes fluc- tuates between the minimum and maximum for any given race. At present two explanations are possible: 1) the tumor cell has an inherent capacity of adapting itself to a foreign host or, 2) the cell possesses fluctuating ‘virulence.’ The first explanation has the support of Bashford, Murray, Haaland, Bowen, Cramer, Woglom, and others; the second that of Ehrlich and Apolant. The arguments for and against these explanations are too well known to be repeated here. We may emphasize, however, that neither theory is entirely acceptable. Each assumption rests on the old idea of the inheritance of acquired character- istics, although several attempts have been made to mask this implication. The conception of virulence has been analyzed into two ele- ments by Apolant, 1) transplantability, determined by the per- centage indication of positive takes, and, 2) proliferative energy, calculated from the rate of growth of the transplanted mass. In the light of modern genetical investigation, both of these terms lose much of their significance. The percentage indica- 74. LEONELL C. STRONG tions of reactions of positive growths is determined not by any characteristics of the tumor cell alone, but by its reaction with the genetic constitution of the strain of mice under investigation. Again, the rate of growth of the transplanted mass is constant provided one is dealing with a constant homogeneous race of individuals. A genetic interpretation of the observed results (that by progressive inoculations into a foreign strain one can increase the percentage of positive indications) would be somewhat as follows: Since all races of ‘market mice’ have had a common origin, they have therefore some genetic factors that relate to cancer in common. Within a single family individuals are more closely related to one another than to individuals from another family. When an investigator found that an individual of the foreign strain grew the transplanted tumor, he would naturally pick out individuals within the family of susceptible mice and eliminate those from other apparently non-susceptible families. There is thus an unconscious tendency to select, from the stock, individuals more susceptible to the transplantable tissue—more susceptible because they approach more nearly the genetic constitution of the mouse that grew the original tumor spon- taneously. An investigator not realizing the full significance of the race factor would believe that he was dealing with only one variable—that of the behavior of the tumor cell itself.2. This explanation is offered not as conclusive proof of what necessarily was involved, but as what may possibly have occurred in the experiments that gave the conflicting results previously obtained by various workers. c. Transitional conditions. Certain investigators believe that a carcinoma may be transformed directly into a sarcoma and vice versa. Several investigators have already foreseen the difficulty encountered in such a transformation from epithelial to. con- nective tissue. Among these may be mentioned Ehrlich, Apolant, Ribbert, and, more recently, Woglom. It has been suggested 2 Of course, healthy and vigorous tumor tissue alone must be used, or varia- tions due to infection will enter in. SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS 75 that the tumor mass under consideration was at the outset a ‘mixed’ tumor, each element being derived from its embryonic anlage (carcinoma from epithelial cells and sarcoma from the connective tissue). Assuming that connective-tissue products can only be produced by connective-tissue elements, we may conclude that if a tumor mass contained these specialized products it must have also contained functioning connective- tissue elements. Some evidence of this nature has been dis- covered by Haaland, Slye, Holmes, Wells, and Woglom. MHaal- and found that in a mixed carcinosarcoma, there were inter- and intracellular fibrils present in the sarcomatous parts. By the discovery that myxomatous changes may occur in the connective-tissue part of a carcinosarcoma, Slye, Holmes, and Wells indicated that the sarcomatous part must have arisen in the stroma. Woglom in a recent communication concludes that, since cartilage is found in a carcinosarcoma of the mouse, the sarcomatous element of the mass must have been derived from preéxisting connective tissue. Il. EXPERIMENTAL - 1. Materials In order to test how far the conflicting results that have been obtained in investigations with transplantable tumors on the lower animals have been due to the use of various market stocks, careful attention has been given to the strain of mice employed. Rigorous inbreeding may or may not produce harmful results. It does produce genetically homogeneous races. Relative homo- zygosity (95 to 99 per cent) is only approached after from eight to ten generations of the most intense method of inbreeding. The third inbred generation by any method can never give an index of homozygosity of more than 87.5 per cent. Counting a generation every three months, this process of inbreeding would consume over two years. The approach toward homozygosity is very slow. The time element and expense are therefore ob- viously too great for the patience and resources of most investigators. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 1 76 LEONELL C. STRONG a. Races used. The common wild house mouse fulfills the requirement of a homogeneous race*® to a marked degree. If collected in the same locality (an isolated group of buildings, a small island, ete.), one may be fairly sure that such stock is homogeneous for several reasons. Strange mice very seldom invade any particular location already occupied by a well- established colony of mice. This is evidenced by the fact that slight variations in color and form tend to be restricted to the same corner of a building, etc. Wild adult female mice will not breed readily in captivity, nor will wild pregnant females (caught wild) usually care for their young when born. It was necessary to find the breeding places (nests) of the wild mice. The young found were reared by foster-mothers from an albino stock. Care must be employed in placing the new young in the nest. The foster-mother must first be removed from the box. After the young have been in the nest long enough for them to acquire the odor of their new surroundings, the foster-mother may be replaced. If this pre- caution is not taken the females may eat the wild young. The mice were kept in a closed room in which the temperature did not vary more than a few degrees throughout the day and night. The wild mice are numbered consecutively, their serial number being preceded in each case by the letter W, so that no confusion would arise as to their origin. The dilute brown (silver fawn) stock. This is a special strain of mice produced by inbreeding during the last eleven years. With the exception of the Japanese waltzing-mouse strain of Mr. Lambert (Boston, Massachusetts), this strain represents, no doubt, the nearest approach to a homogeneous strain of mice employed in cancer research. It has a distinct advantage over the Japanese waltzers in the matter of breeding and of rearing the young. This strain consists of animals containing the three recessive characters for coat color: 1) dilution of pigmentation, 3 Theoretically, there is no question but that homozygosity would be produced by continued inbreeding. In the absence of controlled experimental evidence, it seems better to employ the term ‘genetic homogeneity’ or merely ‘homogeneity’ to express this result. SUSCEFTIBILITY TO TRANSPLANTABLE TUMORS es 2) black (non-agouti) and, 3) cinnamon (brown agouti). The inbreeding was started by Doctor Little while at the Bussey Institution (1909) and is still being continued at the Carnegie Institution Station, Cold Spring Harbor. This is the parent strain of mice, in that it gave rise to the two adenocarcinomata employed in this investigation. We will refer to this strain as the susceptible race. By a susceptible race we mean one in which there is 100 per cent indications of progressive tumor ‘takes’ upon inoculation with a bit of the transplantable tissue. The albino stock. This is one of the non-susceptible races employed. By a non-susceptible race we mean one in which every mouse inoculated with the tissue fails to grow the trans- planted tissue to an appreciable size. This stock has been used as a control on the experiments with the wild mice. The albino strain was obtained from Dr. H. J. Bagg, of Memorial Hospital. He has inbred this strain, brother-to-sister matings, since 1912. Mice of this strain were given serial numbers preceded by the capital letter A. b. The tumors employed. We have used two adenocarcinomata of the mammary gland that arose spontaneously and independ- ently of each other in the pure dilute brown strain. The first arose some three weeks before the other. The first was desig- nated as dBrA; the second, dBrB. Microscopically, the two tumors are indistinguishable, as shown by the accompanying microphotograph (fig. 1, p. 78). The slight difference is not real as might be supposed, but due to a slight defect in the staining of the dBrB tumor We are in agreement, then, with the con- clusion of Dr. James Ewing that these two tumors are histo- logically identical. The preliminary experiments dealing with the inoculation of these two tissues into the pure dilute brown stock were performed by Doctor Little during the spring of 1920. He determined that either tissue grows progressively in 100 per cent of the mice inoculated, regardless of whether the tissue is placed in separate individuals or on opposite sides of the same mouse. ‘There is no appreciable difference in the tumors (rate of growth, etc.). Within limits, there is apparently no effect of one tumor upon 78 LEONELL C. STRONG the other when growing in the same host. They each show identical growth activities, percentage of indications, ete. So far, one is warranted in concluding that the two are identical. There are, however, as outlined in the following pages, other tests that can be employed. Figure 1 SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS 79 2. Methods a. Inoculation and observation. The methods used in trans- ferring the tissue from one host to the other are those commonly employed. Ordinary precautions of asepsis were used. It has already been shown (Crocker Laboratory) that tumor sus- ceptibility is not influenced by the manipulation of the tissue during the transfers. The mouse possessing the tumor was first carefully shaved around the site of the proposed incision. The approximate volume of the mass was then determined by palpation and recorded. The instruments employed were sterilized by being placed for a few moments in boiling water. The mouse was etherized as lightly as was possible consistent with relaxation. A straight incision of about three-fourths of an inch was then made by means of curved scissors. By manipulating two pairs of forceps around the tumor, the con- — nective-tissue strands that anchor the tumor to the skin and body wall of the mouse can be severed. A small amount of hemorrhage usually occurs. The blood may be removed by a moistened piece of absorbent cotton. The operation was performed with as much speed as possible—the ‘time element being one of the important factors of success, especially when the mouse has two tumors to be removed. Complete extirpa- tion of the mass was frequently obtained, this being a distinct advantage, since the mouse can then be used for breeding after its susceptibility has been tested. The tumor mass was then placed in a weighed sterilized Syracuse watch-glass to determine the actual weight of ‘type’ masses. b. Measurement of size of tumor. Several methods have recently been employed in determining the rate of growth of the tumor mass. The older method (used by Bashford, Murray, etc.) was to compute the interval between inoculation and the time at which the tumor reached the inoculating point. The procedure gives a rough approximation concerning the time required to reach a given size when a series of tumors are to be compared. It does not, however, give any evidence of the successive growth points before this end point is reached. By 80 LEONELL C. STRONG this method it is impossible to differentiate between the be- havior of a tumor that developed fast for the first few weeks and then became practically stationary and one that showed a slow initial impulse and a.rapid progressive advance during the latter part of the experiment. A modification of this method has been used by Tyzzer and others. By weighing the tumor mass at definite intervals of time, it is possible to compute the rate of growth, provided that the rate of size increase was pro- gressive. It is, however, a matter of common observation that transplantable (or even spontaneous) tumors do not pro- gress uniformly. This method does not give relatively accurate growth rates. By palpation one can estimate the relative increase (or de- crease) week by week. ‘The method is an advance over the two older methods, but has a few disadvantages. ‘Tumor masses are usually irregular in outline. The time element therefore involved in computing the volume of such a mass is consider- able. The method, moreover, does not take into considera- tion that there may be necrotic or hemorrhagic areas present in the tumor mass. By weighing of ‘type’ masses from 0.01 gram to 10 grams we have endeavored to eliminate the disadvantage of the previous methods. The entire history of the mouse under consideration was kept on one chart of codrdinate paper. By comparing each individual mass (determined by palpation) with the series of type masses, we were able to estimate the approximate weight of the tumor under investigation from week to week. Calcula- tions of rates of growth were only made on tumors which were firm. The method is not as accurate with recurrent masses for the reason that ulceration and hemorrhagic areas are ap- parently more prevalent than in the original inoculated growth. The data thus obtained were used in the determination of rates of growth (both progressive and regressive), studies on viru- lence, ete., as outlined below. The method gives only an approximation to the true state of affairs. By the use of large numbers of mice, however, closer approach toward accuracy can be made than by any other practicable method. SUSCEPTIBILITY TO TRANSPLANTABLE TUMORS 81 The trochar method of implantation was employed through- out the experiment. A small piece of the tumor is packed snugly into the base of the neck of the trochar with the blunt plunger. The assistant holds the mouse firmly with the left hand by the ears and with the right by the tail, by taking hold of the skin of the mouse near the iliac region with the right hand. The instrument is then pushed forward, placing the tissue in the axillar region. The trochar is sterilized in hot water between every inoculation. No ether is necessary for this process. By this method fifty or sixty mice can be inocu- lated in about forty-five minutes. c. Gonadectomy. The term ‘gonadectomy’ signifies the re- moval of the sex glands of either male or female. Wherever ‘castration’ is used in this paper we mean the removal of the male gonads only. ‘Spaying’ will be employed for removal of the female gonads. Gonadectomy should not consume more than three minutes of actual operating time for the male and not more than four minutes for the female. Ordinary aseptic precautions suffce. Castration. The mouse is etherized until almost all volun- tary action ceases, then placed on the table ventral side up. The assistant holds the mouse by pressing two fingers of the right hand down on the hind legs; with the left hand it is possible to manipulate a small etherizing bottle periodically and to keep the mouse stretched out by pressing lightly on one of the front legs. The hair on the posteroventral portion of the mouse can be removed by means of straight scissors. With a pair of forceps, the testes are then pushed back out of the scrotum into the body cavity. All instruments used from this point on should be sterilized in boiling water for several min- utes. A single longitudinal incision about midway between the umbilicus and penis is then made by means of a pair of small curved scissors. This opening need not be more than three-eighths of an inch in length. By pressing the forceps between the outer skin and the body wall, these two layers are separated in the region surrounding the incision. O1 bo 19) (or) PER CENT OF DEATH 5.6 2.2 NUMBER OF DAYS 30 30 34 32 FEEDING PARAMECIUM KNOWN BACTERIA Ua TABLE 8c Summary of results for period III, from December 5, 1920, to December 28, 1920. Food and media as in table 3b AVERAGE Poco ire teaieter| Senna ea ar ee censor || cernioay Picea Cy 1.13 21.6 33.6 4.8 21 Aa! 2.108 59.8 16.3 ot 21 ACA Cr 1.883 50.8 18.2 13 21 M 1.098 23.4 36.3 U2 20 TABLE 3d Summary of results for period IV, from January 12, 1921 to February 5, 1921. Food and medium as above Boa Soleo re eer sue | ienies: eae Oi 0.906 15.5 34.8 i 20 sae Ove 1.303 38 29.3 3.8 21 HE Wid 1.168 28.3 28.3 4.9 21 M 1.058 9.4 10.8 Bila 61 TABLE 3e Summary of results for period V, from February 13, 1921, to March 7, 1921. Food and medium as above wc vies \pacoee parce, | SDC Care gS | REN Ce Se ee | eae Cr 1.254 S2e2 37.4 4.3 23 AC? 2.084 69.1 1522 1 23 ABBA! 1.384 40.2 Goul 10.2 23 M 0.727 23.1 15.3 15.4 3? 1 Died and restarted after a week’s interval, then died again. ? Started three days before end of period. Showed low vitality. Tables 3, a to e, demonstrate that the chance mixture M showed a steady decline in all factors considered. This decline, however, was not evident during the daily course of the work, for, as the graphs show, such fluctuations as occurred would naturally be attributed to normal rhythms, and were not noticeably different from those occurring in the artificial mixtures. It was only when all evidence was collected and the final averages made that 158 RUTH L. PHILLIPS it could be seen that there was in reality a steady decline in the power of the chance mixture M to maintain a normal rate of metabolism in Paramecium, and that the seemingly abrupt failure of this mixture was, after all, only apparent. The streptothrix C’, the only organism of those tested which proved to be a satisfactory food when used in pure culture, is a filamentous organism, the individual filaments of which are too large for Paramecium to ingest. The animal was, therefore, forced to subsist upon the fruiting bodies or upon very young filaments. The questions might be raised: Was not the low metabolic rate of the animals fed with this organism due to an insufficient amount of food? Was the supply of fruiting bodies adequate? It is probable that the food was quite adequate in amount and the increases in activity noted for these animals were not due to any increase in the reproductive power of the streptothrix. It is far more likely, since they occurred synchro- nously with similar increases in other cultures, that they were associated with rhythms and had no direct relation to food. The mean division rate for C’ for the entire series of observa- tions was 1.033. An inspection of table 3, shows that the divi- sion rate fell below this mean three times; at the beginning of the experiments and during the second and fourth periods. During the other periods it was slightly in excess of the mean rate. On the whole, it showed less variation from the mean than the other two artificial mixtures. The fourth period, dur- ing which the rate dropped below the mean, was marked by a fall in metabolic rate in all cultures. It is possible that the greater range in temperature combined with an unsatisfactory condition of M was sufficient to cause the death of the animals in this mixture. When A’C’ is compared with C’, it is evident that the addition of A’ to C’ caused a marked acceleration or stimulation of the metabolic rate of Paramecium. The division rate exceded that of C’ during all the periods. The percentages of high divisions, low divisions, and deaths show an increased metabolism. There was more fluctuation in the death rate for A’C’, and it was greater than for C’ during the third period. It was found in computing FEEDING PARAMECIUM KNOWN BACTERIA 159 the percentages for the three-day intervals that an increased death rate sometimes accompanied an increased metabolism. This suggests that an accelerated rate of metabolism may be paralleled by an excessive mortality, as if the unusual expendi- ture of energy was destructive to weak individuals. With the exception of these occasional increases, the death rate of A’C’ remained less than that for C’. The significance factor for the difference between C’ and A’C’, 9.832, indicates a marked ac- celeration of metabolism in animals fed with A’C’. The analysis of the conditions obtaining in the feeding of the above mixtures is aided by a series of graphs of the division rates averaged for three-day intervals during the five periods of ob- servation. An inspection of these graphs shows the superiority of the A’C’ mixture. It is true that for the first ten intervals, the average division rate, 1.712, was less than that for M, which was in excess of 2 (table 3a). A fission rate greater than 2 is unusual for Paramecium aurelia, yet the rate for A’C’ exceeded 2, twelve intervals out of a total of forty-four. The rate for the mixture A’B’C’, used for a total of forty-two intervals, exceeded 2 nine times, thus approximating A’C’. The division rate of the control, C’, did not exceed 2 in any of the averages for three- day intervals. Although the daily rate rose above this figure or equaled it 174 times between August and March, these high rates did not occur with sufficient frequency to bring any single average for a three-day interval up to 2. Certain minor fluctuations in the rhythms are to be seen on an inspection of the graphs. These fluctuations are independent of those for the three-day intervals and of the occurrence of endo- mixis. They are evidence of an alternating elevation and de- pression of the metabolic rate which seems to be independent of food. They occur in all cultures and are fairly synchronous ir- respective of the type of food used. The temperature of the laboratory was fairly constant; that for the liquid in which the animals were living was necessarily more constant than the air of the room, and this even temperature obtained throughout the course of the work save for a part of the winter when there was some variation. Since these minor fluctuations occurred during 160 RUTH L. PHILLIPS the time of even laboratory temperature as well as when it was more variable, it would seem that they cannot be entirely due to this factor. In the case of a satisfactory food like A’C’, the de- gree of fluctuation tends to be greater than with a food like C’, which is incapable of sustaining a high metabolic level in Para- mecium. Aside from this fact, no definite correlation seems to exist between the occurrence of these fluctuations and the type of food used. They appear to be due to intrinsic factors affecting the metabolism irrespective of food or to slight variations in the environment not under control. It is of interest to note that the chance mixture M, which throughout the first period appeared to be the most satisfactory kind of food used, showed the least degree of fluctuation of any of the cultures under observation during this period. That this mixture was comparable at this time to any laboratory infusion in which Paramecia thrive, there can be no reasonable doubt. That it did not continue to furnish conditions favorable to the continued life of Paramecium has been demonstrated. The_ question arises as to whether the high rate of metabolism of the animals in M during this period was due to a preponderance of favorable types of bacteria which prevented the increase of un- favorable types. Since it is well known that bacteria vary in their resistance to drying, is it not possible that some of the more resistant forms are unsuitable food for Paramecium? The foregoing observations may be summarized as follows: The data dealing with the behavior of Paramecium when fed in a standard 0.1 per cent timothy hay infusion upon a diet consist- ing of either a pure culture of C’, or its mixtures A’C’ and A’B’C’, and of the chance mixture M, demonstrate that it is possible for this animal. to live upon a single article of diet, although the metabolism under such conditions is not so high as when a mixed diet is used. The chance mixture M was very much more satis- factory during the first period than either of the artificial mixtures, but such a chance mixture is not easily kept constant, and under the conditions of these experiments failed to maintain a continued normal rate of metabolism in the animals fed upon it. Of the artificial mixtures, A’C’ proved to be the most satisfactory and 161 FEEDING PARAMECIUM KNOWN BACTERIA “seSsTOGe UO Ss[VAIOJUT ABp-dd1} JO JOGUINU {so}BUIpPIO UO poyyoTd SozVI UOISIATp OSBIOAW *) YOIBTY 0} ET ‘9g ‘A {S ‘Gad OF ZE URL ‘AT £8% ‘00d OF G99 “TIT FZ "AON 0302 “990 “IL +82 "3498 09 8g “SnY ‘7 :SMOT[OF SB pud}xo puv sporiod 07 Jojol S[VIOUINU UBUIOY “TZ6T 2 Wore 04 ‘OZ6T ‘8z Ysnsny ‘Tl pue 4O,Q/V OvV OQ BitopoRq YPM poy UoyA UINTooWBIeT JO LOLAVYO SuiMoys sydviy T “Sty Sens Gime. z 9 3 9 yp % @ OT 6 8 ) b OtT6 8 2 ¥ a 20.7 162 RUTH L. PHILLIPS appears to be capable of maintaining a normal rate of metabolism over an extended period. 2. Change of food of Paramecium without change of medium. This section of the work consisted of two parts. First, Para- mecia which had been fed for a long time upon the bacteria C’, A’'C’”, A‘'B’C’, and M were shifted from one such food to another, as, for example, animals fed upon C’ were washed and then placed in the A’C’ mixture; the presumably unfavorable foods, A’ and B’, were added to M, and other such shifts made as de- scribed below. The second part of the work consisted in an entire change of food. Animals which had been fed upon one of these combinations were washed and placed in pure cultures of bacteria used in the earlier part of the work. Table 4 gives the data for the first type of food change, and in table 5 are the probable errors and significance factors computed from the data of table 4. The original cultures were used as controls, and the change of food is indicated by changing the order of the letters used, e.g., cultures containing animals changed from C’ to a combination of A’ with this bacterium are designated as C’ A’, to distinguish them from A’C’, the control of such a mixture. The culture, C’, serves as a control for all the mixtures used. It is evident from the data in table 4 that the change from C’ to C’B’ resulted in a serious decrease in the vitality of the animals, for they could be kept alive but nine days in this mixture. Animals changed from C’ to C’A’ appeared to experience an increase in metabolism. This increase was too slight to be due to the change in food as is suggested by the significance factor, 1.985. The disturbing effect of the change in environment was not overcome in the time during which the animals were under observation. The division rate failed to reach that of A’C’, and it is evident that in the change from C’ to a food presumably more satisfactory, the mere fact of the change tended to counter- act the effect of the more favorable food to such an extent that the animals failed to show a marked reaction to this type of variation in the environment. The significance factors for all the other changes save MA’B’ tend to show the same thing. In the case of MA’B’, however, the significance factor, 3.965, in- FEEDING PARAMECIUM KNOWN BACTERIA 163 dicates that the addition of A’ and B’ to the chance mixture had a stimulating effect. TABLE 4 Data for the growth of Paramecium aurelia in the foods C’, A' C', A’ B’ C’, and M, with the following changes in food; C’, to C’ A’, C’ B’, C’ A’ B’; A’ C’, to A’ C’ B’; M, to MA’, MB’, and MA’ B’, during the time from December 5, 1920, to December 28, 1920 AVERAGE 4 d TUR FOOD Nope Dairy | ‘"Drvisrowa | Divisions | Dears | Dave CG 1.13 21.6 33.6 4.8 21 ALG? 2.108 59.8 16.3 7.7 21 ACBL GC 1.883 50.8 18.2 11.3 21 M 1.098 23.4 36.3 7.3 20 Gt AY 1.535 50.6 18 10 20 Grp’ 0.794 10.4 44.8 27.6 9 eA’ B 1.880 52.6 15.9 7.9 20 eB 1.936 57.1 14.3 V2 18 MA’ 1.177 29 29 10.1 19 MB’ 1.233 30 30 7.5 20 MA’B’ | 1.504 38.6 26.3 7 16 TABLE 5 Mean division rates with their probable errors, differences in mean division rates with probable errors of the differences, and significance factors for the data summarized PROBABLE FOOD DIVISION Moncey DIFFERENCE IN MEAN DIVISION RATE oF pieFER: “CANCE GOW TS 0.0911 C’and , ts6l 58 21.3 3.6 21 1.868 50.8 13:2 11.3 21 / / / . . SS Tera 46.5 19.4 10 16 “ 1.386 23.4 36.3 rigs) 20 1.45 32.1 8.9 26.8 7% THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 2 166 RUTH L. PHILLIPS TABLE 6c¢ Summarizing the data contained in tables 6a, and 6b, for the time from November 6, 1920, to December 28, 1920 crate Nae Toe pete fire ys wb eu 1.162 19.8 32.9 5.9 39 1.113 22.9 28.4 6.2 39 A'O’ 1.756 49.4 24.7 4.6 39 1.705 49.9 26 4.1 39 A’B'C’ 1.695 45.5 23.2 7.3 39 1.652 43.9 28 6.1 34 M 1.348 22.1 36.9 aks 36 1.478 21.9 20.8 20.2 23 due to a depressing effect of the medium. It is just as probable that the change in environment in a culture which was becoming unsatisfactory was enough to cause the death of the animals. Had the food in the mixture M been as satisfactory as was the case when this was first used, it is not likely that the death rate in the new medium would have been greater. The second infusion tested was made of entirely different material, the dry, uncured, succulent moneywort, Lysimachia nummularia L. It presumably had a different chemical compo- sition than either of the other infusions used. As in the case of the uncured swamp hay, it was tested in connection with controls in the standard 0.1 per cent hay infusion. The effect of using this infusion is shown in table 7. The first impression one gains from a study of this table is that the change from hay infusion to moneywort produced a marked increase in metabolism of the animals thus treated. The signif- icance factors for these changes do not indicate that this was the case save for the mixture A’B’C’. However, since there was a stimulating effect of the medium in this one case, we are justified in concluding that this particular change in medium tended to be stimulating rather than depressing or without effect. FEEDING PARAMECIUM KNOWN BACTERIA 167 During the second period of observation the animals passed through an endomixis. This was accompanied by a lowering of the division rate, as was to be expected, and after recovery the rate for the animals in the new medium remained slightly TABLE 7a Showing results of feeding Paramecium with the bacteria C’, A’ C’, A’ B’ C’, and M in the 0.1 per cent standard timothy hay infusion, and in 0.1 per cent dry uncured moneywort infusion, during the time from January 12, 1921, to February 5, 1921. Data for the moneywort infusion in the second line in each case FOOD Cc A (GH Ay BiG. M P ceeetiieee TE ocean TE commen TT or see AVERAGE NUMBER DAILY DIVISIONS 0.906 1.485 1.303 2.023 1.168 2.184 0.764 0.643 PER CENT HIGH DIVISIONS PER CENT LOW DIVISIONS 20.8 24.6 TABLE 7b PER CENT OF DEATH NUMBER OF DAYS SIGNIFI- CANCE FACTOR Showing the results of feeding Paramecium with the same bacteria in the same media, as in table 7a, during the time from February 13, 1921, to March 7, 1921 FOOD Cv A’C’ AY BoC? M Nea on AVERAGE NUMBER DAILY DIVISIONS PER CENT HIGH DIVISIONS PER CENT LOW DIVISIONS PER CENT OF DEATH NUMBER OF DAYS SIGNIFI- CANCE FACTOR 168 RUTH L. PHILLIPS TABLE 7c Summarizing the data contained in table 7a and b, for the time from January 12,1921, to March ?, 1921 AVERAGE FOOD NUMBER DAILY DIVISIONS PER CENT HIGH | PERCENT LOW PER CENT OF NUMBER OF DIVISIONS DIVISIONS DEATH DAYS c { 1.094 13.5 41.7 6 43 \| 1.899 32.4 34.3 2 43 atc! 1.591 41.5 25.1 3.1 43 1.832 44.4 18.5 7.6 43 Baty, 4, 1.234 23.9 27.5 6.7 44 een 1.916 45.7 7S 9.6 44 x 0.755 9.1 19.7 31.8 9 0.595 3 27 37 9 below that for those in the hay infusion. That this depression was not due to the changed medium is shown by the significance factor for A’C’, the only mixture in which the depression ap- peared to be at all marked. When the data for the two observation periods are combined, it is seen that on the whole this change in medium was without significant effect. In general it may be said that changes in medium which were tried had no appreciable effect over a con- siderable time. The only result of change in medium of any importance was that of a slight transitory stimulation of the metabolism in animals changed to the moneywort infusion. 4. Change in medium accompanied by change in food. In order to determine whether a change in medium would produce an effect upon the metabolism of Paramecium if the food was changed at the same time, animals were washed and placed in 0.1 per cent standard timothy hay infusion and in 0.1 per cent moneywort infusion with the bacteria J, K, Land the mixtures JK, JL, KL, and JKL as food. The culture C’ was used as con- trol. It was found that the animals in the moneywort infusion did not show any significant increase in rate of metabolism, but the duration of life in the moneywort was in most cases slightly longer. In the hay infusion none of the animals fed with the FEEDING PARAMECIUM KNOWN BACTERIA 169 bacteria J, K, L, or their combinations, lived over six days, whereas many of those in the moneywort with the same food lived seven days. There was very little effect which could be ascribed to the change in medium, but the increased length of life mentioned may indicate a slight tendency of the moneywort to stimulate. However, in case this was so, the effect was not great enough to overcome the unfavorable nature of the bacterial food. This experiment indicates that a change of medium in the presence of a food known to be unsatisfactory does not so affect the animals that they are able to utilize such food for any length of time. It would seem, therefore, that whereas the stimulating effect of a changed medium may be an aid in reviving cultures of Paramecium which are not in a thriving condition, the nature of the food is at least as important, and if this be unsuited to the requirements of the animal, no amount of artificial stimulation by altering the medium will prevent the death of such cultures. 5. The effect of sterile media. In order to determine whether the results obtained by feeding certain bacteria which did not support active metabolism in Paramecium were due to the toxic action of such bacteria or merely to the fact that they were for some reason not utilizable for food, certain individuals were thoroughly washed and placed in sterile media. The average length of life in hay infusion was 2.15 days, in uncured swamp- hay infusion, 2.54 days, and in moneywort infusion, 1.72 days. The maximum duration of life for animals in the first infusion was 4 days, for those in the second, 5, and for those in the moneywort, 4 days. The minimum duration of life in all three infusions was the same—less than twenty-four hours. Five lines were tested in each case, and controls carried in C’ during this time showed the normal rate of metabolism for animals fed upon this organism. These tests show that Paramecium aurelia will not live for any length of time in sterile media of the type ordinarily used in the laboratory. It is, therefore, to be assumed that the failure of the animals in the preliminary experiments to live in pure or mixed cultures of the bacteria used, was due in most cases to starvation, 170 RUTH L. PHILLIPS rather than to any toxicity of the bacteria used for food. In such cases as the cultures L’ and K’L’, however, it is very prob- able that the bacteria were toxic, since all Paramecia died within twenty-four hours. HISTORICAL AND DISCUSSION Whatever the title of the article or scope of the investigation, all who have worked with Paramecium have been concerned directly or indirectly with some phase of the life-history of this animal. Earlier workers did not conceive the complexity of function and behavior revealed by later studies of its metabolic processes. The sum total of these results reveals a striking similarity to the vital processes of many-celled organisms. The food of an animal is not the least important factor in determining its behavior, yet this has sometimes been ignored, sometimes obscured by too great emphasis of other factors by workers with Paramecium, and still remains to be clearly understood. The reason for this neglect lies in the fact that since the food of Paramecium consists mainly of bacteria, any study of this factor must necessarily overlap the field of bacteriology. Ac- cordingly, the zoologist has left it alone almost completely, either because of lack of technical skill or because of hesitation to enter unknown territory. Hargitt-and Fray (17) have de- vised a technic which makes possible the study of the relationship of Paramecium to food. The technic of getting pure cultures of bacteria is the only part of the work essentially new to the pro- tozoologist, and this can easily be learned. One of the most striking things met with in reading the litera- ture is that the earliest worker to apply experimental methods to the study of Paramecium (Maupas, ’88) had possibly a clearer idea of the importance of food than any who followed him until a few years ago. He says: ‘Possibly the use of Pasteur’s methods for culturing bacteria would prove to be more suited to the needs of the Ciliates, but I have never made any attempt in this direc- tion.’ It seems strange that this very pertinent suggestion should have been so long overlooked, but again it may be that the technical difficulties of obtaining such cultures discouraged FEEDING PARAMECIUM KNOWN BACTERIA 171 workers from entering this field and forced them to be content with more or less general methods of food control. Meissner (’88) was one of the first to investigate the food habits of Protozoa. His experiments dealt with the digestibility of the various types of food, such as starch grains, oil drops, and particles of albumen. His work is of importance in demonstrat- ing that the Protozoa are not essentially unlike the Metazoa in their power to utilize food. Moreover, Meissner showed that not all types of food are digested with equal ease, and some not at all. These findings are of significance in suggesting that variations in digestibility may exist in the normal food of these animals which are of importance when one is attempting to study their behavior. Environmental factors other than food have received the greater amount of attention of investigators of the Infusoria. Of such factors, the nature of the medium has been perhaps too much emphasized. That the medium is important, there can be no doubt, but if it is of such a nature as to fulfill the osmotic requirements of animals living in it, and to furnish the proper salt content, it would seem that its importance, as far as Para- mecium is concerned, lies in its ability to furnish a proper food for the bacteria upon which this animal feeds. Calkins at first (02 a) thought of the medium as being of main importance, but he soon came to appreciate the relation of bacteria (02 b). His statement that B. subtilis was the chief food of his Paramecia is probably not justified. Wenow know that the variety of bacteria living in an ordinary hay infusion is not confined to a single group. Moreover, bacteriologists have subdivided the group of bacteria known as B. subtilis until it includes a large number of forms. Not all of these will support the life of Paramecium, as is shown by a comparison of my results with those of Hargitt and Fray (17). Calkins was careful to sterilize his media by heating to 90°, but we now know that this temperature is not sufficient to kill many spores. No attempt was made to exclude air- borne bacteria which he assumed furnished the greater part of the bacterial food. He may, therefore, have been using an infusion in which deleterious bacteria came to predominate. 172 RUTH L. PHILLIPS Woodruff (’08) was led to believe that the death of Calkins’ animals might have been due to too constant a medium, and accordingly began the cultivation of Paramecium in what he termed a ‘varied medium.’ He believed that by collecting ma- terial at random, he would provide infusions which would more nearly approximate the natural environment of this animal than a constant medium of hay infusion. The conclusion of the procedure was truly remarkable, resulting as it did in the con- tinuing of a single line for several years without a conjugation and a very complete analysis of the life-history. Later, Woodruff and Baitsell (11 a) were successful in culti- vating Paramecium in a constant medium of beef extract. This work established that mere constancy of medium, provided the fluid used was suitable, did not interfere with the normal course of the life-history of the animal. A constant medium did as well as one which was continually varied. None of these investigators were working with a closely controlled food. They were, therefore, continually dealing with an important unknown factor. With a known food it is possible to test further the effect of medium upon Paramecium. ‘Two types other than the 0.1 per cent standard timothy hay infusion were used in this work. ‘The infusion made with uncured swamp hay differed from that made with cured timothy hay in that it was prepared from a mixture of grasses which had grown in a swampy lowland district, and was presumably different chemi- cally from the standard infusion. The moneywort differed even more. It was uncured when used, was extremely succulent to start with, and much richer in cell sap than either type of grass. The method used in preparing these infusions was the same, so that gram for gram, equal concentrations were used. The result showed that there was no significant difference in effect of the two grass infusions. The moneywort tended to have a slight stimulating effect for a short time, as shown in the case of A’B’C’, and in the fact that when used with such unfavorable foods as J, K, and L, the duration of life of the animals was somewhat greater than in the standard hay infusion. FEEDING PARAMECIUM KNOWN BACTERIA 173 Since the animals fed with C’, A’C’, and A’B’C’ showed no tendency toward diminished vigor for a period of more than six months in the standard hay infusion, it is possible that the di- verse results obtained by Woodruff and Calkins may have been due to fundamental differences in the bacterial content of their infusions rather than to the nature of the media or to the old age of the Paramecia. May it not be that the hay infusion is not so well suited to the maintenance of a favorable mixture of bac- teria as is beef extract, and that this accounts for Woodruff’s results with this medium? Granted that substances do occur in media which are capable of stimulating Paramecia for a time, is it not more likely that the most important factor in the environ- ment is food? The full importance of food in the behavior of Paramecium was first recognized by Jennings (’08). He not only recognized that cultural conditions should be identical for different series of animals, but he took special precautions to make them so. He realized that the nature of the bacteria in a given culture was very important, for he says: “It is not sufficient to attend merely to the basic fluid, the bacteria in the culture must be the same.” His methods were not directed toward a determination of the exact nature of this food, and he was probably correct in maintaining that if precautions were taken as to the method of making cultures, frequent changes of the animals, and the like, a reasonably constant mixture of bacteria would be obtained, in which enough favorable types would be present to effect a normal metabolism in the animals observed. | The first account of which we have record of feeding Parame- cium with a particular bacterium is that of Popoff (10). He speaks of feeding Paramecium caudatum with cultures of B. proteus mirabilis grown upon potato. Popoff’s attention was centered upon the effect of various media on cells. In this case the medium used was ammonia-rich water. He supposed that the food was uniform, but makes no mention of having freed the animals of other bacteria. These Paramecia lived but a few days, and their death was attributed by Popoff to the nature of the medium. Since my work has shown that Paramecium aurelia 174 RUTH L. PHILLIPS will not live upon B. proteus, it would seem that Popoff’s results may have been due to the effect of unfavorable bacterial food rather than the result of an unfavorable medium. Any experiments dealing with feeding Protozoa pure cultures of bacteria necessarily involve a special technique. It is, how- ever, not difficult for the protozoologist to master. Hargitt and Fray (17) were the first to devise a method for rendering Para- mecium bacteria-free, which is very simple and involves no new method of handling the animals. All that is necessary is to have absolutely sterile apparatus and media. Their method con- sisted in a thorough washing of the animals in several changes of sterile water. The bacteriological tests for controlling cultures in the progress of the work are of the simplest, and with the pre- pared media now on the market, take little more time than does the making of ordinary hay infusions. In my experiments I have so shortened the method described by Hargitt and Fray that it is possible to carry a very large number of cultures, and my work has shown that with care as to the sterility of all appa- ratus, one need not fear contamination for some time. This reduces the amount of washing necessary to a minimum—an important factor in economy of time. It would seem, therefore, that there should be no technical obstacles in the way of further investigations concerning food, and that much of the work now under way regarding the effect of glandular extracts and vita- mines on the Protozoa might be made more exact by the use of these methods. The method of making control cultures containing chance mixtures of bacteria is important. Jennings (’08) describes his method for keeping the mixture of bacteria constant. He took Paramecia from vigorous stock cultures and washed them in a large amount of culture fluid. Since the animals were from dif- ferent infusions, a very representative mixture of bacteria war obtained in the washing fluid. This was used to seed the cul- tures in which he wished to grow pure lines of Paramecium. By doing this every few days he was able to keep the bacterial con- tent of his cultures at an optimum efficiency as to food. It seemed to me that a chance mixture even more reliable than this FEEDING PARAMECIUM KNOWN BACTERIA 105 might be had if sterile media were inoculated every day with fresh hay, and the cultures obtained used when twenty-four hours old. Experience demonstrated that this method is not reliable. Cultures prepared in this way failed to support the life of Paramecium after a period of four months. Since bacteria are known to vary considerably in their resistance to drying, it is very probable that in the method used, I was actually dealing with a progressively changing bacterial content rather than a fairly constant one, as I at the time supposed. It is, therefore, probable that some such method as described by Jennings is to be preferred for keeping Paramecium in vigorous condition upon a chance mixture of bacteria. It is possible that the determina- tion and selection of the predominant bacteria in an infusion by pouring agar plates every day or two, and seeding infusions with predominating colonies thus obtained, without resorting to first growing them in pure culture, might give satisfactory mix- tures. This method is, however, open to objection. It is at the best tedious and more time-consuming than the familiar way. Moreover, one is not sure that all types of bacteria found in infusions grow equally well on the ordinary media.. Considerable study would be necessary to determine this, and additional time consumed in finding a suitable medium for the growth of all types of bacteria. The rate of fission is our main index of the metabolic condition of Paramecium. Calkins (’02 a) introduced the method now in use of taking the average division rate for a given number of days as the basis for conclusions regarding the behavior of this infusorian. The number of days chosen in computing the averages is arbitrary and varied with different investigators. Calkins preferred the ten-day interval. Woodruff used the five-day in- terval in his investigations. Since, in dealing with the effect of known food, it has seemed desirable to have a record of all fluetua- tions possible and avoid undue error, a three-day interval was chosen for averaging division rates in this study. Although it is true that the division rate is the only visible index of the progress of metabolism in Paramecium, and it must serve as the basis for all data bearing on the subject, the phe- 176 RUTH L. PHILLIPS nomena indicated by the actual rate of division may be expressed in other ways, such as the percentage of high divisions above the mean rate for a given period, percentage of divisions below this mean, and the percentage of deaths. It is believed that such expressions of the activities of the animals are helpful in interpreting their reaction to food. These percentages have been computed and are included in the tables. One of the newer methods in biometry is the determination of what I have elsewhere termed the ‘significance factor.’ I have used this term as being less clumsy than any phrase describing the mathematical processes involved, believing that its meaning is as clear as a longer expression. Gross (20) and MacDowell (’21) have used this method in interpreting physiological results, but, so far as I am aware, it has not previously been used in experi- mental work with the Protozoa. The use of the significance factor has proved to be the most helpful of any instrument em- ployed in interpreting the facts. It has been possible by its use to decide as to the relative value of different foods or the effects of changes in medium. Division rate alone, even when aided by percentages of high divisions and the like, fails to reveal the entire truth in such cases. For instance, in dealing with changes in medium, the conclusion from an inspection of these data would be that a moneywort infusion was stimulating when first used in all the lines tested. Determination of the signifi- cance factor reveals that the only real evidence of acceleration. of metabolism was in the case of the mixture A’B’C’. We may state, therefore, that although the division rate is the truest index of the metabolism we have yet found for the Protozoa, the interpretation of its meaning is greatly helped by other indices, especially by the use of the ‘significance factor,’ which at once enables one to settle any doubt as to the trend of the metabolism as expressed by the rate of division. Under normal conditions Paramecium feeds mainly upon bacteria. The enormous variety of bacteria in infusions makes it possible for this animal to obtain adequate food under natural conditions. However, since Paramecium has no choice as to the type of food, but must ingest whatever comes in its way, it is FEEDING PARAMECIUM KNOWN BACTERIA 177 entirely unprotected against deleterious bacteria if these pre- dominate in infusions in which Paramecium is living. The ani- mals may die from starvation in the midst of an abundance of unavailable food, or from poisoning should these bacteria be toxic. The lack of exact knowledge concerning the characteristics of saprophytic bacteria is a great handicap in undertaking the study of any bacterial mixture serving as food for Paramecium. Har- gitt and Fray (’17) found it impossible to identify adequately the organisms with which they worked. Miss Watt and I found the same difficulty in the course of this investigation. I have in- eluded cultural characteristics of the organisms used in these experiments with the idea that they might be of some slight help to anyone desiring to undertake similar work, but there is great need for a thorough investigation of the saprophytic bac- teria. Until such a study shall have been made, it is desirable that any satisfactory cultures which may be discovered shall be carefully kept and made available to any investigators who wish to use them. The cultures A’, B’, and C’ are being maintained, and subcultures will be given to those who desire them. For ordinary routine work, a satisfactory chance mixture main- tained at a high state of efficiency is to be preferred to an artificial mixture such as used in this investigation. The behavior of animals fed with M during the first month amply demonstrates this, for this mixture was superior to the artificial ones during this period. However, if one is investigating the behavior of Paramecium with great care, a known bacterial content is necessary in infusions. Deleterious bacteria certainly lower the rate of metabolism or cause an undue percentage of deaths and so modify normal metabolism as to greatly influence the inter- pretation of results. A varied diet is natural for most animals. Although a given environment may be normal for a particular organism, it is not necessarily one in which the optimum of metabolism can be maintained. Variety of food seems to produce an optimum metabolic rate in all cases investigated, largely because no one type of food has been found which combines all the factors neces- 178 RUTH L. PHILLIPS sary to maintain this rate. The question arises, does this generalization hold true for the Protozoa? Will Paramecium thrive upon a single type of food, as illustrated by a pure culture of a bacterium, or is a mixed food better for this animal? The fact that Paramecia were able to live upon the streptothrix C’ for a period of nine months, when the line was discontinued, would seem to indicate that it is possible for them to live upon a single article of diet. Moreover, in the case described, the metabolic rate was more uniform than with any food save the chance mix- ture during the first month. The division rate was not so high among animals fed with C’ as with the unknown mixture before it failed, or the mixtures A’C’ and A’B’/C’. That such organisms as C’, which are capable of supporting the life of Paramecium when fed in pure culture, are not numerous is also indicated. Of the nine cultures of bacteria isolated from infusions and tested, only C’ could be so used. It is also true that satisfactory mix- tures of known bacteria are hard to find. Two only were dis- covered out of twelve tested. There is no question but that the method used in this work, and by Hargitt and Fray, is not suit- able for providing a thoroughly efficient food, but on the other hand it is the only one yet devised whereby the food can be adequately controlled and known types of bacteria included. These bacteria can be determined only by long and tedious tests, and they must first be obtained in pure culture if the food is to be adequately controlled. Pure cultures of bacteria usually fail to support the life of Paramecium. This failure may be due to one of two reasons. Paramecium may not be able to digest them and so utilize their contained energy, or else the bacteria may contain or excrete substances which are toxic to this animal. In the first instance the bacteria are ingested, but are not digested. The animal starves as it would in asterilemedium. In the second ease death comes more quickly. Khainsky (’10) undertook to study the relationship between the structure and the physiological state in Paramecium cauda- tum. He studied the course of the food vacuole and the changes occurring within it by the use of vital stains. It is possible that FEEDING PARAMECIUM KNOWN BACTERIA 179 this technique might prove useful in testing the food value of particular bacteria for Paramecium. The question of toxicity is more complex. In the account of the preliminary experiments of this study, 100 per cent of deaths occurred the first twenty-four hours among animals fed with L’ and K’L’. A wholesale mortality such as this would lead one to suspect that the bacterium L’ was toxic for Paramecium. Yet, when it was combined with J’, itself not a satisfactory food, im- mediate death did not result. Phenomena such as these need further investigation. The evidence presented in this paper seems to show that mix- tures of bacteria furnish the most satisfactory food for Parame- cium. Of all the mixtures tried, the chance mixture M seems to have been the best, could it have been maintained at an optimum of efficiency. The objection to the use of such a mixture in certain types of experimental work is that its exact content is unknown and is subject to daily variation. A mixture such as A’C’, when studied with regard to the division rate and signifi- cance factor, is found to be so nearly the equal of the chance mix- ture during a long period of time, that it may be said to be on the whole as advantageous. It possesses the advantage, more- over, of being known and subject to control. Mention has been made of the stimulating action of mixed cultures of bacteria as contrasted with pure cultures. The word stimulating is not used herein the same sense as in the account of the effect of change in medium. In this latter instance, any acceleration of metabolism noted is probably due to the action of some chemical constituent of the medium less complex than a food. Mixing foods may cause an acceleration of metabolism simply because more energy becomes available from an outside source, whereas the effect of a chemical stimulant is to release energy locked up within the organism. ‘The increase in meta- bolic rate as a result of combining pure cultures of bacteria has been the usual experience during this work. Frequent as it has been, however, but two instances, A’C’ and A’B’C’, were ob- served where this acceleration of metabolism continued for any great time. 180 RUTH L. PHILLIPS Woodruff and Erdmann (’14) showed that what they describe as rhythms in Paramecium are due to the internal reorganization of endomixis. The study of Paramecium with reference to food seems to show that minor fluctuations occur in the course of rhythms which, like these latter, are largely independent of en- vironmental conditions. Such fluctuations have been noted by others, but have been attributed to such unknown factors as variation in food or medium. In my experiments food and medium were as exactly controlled as possible. All the organisms were subjected to the laboratory temperature, but, as has been pointed out, since these fluctuations took place when there was . little change in the temperature, it would appear that this factor had little to do with their occurrence. More carefully controlled experiments are necessary to determine this. There appear, how- ever, to be fluctuations in the metabolism not directly connected with the rhythms of endomixis, but, like them, due to some in- trinsic characteristic of the protoplasm. These fluctuations occur in all cultures irrespective of changes in food or medium and do not appear to be directly due to environmental factors, but rather to be manifestations of protoplasmic changes. This subject needs careful investigation before definite conclusions can be drawn. Having determined that mixtures of bacteria are more satis- factory than pure cultures, one is led to inquire if such a mixture should be kept constant. Such experiments as were performed in testing the effect of change of food showed that under the conditions obtaining, change of food was usually accompanied by so much disturbance in the metabolism that no marked in- crease in division rate resulted, and that with these mixtures at least constancy of diet was preferable to change. ‘This result with a known food supports the contention of workers with chance mixtures, namely, that every effort should be made to- ward maintaining a uniform bacterial content in media used for growing Paramecium. The behavior of Paramecium in sterile media is of interest for two reasons. It demonstrates that these animals are incapable of utilizing for food any substances which may be dissolved in FEEDING PARAMECIUM KNOWN BACTERIA 181 such a medium. For this reason, it is valuable in testing the availability of various bacteria for food. If Paramecia live no longer, or but little longer than in sterile medium, we are justified in assuming that these organisms are unsuitable food; that their energy content is unavailable. Since it has been demonstrated that Paramecium does not utilize the substances dissolved in sterile media of the sort ordinarily used in the laboratory, it would seem that Peters’ (20) contention that Protozoa are capable of saprophytic existence needs further investigation. His experi- ments dealt with the growth of Colpidium in a sterile synthetic medium very different from hay infusion. Colpidium encysts, and so has a means of becoming adjusted to marked environ- mental change. It would seem, therefore, that Paramecium or some other non-encysting protozoan should be tested in this synthetic medium before generalizations can be made regarding the ability of Protozoa to live as saprophytes. SUMMARY The study of the behavior of Paramecium aurelia when fed with known bacteria shows that it is perfectly feasible to control the bacterial content of a medium and that the technique re- quired is not too laborious. In studying such conditions, it has been found that, in addition to the rate of fission, a consideration of the percentages of high and low divisions and the death rate is of value. The application of the significance factor to data of this type has proved exceedingly useful, making possible decisions with regard to effects of food and media which could not other- wise have been reached. The contention of Hargitt and Fray that pure cultures of bacteria are as a rule unsatisfactory food for Paramecium has been sustained in this work. Moreover, in the single instance in which a pure culture could be used over a long period, the meta- bolic rate was consistently lower than that of any mixture em- ployed. Mixtures of bacteria would then appear to be the most satisfactory food for Paramecium. Of all the artificial mixtures tested, but two were found which furnished adequate food over 182 RUTH L. PHILLIPS a long period of time. These two artificial mixtures were nearly as satisfactory as the usual chance mixture. When Paramecia are fed with known mixtures of bacteria, it is found that minor fluctuations in division rate occur which are independent of endomixis, and like it do not seem to be greatly influenced by environment. The evidence gathered tends to favor constancy of food rather than frequent change. The in- fluence of ordinary media is practically without effect. Para- mecium is unable to utilize food substance dissolved in such media. LITERATURE CITED Caukins, Gary N. 1902 a Studies on the life history of the Protozoa. I. The life history of Paramecium caudatum. Arch. f. Ent-Mech., Bd. 15, S. 139-186. 1902 b Studies on the life history of the Protozoa. III. The six hundred and twentieth generation of Paramecium caudatum. Biol. Bull., vol. 3, pp. 192-205. Cuester, F. D. 1914 A manual of determinative bacteriology. The Mac- millan Company. GituesPig£, L. J. 1920 Colorimetric determination of the H-ion concentration without buffer mixtures, with special reference to soils. Soil Sci., vol. 9, pp. 115-136. Gross, A. O. 1920 The feeding habits and chemical sense of Nereis virens Sars. Jour. Exp. Zo6l., vol. 32, pp. 427-442. Harairt, G. T., anp Fray, W. W. 1917 The growth of Paramecium in pure cultures of bacteria. Jour. Exp. Zodl., vol. 22, pp. 421-455. Huntinaton, KE. J. 1918 Handbook of mathematics for engineers. McGraw Hill Book Company. JENNINGS, H. S. 1908 Heredity, variation and evolution in Protozoa. III. Proc. Amer. Phil. Soc., vol. 47, pp. 393-546. 1910 What conditions induce conjugation in Paramecium? Jour.. Exp. Zo6l., vol. 9, pp. 279-299. Kuartnsky, A. 1910 Physiologische Untersuchungen iiber Paramecium cauda- tum. Biol. Centr., Bd. 30, S. 267-278. MacDowe tu, E. C., anp Vicari, E. M. 1921 Alcoholism and the behavior of white rats. I. The influence of alcoholic grandparents upon maze behavior. Jour. Exp. Zool., vol. 33, pp. 209-291. Maupas, E. 1888 Recherches expérimentales sur la multiplication des in- fusoires ciliés. Arch. de Zoédl. Exp. et Gén., Ser. 2, T.6, pp. 165-277. Meissner, M. 1888 Beitrige zur Ernihrungsphysiologie der Protozxen. Zeit. f. Wiss. Zool., Bd. 46, S. 498-516. FEEDING PARAMECIUM KNOWN BACTERIA 183 Perers, R. A. 1920a Nutrition of the Protozoa. The growth of Paramecium in sterile culture medium. Jour. Physiol., vol. 53; Proc. Physiol. Soc., Feb. 21, 1920, eviii. 1920 b Nutrition of the Protozoa. 2. Carbon and nitrogen com- pounds needed for the growth of Paramecium. Jour. Physiol., vol. 54; Proc. Physiol. Soc., Oct. 16, 1920, i. Pororr, M. 1910 Experimentelle Zellstudien. III. Ueber einige Ursachen der physiologischen Depression der Zelle. Arch. f. Zellf., Bd. 4, S. 1-48. Winstow, C.-E. A., AND oTHERS 1920 The families and genera of the bacteria. Final report of the committee of the Society of American Bacteriolo- gists on characterization and classification of bacterial types. Jour. Bact., vol. 5, pp. 191-229. Wooprvurr, L. L. 1908 The life cycle of Paramecium when subjected to a varied environment. Am. Nat., vol. 42, pp. 520-526. 1911 Evidence on the adaptation of Paramecium to different environ- ments. Biol. Bull., vol. 22, pp. 60-65. Wooprurr. L. L., anp Baitspuy, G. A. 1911 The reproduction of Paramecium aurelia in a ‘constant culture medium’ of beef extract. Jour. Exp. Zo6l., vol. 11, pp. 185-142. Wooprurr, L. L., Aanp ERpMANN, R. 1914 ay is ., path ee So . ; * : Coys ( _. i. i 4 : 4 i pat} a ms 1 ' > ~~ A’. ate ¥ iy 4 ast Pi’ f £8) 2 te ban ¢ i * we ~ 5 See : ‘ . 4is% a. . ‘ ea ea ; i , ee ; ’ Poy + mi, ° ‘ iJ ‘ My ‘ \ an ee ; . } , 4 OT ial a . ‘ > th Lay ; , ono ar .* 3 , " ' - , P| : ia . at k Pau OA ee “se Pd . a a vos a4 pe i 1 ne . 7 ae * ir q i ie R 1 “a. | ti 7 +" i] ye ; ; ee = rg ve Pe a mee aly. t ~ J wae i eae i ee z 2 “St yr 4 tj te wwe. Bs nt ; j ; hay . ; i —_ FeLi Di ¥ pe a ; ; are +; dint | iy Pris ca We es o's aes ae iy hint (nore ‘ Pri) ee ae)" ee gas wet 7 7 s Md in) af? i PROMPT PUBLICATION The Author can greatly assist the Publishers of this Journal in attaining prompt publication of his paper by following these four suggestions: 1. Abstract. Send with the manuscript an Abstract containing not more than 250 words, in the precise form of The Bibliographic Service Card, so that the paper when accepted can be scheduled for a definite issue as soon as received by the Publisher from the Editor. 2. Manuscript. Send the Manuscript to the Editor prepared as described in the Notice to Contributors, to conform to the style of the Journal (see third page of cover). 3. Illustrations. Send the Illustrations in complete and fin- ished form for engraving, drawings and photographs being pro- tected from bending or breaking when shipped by mail or express. 4. Proofs. Send the Publisher early notice of any change in your address, to obviate delay. Carefully correct and mail proofs to the Editor as soon as possible after their arrival. By assuming and meeting these responsibilities, the author avoids loss of time, correspondence that may be required to get the Abstract, Manuscript and Illustrations in proper form, and does all in his power to obtain prompt publication. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO.3 OCTOBER, 1922 Resumen por el autor, Edgardo Baldi Investigaciones sobre la fisiologia del sistema nervioso de los insectos. II. Movimientos circulares de los coleopteros. El autor estudia en algunos géneros de coleépteros (Blaps, Pimelia, Carabus y otros) aquellos movimientos circulares causados por lesiones de la regién lateral de los ganglios supraeso- figicos. Después de haber investigado la naturaleza de la alteraciOn necesaria para producir tales movimientos y de haber descrito el mecanismo de la locomocién normal de los coleép- teros, afirma que los movimientos circulares estan causados, sobre todo, por un mayor grado de flexién en todos los grupos musculares del lado del organismo opuesto ‘al de la herida cere- bral, especialmente por el mayor grado permanente de la con- traccion de los mtisculos adductores de las patas (articulacion tibio-femoral). También discute basdindose en este punto de vista mas general, las conclusiones de otros autores que le han precedido, especialmente las de Bethe. Indica como una herida cerebral produce una serie de alteraciones generales de la muscu- latura, las cuales afectan a todo el cuerpo del insecto y no estan localizadas, conforme han creido otros muchos autores, en una mitad separada del organismo. Basdndose en estas observa- ciones y en un estudio cuantitativo de la disimetria muscular (empleo de los reogramas) propone una teoria de los movimientos circulares de los coleépteros que se diferencia de todas las pre- cedentes en el hecho de fundarse solamente en datos experi- mentales sin recurrir a las expresiones vagas y ambiguas de “inhibicion’” y “tono. ’ Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, SEPTEMBER 11 STUDI SULLA FISIOLOGIA DEL SISTEMA NERVOSO NEGLI INSETTI II. RICERCHE SUI MOVIMENTI DI MANEGGIO PROVOCATI NEI COLEOTTERI EDGARDO BALDI Istituto di Zoologia, Universita di Pavia QUARANTA FIGURE INDICE 1. Introduzione. Moti di rotazione e moti di maneggio.................... 211 2. Supposizioni sulla determinazione del moto di maneggio................. 218 3.7 ba locomozionemermale der icoleottert.o. 0... ..4/./..o.9 «2 aon ooee vos Lele 4. Liinserizione graticader iatt1 locomotoril.. 262.450... « neediness eeese 231 5. Le alterazioni della deambulazione normale nel maneggio............... 239 6. La riproduzione sperimentale dei moti di maneggio..................2.-. 259 7. Aspetti del maneggio. L’orientamento del corpo. La variazione dei TTB GUE sere ETE ic Sie aye orci ore oka Aas RRR, Sate Pies eaceeSyNe evel hno oe weaver 266 $2 ll-determinismo dei moti di maneggio..%..2.2.4- 400/22. os sos eoe-ce sare dce cls 274 1. INTRODUZIONE. MOTI DI ROTAZIONE E MOTI DI MANEGGIO Prima di accingerci allo studio descrittivo ed interpretativo dei fenomeni di maneggio, e pure attendendo da questo studio luce che ci possa permettere di afferrarne il significato e quindi di trovare loro un posto tra le altre manifestazioni dell’attivita fisiologica degli insetti studiati,! converrd tentare di circoscrivere il concetto dei movimenti di maneggio, cosi come li studieremo, trovando per essi un approssimativo collocamento tra gli altri fenomeni consimili, di andamento normale od anormale, che ci sono presentati dagli organismi animali, dagli insetti in ispecie. 1 Delle specie che mi hanno interessato ho fatto cenno in una precedente nota, alla quale rimando come ad una informazione preliminare ed in certo modo neces- saria alla comprensione di talune pagine della presente: “‘Ricerche sulla fisiologia del sistema nervoso negli insetti. I. L’influenza dei gangli cefalici sulla locomo- zione dei coleotteri.’? Atti Soc. ital. Scienze Naturali, vol=40, Milano, 1921. 211 Al be EDGARDO BALDI I moti di rotazione compiuti da un organismo non sedentario e che interessino l’intero suo corpo possono effettuarsi attorno ad un asse che passi per una regione del corpo, oppure attorno ad un asse che cada fuor d’esso corpo. I primi non sono evidente- mente legati ad una trasposizione spaziale dell’organismo in regione sufficientemente lontana dall’iniziale: l’organismo, cioé, vi pud obbedire rimanendo in situ, mentre 1 secondi causano un suo trasporto attraverso lo spazio. I due moti possono essere abbinati nella loeomozione ad elica od a cavaturaccioli, consueta ad aleuni animali inferiori e provocabile in altri mereé opportune lesioni ai gangli cefalici. Anche moti di rotazione attorno ad assi cadenti fuori del corpo possono presentarsi non anormalmente. Cosé dell’Agromyza scrive lo Zetterstedt che sia ‘‘motu valde agilis, inquieta, per folva fere in circulo currens.”’? Né é raro osservare, anche in insetti comunissimi—io |’ho vistain pit generi di ditteri—l’apparizione sporadica di moti in circolo, in ambiente naturale e probabil- mente in particolari condizioni di illuminazione. Tali moti per6 sono occasionali e di breve durata; né molto difficile sarebbe il riunire qui altri esempi di moti rotatorii in senso lato, generica- mente presentati da organismi illesi, in ambiente normale. Ma non é di essi—fenomeni fuggevoli e di vario ed impreciso signi- ficato—che noi intendiamo di occuparci. Noi ci riferiremo infatti, in queste nostre ricerche, all’individuo singolo e terremo sopratutto conto, nello studiare 1 fenomeni di giro in circolo, in esso artificialmente provocati, come delprinci- pale loro fattore, del grado e della natura della dissimmetria sen- soria e locomotoria ch’esso individuo presenta. E, per chiarirei il concetto del moto in circolo, considerato come conseguenza di una alterazione nella simmetria delle condizioni che regolano Vattivit’ normale dell’organismo dell’insetto, considereremo quest’ultimo come segue. Nell’animale normale i gruppi muscolari che presiedono, con fenomeni di contrazione attiva e di tono, alla dirittezza della 2 Citata da E. Corti ‘““Contributo alla teratologia dei ditteri’’ (Lavori dell’ Isti- tuo Zoologico dell Université di Pavia, No. 14, 1913) secondo i “Diptera Scan- dinaviae disposita et descripta’”’ dello Zetterstedt (VII. 2729). MOVIMENTI DI MANEGGIO NEI COLEOTTERI Dil deambulazione ed alla conservazione di un certo orientamento nello spazio, sia dell’intero organismo, sia di singole sue regioni, sono simmetricamente distribuiti ai due lati del piano sagittale. Cosi avviene ad esempio, dei muscoli che si inseriscono a parti chitinizzate mutuamente mobili, come i segmenti del corpo ed i diversi tratti degli arti ed in genere alle appendici pari dell’ organi- smo. Per riassumere in una espressione sola questo tipo di disposizioni, potremo dire che sussista, nell’organismo dell’insetto, uno “scheletro motorio” simmetrico. Ma questa simmetria delle disposizioni e delle attivité muscolari é sorretta e guidata in certo senso e dentro certi limiti, da un’analoga disposizione ed attivita delle regioni sensorie alla superficie dell’organismo. Quelle regioni infatti della superficie esterna dell’organismo che sono devolute all’ufficio di zone concentratrici e trasmettitrici delle azioni del mondo ambiente, le regioni sensibili, cioé—e, fra esse, gli organi di senso ed 1 sensilli—sono del pari e ad un dipresso distribuiti in maniera simmetrica sui due antimeri dell’animale. I pit vistosi organi di senso, gli occhi, le antenne, i palpi, sono pari e simmetrici; gli eventuali organi impari, taluni ocelli, ad esempio, sono contenuti nel piano sagittale, e per i nostri fini, possono venir considerati come due organi pari infinitamente vicini. Volendo indicare con una nuova espressione questa disposizione di cose, potremo dire che, accanto ad uno scheletro motorio esiste, nell’organismo di molti insetti uno ‘‘scheletro sensorio.”’ Fra questi due scheletri simmetrici, come li abbiamo chiamati, per amore di schematicita’, intercedono definite rela- zioni. Si pué ammettere che, in maggiore od in minore grado, l’insetto venga guidato, nella sua deambulazione, dalla azione di agenti esterni, dalla luce, dagli effuvii chimici, dalla temperatura, da particolari direzioni di spostamento del fluido ambiente e cosi, via. Le percezioni di tali diverse qualita di stimoli, aventi valore motorio, avvengono mercé quelle gii nominate regioni sensorie ’ Fenomeni che nella teoria dei tropismi assumono ordinatamente il nome di fototropismo, di chimiotropismo, di termotropismo, di reotropismo ed anemo- tropismo ecce. 214 EDGARDO BALDI della superficie dell’organismo e vengono incanalate come stimo- li attraverso vie di conduzione che talora é comodo supporre —aeli effetti fisiologici—mantengano un andamento antimerico connesso con la disposizione simmetrica degli organi percipienti. Eeco quindi come siano legate in certo modo la simmetria sen- soria e la simmetria motrice dell’organismo. Comunque si immaginino costrutte e disposte le relazioni fra le due, quest’é certo, che in determinati casi un disturbo di quella prima summetria st traduce in un’alterazione della seconda. Occorre qui segnare quali note differenziino questo nostro modo di esprimere la determinazione dell’orientamento del piano sagittale dell’organismo in un ambiente stimolatore, dalla nota concezione del Loeb.* Per il Loeb, l’organismo é pure una superficie simmetricamente sensibile; l’ambiente che la circuisce é considerato come un campo di forze nel senso rigidamente fisico, anzi, faradayano, dell’espressione. L’incontro di un tubo di flusso con la detta superficie vi provoca mutamenti prevalente- mente e presumibilmente di ordine chimico, di entité propor- zionale al valore dell’intensita del flusso. E in tali mutamenti che risiede la ragione dell’eccitazione che la regione sensibile trasmette, ad esempio, ad un gruppo muscolare, il quale verra posto cosi in attivit’ con una intensita proporzionale ancora allintensité del flusso. E l’organismo, in forza di questa destata attivit’ muscolare, si orienta rispetto alla direzione delle lnee di forza dell’agente stimolatore in modo che Veccitazione si uguagli sulle due regioni simmetricamente sensibili. Tende cioé, in definitiva, a disporre il proprio asse longitudinale nella dire- zione delle linee di forza del campo od in quella delle risultanti dalla composizione delle linee di forza di due campi che si com- penetrino.® Cosi si pué formulare in breve la teoria dell’orientamento quale si puo trarre dalle interpretazioni loebiane dei fenomeni di tro- 4 Veggasi il cenno bibliografico e per una moderna e pur fedele esposizione del pensiero di Loeb, veggasi Bouvier. La vie psychique des insectes-Flammarion. Paris 1920 e Kiihn. Die Orientierung der Tiere im Raum. Fischer, Jena, 1919 ove, con alquanto diversa forma, sono esposte vedute che si accostano. a quelle discusse nella presente nota. 5 Particolare sul quale ha insistito dettagliatamente il Bohn. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 215 pismo. La differenza fra di essa ed il nostro punto di vista é di earattere sopratutto logico; in realta il biologo, nel considerare un organismo che risponda a variazioni delle condizioni di ambiente (stimoli) alterando comunque il proprio comportamento, non saprebbe risolvere se—nel decidere del senso e del modo di quelle alterazioni—prevalga il fattore puramente fisico e chimico, se cioé l’organismo sia in piena balia del mondo stimolante, oppure se tale decisione dipenda da una destata connessione di attivitd proprie all’organismo e vincolate agli effetti fisiologici della sua sensorieta, se cioé vi sia in esso ed in quale misura, una spontaneita psicologica. La fisiologia non ci da modo di risolvere il pro- blema e l’aderire all’una piuttosto che all’altra spiegazione, alla psicologistica piuttosto che alla chimicofisica, non é gid conclu- slone, ma presupposto pit o meno necessario, della ricerca. Alla stregua quindi dei dati di fatto, ’una concezione non é pit infon- data dell’altra. Ora quel modo, che abbiamo detto, di rappresentarci le con- nessioni fra l’azione degli stimoli del mondo fisico e la reazione motoria dell’organismo, evita entrambi quegli scogli, in quanto non si riferisce tanto alla distribuzione degli stimoli nello spazio ambiente, non tanto, cioé, allo stimolo considerato a sé, quanto alla distribuzione della loro azione sulla superficie sensoria dell’ or- ganismo. Cioé a quello che—per estensione—si potrebbe chia- mare la percezione dello stimolo da parte dell’insetto, ove della percezione si tenga sopratutto presente l’effetto fisiologico. L’ambiente viene cosi, strettamente e continuamente, riferito all’organismo e quasi percepito attraverso ad esso; posizione del ricercatore, cui sono strumento, beninteso, le convenzioni che porge la fisiologia comparata dei sensi, nonché le acquisite nozioni sull’anatomia dell’organismo. Questo punto di vista permette senz’altro una analogizzazione ed una generalizzazione di certo interesse. Si identificano per esso, ad esempio, le alterazioni della simmetria in quello che abbiamo chiamato lo scheletro sensorio, provocate per altera- zione della distribuzione degli stimoli fisici nello spazio ambiente e quelle provocate per alterazione della distribuzione delle zone sensorie alla superficie dell’organismo. Cosi possono apparire 216 EDGARDO BALDI intimamente connessi 1 fenomeni descritti dal Bohn, di giro in circolo, in taluni decapodi, per diseguale illuminazione delle super- fici oculari e quelli descritti, ad esempio, da Dewitz, dal Parker, dal Dolley, di giro in circolo in un campo luminoso uniforme, per opacamento di una cornea dell’insetto cimentato. Né meno interessante é la connessione che si pud istituire fra queste altera- zioni e quelle che il Dubois provocava nel Pyrophorus noctilucus, otturando con cera annerita un degli organi luminosi laterali. L’animale deviava in circolo verso il lato illuminato. II Dubois anzl supponeva che Jilluminamento effettivamente servisse all’animale per Vorientamento. Nelle tre alternative: esperi- menti con ischermi opachi, opacamento delle cornee, soppres- sione della luminescenza laterale, vi 6 sempre un tratto comune, che é rappresentato dalla soppressione della distribuzione sim- metrica delle ‘““percezioni luminose.”’ Infine, passando dalla considerazione della pura sensorietaé dell’ organismo a quella delle vie anatomiche di essa sensorieta, dovremo comprendere entro gli elementi di quel primo nostro “‘scheletro,’”’ lo stesso sistema nervoso centrale il quale é appunto costruito, generalmente, con un’architettura simmetrica. Altro puntosul quale é utile soffermarei qualche poco, e che sembra militare in favore del concetto dei moti in circolo, pro- vocati per generica alterazione della simmetria sensoria dell’ani- male, si é quello della specificita delle reazioni dell’ organismo rispetto agli stimoli in azione. L’aver mutilato un animale di un dato organo di senso, l’averne cioé lesa la simmetria sensoria, non é sufficiente a provocare sempre e comunque nello scheletro motorio tale dissimmetria, che ne sia determinato un moto in circolo. Perché questo compaia, occorre effettivamente che la sensibilita dell’animale venga eccitata, ma non una qualsiasi forma di sensibilita, bensi, ed eventualmente, quella di cui é stru- mento l’organo destinato all’amputazione unilaterale. Ad esem- pio, 1 maschi del bombice del gelso cui sia stata amputata una antenna, compiono moti in circolo solamente allorché siano stati eccitati dagli effuvil odorosi che emanano dal corpo della fem- mina.® Analogamente gli individui della Drosophila ampelo- ® Kafka. op. cit. Vo. 6. (Confermato da Kellogg nel 1907). MOVIMENTI DI MANEGGIO NEI COLEOTTERI DAW phila, amputati dell’articolo terminale di una antenna, com- piono moti in circolo solamente alloché vengano eccitati dalle emanazioni degli alcoli, degli eteri, degli acidi risultanti dalla fermentazione delle frutta.? I] Radl ha osservata una Dexia girare in circolo attorno ad un lume solamente allorché le era stata resa opaca una cornea.’ Dolley e Parker hanno riferito di fatti simili vella Vanessa antiopa, né sarebbe difficile trovare, nella bibliografia, altri esempi di connessione specifica fra la alterazione motoria e l’alterazione di qualche elemento dello scheletro sensorio. I risultati di esperienze consimili non sono quindi facilmente generalizzabili ed occorre in essi tenere rigoroso conto delle con- dizioni dell’ambiente fisico, degli stimoli che cioé possono avere agito sull’organismo. L’aver constatato, ad esempio, che |’a- blazione di una antenna in un carabo non produce alcuno squili- brio della locomozione in condizioni normali (condizioni che generalmente si riassumono nella presenza di un campo luminoso uniforme), non implica che tale ablazione debba per sempre ed assolutamente rimanere senza influenza aleuna sulle attivita dell’animale, ad esempio, sulla sua deambulazione. Se siesperi- mentasse infatti con uno stimolo cui l’antenna dell’animale fosse sensibile, probabilmente anche in esso si dimostrerebbero squili- brii di qualche sorta nel comportamento. Nelle nostre specie non abbiamo mai osservati fenomeni consimili, che potessero sovrap- porsi a quelli di maneggio genuino. Riservandoci di esaminare in seguito quanto é stato visto da altri autori in proposito e di discutere la portata delle loro interpretazioni, prenderemo in esame quei fenomeni di rotazione che si accompagnano a lesioni unilaterali dei gangli sopraesofagei e che—come abbiamo indi- cato—sl possono considerare come interessanti ad un tempo lo scheletro motorio e lo scheletro sensorio. 7 Kafka. ibidem. Bouvier op. cit. IJ. Barrows, op. cit. 1907. 8 Loeb. Die Tropismen, cit. 218 EDGARDO BALDI 2. SUPPOSIZIONI SULLA DETERMINAZIONE DEL MOTO DI MANEGGIO Esporremo con maggior dettaglio altrove 1 procedimenti che abbiamo posti in atto per gli interventi sperimentali sui nostri coleotteri, analizzandone le particolari modalité. Le speciali con- dizioni di esperimento non permettendoci altra tecnica pit delicata, abbiamo aggrediti i gangli sopraesofagei indirettamente, attraverso la cuticola chitinosa della volta cranica, servendoci di aghia manico. Poiché il procedimento pué sembrare grossolano e dare appiglio a giustificati dubbi circa la portata della lesione, ci preoccuperemo ora di eliminare questi dubbi, allo scopo di appianare la via alla ulteriore esposizione. Il dubbio pué essere formulato cosi 1 moti di maneggio osser- vati nelle nostre esperienze dipendono effettivamente dalla lesio- ne di una regione nervosa? E cominceremo da un’obiezione, da taluno mossami, che si puoé tradurre nel concetto che il moto di maneggio dipenda es- clusivamente da una sorta di “fuga continuativa” del dolore, causata dalla lesione. Non discuter6 ora dell’opportunita di introdurre in fisiologia quest’ambigua nozione del dolore, accon- tentandomi di ricordare qui la nota esperienza del lombrico di- mezzato, riportata dal Loeb nel gid citato libro;’ ma ritenendo pure che ad una simile sensazione sia dovuta, ad esempio, quella retrazione degli arti che segue ad un toccamento dei tarsi e che in definitiva porta ad un incurvamento nell’opposto senso della deambulazione dell’animale, oppure quella serie di moti degli arti che per toccamento di un’antenna sorte medesimo effetto e cosi via, potrebbe sembrare che una persistenza di stimolo, quale deve essere legata ad una grave lesione di una mets dei cerebroidi sarebbe capace di determinare una persistente analoga reazione. 9 La quale esperienza é peré diW. Normann (che il Loeb non cita) e fu compiuta sull’Allobophora ealiginosa. Pfliiger’s Archiv, 1897. Un’idea analoga ci sembra aver visto espressa, in altra forma, dal Mast a proposite dei moti di rotazione pre- sentati dalla Planaria torva. Riferisco le stesse parole dell’Autore: ““Planaria with one eye removed turns continuously from the wounded side for some time, evi- dently owing to the stimulations of the wound’? (Mast. Preliminary report in the reactions to light in marine turbellaria. Carnegie Inst. Yearbook. Vol. 9. Cit. sec. Taliaferro Reactions to light in Planaria maculata. Journal of exp. Zool. luglio 1920.) MOVIMENTI DI MANEGGIO NEI COLEOTTERI 219 Ma non si comprende allora perché la sensazione dolorifica do- vrebbe essere esclusivamente legata ai gangli cerebroidi; qualsiasi altra grave lesione, infatti, di qualunque altra regione sensibile e laterale, dovrebbe trarre seco analoghe sensazioni dolorose ed analogamente produrre moti in circolo, il che non ayviene. Pud sorgere il dubbio che il moto di maneggio dipenda dalla lesione di qualche altro apparato o sistema che non sia il nervoso, contenuto nel capo e nei pressi dei cerebroidi. Sollevando in- fatti la chitina dalla fronte all’occipite, accanto ai ciuffi muscolari laterali e sopra il ganglio, viene messa allo scoperto una porzione di corpo adiposo e, sotto questo, un brillante plesso di ramuscoli tracheali. Fini diramazioni tracheali si estendono anche attorno e sopra ai gangli, avvolgendoli come in una lassa rete, in genere superficiale e talora qualche poco approfondita nello strato cor- ticale del ganglio, che, per la sua trasparenza, la lascia intravve- dere al fuocheggiamento. In taluni casi tale rete si fa tanto cospicua da attirare vivamente l’attenzione e da affacciare con essa un sospetto. I processi di ossidazione della biomolecola nervosa infatti non ne debbono essere indipendenti, poiché la tra- chea é il veicolo dell’aria respirata. La recisione di qualche ramo collettore tracheale, operata dall’ago introdotto nel capo potrebbe aver compromessa la normale ossigenazione di qualche regione del cervello ed aver causati con cid in esso disturbi funzionali tali da tradursi in alterati comportamenti dell’intero organismo. Ma poiché lo squilibrio dinamico del corpo qual’é espresso nei moti di maneggio, deve riposare sul turbamento di una simmetria funzionale, occorrera, per render responsabile la lesa tracheazione, che questa normalmente si effettui secondo dispositivi simmetrici. Ora, da apposite ricerche sul cervello delle Blaps e delle Pimelia tale simmetria risulta confermata (fig. 1); né il decorso delle diramazioni tracheali segue, nella specie, vie costanti per tutti gli individui, bensf individualmente alquanto variabili, nella rete cerebrale. Solamente nella Pimelia é accennata una distribu- zione grossolanamente simmetrica (fig. 2): da un grosso nodo tracheale postcerebroidale, ove per breve tratto si fondono due cospicui tronchi dando origine a tasche tracheali ed a nuove diramazioni, si dipartono tre ramuscoli distintamente, i quali 220 EDGARDO BALDI Fig.1 Tracheazione dei gangli sopraesofagei di Blaps mortisaga. Fig.2 Tracheazione dei gangli sopraesofagei di Pimelia undulata, Si osservi la distribuzione grossolanamente simmetrica dei 3 rami tracheali. MOVIMENTI DI MANEGGIO NEI COLEOTTERI Don mandano diramazioni rispettivamente alla metdé interna della superficie superiore della porzione sinistra del sopraesofageo— al’avvallamento centrale che segna nel ganglio una sorta di strozzatura mediana, con fini diramazioni laterali—alla meta interna della superficie superiore della parte destra del ganglio. Vi sono poi altri plessi di minor entit’, che discendono lungo le commissure con il sottoesofageo. Ma checché si pensi di questa dubbia simmetria, il fatto che alla superficie del cervello, i ramuscoli tracheali, per distinte frequentissime anastomosi sono in mutua comunecazione, é sufficiente a far porre da parte anche questa interpretazione dei Seo re ae ; ie f ii—— j 5 f a ‘ / je : f 5 \ y A 2 a gee eee ay VF ‘A i j \ Loe \ ) +i K~ Ls T= ; es to. | \ \ i C= | ft . fo \ i } . } * i ; { yt Celt \A> y / NA | % 4 ; xe \ } / Fig.3 Distribuzione dei ramuscoli tracheali nella cortica dei gangli sopraeso- fagei di Pimelia. moti di maneggio (fig. 3). Perché la recisione di un ramo tra- cheale, anche se laterale, possa infatti dar luogo agli effetti sup- posti, é d’uopo che le sue diramazioni conservino un’autonomia ed una mutua indipendenza che mantengano valore alla sua disposizione simmetrica, la quale altrimenti diviene un puro fatto morfologico senza grande significato fisiologico. Vi 6, infine, un’ultima possibilita degna di venire presa in con- siderazione: un coleottero leso che descriva un moto durevole di maneggio presenta il capo volto da quella banda da cui gira. Non potrebbe darsi che l’introduzione dell’ago nel capo avesse lesa la muscolatura del collo cosi da obliterarne la funzionalita da un lato e da sottoporre il capo all’unica azione dei muscoli dal lato opposto che lo costringano in quella posizione? E non po- 222 EDGARDO BALDI trebbe indi essere che—locomovendosi un organismo, ad un di- presso secondo le idee del Loeb, in un campo di forze stimolatrici nella direzione del piano sagittale del capo (del piano mediano, cioé, fra le superfici fotosensibili recettive) tale deviazione pura- mente meccanica del capo fosse sufficiente a determinare il moto di maneggio? Ma tale abnorme disposizione del capo pud venire provocata ad arte mercé la resezione della muscolatura del collo dall’un dei lati, previa asportazione di un tratto di chitina dal corsaletto. Orbene, un animale cosi operato ed in cui il capo presenta una costante piegatura da un lato senza che i gangli cefalici siano stati toechi—si muove sicuramente e coordinata- mente secondo una traiettoria contenuta nel piano sagittale, non del capo, ma del rimanente corpo. Risultato che non solamente esclude che ad un tale obliquamento del piano interoculare sia dovuto il moto in circolo, ma che é tale da infirmare forse lo stesso concetto del Loeb, nella sua applicazione almeno a questo organismo. Altre lesioni inferte ad altre regioni della muscola- tura degli arti e praticate lungo le pareti laterali del torace, allo scopo di disturbare la motilita degli arti da un lato non hanno avuto altro effetto che una parziale soppressione dell’attivita motoria degli arti interessati, la quale non ha mai condotto a moti in circolo, ma all’assunzione di atteggiamenti anormali, per la soppressione dei fattori meccanici dell’equilibrio. E’ lecito concludere, da quanto precede, che il maneggio non é vincolato ad una alterazione statica della simmetria muscolare, cioé alla inattivita di determinati gruppi di muscoli. Dipende esso forse, in prima analisi, da una alterazione dinamica di essa simmetria—da una alterazione, cioé, della funzionalita simmetrica degli arti? 3. LA LOCOMOZIONE NORMALE DEI COLEOTTERI Per poterne giudicare, é necessario ricordare come avvenga, nei coleotteri delle nostre esperienze, la locomozione normale. La deambulazione delle imagini degli insetti, vincolata alla presenza in essi di sei arti contemporaneamente attivi, si attua in uno specialissimo modo, che, nelle sue linee generali ed in modo sufficientemente preciso, era noto gid al Weiss e che indi é MOVIMENTI DI MANEGGIO NEI COLEOTTERI Tapes stato accuratamente studiato da Paul Bert, dal Graber, dal Plateau, dal Dahl. Tale deambulazione implica un complicato mececanismo di regolazione dei moti, meccanismo accuratamente studiato, nei suoi particolari statici e cinematici, da Jean Demoor. Secondo la precisa ed espressiva designazione del Graber, l’insetto in cammino si pud considerare come un doppio treppiedi ambu- lante e gli arti in moto possono venire raggruppati in due terne, ciascuna costituita dall’anteriore e dal posteriore degli arti di un antimero e dall’arto medio dell’antimero opposto, terne le quali si alternano nelle fasi di moto e di apparente riposo. Mentre una terna d’arti, quella ad esempio, rappresentata dal primo sinistro, dal secondo destro e dal terzo sinistro, é sollevata da terra e sta descrivendo un’arcata per prender terra poco pit avanti, la seconda terna, rappresentata dal primo e dal terzo destro e dal secondo sinistro posa a terra e costituisce supporto al corpo dell’ animale durante il ‘‘passo.”” Essa a sua volta si porrd in moto e deseriverd un altra arcata all’avanti, quando la prima si sara posata a terra. L’arcata viene descritta separatemente da cia- scuno degli arti che costituiscono la terna ed il cui moto é contem- poraneo o quasi. In linea generale non si da per6é contemporaneo moto degli arti appartenenti alle due terne. Al loro turno, gli arti simmetrici compiono regolarmente escursioni di uguale am- piezza, cosicché, uguagliandosi gli effetti di trazione e di pro- pulsione da entrambi i lati dell’animale in moto, viene deter- minata la marcia rettilinea. Designeré come ‘‘coordinazione normale’ dei moti locomotorii questo ritmico alternarsi di condizioni di attivita e di quiete fra i sei arti avendo sopratutto riguardo al criterio cronologico, cioé al tempestivo e reiterato intervento di ogni arto nella locomozione e non tenendo conto delle particolari modalité di impiego di ogni arto, modalité connesse alla loro morfologia. La coordinazione dei moti non interessa quindi la velocité dei moti medesimi, né puod influire sulla forma della traiettoria descritta dall’animale o sulla velocita’ del moto lungh’essa. Il discorrere del particolare impiego di ciascun arto nella loco- mozione, come di fenomeno intimamente legato alla morfologia dell’arto stesso—mentre le osservazioni precedenti valgono ad un 224 EDGARDO BALDI dipresso per la locomozione degli insetti in genere—ci conduce a limitare la nostra esposizone ai soli coleotteri, riferendoci a quanto direttamente abbiamo potuto osservare sugli esemplari avuti in esame. Fig.4 Schema di un arto di insetto (modificato dal Dahl). Un arto di coleottero adulto é un insieme di parecchie leve (fig. 4) le une vincolate alle altre in modo ben determinato, cosi da limitarne le possibilita di spostamento nello spazio. In modo approssimativo e schematico possiamo riferire questi movimenti a due piani perpendicolari (fig. 5) nei quali tipicamente hanno luogo 1 movimenti delle due leve principali dell’arto: il femore e la tibia. Porremo l’un piano orizzontale e tangente alla superficie sternale del segmento nel suo punto mediano, l’altro, normale al MOVIMENTI DI MANEGGIO NEI COLEOTTERI 225 primo, orientato verticalmente ed individuato dal suddetto punto sternale e dall’asse della tibia. Nel primo piano si com- piono i moti del femore, nel secondo quelli della tibia; general- mente, gli spostamenti di questa sono compresi entro un angolo massimo, determinato in valore dalla morfologia dell’ articolazione tibiofemorale. In realti& le cose non istanno cosi semplicemente: il piano dei femori non é orizzontale che in particolari casi ed in genere é in- clinato cosi da riuscire (mediante opportuni trasporti paralleli) tangente, non alla linea mediana degli sterniti, ma ad un punto della superficie del corpo pit o meno presso le pleure. I] piano tibiale non mantiene nello spazio un’orientazione costantemente Fig.5 Piani di riferimento dei moti del segmenti degli arti medi nella deam- bulazione (vedi testo). verticale, bensi spesso ruota leggermente e ritmicamente intorno ad un immaginario asse orizzontale, contenuto nello stesso piano. Infine la serie di leve ch’é rappresentata dalla linea dei tarsi ha spesso una meccanica propria e variabile da specie a specie. Ma, attenendoci a quello schema generale, caratterizzeremo l’impiego delle tre paia d’arti, constatando che i moti delle leve del primo paio avvengono tipicamente in un piano B e solo secondariamente in un piano A (fig.5). Occorrerd assegnare un verso al moto con cui sono descritti gli angoli tibiali nel piano B; e chiameremo per6é positivo il verso del moto descritto per adduzione della tibia sul femore e negativo quello descritto per abduzione del primo seg- mento sul secondo. Nella locomozione, il moto dei primi arti é tipicamente efficiente per essere descritto nel piano B con verso THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 226 EDGARDO BALDI positivo; il moto degli arti medii é invece caratterizzato dal pre- ralere di spostamenti nel piano A e dalla subordinata importanza (almeno nella marcia rettilinea) degli spostamenti tibiali nel piano Bb. Il moto degli arti posteriori si avvicina a quello degli arti anteriori: sono cioé particolarmente efficienti in esso gli spostamenti nel piano B, ma descritti con verso negativo. Supposto, per comodita, che tutto il sistema di leve dell’arto sia contenuto in un unico piano, nel piano B, ad esempio, é facile constatare come, per le singole paia di arti, questi plani non siano ugualmente inclinati sul piano sagittale; ad un dipresso ortogonale gli é quello dei medii, mentre quello dei primi gli é obliquo e diretto all’avanti; parimenti obliquo, ma diretto all’indietro é quello dei terzi arti. Riferendoci ad un asse orientato, rappre- sentato dall’asse sagittale dell’animale, diretto dall’addome al capo, l’angolo compreso fra di esso e la traccia del piano B nel primo paio di arti é minore di un retto, é prossimo ad un rettoper il piano mediano, é molto maggiore di un retto ed in taluni casi prossimo ad un piatto per il terzo paio. Possiamo ora in altre parole esprimere la diversa funzione di ogni arto nella locomozione; gli arti medii hanno un’azione emi- nentemente propulsiva, sospingono cioé il corpo all’avanti, de- scrivendo una doppia serie diarcate: arcate sollevate ed all’avanti per ‘‘compiere il passo”’ e prendere indi terra—arcate in posizione di appoggio al terreno e dirette all’indietro, per sospingere il corpo. I terzi arti pure compiono un’azione schiettamente pro- pulsiva’® contraendo la tibia sul femore dapprima perché 1 tarsi e l’estremo tibiale prendano terra all’avanti ed estendendo indi la tibia sul femore, cosi da impellere il corpo verso l’innanzi. Vi é dunque fra gli arti del secondo e del terzo paio una parziale somiglianza d’impiego, ottenuta con diversi mezzi, poiché in quelli il principale lavoro é sostenuto dall’articolazione coxo- femorale ed in questi dalla femorotibiale. Una funzione ben carat- teristica spetta agli arti del primo paio; essi, alquanto rivolti verso l’avanti, hanno un’azione prevalentemente attrattiva sul corpo, si stendono, di afferrano al substrato con i tarsi, indi con- 10 Poiché i secondi in condizioni che non siano quelle della marcia rettilinea entrano pure in gioco con azioni attrattive nel piano B. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 227 traggono la tibia sul femore e avvicinano il corpo al punto di aggrappamento dell’ultimo articolo tarsale. A questo moto che avviene nel piano B con verso positivo e che caratterizza l’impiego degli arti del primo paio si accompagna generalmente un moto ad areata, operato nel piano A, il quale peré non propelle il corpo, ma modifica il valore del moto di pura trazione esercitato dalla contrazione tibiofemorale suddescritta. I primi arti godono, rispetto ai successivi, di un certo grado di liberta e di movimenti, che fa veramente di essi—come gid da tempo si é detto—i timoni dell’animale. Potendosi essi aggrappare nell’atto della loro estensione, in posizioni alquanto laterali, possono causare oscil- lazioni e deviazioni della deambulazione dalla linea retta. Gio- vera notare peré che anche per gli stessi coleotteri, ’impiego dei primi arti pud mostrarsi nel dettaglio, abbastanza vario, cosi come varia la loro morfologia, dalla snellezza e dalla elegante sagomatura degli arti dei carabici alla tozzezza della tibie appiat- tite e dentate dei Copris, dei Gymnopleurus, degli scarabei in genere, in cui la linea dei tarsi si riduce e pud anche mancare com- pletamente come negli Ateuchus, Vinsetto servendosi allora per la locomozione delle estremita distali dei femori. Se queste sono le linee generali della locomozione dei coleotteri, é d’uopo convenire che la meccanica ne é assai plastica e che animale, di fronte ad amputazioni gravi dei tarsi ed anche di parte delle tibie od a svariati disturbi meccanici della morfologia dell’arto, reagisce con fenomeni che potrebbero essere detti di autoregolazione, mercé i quali viene consentita la prosecuzione della marcia. Noi non abbiamo tracciato quindi—per quanto in modo molto sommario—che lo schema della locomozione di un coleottero morfologicamente integro in condizioni normali di funzionalita, procedente in linea retta. Intervengono fatti nuovi, allorché il coleottero in marcia cambia di rotta. Il che agevolmente si pué ottenere premendo leggermente con una can- nuccia ed anche sfiorando leggermente con un pennello uno dei tarsi, in ispecie delle due prime paia di arti; allo stimélo l’animale risponde deviando dal lato opposto da quello stimolato, allon- tanandosi cioé dallo stimolo. L’arto stimolato si é vivamente retratto (come accade sempre, anche allorché |’insetto sia in quiete 228 EDGARDO BALDI od impossibilitato per particolari condizioni fisiologiche, alla loco- mozione) posandosi indi a terra in un punto diverso da quello che avrebbe occupato nello stabilirsi normale della terna di riposo. Passando da questa nuova posizione alla terna di moto, poiché il punto d’appoggio dell’arto al terreno é pit presso al piano sagit- tale di quello che sarebbe stato il normale, l’impulsione fornita dalla distensione dell’arto stimolato é pit intensa di quella for- nita in una terna precedente dall’arto simmetrico, oltrecché diversamente applicata. L’equilibrio degli sforzi propulsivi dai due lati del corpo viene turbato ed il piano sagittale ruota cosf da indirizzare la marcia verso quel lato da cui la somma di detti sforzi é rimasta inalterata e quindi minore, cioé dalla parte non stimolata. Ma Vinsetto pu6 girare anche “‘volontariamente”’ su sé stesso, cioé in seguito all’azione di stimoli non direttamente applicati dall’osservatore. La rotazione pu6 avvenire da fermo ed essere operata da una serie di piccoli spostamenti eseguiti principal- mente dagli arti del primo paio cui secondano i successivi con piccoli moti di aggiustamento; tali spostamenti consistono, nel caso che l’animale volga, ad esempio, a destra, nell’esagerarsi dell’attivita delle leve femorotibiali, le quali compiono dal lato destro e lateralmente, moti di aggrappamento e di adduzione, dal lato sinistro e pure lateralmente, moti di abduzione, cosi che il corpo, per l’azione combinata di queste attivita antagoniste viene ruotato verso destra, finché l’animale non riprenda la deambulazione rettilinea. La nuova rotta, fenomeno caratteris- tico di questo tipo di rotazione, forma un angolo ben deciso con la vecchia direzione (fig. 6e 7). Avviene talora, invece, che la curva abbia pit grande raggio e che non si palesi fra le due di- rezioni un brusco mutamento (fig. 8). Ma lo studio del mec- canismo di questo secondo tipo di svolta richiede che ci rifac- ciano ai procedimenti di inscrizione grafica degli atti ambulatorii dei coleotteri. Noteremo per6é ancora che la coordinazione dei moti durante la locomozione non tralascia mai di dimostrarsi nella marcia normale; il meccanismo testé descritto,*e che im- pegna pochi arti dell’insetto, prevalentemente due arti simmetrici, potrebbe sembrare una contraddizione. Ma le svolte ad angolo MOVIMENTI DI MANEGGIO NEI COLEOTTERI 229 retto si producono solamente allorche’ l’animale abbia sospesa la locomozione progressiva e sia quindi in quiete. Non é per6 il caso di parlare di coordinazione, poiché ogni arto—salvo le de- Fig.6 Reogramma di Blaps con svolte ad angolo (rid. 1/2). bite eccezioni—puo venire usato indipendentemente dagli altri (nei moti di pulizia, ad esempio) in tutti quei movimenti che non siano di locomozione. DGARDO BALDI 7 u E 0 9 “ao *(¢/Z° pit) 99[Op VYOAS U0d Hj092L0)f D1U0}9,) Ip BUIWIBIBOOY § “ST ‘(¢/Z pl) 9 °SY Vi[[OU 9YO OFNNv OUDUI O[OSUB pv vY[OAS MOD sdy7g Ip VUIUILATONY 1°31 MOVIMENTI DI MANEGGIO NEI COLEOTTERI 231 4, L?INSCRIZIONE GRAFICA DEI FATTI LOCOMOTORII Onde poter praticamente applicare il criterio di fare dell’arto uno strumento di segnalazione, che con la sua attivitaé normale od alterata designi l’andamento dei fenomeni che si svolgono nell’intimit’ dell’apparato neuromuscolare interessato, occorre trovar modo di registrare continuativamente i fatti della marcia dell’insetto. L’inchiostratura diretta degli arti non sorte buon effetto e per l’untuosita caratteristica della cuticola dei coleot- teri che impedisce un regolare deflusso della vena d’inchiostro sul sottostante foglio di registrazione, e per l’inomogeneita del tratto e per la troppo breve durata. Demoor afferma di aver usato un simile procedimento, ma non da particolari di tecnica. IE pre- feribile trasportare dal tamburo di Marey sul tavolo registratore il procedimento tanto usato in fisiologia, di inscrivere i moti su di un foglio di carta affumicata. Di questo procedimento gid aveva usato il Dubois, il quale pubblicava nelle sue “‘Lezioni di fisiologia generale” alcuni clichés di grafici consimili, da lui pero eseguiti occasionalmente e non istudiati sistematicamente ed in dettaglio. I coleotteri in particolare si prestano bene a simili inscrizioni. Lascarsa reattivita delle nostre specie a stimoli fotici faceva si che esse non rimanessero gran che turbate dalla presenza di una superficie nera sotto il loro corpo. Molteplici sono i vantagegi offerti da siffatti reogrammi. Ri- conosciute le relazioni fra le tracce ed i moti corrispondenti degli arti che le hanno descritte, losservatore pud studiare, con tutto suo comodo a tavolino, una serie di manifestazioni che mal si lasciano cogliere a volo nell’affaccendato susseguirsi degli arti nella locomozione ed i dati ricavati dalle due forme di esperienza mutuamente si possono integrare. Un reogramma permette confronti precisi tra due fasi successive di una medesima locomo- zione e consente l’effettuazione di misurazioni, l’introduzione, cioé, di un criterio quantitativo nell’indagine dei fenomeni. La comparazione di reogrammi ottenuti da diverse specie, permette l’immediato riconoscimento di una caratteristica, al- meno specifica, della locomozione; ogni specie fornisce un suo tipo di reogramma, dotato di certa costanza. La successione di tracce brevi e distanziate di una Blaps (fig. 9, 10), in cui i graffiti 232 EDGARDO BALDI ro: : ¢ 4 } Ps / i \ . * ; ~ 10 Fig.9 Reogrammidideambulazione rettilinea di Blaps (rid. 2/3). Fig. 10 Schema della disposizione delle tracce deambulatorie per |’inter- pretazione dei reogrammi di Blaps. Le traece esterne ed oblique all’indietro sono degli artimedi. Le interne doppie a semiluna, degli arti posteriori. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 233 dovuti alle estremita dei tre arti si trovano ravvicinati in grup- petti ben distinti, mentre rivela la leggerezza e la sveltezza della marcia dell’animale, nettamente la distingue, ad esempio, dalle tracce pesanti, spesseggianti, continuative di un Osmoderma (fig. 11), le quali sembrano gid rivelare la tozzezza dell’animale e la lentezza impacciata dei suoi moti. Né, per quanto superficialmente si rassomiglino, si potranno confondere le tracce di un Carabus (fig. 12-13-14) con quelle di una Aromia (fig. 15). Le strisciature faleate del primo, dovute al rapido moto delle leve scriventi, sono ben diverse dalle tracce bene impresse dell’Aromia, le quali talora permettono di distin- guere il numero degli articoli tarsali appoggiati a terra e che denotano un posamento lento, preciso, durevole, degli arti sul terreno. Né sarebbe difficile continuare per altri tipi in inter- pretazioni del medesimo genere. Gia dunque di primo acchito il reogramma dice qualcosa, circa la locomozione e la motilita in genere del coleottero (fig. 16—-17—18-19). Anche pit dice un suo esame dettagliato. Ben evidente é il trascinamento dell’estremo dell’addome lungo una linea sinuosa, trascinamento gia’ notato con precisione dal Demoor e di cui lUexkiill ha indicate le ragioni meccaniche. Riprendiamo sui reogrammi lo studio dei mutamenti di rotta ad incurvamento dolce che abbiamo distinti dalle svolte da fermo e riferiamoci ad un simile grafico desritto da una Blaps mortisaga normale; 1 eruppi di tracce nella deambulazione normale, lasciati da tutti gli arti da una banda, sono disposti alternatamente dalle due parti del piano mediano, cosicché ognuno di essi gruppi corris- ponde all’intervallo fra due gruppi attigui, segnati dagli arti dell’antimero opposto. Le due fascie di tracce, destra e sinistra, lasciano fra di loro una zona vuota di segni, e sinuosamente ed interrottamente rigata dalle tracce dell’ addome che a tratti viene a strisciare sul terreno; zona la quale corrisponde alla proiezione sul piano della zona di spazio occupata dal corpo dell’animale. I] margine esterno delle fascie laterali é segnato dalla successione dei colpi di unghia degli estremi tarsali dei medii arti, punti facilménte reperibili. _Misurando le distanze che li separano per i due lati del tratto di marcia che presenta curva- tura, si ottengono, in decimi di millimetro, i dati che seguono: EDGARDO BALDI Arti destri Arti sinistrt 150 170 115 190 158 160 180 Fig.11 Reogramma di Osmoderma heremita (rid. 2/3). Fig.12 Reogramma di Carabus morbillosus (rid. 2/3). ge £ / MOVIMENTI DI MANEGGIO NEI COLEOTTERI 235 Fig. 13 Schema della disposizione delle tracce nei reogrammi di Carabus. Le tracce marginali trasverse sono degli arti medi, quelle faleate dei posteriori. Le tracce interposte alle faleate sono dei primi arti. Allorché si tenga presente che la distanza fra due punti omo- topi dei gruppi di tracce corrisponde all’ampiezza del colpo d’arco che l’arto relativo ha data al proprio moto propulsivo—e si pud quindi ritenere proporzionale all’intensitd stessa dell’impulso che Varto ha trasmesso al corpo nell’atto locomotorio—appare evi- dente come l’animale, per isvoltare a destra abbia raccorciate le ampiezze dei colpi d’arco dal lato destro, abbia cioé diminuita Vintensité degli impulsi motorii degli arti impressi al corpo da 236 EDGARDO BALDI Fig. 14 Reogramma di Carabus morbillosus, di tipo diverso dalla fig. 12 (rid. 2/8). Fig.15 Reogramma di Aromia moschata (rid. 2/3). Fig.16 Reogramma di Pimelia undulata (rid. 2/3). quella banda, mentre ha esagerate le ampiezze dei moti e le intensit’ degli impulsi dal lato sinistro. L’intervallo normale, infatti, tra due posizioni omotope nella serie locomotoria, rileva- bile da altri tracciati di Blaps, é di quindici millimetri. L’analisi che compiremo in seguito con 1 medesimi mezzi sul traeciati dei movimenti di maneggio ci dimostrera’ una certa MOVIMENTI DI MANEGGIO NEI COLEOTTERI 231 lee 8 wT ? 4 & o % t cad satiaee i 9 % | Pa fae As & a 4 od t e,.. | ia 623 ES « 3 4 r @ of 17 Fig.17 Schema della disposizione delle tracce degli arti nella deambulazione di Lamia. Fig.18 Reogramma di Oryctes griphus (rid. 2/3). somiglianza qualitativa fra di essi ed i fenomeni di svolta “volon- taria,’ somiglianza sulla quale ritorneremo. L’esame diretto dei fenomeni della deambulazione puo chiarire qualche dettaglio dei tracciati reografici. Cosf, nella Blaps mor- tisaga normale tutti e tre gli arti poggiano sul terreno con la spina della estremita distale della tibia e con le unghie dell’ultimo 238 EDGARDO BALDI anello tarseo. ‘Talora anche, per un meno pronunciato incurva- mento della linea dei tarsi, il substrato é sfiorato anche dalle apofisi dei rimanenti articoli tarsali, col che viene inscritta sulla carta affumicata la serie di piccoli tratti che qualche volta si scorgono dietro il graffio delle unghie. Graffio che nei primi arti ha spesso una caratteristica forma lunata. Infatti l’azione degli arti del primo paio si puo scin dere in tre momenti successiv1: 1. I tarsi unghiati, aggrappandosi al substrato, esercitano una trazione dovuta alla tibia che viene addotta verso il femore; 2. la tibia avvicinatasi maggiormente al corpo fa forza sulla sua spina distale puntellandosi molto obliquamente all’indietro: Fig. 19 Reogramma di Oryctes griphus. Le tracce dei terzi arti sono pit strisciate che nella fig.18 (rid.2/3). 3. frattanto la linea dei tarsi, facendo perno su detta spina, descrive un piccolo angolo all’indietro tracciando sul terreno le areature dell’unghia ed i trattini delle apofisi tarsee, che si rile- vano nel reogramma. La meceanica dei secondi e terzi arti nella Blaps si conforma ad un dipresso alle regole generali che abbiamo date pi su. Né molto se ne discosta la locomozione della Pimelia undulata, in cui i moti dei primi arti nel piano B sono meno evidenti che il consueto ed in cui tali arti descrivono con la linea tibiotarsale brevi arcate quasi rettilinee ed orientate parallelamente all’asse sagittale del’animale. Nella Pimelia é invece spiccata la carat- teristica meccanica di propulsione all’indietro secondo lo schema descritto, degli arti dell’ultimo paio. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 239 I. earatteristica delle specie, buone e rapidi camminatrici,— earabi e blapsidi, ad esempio,—una molto precisa coordinazione dei moti ambulatori, laquale spessosiesprime nel raggruppamento e nel distanziamento delle traccie locomotorie sui reogrammi. Con minor precisione essa si effettua nelle specie che camminano malamente; in esse, come nelle Cetonia e negli Osmoderma la contemporaneita nel moto degli arti di una terna é rotta da un regolare ritardo di fase degli arti posteriori, 1 quali iniziano il loro moto allorché i primi sono gid presso al termine della loro escur- sione. In tutte le specie che ho cimentate, vi é, del resto, assai piti assieme; sussistono, cioé, connessioni funzionali assai pit rigide fra gli arti delle prime due paia che fra questi ed i terzi, 1 quali, nei riflessi complessivi dell’organismo, godono di una certa autonomia. Accennato appena all’esistenza di una deambulazione “‘sol- levata’”’ non ci dilungheremo oltre sulle modalita della locomo- zione in tutte le specie che ci hanno servito nelle nostre ricerche, poiché nell’esporle, ci fonderemo sopratutto sui dati che ci hanno fornito le Blaps, i Carcbus e le Pimelia. (a9 5. LE ALTERAZIONI DELLA DEAMBULAZIONE NORMALE NEL MANEGGIO Lasciando per ora da parte il problema delle cause intrinseche del movimento di maneggio nei coleotteri—in qual misura debba cioé esso moto venir riportato all’alterazione di un’attivita del sistema nervoso centrale—non occupiamoci che della questione del determinismo suo immediato. Con quali modificazioni dell’- attivita locomotoria normale viene effettuato un continuativo giro in circolo? Scindiamo senz’altro il procedimento di risoluzione del pro- blema in due parti. Esamineremo dapprima lo spostamento delle relazioni dinamiche fra l’attivitaé dei singoli arti, rispetto alla traslazione complessiva del corpo, esamineremo cioé, la proie- zione di detti moti sul piano di deambulazione, servendoci dei reogrammi. In un secondo tempo passeremo dal piano nello spazio esaminando—per cosi dire—la morfologia delmovimento—, 240 EDGARDO BALDI Fig.20 Maneggio di Osmoderma heremita (grandezza naturale). Fig.21 Maneggio di Osmoderma heremita (grandezza naturale). MOVIMENTI DI MANEGGIO NEI COLEOTTERI 241 esaminando cioé come si scomponga l’attivita globale di ogni arto in quella delle singole leve che lo compongono. Un primo evidente segno di una dissimmetria locomotoria nel movimento di maneggio si rileva nei relativi reogrammi, dall’es- sere il tracciato relativo nettamente distinguibile in due meta, una concentrica all’altra, talora visibilissimamente separate dallo striscio lasciato sulla carta affumicata dall’estremita poste- riore dell’addome (fig. 20-21-22). Ciascuna di queste due zone concentriche, descritte rispettivamente dagli arti di ogni anti- mero, ha un suo proprio carattere; qualunque sia il senso in cui il giro é descritto la fascia esterna del reogramma corrisponde agli arti dalla banda lesa, l’interna a quelli della banda sana. Ora, la zona esterna ha sempre una larghezza maggiore dell’interna, particolare che gioverd ritenere poiché ne troveremo altrove la spiegazione. Tale differenza pud agevolmente venire tradotta in cifre. Reogramma fig. 22 LARGHEZZE MINIME IN MILL. LARGHEZZE MASSIME IN MILL Larghezza Larghezza Larghezza Larghezza Larghezza Largh. totale fascia int. fascia est. totale. fascia int. fascia est. a ae, G0 115),(0) ay 25 Seo 16,5 b 21 6,5 14,5 oy? De 8,0 14,0 (Go c 23 Oa) 11659555 e238 9,0 14,0 Riferendo tali cifre alla larghezza totale della fascia, assunta come unitd, si ottengono 1 seguenti rapporti, che dimostrano la relativa estensione della fascia interna. 6, = iL/B, 14! i? = i/o lI) = N/R 2 ba sD Ce i306 Coane 2a) Cioé, nel citato reogramma, la fascia interna ha approssimativa- mente l’estensione di un terzo della fascia totale; supera tale valore nei punti di strozzamento del reogramma, gli é inferiore in quelli di allargamento. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 242 EDGARDO BALDI 23 Fig.22 Maneggio di Cetonia aurata rand. natur.). ee Fig.23 Maneggio di Blaps mortisaga (grandezza natur.). MOVIMENTI DI MANEGGIO NEI COLEOTTERI 243 Reogramma fig. 23 PUNTI LARGHEZZA TOTALE FASCIA INTERNA FASCIA ESTERNA a 28 10,5 17,5 b 27 10,5 16,5 c 26 9,5 16,5 d 25 8,0 17,0 e 25 8,5 16,5 Donde, calcolando come dianzi i rapporti della larghezza della fascia interna alla larghezza totale: a = 1/2,66;b = 1/2,57;¢ = 1/2,73;d = 1/2,12;e = 1/2,94 Anche qui, quindi, l’ampiezza della zona interna si approssima ad un terzo dell’ampiezza totale. Altri reogrammi di Blaps (fig. 23) mostrano ad un dipresso le medesime relazioni. Un valore pure prossimo ad un terzo della larghezza totale della fascia assume la larghezza della zone interna del reogramma di un carabo (fig. 24) Fig. 24 Maneggio di Blaps mortisaga, con amputazione delle tibie sinistre (grand. natur.). 244. EDGARDO BALDI laddove, almeno essa é misurabile. Tale larghezza della fascia ha il suo significato; infatti 1 margini che la determinano segnano i punti in cui 1 tarsi, o comunque, la parti distali degli arti si sono posati a terra, per sospingere od attirare il corpo. Nella marcia normale, eguagliandosi l’ampiezza delle due fascie dai due lati, le impulsioni trasmesse attraverso due bracci di leva di uguale lunghezza, cioé attraverso agli arti in normale esten- sione, parimenti si uguagliano. Una diminuzione della lunghezza di un braccio di leva da un lato, dato che l’intensita degli sforzi motoril si mantenga simmetrica, dovra tradursi in una diminu- zione di impulso ivi, l’organismo dovra volgersi da quella banda. Vedremo quanto vi sia di attendibile in questa interpretazione suggerita dalla minore ampiezza della fascia interna del reo- eramma. Inoltre tale fascia interna si distingue talora dall’e- sterna anche per il carattere dei tratti che la costituiscono. Men- tre la fascia esterna presenta una successione di gruppi di tracce ben distanziati, non dissimile da quella che si osserva nei reo- grammi della deambulazione normale, |’interna si mostra co- stituita da traecce continue, da ghirigori allungati e continuantisi, quasi che le arcate descritte dagli arti corrispondenti, anziché elevarsi ritmicamente dal suolo, per ritornarvi in punti decisi, si fossero schiacciate, appesantite e l’arto talora si trascinasse sul terreno. Tale sintomo di abbassamento dell’attivita loco- motoria degli arti 6 specialmente manifesto nei posteriori. La misurazione delle distanze fra 1 punti omotopi di gruppi succes- sivi di tracce da altresi modo di poter misurare le differenze di attivita fra gli arti dei due antimeri, una quantita proporzionale, cioé, allo squilibrio dinamico fra di essi. Un esame superficiale del reogramma (fig. 23) descritto da una Blaps in movimento di maneggio, mostra come la curvatura del moto non sia costante, ma come la traiettoria sia approssimativamente una ellissi. La curvatura ne é minima (cioé il moto é pit rettilineo) nelle vici- nanze della piccola freccia, massimo nella regione che ne dista di un arco di circa 90° gradi, il che é bene osservabile nella zona interna descritta dagli arti sinistri. Passando dal tratto pit curvo al meno curvo, ecco, in decimi di millimetro, le distanze fra i colpi d’unghia degli arti medii: MOVIMENTI DI MANEGGIO NEI COLEOTTERI 245 Arti sinistri Arti destri 30 170 50 170 50 180 60 180 70 190 Fig. 25 Schema per la interpretazione delle tracce periferiche del reogramma fig. 24 (grigio = primi arti—bianco = secondi arti—nero = terzi arti). Da esse risulta non solamente l’altissima differenza di attivita fra gli arti dei due antimeri, ma anche che, essendo le due curve su di esse costruibili diversamente inclinate sull’asse dei tempi tale differenza é minima per il tratto iniziale, meno curvo, del reo- gramma ed é massima per il tratto che nel reogramma si presenta come piti curvo. E poiché la curva dei destri é molto meno inclinata di quella dei sinistri, ne segue che lessersi fatto piu stretto il maneggio 6 effetto di una ulteriore diminuzione di attivitd dei sinistri, pii che di un aumento di attivité dei destri, allorché beninteso, si considerino le componenti delle prestaziont locomotorie degli arti nel piano di deambulazione e non nello spazio. Il reogramma (fig. 24) si presta abbastanza bene alla misura per essere stato descritto da una Blaps, cui, per altri fini, erano stati recisi i tarsi degli arti sinistri, il che ha contribuitoadau- mentare la schematicité e la nettezza delle tracce. I] maneggio é destrorso; le tracce della fascia interna sono facilmente interpre- tabili grazie allo schema di cui alla fig. 10 (deambulazione nor- male). Le tracce periferiche alterate si scindono come mostra lo schizzo a fig. 25. 246 EDGARDO BALDI Riferiamoci quindi alle tracce dei primi arti, facilmente indi- viduabili e misuriamo le distanze da gruppo a gruppo dalle due parti in decimi di millimetro. Arti sinistri Arti destri 110 90 100 90 120 90 120 90 120 90 100 90 105 80 100 70 105 80 110 85 125 90 120 95 115 100 130 95 120 Sussiste fra le arcate dei singoli arti la costante differenza che gid altrove abbiamo notata. Analogo computo possiamo compiere sulle arcate dei secondi arti. Arti sinistri Arti destri 110 90 105 70 120 70 MOVIMENTI DI MANEGGIO NEI COLEOTTERI 247 Arti sinistri Arti destri 125 70 120 85 125 70 105 80 100 55 95 50 115 ? 110 55 120 65 130 10 122 70 120 70 120 Ripetiamo infine la misura per le tracce dei terzi arti Arti sinistri Arti destri 100 80 110 80 115 100 i115} 80 105 85 95 75 85 60 105 80 80 bo iS GO EDGARDO BALDI Arti sinistri Arti destri 110 80 120 80 120 90 125 90 115 80 115 70 125 90 Gioverdi notare che l’ampiezza delle arcate destre e sinistre é anche attenuata dal fatto dell’amputazione delle tibie sinistre, le quali, cosi mozze, diminuiscono la lunghezza della leva arto e quindi ’ampiezza della arcate sinistre. A tale causa sono pro- babilmente dovuti taluni levi searti rilevabili nella tabella. Le cifre relative all’attivita degli arti destri sono per6é costantemente inferiori a quelle misurate perisinistri. Su tali dati é costruito il diagramma fig. 26. Ad una prima ispezione le curve del diagramma rivelano nelle frequenti irregolarita di ciascuna la presenza di qualche fattore secondario disturbatore del loro regolare andamento, che crediamo in parte almeno poter riferire alla inevitabile imprecisione di una misurazione su di un tracciato eseguito daartiinegualmentemozzi, indi al numero non sufficiente di misurazioni, indi a condizioni interne della locomozione, di apprezzamento non immediato. Comunque, appare netta la distinzione fra il gruppo superiore, che traduce l’attivita degli arti sinistri e l’inferiore, relativo ai destri. Il gruppo superiore mostra indi un certo parallelismo nell’anda- mento delle sue tre curve (e segnatamente nella seconda meta del diagramma) che é buon segno della normalita di funziona- mento degli arti relativi. Vi sono in esso oscillazioni d’assieme, cui le tre curve contemporaneamente obbediscono e che provano, al piu, una obbedienza complessiva degli arti dell’antimero alle condizioni che hanno determinate quelle oscillazioni. Verosi- milmente, se il reogramma fosse stato descritto da un animale MOVIMENTI DI MANEGGIO NEI COLEOTTERI 249 dotato di arti illesi, dalla banda sinistra esso gruppo di curve si sarebbe spostato anche pit in alto, segnando piu nettamente la sua distinzione dall’inferiore. Il gruppo inferiore di curve, rela- ? tivo agli arti destri, 6 meno unito nelle sue oscillazioni, come wee Atti destri ous Arti sin, Fig. 26 Diagramma dell’attivité degli arti nella Blaps di cul alla fig. 24, Vedi testo. (dall’alto in basso: 2° sinistro, 1° sinistro, 3° sinistro = 1° destro, 2° destro, 3° destro.) Fig. 27 Diagramma delle medie per i due fasci del precedente (Cfr. testo). risulta dal diagramma fig. 27, nel quale sono prese per ordinate le medie fra le ordinate di punti di uguale ascissa nel primo dia- gramma, per ciascuno dei due fasci di curve. Consideriamo, per chiarircene l’andamento, le relazioni fra le singole coppie di 250 EDGARDO BALDI curve nei due fasci, dovute al medesimo paio di arto. Appare manifesto un parallelismo fra le curve relative all’attivité dei terzi arti, curve che mantengono ad un dipresso lo stesso anda- mento, maggiormente scostandosi verso la fine del diagramma. Ci6 significa che la differenza di attivitd fra i terzi arti si mantiene ad un dipresso costante, accentuandosi un poco nei punti di minima curvatura del reogramma. Non é dai terzi arti, quindi, come insegna anche |’osservazione immediata, che principalmente dipende il meccanismo del maneggio. Una constatazione ana- loga suggeriscono le curve relative all’attivita dei primi arti, le quali mantengono un andamento grossolanamente parallelo e sprovvisto persino di quella divergenza terminale che abbiamo rilevata nelle curve per i terzi arti. Ma nella meccanica dei primi arti intervengono altri fenomeni non rilevabili da un reo- gramma (in quanto prevalentemente si svolgono nello spazio) e che loro assegnano una parte principale nei moti di maneggio. E’ da notare, peré, nel reogramma, l’avvicinamento delle tracce relative alla linea mediana del tracciato, nella quale talora esse penetrano. Il piti cospicuo grado di dissimmetria é porto dalle curve rela- tive agli arti del secondo paio, le quali, con la loro reciprocita di andamento, ricordano diagrammi precedentemente costruiti—e precisamente su dati di arti mediani. Conviene riconoscere che, per quanto riguarda l’entita degli impulsi propulsivi in wn piano parallelo a quello di marcia, la parte principale del moto di maneg- gio, in quanto squilibrio dinamico, viene assunta dagli arti me- diani. Fatto che si pu6 osservare evidentemente trascritto nel diagramma fig. 28, nel quale sono riavvicinate le curve del secondo arto sinistro e la curva delle medie degli arti sinistri, esprimente cioé la globale attivita dell’antimero sinistro. Ora, tra le singole curve, le cui medie hanno servito alla costruzione del predetto diagramma (fig. 27) quella che piu si accosta alla forma della curva di media, quella che, cioé, in certo senso, le conferisce il suo proprio carattere, 6 precisamente la curva dell’iso- lato secondo sinistro, come il nuovo diagramma appunto mostra. Dal primo diagramma si possono inoltre ricavare le differenze fra ordinate di punti corrispondenti appartenenti alle due curve MOVIMENTI DI MANEGGIO NEI COLEOTTERI Zou degli arti di una coppia simmetrica: dei due primi, delduesecondi, deidueterziarti. Tali differenzespecificatamente e continuativa- mente indicano i valori della dissimmetria dinamica esistente 29 Fig.28 Veditesto. Fig.29 Vedi testo. fra gli arti della coppia e, prese complessivamente, il valore della dissimmetria dinamica esistente fra i due antimeri. Con tali differenze, ordinatamente calcolate per ogni coppia di arti, é stato costruito il diagramma fig. 29, dal quale evidentissima- 252 EDGARDO BALDI mente risulta il maggior valore che tale differenza assume per eli arti del secondo paio, rispetto a quelli del primo e dell’ultimo paio. La curva dei secondi arti abbraccia pressoché completa- mente le altre due. Sarebbe ardito pretendere di voler ricavare altre conclusioni dalla analisi dei reogrammi. Un/’ultima occhiata al secondo— diagramma di medie—mostra la netta distinzione delle curve relative ai due antimeri ed il loro divergere nei punti di maggiore curvature del reogramma. In questo la disposizione alternata dei gruppi di tracce ancora ben riconoscibile, prova che la coordi- nazione non é andata perduta, durante il maneggio. In altri reogrammi, questo particolare non é nettamente visibile, la fascia interna peré presenta una sorta di festonatura, in cul gli apici ed altri punti di facile riferimento cadono fra i corris- pondenti gruppi della fascia esterna. Altri reogrammi, sui quali, piti o meno agevolmente, si possono istituire computi analoghi, mostrano diversi tipi del moto di maneggio, sui quali ritorneremo dicendo delle particolari condi- zioni in cui, di volta in volta, si attua quella meccanica tipica del maneggio, che ora schematizziamo. Con la convinzione che ulteriori misure di reogrammi netti e significativi piu di quelli che noi possiamo considerare qui possano condurre ad interessanti constatazioni sulla dinamica compara- tiva degli arti nel maneggio, riassumeremo le conclusioni che si possono trarre dall’esame di quelli che abbiamo sott’occhio. 1. L’attivitd degli arti dei due antimeri si mostra dissimmetrica, per essere la fascia descritta dagli arti del lato sano pit ristretta dell’opposta di cirea un terzo dell’ampiezza totale. 2. E per essere ivi le tracce talora altrimenti disposte che nei reogrammi normali e nella fascia opposta. 3. Per essere talora la fascia interna costituita di tratti conti- nuativi e non bene differenziati in gruppi. 4. Per essere sopratutto le arcate descritte dai singoli arti assal meno ampie dal lato integro che da quello leso. 5. Tutti segni—questi—di una minore attivitd degli arti dal lato sano, rispetto a quella degli arti dal lato leso. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 253 6. Parte preponderante in questo disquilibrio dinamico della locomozione hanno gli arti medi. 7. V’é fra gli arti degli antimeri una certa dipendenza fun- zionale, per cui quella proporzionalita fra le attivita di ciascuno si mantiene grossolanamente costante, oscillando l’inferiorita degli arti dal lato sano rispetto all’attivité degli arti opposti da un terzo ad un mezzo di quest ultima. Gioverda soffermarci un istante sul punto segnato con il numero 5. Nel caso della Blaps abbiamo gid visto come l’intervallo normale fra due gruppi di tracce locomotorie sia di 150 decimil- limetri in media. Poiché un moto in circolo é genericamente provocato da nua differenza di attivita, vediamo se nei nostri reo- grammi esso sia dovuto ad una eccedenza sulnormale dell’attivita’ degli arti esterni oppure adunadeficienzadegliinterni. Scegliamo in ultimo un esempio nel reogramma fig. 30 descritto daun Carabus (veggasi anche fig. 31). La media distanza fra i gruppi di tracce in esso non supera i 27 o 28 millimetri dal lato leso, valore pro- prio alla deambulazione normale retta e non lenta degli individui congeneri sani. Un confronto fra i valori delle distanze in punti di diversa curvatura si pué istituire sul medesimo reogramma, laddove il tracciato momentaneamente si allarga, assumendo curvatura molto minore. I seguenti dati sono misurati in esso lungo il margine esterno e relativamente alle tracce degli arti medii. alam 25 h. mm. 25 mm. 23 te mm. 25 Gy iahenl,, 78} 1. mm. 24 d. mm. 24 m. mm. 24 e. mm. 24 n. mm. 24 it, 1oauaayy 2A5) OD, aaa, PA} [eescavaalyy ii Il divario é pressoché insensibile. Nell’ultima parte del trac- ciato, laddove ne comincia la diminuzione di curvatura, € pos- sibile una sommaria misurazione tra le insenature manifeste nella curva lasciata dal terzo arto sinistro, la cui sinuosita continuativa permette di seguirla per un tratto entro il reogramma stesso. Eccone i dati paragonati a quelli per il corrispondente arto destro. EDGARDO BALDI 254 ‘O[IGVIUVA BINZBAINO B SNSOT[IGLOUL SNgBiT OTporssouvyy TE ST ‘Q[IQUIIVA BINZVAIN B SNSO][IGIOU SNqVIVD Ip orssouvyy Og "Sly MOVIMENTI DI MANEGGIO NEI COLEOTTERI 255 Arti destri Arti sinistri 110 210 130 225 150 230 150 230 175 235 170 220 175 220 180 La curvatura della traiettoria va continuamente diminuendo dall’alto al basso della tabella. II] grafico fig. 32 traduce in curve Fig.32 Vedi testo. 256 EDGARDO BALDI questi valori. Da esso risulta evidente la differenza di anda- mento delle due curve. Mentre quella degli arti destri non di- scende che leggermente verso sinistra, cioé verso il punto di maggiore curvature della marcia, la curva degli arti sinistri nella seconda meta precipita rapidamente, dimostrando come la mag- giore strettezza del giro sia dovuta pit ad una diminuzione di attivita nei sinistri, che ad una esaltazione di essa nei destri. Si noti, infine, come le curve si riferiscano ad una coppia di terzi arti che gia nella Blaps abbiamo visto non avere la parte princi- pale nello squilibrio dinamico del maneggio. Passiamo ora all’ e- same del moto degli arti nello spazio. Descriver6 la meceanica degli arti nel maneggio tipico, riferen- domi specialmente alle Blaps che ne hanno fornito i migliori casi. Supporro’, per comodita, nelle considerazioni che seguono, che Vanimale descriva un maneggio sinistrorso; parlando cioé di arti sinistri e destri, intenderé riferirmi rispettivamente agli arti dal lato illeso ed a quelli dal lato leso. Le differenze fra l’attivita de- gli arti destri e quella dei sinistri risulta a prima vista dall’osser- vazione che, mentre nel descrivere le relative arcate, gli arti destri si raggiungono o quasi, nelle posizioni estreme, i sinistri rimangono costantemente lontani gli uni dagli altri. Gli arti destri, in forza delle pit ampie traiettorie descritte linearmente, sembrano animati da maggiore velocita’. In realta, la coordina- zione non viene gran che menomata; persistono, cioé, le relazioni fra i tempi di inizio dei moti per ciascuno degli arti, in valori uguali o proporzionali a quelli normali. Inoltre gli arti sinistri sono tutti—e specialmente i primi—piw’ flessi dei corrispondenti arti destri. Ossevando l’animale dal- Valto si veggono cosi gli arti destri sporgere dal corpo di un tratto maggiore che i sinistri; infine il corpo presenta verso sinistra una inclinazione pii o meno accentuata. Nell’arto medio si pud talora osservare chiaramente la disposizione che causa tale ab- bassamento in pari tempo che tale flessione (fig. 83). Rispetto ai destri, i femori sinistri sono maggiormente flessi sulle coxae cioé pit aderenti alle pareti del corpo; in pari tempo le tibie sono maggiormente flesse che nel normale, rispetto ai femori ossia si sono ad un tempo fatti pid acuti i due angoli cox ofemorale e MOVIMENTI DI MANEGGIO NEI COLEOTTERI 257 femorotibiale. Poiché, comunque il punto d’inserzione della tibia all’estremo distale del femore viene sollevato nello spazio rispetto a quella che é l’orientazione normale del corpo (esprimentesi neila verticalit’ del piano sagittale) e poiché, d’altro canto, é forza che l’estremo distale della tibia o taluno fra i primi articoli tarsei toechi terra, tale orientazione viene menomata ed il corpo é obbligato ad obliquare verso sinistra. I] piano sagittale non 6 pit coincidente con un piano verticale, ma fa con esso un angolo sulla sinistra, pii o meno acuto. Fenomeni analoghi, se forse non cosi schematici, si osservano nel primo e nel terzo arto. La meceanica dei primi arti conserva loro ancora un compito direttivo nella marcia ed €é percié particolarmente interessante. Fig.33 Meccanismo dell’ inclinazione del corpo nel maneggio. Nel primo arto destro é sopravvenuta una modificazione nell’im- piego delle singole sue leve che lo raccosta all’arto destro del secondo paio. L’arto descrive cioé ampie arcate in cui il moto anteroposteriore del femore ha parte capitale; il moto d’attrazione della tibia sul femore, caratteristico nell’animale normale, ha perduto d’importanza. Ove esso sussista (poi che talora é affatto obliterato) agisce in opposto senso, non con il risultato attrattivo, cioé, di un moto flessorio, ma con il risultato impellente di un moto estensorio. La tibia, mentre il femore compie la sua arcata d’avanti in addietro, si viene estendendo sul femore stesso, cost da trovarsi estesa sulla sua linea allorché il femore abbia raggrunta V’estrema sua posizone. Si vede come in tal caso la linea d’azione dell’arto traversi diagonalmente il corsaletto dell’animale. Ma l’azione tipicamente impellente del primo arto destro si accom- pagna e si coordina ad un’azione attrattiva del primo sinistro, la quale rappresenta del pari un’alterazione dell’impiego normale. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 258 EDGARDO BALDI In tale arto sono grandemente ridotti i moti ad areata, moti im- pellenti in senso anteroposteriore; talora essi sono affatto scom- parsi; il femore non ha che piccole oscillazioni in un piano orizzon- tale e la maggiore attivita viene esplicata dalla tibia che ha esagerato il suo moto contrattivo sul femore. La tibia, e con essa la linea dei tarsi, pud essere paragonata ad un uncino che si ag- erappa e si ritrae. Il suo moto avviene pressoché completa- mente entro il piano di simmetria del segmento femorale; il fe- more pud essere pit o meno inclinato sul piano sagittale; spesso la sua inclinazione é uguale in valore a quella dell’arto simmetrico all’estremo della sua corsa. Cosi che le due azioni, impulsiva a destra, attrattiva a sinistra, si sommano lungo una medesima direzione, raggiungendo un effetto massimo. I secondi arti rivelano poco pit attenuato uno squilibrio simile nel loro impiego; l arto destro continua od esagera nello stesso senso la sua atti- vitd normale: da grandi colpi di areata dall’innanzi all’addietro, cui prendono parte tutti 1 segmenti dell’arto, dal femore in git. In esso sono scomparsi 0 ridottissimi i fenomeni di flessione della tibia sul femore. Dal lato sinistro predominano invece tali azioni flessive; i moti ad areata del femore sono di molto ridotti (raramente scompaiono del tutto). I moti della tibia collaborano con quelli del femore. I] piano, cioé, in cui muovesi la tibia, é obliquo rispet o al piano di simmetria del femore; entrambi 1 piani della locomozione vengono trasportati in una direzione ad un dipresso normale a quella del piano di simmetria del femore (moto anteroposteriore del femore) il che fa credere che la tibia compia moti ad areata pit cospicul del reale. Aggiungasi che le linee tarsali degli arti destri descrivono quelle rotazioni che abbiamo rilevate nell’individuo normale, mentre i tarsi di sinistra vengono spostati parallelamente a se stessi. Nei terzi arti infine, nei quali le squilibrio é meno visibile, data la loro funzione eminente- mente impulsiva, si pué notare che gli angoli massimi e minimi (di contrazione e di estensione) fra la tibia ed il femore hanno valori superiori nell’arto destro, il quale veramente ‘‘impelle.”’ L’arto sinistro o da deboli impulsioni, oppure permane in uno stato di flessione pressoché costante, spostandosi all’avanti a suo turno, di quel tanto che é richiesto dallo spostamento generale del corpo senza esercitare impulsione veruna. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 259 Rapidamente abbiamo cosi tratteggiate le linee di impiego degli arti nel moto di maneggio, ponendone in luce due momenti fondamentali che per ora daremo come fatti di osservazione, senza occuparci della loro interpretazione: 1. Una differenza quantitativa di attivit&é muscolare fra gli arti dei due antimeri. 2. un predominio dal lato illeso di atteggiamenti flessivi delle leve dell’arto. Riteniamone la nozione di una dissimmetria dinamica fra gli antimeri, intesa come differenza della complessiva quantita di la- voro fornita dagli arti di ciascuna banda e domandiamoci: é tale dissimmetria come puro fatto meccanico condizione sufh- ciente a determinare il maneggio? Non meglio si pué tentare di rispondere a tale problema, che studiandosi di provocare, in animali integri, 1 moti in circolo, per alterazione delle condizioni meccaniche della loro locomozione. 6. LA RIPRODUZIONE SPERIMENTALE DEI MOTI DI MANEGGIO Esperienze di questo tipo meriterebbero di venire riprese ed analizzate dettagliatamente; se ne guadagnerebbero forse interes- santi notizie sui processi di autoregolazione dell’organismo. Dir6é rapidamente delle poche che io ho allestite. Gid esse, nel loro sistematico fallire, ci provano quel ch’era di leggieri prevedi- bile; come l’organismo in locomozione non sia una semplice macchina in moto, ma bensi qualeosa di pit delicato e complesso, Il che—beninteso—non tange i procedimenti della ricerca, ma pud solamente distribuirli in una seriazione le cui gradazioni corrispondono ad una diversa approssimazione alla complessita reale delle relazioni. Poi che il momento pit appariscente del maneggio sembra consistere nella differenza di attivita degli arti dei due antimeri abbiamo tentato, ricordando anche le con- clusioni del Bethe, in Pimelia ed in Blaps normali, di provocare un simile squilibrio, operando l’immobilizzazione degli arti da un lato, mediante legatura loro, in un solo assieme. Ma con questo grossolano procedimento vengono turbate le condizioni generali di equilibrio dell’animale nello spazio, in tal modo che esso, nonché locomoversi, neppure riesce a mantenere la stazione eretta. 260 EDGARDO BALDI Poiché non sembrava praticamente possibile, in un individuo normale, altrimenti sopprimere l’intera attivita di un antimero, abbiamo provveduto ad abbassare il valore locomotorio, accor- ciando la lunghezza delle leve che, puntellandosi al terreno, tra- mettono il movimento al corpo. Abbiamo mozzati, cioé, i tarsi e parte delle tibie sino a due terzi della loro totale lunghezza agli arti da un lato, cosf che da quel lato essi incontrassero il terreno in regioni assai piu prossime al corpo che dall’opposto., tentando eosi di riprodurre all’incirca una delle condizioni occorrenti nel maneggio e dicuiabbiamo detto. In seguito alla mozzatura degli arti sinistri era quindi preveduto un maneggio sinistrorso. Pre- visione non confermata dall’esperienza; l’animale ha tenuta marcia variamente orientata ed eccitata, senza tracce di giro in circolo, verosimilmente supplendo al disturbo arrecatogli con una pit intensa impulsione agli arti sinistri e con una maggior distensione di questi e retrazione degli opposti. In un Blaps che seguiva maneggio destrorso, una simile operazione non ha che allungato insensibilmente e fugacemente il circolo descritto. Sempre al medesimo fine, in altro Blaps abbiamo immobiliz- zate tutte le articolazioni degli arti destri mediante un avvolgi- mento spirale attorno all’arto, di un sottile e robusto fil di ferro, cosi da irrigidire l’arto in una posizione di anormale estensione. Analogamente ed inversamente abbiamo agito con una simile legatura che costringesse l’articolazione tibiofemorale sinistra in un angolo fisso minore del normale. In entrambi i casi era pre- veduto un giro in tondo a sinistra ed in entrambi l’animale si é invece faticosamente mosso sia in linea retta, sia descrivendo svolte varie in diverso senso. E’ notevole come animali siffatta- mente operati mostrino spesso nella deambulazione periodi di netta incoordinazione dei moti e—naturalmente— periodi frequenti di sosta e di immobilita’. La slegatura degli arti ripristina 1m- mediatamente la deambulazione normale. Per non inceppare immediatamente i moti delle leve dell’ arto, abbiamo indi ag- giustati alle tibie destre di una Pimelia tre leggeri fuscelli, di lunghezza appropriata, allo scopo di allontanare dal corpo il punto di applicazione dello sforzo motorio. Talora animale ha dato cenn di una certa tendenza ad obliquare sulla sinistra ed MOVIMENTI DI MANEGGIO NEI COLEOTTERI 261 ha descritto pure qualche largo giro sinistrorso, ma senza co- stanza aleuna. I periodi di immobilité abbondano ed é sufficiente una eccitazione per pressione sulle elitre, per provocare marcia in linea retta, talora non coincidente con l’asse sagittale, ma in direzione ad esso obliqua sulla destra. Qualche risultato positivo é possibile ottenere legando fra di loro le estremitd delle allunghe assicurate alle tibie del secondo e terzo arto destro. In tal modo non viene gran che compromesso Fig.34 Maneggio artificialmente provocato in Pimelia. Si notila fisionomia molto differente da quella dei maneggi naturali. lequilibrio generale del corpo, inoltre vengono completamente paralizzate le attivité dei due arti, medio e posteriore, di destra. Grazie all’attivité, ora isolata, degli arti di sinistra, allorché essi si muovono, il corpo viene sul posto o con lievi spostamenti, ruotato sulla destra. Il primo arto destro, che é rimasto libero nei suoi movimenti, ora compie tentativi di raddrizzamento della marcia, arrancando nel modo che gli 6 consueto ed ora inverte il senso del proprio moto ed eseguisce una vera marcia indietro (fig. 34). FE’ notevole la difficolté che si incontra a porre in moto l’animale cosi obbligato, anche con violenti stimoli di percussione sulle 262 EDGARDO BALDI elitre. Sembra cioé, che la soppressione delle possibilitaé mec- eaniche della locomozione coordinata (legatura di due arti conti- gui e soppressione della loro indipendenza di moto) tragga seco difficolt’ generali nella stessa generica deambulazione. Tali esperienze in genere negative, tranne che in un caso banale, mostrano come nel maneggio uno squilibrio appunto dinamico delle condizioni delle locomozione non esaurisea il fenomeno e—se pure ne costituisca un momento necessario—non sia peré sufficiente a determinarlo. D/’altronde gid dalla pura descrizione della meccanica locomotoria degli arti abbiamo appreso come a quel momento ‘‘meccanico’’—per cosi dire—si ageiunga e mi si passi l’espressione—un momento “‘fisiologico,”’ con che intendo accennare alle generali e fini alterazioni dell’im- piego di ciascun arto, esplicantisi sopratutto nel predominare di attivita flessorie nell’antimero illeso. Ora, non v’é che un si- stema dell’organismo la cui lesione sappia produrre alterazioni tanto dettagliate e generali dell’attivita muscolare di quello. Ed é il sistema nervoso. In particolare, poi che la lesione é inferta al capo, il sistema nervoso cefalico. Riserveremo quindi il nome di maneggio ai fenomeni di altera- ta motilita, implicanti una rotazione dell’organismo attorno ad un asse qualsiasi, passante o non per il corpo, causati da una alterazione patologica delle regioni nervose cefaliche, avvertendo che l’alterazione del cervello pué non essere morfologicamente evidente; é per6 sufficiente che essa sia un’alterazione della sua simmetria funzionale. L’espressione di maneggio é stata imfatti diversamente usata dai varii autori e ad opera di taluno ha subito notevoli e—a parer nostro—non ben giustificate estensioni. Passiamo rapida- mente in rivista quello che gli autori hanno appunto osservato in proposito. Tra le alterazioni della simmetria sensoria che non interessino direttamente i centri nervosi, abbiamo gid elencate le esperienze di opacamento delle cornee compiute dal Dolley (’16), dal Parker, analoghe a quelle escogitate e praticate su varie specie di artro- podi dallo Holmes (01-05) da Brundin e da McGraw (13) suoi MOVIMENTI DI MANEGGIO NEI COLEOTTERI 263 allievi, da Carpenter (18) sulla Drosophila, con risultati incos- tanti e similmente dal Radl sulla Musca domestica (03). Nel 1901 il RAdl aveva gid pubblicate le osservazioni di moto in circolo relative all’idrofilo, per estirpazione di un occhio. Lo Hadley, nel 1908, distruggendo nei gamberi la cornea di un occhio, otteneva, oltre a movimenti in circolo, anche rapide. rotazioni intorno all’asse longitudinale dell’animale. Un accoppiamento dei due moti: di traslazione circolare e di rotazione, aveva gid osservato il Demoor nel 1891 in un Palaemon serratus, per lesione, non di un organo ricettivo, ma di una porzione laterale del cer- vello. Rimanendo sempre nel campo delle alterazioni della simmetria sensoria, ricorder6, oltre a quelli gia citati, di Barrows e di Kellogg, i moti in circolo per amputazione di un’ antenna, osservati dal Dubois (’86) nel Pyrophorus, previa asportazione bilaterale degli oechi. Sembra che anche gli otocisti possano determinare, se aspor- tati unilateralmente, fenomeni di moto in circolo e di rotazione introno all asse longitudinale, secondo riferiva Yves Delage nel 1887 a proposito della Mysis. L’Herrera, nel 1893, presentava alla Société zoologique de France una breve nota, in cui, con molta parsimonia di dettagli e di documenti, riferiva diaver provocati moti in circolo in mosche ed in altri insetti (sic!) introducendo nel corpo dell’animale, per una ferita laterale, qualche cristallino di bromuro di potassio, la cui azione ipostenica era per6é di breve durata. Il medesimo autore avrebbe ottenuti maneggi anche per alterazione delle condi- zioni puramente meccaniche della locomozione: ponendo a caval- cioni dell’insetto una sorta di bilanciere fatto con un ago incurvato e lateralmente caricato di un peso (di una pallina di cera) ed anche amputando completamente da un lato gli arti dell’animale. Quanto noi stessi abbiamo visto sui nostri coleotteri, ci fa al- quanto dubbiosi circa la semplicité dei mezzi e la facilitaé dei risultati delle esperienze dello Herrera. Anche Demoor ri- ferisce (’90) di un moto in circolo osservato in un Carabus monilis var consitus per deficiente impiego di un arto, l’anteriore destro. Tale deficienza non era sperimentale, ma dovuta a condizioni patologiche spontaneamente insorte nell’animale. 264 EDGARDO BALDI Ma i moti di rotazione osservati da pit antica data e verificati di maggiore costanza, sono quelli dovuti ad alterazioni speri- mentalmente apportate al sistema nervoso centrale e particolar- mente ai gangli sopraesofagei. I dati relativi al ganglio sottoeso- fageo sono infatti scarsi, l’aggressione di quest’ultimo ganglio essendo in realté cosa assai pit complessa ed irta di difficolta tec- niche, che nol sia la lesione dei gangli dorsali all’esofago. De- moor riferisce in proposito (91) una esperienza del Faivre in Fig.35 Maneggio di Aromia, cui la lesione unilaterale del sottoesofageo nel ditisco ha pro- dotto un breve maneggio. Si pué dubitare, in base ai fatti esposti in altra nota di chi scrive, se tale breve maneggio sia effettivamente legato ad una lesione nervosa. La bibliografia cirea i moti in circolo per lesioni unilaterali dei sopraesofagei é assai vasta; la si pud vedere nei lavori citati onde sarebbe inutile che io la riportassi. Gl autori hanno descritti diversi tipi di rotazioni: 1. in un piano secondo curve chiuse o spirali o ad ansa (moti di maneggio propriamente detti) (fig. 35). 2. moti di rotazione dell’organismo intorno all’estremo cefa- lico od all’estremo aborale od attorno ad assi passanti per regioni MOVIMENTI DI MANEGGIO NEI COLEOTTERI 265 del corpo prossime a queste, moti cui il Demoor aveva assegnato (91) ilnome di moti a raggio di ruota (en rayon de roue), togliendo a prestito l’espressione al Beaunis, che precedentemente l’aveva Fig. 36 Due maneggi della medesima Blaps mostranti il passaggio dal ma- neggio p.d. al maneggio in posto. Fig.37 Maneggi in posto di Carabus. usata per i vertebrati e che pitii recentemente e pittorescamente furono dagli autori francesi chiamati moti di valzer (fig. 36-37). 3. moti di rotazione intorno all’asse longitudinale in entrambi 1 Versi. bo =P) or) EDGARDO BALDI 4, ed infine moti che il Demoor (’91) ha chiamati di culbute, effettuantisi secondo curve chiuse contenute in un piano verticale e da lui osservati nel Palaemon serratus. Una complessiva denominazione di moti di maneggio assegna il Bohn ai moti rotatorii che egli provoea per disuguale illumina- zione oculare in aleuni organismi marini e dei quali abbiamo gid fatto cenno. Vanno ricordati infine, benché non siano propriamente moti di rotazione, i movimenti a rinculoni segnalati gidé dal Dubois nel 1886 per l’emisezione sagittale dei cerebroidi e per lesioni in regioni prossime, sui quali é tornato il Comes, che crede di averli osservati per il primo negli artropodi decapitati. Quest’ultima forma, come, noi stessi abbiamo osservato nei Carabus e nelle Blaps si pu6 combinare al moto in circolo, dando i maneggi a rovescio, 0 retrogradi. Il quadro, vasto, di simili moti di rotazione, offre certi tratti comuni in tutto il tipo degli artropodi, tratti comuni che non mi sembrano ancora ben saldi rispetto al loro condizionamento ana- tomico e fisiologico. Espressamente ho taciuto dei casi simili osservati, ad esempio, per lesioni dell’orecchio interno nei vertebrati, onde non ista bilire connessioni che possano essere illusorie. 7. ASPETTI. DEL MANEGGIO: L’ORIENTAMENTO DEL CORPO. LA VARIAZIONE DEI MOTI Ci é qui impossibile dettagliatamente esporre tutte le varianti e gli aspetti speciali che il moto di maneggio assume nei singoli casi. Tale studio analitico e minuzioso esige separata tratta- zione ed io la intraprenderé altrove, esaminando altresi il pro- blema delle relazioni che intercedono fra l’ubicazione topografica el’entitd della lesione ai sopraesofageie la reazione motoria dell’ or- ganismo. I dati schematici che ho precedentemente esposti ci saranno sufficienti per un tentativo di interpretazione generale dei movimenti di maneggio nei coleotteri. Nell’insetto che sta compiendo un movimento di maneggio, non solamente il portamento degli arti 6 soggetto ad un’anormale dissimmetria, bensf quello di tutte le parti dell’organismo su- MOVIMENTI DI MANEGGIO NEI COLEOTTERI 267 seettibili di. reciproca mobilité. Prescindiamo dalle antenne; Vafflosciamento dell’antenna dalla parte lesa é sovente in diretta connessione con la lesione, sia dello stesso nervo antennario,sia della sua inserzione al cerebron (l’antennario si diparte poco avanti e poco sotto all’inserzione del lobo ottico), sia dei muscoli propril dell’antenna. ‘Tale immobilizzazione non é del resto, fenomeno costante. Pochi casi ho osservati in cui le antenne mostrassero un portamento diverso. tra di loro, senza che aleuna lesione dei tessuti suindicatiifosse palese..» Il-capo é invece, in tutte le specie cimentate, volto da quella parte da cui si effettua il maneggio e talora obliquamente piegato dalla medesima banda. Tale piegatura aumenta di valore allorché il giro di maneggio si faccia piu stretto e si stabilisce allorché l’insetto passa da una deambulazione rettilinea ad un moto in circolo. Ho mostrato per6 come tale fenomeno non sia da annoverarsi tra le cause efficienti del maneggio. Esso é semplicemente un fatto concomi- tante. Allorché il corsaletto sia alquanto mobile sull’addome, come nei carabi, esso pure si mostra incurvato nel medesimo senso del capo, rispetto all’asse sagittale dell’addome. Anche tale curva- tura si stabilisce e si accentua durante il maneggio, analogamente a quella del capo. Ci sono ormai note le flessioni cui sono stati sottoposti gli arti dal lato illeso: maggiore accostamento dei femori alle pleure e delle tibie ai femori. Gli arti dal lato illeso sono relativamente pit flessi e piu retratti verso il corpo degli opposti. In conseguenza di cio’, l’orientamento stesso di tutto il corpo nello spazio viene turbato; se la lesione sia stata praticata a destra, il corpo si mostra piti o meno sbieco sulla sinistra. Tale abbattimento laterale del corpo non é legato alla lesione di qualche organo di senso statico od a quella di qualche apposita funzione nervosa, bensi alla sola condizione meccanica della maggiore flessione degli arti da un lato. Nei carabi normali l’orientamento generale del corpo sembra vincolato a quello del piano anteroposteriore che passa per gli equatori delle cornee. Ledendo infatti la muscolatura del collo ad un carabo, cosi da impedire al capo movimenti spontanei e da poterlo per qualche tempo obbligare in posizioni determinate, 268 EDGARDO BALDI ho osservato che l’abbattimento del corpo sulla destra era dovuto ad una rotazione del capo sulla sinistra. Rotazione, cui l’ani- male, privato del controllo muscolare sul capo, non poteva altri- menti reagire. Per effetto di tale rotazione, il piano oculare veniva ad essere obliquo sulla sinistra; l’evidente conato dell’ani- male di riportarlo orizzontale causava l’abbattimento a destra del corpo. Abbattimento che infatti cessava, cosi che l’animale riprendeva la postura normale, allorche’ io ruotavo il capo in senso inverso orientandolo come negli individui integri. Che peré tale reorientamento automatico, del quale gid numerosissimi e svariati esempi offre la bibliografia in argomento, fosse dovuto ad una autoregolazione in certo senso cenestetica, se non Visiva, Fig.38 Vedi testo. ma non ad un ipotetico senso autonomo della spazialitéa (quale sembrano indicare, ad esempio, le esperienze del Cornetz sulle formiche) dimostra il fatto che l’animale posto su di un piano inclinato non si muove secondo una parallela al lato d’appoggio e nel senso che meglio serva a correggere l’obliquita del piano oculare, cioé secondo la direzione r della fig. 38, ma indifferente- mente secondo le direzioni r’ ed r’’ in cui |’osservatore lo ponga. I] reorientamento sembra quindi legato qui a sole percezioni cenestetiche, interne all’animale, in discordanza con le conclu- sioni di Lyon ed Uexkiill. Un caso piti complesso e pit difficile da analizzare mi é stato offerto da un ditisco leso al sopraesofageo destro. Adunanatazionesinistrorsa si accompagnava un distinto obliquamento del corpo all’avantied in basso ed uno sbandamento, MOVIMENTI DI MANEGGIO NEI COLEOTTERI 269 pure nettissimo, del corpo sulla sinistra, il quale ritmicamente ed a non lunghi intervalli si mutava in una rotazione dell’animale intorno all’asse sagittale, sinistrorsa per chi guardasse l’animale a posteriori. Che tale rotazione non fosse dovuta che ad alterati moti degli arti, 1 quali non ho per6é potuti individuare, é dimo- strato dal fatto che una successiva lesione alla parte sinistra del cervello ha riequilibrato ’animale. Neppure qui, dunque, trat- tasi dell’dlterazione di un senso specifico. Tutte le anomalie che abbiamo cosi riassunte si lasciano ricon- durre ad un’unica espressione; predominio nell’antimero illeso delle attivita muscolari flessorie ed in genere dei gruppi muscolari adduttori e contrattori. La flessione del capo e del corsaletto, la flessione degli arti, sono diverso aspetto di una medesima condi- zione fisiologica; lo stesso addome, ove non fosse costretto entro una capsula chitinosa, probabilmente seguirebbe quelle medesima flessione dell’asse sagittale che fa il corpo curvo verso il lato illeso. E qui ci aiutano i risultati delle esperienze del Matula sulle larve di Aeschna, larve fornite di un tegumento debolmente chitinizzato ed articolato, sui segmenti dell’addome. ‘Tali larve, per estir- pazione di una metd laterale del cervello, mostrano l’addome piegato cosi che la parte coneava guarda dal lato illeso del capo. Di pid, in tali organismi, il citato Autore ammette che lo scer- vellamento produca un aumento di tono dei gruppi muscolari flessori all’indietro ed una diminuzione dei flessori all’avanti, Ossia un meccanismo analogo a quello che io suppongo nei co- leotter1 lesi ad una regione laterale del cervello. Né é molto diffcile agire sulle condizioni generali del muscolo, anche prescindendo da lesioni del sistema nervoso, in modo da provocare simili esclusivismi di dati gruppi muscolari. Loeb, Garrey e Maxwell, ad esempio, studiando le reazioni galvanotro- piche dell’Amblystoma e del Palaemonetes hanno trovato che ’animale, in un campo percorso da una corrente elettrica, tende a spostarsi verso il catodo o verso l’anodo, con alterazioni caratteris- tiche del portamento degli arti, cui sono impediti 1 movimenti in un senso determinato, per il predominare dell’azione di gruppi di muscoli antagonisti. 270 EDGARDO BALDI Ma vi é tutta una serie di fatti assodati dalla fisiologia della innervazione muscolare e che va sotto il nome di innervazione reciproca dei muscoli antagonisti, la quale ci pudé dare ancora qualche lume per l’interpretazione dei fenomeni di maneggio. Alloreché una regione mobile dell’organismo sia sollecitata da muscoli antagonisti, tali cioé che la loro contrazione induca in essa movimenti reciproci, allorché l’uno dei muscoli é in contra- zione, l’antagonista si mostra rilasciato. Tale rilasciamento non é puramente passivo e dovuto sola- mente alla distensione operata sul muscolo dall’azione dell’antago- nista, ma la condizione di rilasciamento viene determinata in esso per via riflessa e sussiste anche allorché le connessioni mec- caniche fra 1 due antagonisti vengano obliterate. Cosi’ che en- trambi i processi, nel gruppo antagonista, sono, in certo senso attivi. Ed il fatto é stato sperimentaménte provato dallo Sher- rington sui muscoli retti del globo oculare di rana. Fenomeni simili sembrano largamente diffusi, benché il Dubois*Reymond ne abbia infirmata una facile generalizzazione. I] Frohlich ha con- statata anche nella muscolatura dei cefalopodi la contemporaneita della contrazione e del rilasciamento nei gruppi antagonisti. I] fenomeno si verifica tanto eccitando il centro nervoso relativo, quanto eccitando il nervo lungo tutto il suo decorso. Lo Sher- rington ha supposto che il rilasciamento riflesso sia dovuto ad un arco destato dalla contrazione dell’un muscolo negli organi mus- colo tendinei del Golgi e deprimente il tono del muscolo antago- nista. Il Verworn é riuscito a tradurre in tracciati i fenomeni descritti dallo Sherrington. Ora, negli arti flessi di un insetto che descriva un moto di maneggio, ci troviamo indubbiamente in presenza delle medesime relazioni, poi che i segmenti dell’arto dell’insetto sono posti in movimento da una serie di coppie di muscoli antagonisti che regolano il loro spostamento nei due opposti sensi (fig. 4). In esso insetto 1 muscoli contrattori degli arti dal lato illeso saranno in condizione di esaltato tono e gli estensori loro antagonisti in condizioni opposte di tono depresso. Ossia, le condizioni com- plessive della muscolatura dell’antimero illeso sono alquanto complesse. Come si pud quindi genericamente parlare di una MOVIMENTI DI MANEGGIO NEI COLEOTTERI 271 esaltazione globale del tono muscolare, in un antimero, per lesione unilaterale di centri nervosi, come della principale causa del moto di maneggio—cosi come é nel pensiero, ad esempio, del Bethe? Tutto questo insieme di alterazioni a carattere dissimmetrico, che accompagna l’effettuarsi del moto di maneggio, dipende anch’esso dal sistema nervoso, indubbiamente, ma non sembra vineolato sempre ed esclusivamente al maneggio, benché sia du- rante il maneggio appunto che esso trova la sua pit netta mani- festazione. In taluni casi—i pii— in cui il maneggio non abbia carattere di continuita, le vediamo definirsi allorché il maneggio interviene; in altri, in cui il maneggio manea, le vediamo abboz- zarsi, accennarsi. Sono frequenti infatti gli individui lesi, che, pur non girando nettamente in tondo, manifestano leggere curva- ture del corpo e del capo da quel lato ove gli arti si mantengono un poco piu flessi, senza che questi lievi sintomi si acuiseano tanto da passare ad un vero moto dimaneggio. In altri casi le vediamo persistere, affievolendosi, qualche tempo dopo~che l’animale, trascorso un periodo di passeggero maneggio, ha ripresa la deam- bulazione normale. Sembra cioé che la loro determinazione non sia rigorosamente localizzata nella medesima regione nervosa, la cui lesione provoca il maneggio, ma che l’alterazione di zone circostanti sia anche sufficiente a destarle, in forme, peré, meno precise. Non si tratta, beninteso, che di una supposizione. Ma cid che é sopratutto da ritenere si 6 che esse appaiono con varia nettezza tutte le volte che un moto di maneggio interviene, pit spiccate nei maneggi tipici, meno nei transitorii ed atipici. Sono, queste, manifestazioni tali, crediamo, che una teoria dei ma- neggi non possa prescindere dalla loro considerazione. Gli autor1 che hanno descritti moti di maneggio li hanno un poco dipinti come qualcosa di fatale come un ananke rotatorio che sos- pinga incessantemente l’animale su di un binario circolare sino alla consumazione dei suoi giorni. Solamente la Drzewina, ch’io mi sappia, ha riferito di un ripristinamento della deambulazione normale, contraddicendo il Bethe, nel Carcinus moenas e nel Pachygrapsus marmoratus, in cui la guarigione avviene dopo pit che una decina di giorni. La Lygia oceanica ed una specie di 272 EDGARDO BALDI Palaemon le hanno dato risultati analoghi, benché meno precisi. Ma la Drzewina non da particolari suff.cienti sulla teenica dell’ ag- gredimento del ganglio, né ha compiuti controlli anatomici sui suol esemplari. In realta—ed il fatto risulta evidente dai dati relativi ai singoli casi di maneggio che altrove dettagliatamente esporré, il moto di maneggio é ricco di sfumature e di varianti. Raramente, anche prescindendo dai casi di ripristino temporaneo e totale della locomozione retta, esso maneggio 6 ininterrottamente continua- tivo; sempre offre una curvatura variabile che va dai giri in posto ad ampie circonferenze. In un esemplare leso e lasciato a se medesimo, non é raro osservare di giorno in giorno e taloradi ora in ora qualche variazione, sia della forma che dell’ampiezza e della velocita e talora persino, quando turbazioni secondarie si sovrappongano alla lesione originaria, del senso del maneggio. In casi atipici si possono osservare i moti di maneggio insorgere spon- taneamente ad interrompere una deambulazione retta od un diverso comportamento anormale. In casi parimente atipici si puo pure osservare la cessazione definitiva di maneggi durati pit o meno a lungo, talora momentaneamente ripristinati da appositi stimoh, il che ha osservato anche la Drzewina nelle citate specie dicrostacei. In casi di mancata lesione del cervello, ma di lesione di parti finitime—lesione mancata volontariamente od involonta- riamente da parte dello sperimentatore—l’organismo pud rispon- dere al trauma con pochissimi 0 con un sol giro dimaneggio, talora con alcuni giri suecessivamente descritti con opposto verso. Ksiste, negli organismi lesi, una rigenerazione fisiologica? I] problema ha diverso senso a seconda del modo con cui lo si im- posta, a seconda cioé che l’attenzione si porti esclusivamente sulla presenza di un tipico moto di maneggio o sull’insieme delle reazioni dell’organismo leso, nelle quali il maneggio pud rappre- sentare un momento pii o meno duraturo. Fra i numerosi casi tipici di durevole maneggio che ci si sono offerti nelle nostre es- perienze, non ne possiamo annoverare che due, nei quali vi sia qualche cenno, non di rinormalizzazione del comportamento, ma di desistimento dal giro in cirecolo. Né i casi analoghi si pre- sentano del tutto scevri da dubbio. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 273 Nei quadri sintomatici piti complessi dei casi atipici, invece, per una lesione caduta fuor della regione frontale del cerebron, non é raro il trovare, a seadenza piti o meno lontana, ristabilita la deambulazione normale. Dall’insieme dei nostri dati ci sem- bra poter per ora concludere che nei coleotteri da noi presi in esame e per le durate medie dei periodi di osservazione, non segua rigenerazione fisiologica ai casi di lesioni bene localizzate e cor- rispondenti ad un quadro costante di alterazioni locomotorie (maneggio tipico). Ma quale valore pud avere allora il rinor- malizzarsi del comportamento in casi di ferite anche piu gravi, ma non frontali? Quale meccanismo nervoso gli presiede? Il problema é aperto, né certamente é dei meno interessanti. che offra la fisiologia del sistema nervoso in questi insetti."! Riassumendo, per quanto ce lo permette, di fronte alla com- plessitdé ed alla imponenza del problema, la frammentarieta di molti lati delle nostre osservazioni, ci sembra che ad una lesione unilaterale generica del cervello, l’organismo risponda con un vario e complesso assieme di fenomeni fra i quali prende posto anche quella serie di rotazioni nello spazio che abbiamo convenuto di chiamare maneggio. Serie, la quale offre di molte sfumature e di molte varietd, cos{ che il maneggio tipico ne sembra a sua volta, un caso particolare, legato a certa gravité della lesione ed al concorrere di certi particolari ed anormali atteggiamenti del corpo e degli arti. La differenza fra il punto di vista abituale agli autori ed il mio sta appunto qui, nel considerare il maneggio, non come la risposta obbligata o la risposta in certo senso normale, regola- mentare dell’organismo, ma come un particolare punto ed un particolare aspetto fra gli anormali sintomi che seguono alle lesione. E’ lecito domandarci: cos{ come cisembra che il maneg- gio corrisponda, nei casi tipici, ad una ben delimitata lesione di una regione del cervello, non potrebbo essere che ciascuno di quegli aspetti anormali del comportamento che costitiscono il quadro morboso dei casi atipici, cioé della maggioranza dei casi, corrisponda ad una diversa e parimenti localizzata lesione di 11 Questi accenni non posson essere chiariti senza uno stretto riferimento alla conoscenza di dati relativi a ciascuno dei casi che ho presi in esame. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 274 EDGARDO BALDI una regione nervosa? La localizzazione della regione lesa varierd sempre e conformemente al variare del comportamento anormale? Lo potranno eventualmente dire ulteriori ricerche, ma é nostro concetto, genericamente confortato anche da recenti conclusioni della neuropatologia umano di guerra, che la considerazione delle localizzazioni non debba andare disgiunta da quella delle con- dizioni fisiologiche generali dell’organismo. Ma, senza misconoscere al sistema nervoso le sue attribuzioni di unificazione funzionale dell’organismo, poniamo sopratutto at- tenzione, come ad un immanente condizionamento dell’estrinse- carsi della sua attivitdé a quello che nell’organismo i francesi chiamano ‘‘état physiologique”’ .e che, come assieme, é determi- nato dalle attivita e determina le attivita di ogni sua regione. Come é possibile, su queste basi, una teoria del maneggio? Poi che essa non pué che limitatamente tener conto di questo complesso, singolare ed ancor troppo poco noto condizionamento, non pud essere una teoria del maneggio-tipo, che nella schemati- citaé costante delle manifestazioni che costituiscono quest’astra- zione di fenomeno, tenti di fissare e di interpretare le ragioni del permanere di una data alterazione nell’organismo. KE’ quello che ora vedremo, esaminando quanto possano con- tribuire alle ipotesi gid enunciate da altri autori, le nostre osservazioni. 8. IL DETERMINISMO DEI MOTI DI MANEGGIO Cié che ha sopratutto colpiti gli osservatori che hanno indagati e teorizzati 1 moti di maneggio, si é l’esistenza di una dissimmetria quantitativa, se pure non misurata, nelle prestazioni degli arti dei due antimeri, piti che quella di, una dissimmetria di impiego, d’una dissimmetria, vorrei poter dire, morfologica e qualitativa negli atti degli arti. Convien ricordare che le nostre conclusioni non vogliono che riguardare le specie di coleotteri che abbiamo studiate, né noi sapremmo generalizzarle, né eventualmente precisare l’estensione di una loro generalizzazione. Poi che se una dissimmetria quantitativa nel lavoro muscolare di due parti del corpo in quanto si riduea ad una differenza di attivita nel tessuto muscolare nelle due regioni e sia indipendente dalla con- MOVIMENTI DI MANEGGIO NEI COLEOTTERI 215 formazione delle parti mobili cui il muscolo si inserisce é pit comprensibilmente generalizzabile, l’impiego dell’arto é cosa tanto strettamente connessa alla sua morfologia, da essere necessarie constatazioni singole e casuistiche per definirne le condizioni. Il Dubois, nello studiare i moti in circolo descritti dal Pyro- phorus per lesione laterale dei gangli cerebroidi, aveva netta- mente affermato il carattere puramente “relativo’’ della dissim- metria locomotoria e propendeva anzi a riferirne il valore ad una diminuita attivité degli arti dal lato sano, rispetto all’attivita normale degli arti dal lato leso. Egli dice infatti ““L’ observation directe et l’examen des graphiques montrent clairement que les membres du cété opposé A celui de la lésion ne sont pas paralysés, ils sont seulement atteints de parésie: les mouvements ont moins d’amplitude et leur énérgie étant moins grande, l’action des membres du c6té opposé devient prédominante; l’insecte est alors posé du cété le plus faible.” Il Dubois non accenna a diversitdé nell’impiego stesso degli arti, ma la interpretazione che egli propone é corretta, anche da un punto di vista assai generale, poiché egli non ha alcuna difh- eolté ad ammettere che la lesione di una regione laterale dei cerebroidi possa riflettersi sulla regione laterale opposta del corpo ed esplicitamente parla di un incrocio delle azioni fisiologiche, corrispondente ad un decorso chiasmatico delle fibre nervose, alludendovi con l’espressione di “‘paralysie croisée.”’ Poiché dalle ricerche dello Yung in poi (’78) era stato sostenuto il concetto che la trasmissione delle azioni nervose fosse stretta- mente laterale e che, rispetto alla loro innervazione, i due anti- meri dell’artropodo si comportassero in modo affatto indipendente ed autonomo, non solamente rispetto alla trasmissione attraverso la catena subintestinale, m rispetto allo stesso cervello. “‘Aucun fait—diceva lo Yung—ne permet de supposer un entrecroise- ment de fibres dans le cerveau.”’ Concetto sul quale insisteva anche il Demoor nella sua memo- ria del 1891. Entrambi questi autori hanno tratte le loro con- clusioni da ricerche eseguite sui crostacei decapodi. Certamente negli insetti una tale affermazione non potrebbe venire sostenuta, benché riflessi di quella idea, di una azione omoantimerica, cioé delle lesioni cerebrali, si ritrovino anche nella teoria del Bethe. 276 EDGARDO BALDI L’Herrera, che, indipendentemente dal Dubois, aveva com- piuto su insetti le varie esperienze di cui ho sommariamente riferito, proponeva pure (’93) una molto schematica teoria del maneggio, tutta meccanica, di cui fard parola per dovere di imparzialita. Avendo constatato nell’insetto leso un abbassa- mento del corpo verso il lato della lesione, ed assumendo che a tale posizione sbieca sia dovuto l’avvicinamento al corpo degli arti dal lato sano, egli opina che la combinazione di questa forza attrattiva laterale degli arti sul corpo con la spinta in avanti di cui un corpo stesso é dotato, sia sufficente a chiarire la com- parsa dei moti di maneggio. L’osservazione che la somma di due vettori ortogonaliin un piano, rappresentanti due spostamenti- rettilinei ed uniformi in moto, non pud essere una traiettoria circo- lare, bastera a far porre da banda l’interpretazione dell’ Herrera. Ometter6d di ricordare qui le interpretazioni centriste e psico- logiche anteriori, di Faivre, Burmeister, ecc., giudicandole in- terpretazioni non fisiologiche. I] Binet, nel 1894, si acconten- tava di una espressione piti generica, assumendo che i moti in circolo fossero dovuti ad una ineguale eccitazione degli arti dai due lati. Né accenner6é alla sommaria interpretazione del Matula, che per la sua particolarit’ ed incompletezza non si presta ad una discussione generale. Occorre giungere sino al Bethe per trovare un’espressione pit precisa del determinismo dei moti di maneggio, espressione, la quale traduce in veste teoretica quella visione sopratutto quantitativa del moto di maneggio che abbiamo visto soffermarsi come sul momento fondamentale del moto in circolo, sulla dissimmetria delle attivité propulsive degli arti dalle due metd del corpo. Non che il Bethe, accurato osservatore, non abbia notate differenze nell’impiego degli arti, poi che egli es- pressamente rileva nei suoi esemplari una positura anormale degli arti stessi, provocata da una disuguale tensione dei flessori e dei rotatori, ma egli non la pone in istretta relazione con il maneggio, né con la lesione laterale del cervello (egli parla di Ausschaltung des Gehirns) e neppure accenna all’influenza che essa ha sul compimento dei moti locomotorii degli arti. Il Bethe . ammette bensf che il cervello eserciti una generica azione inibi- MOVIMENTI DI MANEGGIO NEI COLEOTTERI Are | toria sugli automatismi e sui riflessi segmentali dei metameri dell’organismo posteriori a quelli cefalici. Esso cervello inoltre agisce come tonificatore dei muscoli; la sua azione si esercita—ed in questo il Bethe si collega alle vedute dello Yung e del Demoor— con uno spiceato lateralismo: ogni meta del cervello inibisce e tonifica la muscolatura del corrispodente antimero. Gli impulsi che partono dal cervello sono trasmessi all’indietro senza sotto- stare a smistamenti od a deviazioni chiasmatiche. Per lesione di una porzione laterale dell’organo inibitore viene tolta questa sorta di controllo ai muscoli degli arti dal lato corrispondente, i quali, movendosi senz’essere inibiti, dispiegano un’attivitd mag- giore di quella degli arti dal lato opposto; questo disquilibrio di attivita produrrebbe il moto in circolo dal lato sano. Keco le parole testuali del Bethe: ‘Der Kreisgang, nach der gesunden Seite ist lediglich auf die Ungehemmtheit der operierten Seite zurtickzuftihren.”’ Ne segue—ed il Bethe infatti lo afferma—che il moto in cir- colo non sia un moto coatto: compensando in qualche modo questa deficienza di inibizione dal lato leso, si deve poter raddriz- zare la deambulazione. Ossia, il Bethe inverte quella che era la posizione del Dubois, il quale indeboliva il lato dell’animale op- posto alla lesione. Mentre il Bethe, per assenza di tonificazione indebolisce il lato corrispondente. Riferendoci sempre a quanto abbiamo veduto sulle nostre specie di coleotteri, confesseremo che l’interpretazine del Bethe ci sembra contenere bensi’ elementi di veritd e rispondere in parte alle condizioni che effettivamente si verificano nel maneggio,ma sembraci pure che essa non descriva che un aspetto del fenomeno e non possa rappresentare una teoria completa del moto in cir- colo. Eccone le ragioni. Anzitutto, constatazioni anatomiche compiute senz’aleuna preoccupazione fisiologica da morfologi puri, il Viallanes, il Cuc- cati, il Berlese, hanno assodato nel cervello degli insetti la pre- senza di chiasmi. Nelle fig. 39-40 riproduciamo taluni disegni del Berlese, togliendone il decorso di taluni fasci che s’incrociano prima di partirsi dal cerebron per incanalarsi nelle commissure della catena subintestinale. bo ~J 10/2) EDGARDO BALDI 40 Fig.39 Decorso di fasci chiasmatici nel cerebron degli insetti (da Berlese) Fig.40 Il chiasma ottico olfattivo nel cerebron degli insetti (da Berlese) MOVIMENTI DI MANEGGIO NEI COLEOTTERI 279 Il fascio indicato con il N° 37, si diparte dalla superficie del protocerebron da un lobulo di cellule gangliari, si incrocia con il simmetrico posteriormente alla sutura dei lobi protocerebrali e si recaailobi dorsali del deutocerebron. I morfologilo hanno espres- samente designato come cordone chiasmatico. Vi é ancora il chiasma ottico olfattivo, che abbiamo schematizzato nella fig. 40 e che dipartendosi dai calici si suddivide in tre rami di cui uno va al corpo centrale, l’altro al deutocerebron dalla propria parte ed il terzo, incrociandosi con il simmtrico, molto dietro il corpo centrale, al deutocerebron dalla parte opposta. Ma vi sono fasci che dal deutocerebron passano direttamente al tritocerebron e di qui alle commissure longitudinali della catena subintestinale. Uno di essi fu deseritto da Cuccati, altro, denominato fascio chiasmatico, parte dal deutocerebrom, indi si biforca e mentre uno dei rami incrociandosi con il simmetrico, termina nel deutocere- bron dalla parte opposta, l’altro, pure dopo essersi incrociato, va al tritocerebron e passa nelle commissure sottoesofagee. Ve n’ é ancora uno, proveniente dai lobi ottici, che, radendo il confine tra la sostanza punteggiata e la sostanza corticale passa al tritocerebron dalla banda opposta. Tutte le regioni laterali del cerebron sono quindi in mutua rela- zione anatomica. In realtd, l’effetto fisiologico della lesione laterale non é ri- stretto all’antimero corrispondente, come provano le informazioni che abbiamo precedentemente esposte e che dipingono un quadro generale di lesioni cui nessuna parte del corpo sfugge e che in ispecie per quel che riguarda l’impiego degli arti nella locomo- zione si accentua negli arti situati dalla parte opposta a quella della lesione. Né é chiaro, fondamentalmente, il concetto mede- simo di inibizione. [1 fatto capitale e pit volte verificato si é che |’assenza del cervello e la sua lesione provocano la comparsa di moti pendolari degli arti, di automatismi, di gesti ritmici e continuativi. Ma sappiamo noi in qual misura si dividano il determinismo di questi fenomeni, il sistema nervoso centrale e le disposizioni neuromuscolari del rimanente organismo? Ed il parlare di centri e di inibizione puéd anche essere giustificato, allorché, con esse espressioni, per mancanza di una migliore cono- 280 EDGARDO BALDI scenza e di una pitt adeguata interpretazione dei fatti, si intenda designare un complesso di reciproche influenze fra le parti inner- rate, le cui relazioni, traducentisi in un definito comportamento, vengono alterate in un deteminato senso dalla mancata fun- zionalita di un anello della catena, del ganglio, cioé. Altri fenomeni di ordine analogo a quelli che hanno suggerito il concetto di una inibizione del cervello sui gangli della catena, hanno condotto infatti al concetto simmetrico di una inibizione della catena sui gangli cerebrali, concetto gid accennato dal Bethe e che, nell’interpretazione del Comes (712) (‘‘inibizione reciproca dei gangli’”’), ha assunto importanza ed ampiezza pari a quella del primo. FE benché noi non mettiamo in dubbio il valore dell’interpreta- zione per dimostrare come all’espressione di inibizione vada asse- enato anzitutto un valore schematico e simbolico, ritorneremo un istante sulla nota esperienza del Normann, che tagliata trasver- salmente una Allobophora, osservava come la meta anteriore, ‘‘inibita’”’ dal cervello tuttora presente, non alterasse il compor- tamento proprio all’individuo integro, mentre la meta posteriore non pit inibita nei suoi riflessi, si divincolava incessantemente. Ripetuta l’operazione sulla meta anteriore, il fenomeno si ripete identicamente. Ma identicamente si ripete pure operando simil- mente sulla meta posteriore, per la quale non pud pit venire invocata lazione inibitoria di un cervello. Vi é forse infine una contraddizione nel modo con cui il Bethe descrive le funzioni del cervello negli artropodi. Esso é ad un tempo organo tonificatore ed inibitore. Quindi gli arti dal lato leso, benché apparentemente godano di una esaltata atti- vité, dovranno avere minore efficacia dinamica, minore energia, almeno in quanto energia é attitudine alla produzione di lavoro meccanico, mentre gli arti dal lato illeso dovranno avere con- servato la loro primitiva efficienza. Il corpo dell’insetto do- vrebbe quindi essere pit debolmente sorretto e guidato e spostato dal lato leso che da quello sano, e, se un disquilibrio vi é e se tale disquilibrio é sufficiente a turbare la dirittezza della deambula- zione, cié.dovrebbe avvenire in senso inverso a quello che é indi- cato dai fatti ed é preveduto dalla teoria del Bethe. Né ci si MOVIMENTI DI MANEGGIO NEI COLEOTTERI 281 obbietti che basti il solo fatto esteriore della maggiore distensione delle arcate dal lato leso ed il maggiore distanziamento dei punti di appoggio, ivi, degli arti sul terreno, per provocare il moto in circolo. Poiché si é visto come l’insetto sappia autoregolarsi e correggere disquilibrii puramente meccanici analoghi e di valore di gran lunga superiore a quelli naturalmente provocati da un esaltato divaricamento degli arti. Ma ci si pué chiedere se tale mancanza di inibizione realmente esista, se cioé esista una reale esaltazione della motilita limitata agli arti dell’antimero che ha sofferta la lesione. I] Bethe pone Vaffermazione senza suffragarla di dati numerici, frutto di es- perienze quantitative. Ma a noi sembra, benché anche per noi Vesecuzione di simili misurazioni sia programma di ulteriori ricerche, che ben poco valore debbasi accordare all’osservazione immediata di un insetto che compia un moto di maneggio. I] nostro occhio é sopratutto giudice, in tale osservazione, non di velocitd angolari, bensi di velocitd lineari (moti degli estremi degli arti, rispetto al centro di curvatura del maneggio). Ed in realta non si saprebbe decidere qual sia la pii adeguata fra le due que- stioni; se l’insetto si muova in circolo perché gli arti esterni sono dotati di maggiori velocitd lineari degli interni, o se detti arti esterni siano dotati delle suddette maggiori velocita perché Vinsetto si muove in circolo. Né la questione potrebbe essere detta oziosa. D’altro canto, cid che nella considerazione delle azioni dinamiche cui bilateralmente é sottoposto l’organismo, ha importanza, non é il valore assoluto di esse dall’uno dei lati, bensi il rapporto di quelle da un lato a quelle dall’altro. Molti- plicando per un uguale fattore i valori degli impulsi forniti al corpo dagli arti dei due antimeri, muterd la velocitaé di trasporto di esso corpo, ma non la forma della traiettoria. Ora, osservando un insetto che compia il maneggio, siamo impossibilitati ad isti- tuire un simile confronto fra gli arti dal lato leso e quelli dal lato illeso, poiche gli arti vengono, dai due lati, impiegati diversa- mente e poiché un possibile aumento della motilita—che negli arti dal lato leso si traduca in una maggiore ampiezza delle ar- cate in senso anteroposteriore, piti facilmente apprezzabile ad un’osservazione dorsale—dal lato illeso pué manifestarsi, ed in 282 EDGARDO BALDI realta si manifesta, come ho detto a pit riprese, con una maggiore ampiezza delle arcate attrattive tibiofemorali, in un piano tra- versale al corpo dell’animale. L’aumento della motilit’ pur essendo esteso a tutto l’orga- nismo, non verrebbe quindi apprezzato che come aumento della motilit’ in senso anteroposteriore degli arti posti esternamente. Se poi tali variazioni realmente corrispondano, 0 meno, a varia- zioni dell’energia intrinseca, del tono e delle condizioni fisiolo- giche del muscolo, non potra essere deciso che da ricerche ergogra- fiche istituite sui muscoli dei due antimeri. Misurando infine l’ampiezza delle arcate sui reogrammi, cioé il valore numerico di una quantita proporzionale alla dissimme- tria propulsiva, ai due lati del corpo, abbiamo visto che in taluni casi—senza peraltro generalizzare—allo stabilirsi di essa dissim- metria maggiormente contribuiscono le diminuite attivita pro- pulsive dal lato illeso, che un loro aumento dal lato oposto. Ancora un’osservazione a proposito dell’inibizione cerebrale sui gangli della catena. Per toccamento elitrale le Pimelia presen- tano la nettissima reazione di una immobilizzazione riflessache deve essere interpretata come una inibizione energica del cervello su ogni impulso motorio coordinato dei gangli della catena. In un animale normale, tale riflesso é simmetrico, cioé, si esercita contemporaneamente ed ugualmente su tutti gli arti del corpo. In un animale privato dei gangli sopraesofagei e quindi delle azioni inibitorie sulla corrispondente meta del corpo, tale immo- bilizzazione riflessa non potra stabilirsi normalmente che nell’anti- mero illeso. Od almeno, fra i due antimeri dovra potersi notare qualche divario nell’assunzione della posizione di immobilita e nella sua durata. Tale divario, in realtdé, non si nota, come se, cioé, non si fosse verificato aleun fatto di inibizione mancata. D’altro canto l’insufficienza dell’interpretazione del Bethe, che uno squilibrio propulsivo sia bastevole ad originare e ad intrat- tenere il moto di maneggio, é gid stata dimostrata nella mancanza di simili moti in quelle esperienze in cui tale disquilibrio era stato artificialmente provocato, prescindendo da lesioni nervose. Vi 6 ancora una difficolts e sta nel concepire le modalita della distribuzione della ‘‘mancanza di inibizione’’ nell’organismo. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 283 Essa dovrebbe infatti colpire tutti i gruppi muscolari ugualmente il che non si verifica, poiché abbiamo visto come, a prescindere dai muscoli degli arti, il capo, lo stesso asse sagittale del corpo siano flessi cosf da formare concavitd verso il lato illeso. E perché nell’antimero illeso, in cui, in grazia della lateralita dell’-a zione cerebrale ammessa dal Bethe, nessuna condizione nuova avrebbe dovuto insorgere, dovrebbero invece predominare i eruppi di muscoli flessori che originano quelle curvature? E per quanto riguarda la muscolatura degli arti, gid abbiamo fatto osservare come essa sia costituita di muscoli antagonisti. Come si potrdé concepire in questi una distribuzione della mancata inibizione? Entrambigliantagonisti dovrebbero essere ugualmente non-inibiti e la loro azione dovrebbe liberamente ed accentuata- mente esplicarsi in ogni possibile senso. Come si interpreteranno allora quelle particolari modificazioni d’impiego e quelle deter- minate ed obbligate direzioni assunte nello spazio dai moti degli arti, che abbiamo particolareggiatamente descritte? Sono, queste, altrettante difficolta che offre l’interpretazione del Bethe e che forse non si oppongono all’ interpretazione che mi ha suggerita l’osservazione dei moti di maneggio nelle note specie di coelotteri. La esporré per sommi capi, senza dettagliarla, come concezione d’assieme. 1. La lesione di una regione laterale dei gangli sopraesofagei trae seco un’alterazione nel portamerto dell’animale, che, in armonia con le disposizioni chiasmatiche osservate nei fasci nervosi cerebrocatenali non é ristretta ad una meta laterale del corpo, ma interessa l’intero organismo e con particolare evidenza si rivela nell’antimero opposto a quello della lesione. 2. Detta lesione provoca molto probabilmente un accresci- mento generale della motilité, particolarmente visibile negli arti dal lato leso. 3. Provoca infine nell’organismo uno squilibrio nel normale uso della muscolatura, predominando in essa l’attivité dei muscoli flessori nell’antimero illeso, attivitdé resa manifesta dalla perma- nente maggiore flessione degli arti illesi e dall’incurvamento del corpo verso il lato sano. 284 EDGARDO BALDI 4. Tali abnormi condizioni fisiologiche della muscolatura in- fluenzano il normale svolgersi dei moti locomotorii, cosi da gene- rare il movimento in circolo. 5. Il moto in cireolo é dovuto prevalentemente—(ma non esclusivamente, poi che esso 6 un movimento cui attivamente partecipa tutto l’organismo)—alle azioni attrattive predominanti dei due primi arti del lato illeso, variamente orientate, rispetto alla direzione dell’asse sagittale. E’coadiuvato e facilitato dai moti propulsivi ad areata, caratteristicamente proprii di tutti gli arti dal lato leso e particolarmente dei primi due. 6. Che tali azioni flessive siano il movente principale del giro in circolo é provato | a) dal’ impossibilité di provocare moti in circolo per esagera- zione artificiale delle attivita propulsive da uno dei lati. b) dal ristabilirsi di locomozione retta od oscillante, allorché, per l’amputazione dei segmenti articolati degli arti, venga resa impossibile l’esecuzione dei moti attrattivi. c) dal permanere del moto di maneggio allorché siano impeditii soli moti propulsivi ad arcata degli arti dal lato leso. 7. Il moto di maneggio risulta quindi bensi da un’alterazione delle condizioni generali di simmetria dell’organismo, come ave- vamo assunto dapprincipio, ma da tale alterazione che interessi tutto l’organismo stesso, assumendo vario aspetto nelle diverse sue regioni. Quest’interpretazione dei moti di maneggio, che crediamo pit adeguata alla complessa realtaé dei fatti, non contiene alcuna ipotesi, né va al di 14 dei fatti medesimi, opportunamente coordi- nati e collegati. Essa quindi non ci dice nulla di preciso circa le modalita dell’al- terata influenza del sistema nervoso sui gruppi muscolari. Né peraltro attribuisce a gangli condensazioni verbalistiche di pro- priet’ non altro che supposte. Essa, per quanto non sia che un’approssimazione a quella che dovraé poter essere la teoria dei moti di maneggio, offre per6é ancora alcuni vantaggi da questo punto di vista, dai quali si potra trar partito, indagando, come é in animo di chi scrive, le alterazioni istologiche del sis- tema nervoso che accompagnano le alterazioni fisiologiche del comportamento. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 285 L’ammettere infatti la propagazione di una inibitivit’ e di una azione tonificante dal complesso cerebrale alla muscolatura ed alla sua innervazione é concetto che, ove anche fosse esatto, sarebbe troppo generico per prestarsi a precisazioni anatomiche. Né il considerare solamente la soppressione dell’inibizione e del tono (di essi, infatti non si pud pensare, al pid, che una graduazione di intensit’) pud rendere conto degli svariati effetti che la diversita della localizzazione lesiva induce come sperimentalmente abbiamo constatato accadere. Converra invece esaminare il decorso dei fasci e veder di porre in relazione la rottura di continuitdé anatomiche e la soppressione di contiguitaé funzionali oppure la deviazione di archi riflessi, che possano render ragione dele particolari condizioni di innervazione che rivela la fisiologia del maneggio. E basterebbe, a provare V’insufficienza di quel concetti, la constatazione di lesioni laterali del cerebron non accompagnate da moti di maneggio. Siamo ben lungi dal poterne precisar le ragioni, ma noi non vediamo altra via—per quanto questa sia irta di difficolta—per giungervi. Riuscirad pure chiaro come nella nostra interpretazione, altret- tanto bene come in quella del Bethe, possano rientrare i moti di maneggio provocati per sezione unilaterale delle commissure fra sopra e sottoesofageo cosi come eventuali maneggi provocati da lesioni del sottoesofageo stesso. Ma conviene lasciare libert&’ completa di indirizzo alla futura ricerca sperimentale e non costringerla entro predeterminate linee suggerite dalla teoria. La quale potrebbe anche venire contraddetta da nuovi fatti, fors’anche per avere troppo voluto rimanere aderente ai fatti medesimi. SUMMARY OF CONCLUSIONS 1. Injury to one side of the supraoesophageal ganglion brings about a change in the behavior of the animal (beetle), which, in harmony with the chiasmatic arrangement of the fibers running from the brain to the lower ganglia, is not restricted to one lateral half of the body but involves the whole organism and 286 EDGARDO BALDI reveals itself with special clearness in the antimere opposite to that of the lesion. 2. This injury provokes very probably a general increase of movement which is manifest particularly in the appendages of the injured side. 3. The final effect is to produce a disturbance of equilibrium in the normal functioning of the musculature, in which the activity of the flexor muscles of the uninjured side predominates, an activity rendered manifest by the greater permanent flexion of the uninjured appendages and by the bending of the body toward the uninjured side. 4, These abnormal physiological conditions in the muscula- ture so influence the normal course of the locomotor movements as to produce movement in a circle. 5. The movement in a circle is due for the most part (but not exclusively, since it is a movement in which the whole or- ganism participates actively) to the traction predominatingly exercised by the first two appendages of the uninjured side, this traction being variously directed with respect to the sagittal plane. It is helped and facilitated by the propulsive arcuate movements, characteristic of all the appendages of the injured side particularly of the first two. 6. That the movements of flexion are the principal cause of circus movements is proved: a) By the impossibility of eliciting such movements by the artificial exaggeration of the propulsive activity of one side. b) By the re-establishment of straight or oscillatory locomo- tion, when, by the amputation of the segments of the leg, the execution of the movements of traction is rendered impossible. c) By the persistence of circus movements where only the propulsive arcuate movements are prevented in the legs of the injured side. 7. Thus circus movement does indeed result from an altera- tion of the general conditions of symmetry of the organism, as we have assumed from the beginning, but from such an altera- tion as affects the entire organism, assuming various aspects in different regions. MOVIMENTI DI MANEGGIO NEI COLEOTTERI 287 GLI AUTORI CITATI Bacuionr Die Grundlagen der vergleichenden Physiologie des Nervensystems und der Sinnesorgane. Winterstein’s Handb. d. vergl. Physiol., 4er Bd. Fischer, Jena, 1913. Physiologie des Nervensystems. Ibidem. Barrows The reactions of Drosophila ampelophila to odorous substances. Jour. Exp. Zodél., vol. 4, 1907. BauprRimont Note sur la marche des insectes. Procés Verbaux Société Linn. Bordeaux, T. 65, 1911. Betue Vergleichende Untersuchungen iiber die Funktionen des Centralnerven- systems der Arthropoden. Pfliiger’s Archiv., 68. Bd., 1897. Bonun Mouvements de manége en rapport avec les mouvements de la marée. C.R. Soc. de Biologie, Paris, 1904. Mouvements rotatoires chez les larves des crustacés. Ibidem, T. 59, 1905. Mouvements rotatoires d’origine oculaire. Ibidem, T.58, 1905. L’éclairement des yeux et les mouvements rotatoires. Ibidem, T.59, 1905. Brunvin Light reactions of terrestrial amphipods. Journal Animal Behavior, vol. 3, 1913. CARPENTER Some reactions of Drosophila with especial references to convulsive reflexes. Comes Sui movimenti di maneggio e sul loro significato nella teoria segmentale. Biologisches Centralblatt, Bd. 30, 1910. Effetti della decapitazione in Calotermes flavicollis ed in altri artro- podi. Ibidem, Bd. 32, 1912. Cornetz Les explorations et les voyages desfourmis. Flammarion, Paris, 1914. Detace Sur une fonction nouvelle des otocystes. Arch. de Zool. exp., IX série, T.7, 1887. Dremoor Recherches sur la marche des arachnides et des insectes. Arch. de Biol., T. 10, 1890. Etude des manifestations motrices des crustacés au point de vue des fonctions nerveuses. Arch. de Zool. exp., Ile série, T.9, 1891. Dotitey Reactions to light in Vanessa antiopa with special references to circus movements. Jour. Exp. Zodl., vol. 20, 1916. The rate of locomotion in Vanessa antiopa in intermittent and in con- tinuous light of different illuminations and its bearing on the con- tinuous theory of orientation. Ibidem, vol. 23, 1917. DrzewiInA Mouvements de rotation et retour 4 la marche normale aprés section unilatérale du systéme nerveux. C.R.Soc. de Biol., T.65, 1908. Dusors Application de la méthode graphique 4 |’étude des modifications im- primées 4 la marche par les lésions nerveuses expérimentales chez les insectes. Bull. Soc. Biol. (8), I, 1885. Les élatérides lumineux. Bull. Soc. Zool. de France, T.11, 1886. Lecgons de physiologie générale et comparée. Carré et Naud. Paris, 1898. Dirxen Experimental-Zoélogie. Springer, Berlin, 1919 288 EDGARDO BALDI yaRREY Light and the muscle-tonus of insects. The heliotropic mechanism. The Journal of General Physiology, 1918. Haputey Reactions of blinded lobsters to light. American Journ. Physiol., vol. 5, 1901. Herrera Sur le mouvement de manége chez les insectes. Bull. Soc. Zool. de France, 1893, vol. 18. Houmes' Phototaxis in the amphipoda. . American Journ. Physiol., vol. 5, 1901. The reactions of Ranatra to light. Jour. Comp. Neur., vol. 15, 1905. Karka LEinfiihrung in die Tierpsychologie auf experimenteller und ethologischer Grundlage. Barth Leipzig, 1914. Kettoaga Some silkworm moths reflexes. Biol. Bull., vol. 12, 1907. Lors Die Tropismen. Wintersteins Handb. der vergl. Physiol., Bd. 4. Fischer, Jena, 1913. Fisiologia comparata del cervello e psicologia comparata. Sandron, Palermo (1908). Lyon A contribution to the comparative physiology of compensatory motions. Americ. Journ. of Physiology, vol. 3, 1900. Martuta Untersuchungen iiber die Funktionen des Centralnervensystems bei Insekten. Pfliiger’s Archiv., Bd. 138, 1911. McGraw AND Hotmes Some experiments on the method of orientation to light. Journ. Anim. Behavior, vol. 3, 1913. NorMANN Diirfen wir aus den Reaktionen niederer Tiere auf das Vorhanden- sein von Schmerzempfindungen schliessen? Pfliiger’s Arch., 67. Bd., 1897. PARKER AND Patren. The physiological effect of intermittent and continuous light of equal intensities. Americ. Journ. Physiol., vol. 31, 1912. Rapti Untersuchungen iiber die Lichtreaktionen der Arthropoden. Archiv. fir die gesamte Physiologie, Bd. 87, 1901. Ueber den Phototropismus einiger Arthropoden. Biol. Centralbl., 21 Bd., 1901. Untersuchungen ueber den Phototropismus der Tiere. Leipzig, 1903. Yune Recherches sur la structure intime, etc. Arch. Zool. expér., T.7, 1878. ot et See ' . hoa vt ts dal Ane ov cme Ley Rigtiarl ad fet Da Hah. divtoben Ht fosdgolntal GA yothigis ‘ Ps as . Lb. 7 1? ha: . : i 410 : ; : ino) oul) lo Y ail nM cones cole’ " ait: Serine. ute oak > pA Lite Solty ov 7 eal oe us dysyiy nit bey a ye Msg “ r i om i balyrtt Lehi) 6 sei es Cr sesh ay ‘testinal: 1h ie See Maso iy wt Lefer oey ig : 4 y yy a} & vit hy Latter ajest geben tical PaO 1, far ea ‘ let re ’ “We lt Med ” i eae MAG EPA ai Me) seit ‘hasipeney + DUE sa A) AO Ea As TT a a j : wes git pT taeE car Po hehen bi sat IEEE no: or ose age Ds Minis Ht ry iting Lodadas wl Me abt ea vent ulate at ee ris SAE, INR os a q Te Hany 17 os jy ag tie ot PAdt? a PMS bev entre Pwp cin echt igdiruth Thhapinngte Ay EASA | Han nt Jl HAs 4 Ber Lara 4 ult var theo : 5 b | gia Ma seni n eaiulenennssod. ain cena ca Smarr out aan Re Athi: La nde Seah le Lone ease wo Ma oppor! wat In vit ait id. Any siRire Bole to ‘i at kee ql nay } 7% ais | i}, * 4 - q i ve ry Wom: : a5 Lal as +, He ag aia § fy rhe = Sf} ata Pye peut) P by (ty tf sonistattb iplen ciate ixoy 2 rv ri ety} a . ral . . c _ Bebab it T ets-94; me 7o Sad aire i il Abstracted by Francis B. Sumner and Henry H. Collins, authors Scripps Institution for Biological Research, La Jolla, California. Further studies of color mutations in mice of the genus Peromyscus. Three recessive color mutations (already discussed in previous papers) are more fully described and the behavior of these in hybridization considered. The mutant races are characterized as ‘yellow,’ ‘pallid,’ and true albino. The first and last of these appeared in stock belonging to pure subspecies, while the other appeared after a subspecific cross. The three depend upon changes in distinct genetic factors. Any two give the wild type in the F, generation, with the wild type and both mutants in the F;. In respect to relative numbers, these last follow dihybrid ratios, at least in some cases. As regards the yellows, however, irregularities are found, both in respect to the proportions which emerge from dihybrid crosses and the tendency of certain yellows to produce offspring which intergrade with the wild type. The causes of these irregularities have not yet been cleared up. Also, at least two distinct strains of yellows have been encountered, which differ from one another in respect to their mean color values. ‘The differences between these are hereditary and crosses between the two give an intermediate condition. The pelage of these various mutant races, as well as individuals of the wild type, has been subjected to color analysis by means of the Hess- Ives tint-photometer. This has rendered possible a fairly exact quantitative expression of the various color differences concerned. Three colored plates of skins are included. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JULY 24 FURTHER STUDIES OF COLOR MUTATIONS IN MICE OF THE GENUS PEROMYSCUS F. B. SUMNER AND H. H. COLLINS TWO PLATES (NINE FIGURES) INTRODUCTION The several sports or mutations to be considered in the present paper have already been mentioned or discussed in previous papers by the senior author (Sumner, 717, 718, ’20, ’22), and two of them—the ‘yellow’ and ‘pallid’ races—have been rather fully described. We shall here report the results of later observa- tions and breeding experiments upon these mice. Crosses have been made between the chief mutant races, and their ‘gen- etic behavior’ tested according to customary mendelian methods. More accurate color determinations have been rendered possible through the purchase by the Scripps Institution of an efficient colorimeter. In addition to this, we have thought it desirable to publish for the first time colored illustrations of several of the mutant strains. 1 Reference is made to the Hess-Ives tint-photometer. The use of this instru- ment for determining the color values of mammalian pelages has already been briefly discussed by Sumner (’21). In using this apparatus, light reflected from the object to be examined is viewed in juxtaposition to light from a pure white block of magnesium carbonate, the two being seen through the same color screen. Three of these color screens are employed in succession, these being of such wave lengths as would give pure white light (or neutral gray) if the transmitted rays were combined. In making the reading with each of these screens, the light reflected from the ‘magnesia’ block is cut down by a diaphragm to a point at which its intensity is exactly equal to that of the object to be examined. At this point the two halves of the visual field are of equal illumination, and likewise (owing to the color screen) are of the same color. The illumination of the entire field is rendered homogeneous by a special series of rapidly rotating lenses, in consequence of which the area of pelage under examination appears of an ab- solutely uniform tint. Specially prepared flat skins are used, these being first thoroughly cleaned in benzine to remove grease. 289 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL, 36, NO. 3 290 F. B. SUMNER AND H. H. COLLINS The situation as regards one of the color varieties—the ‘yel- lows’—has proved to be less simple than was at first supposed. This variety has appeared in several independent descent lines, and two of these lines seem to differ from one another character- istically in respect to the exact shade of ‘yellow’ which is mani- fested. Furthermore, neither of these last-named races appears to behave strictly like a monohybrid recessive. It seems likely either that there are independent ‘modifying’ factors concerned, which segregate according to principles not yet ascertained by us or that the ‘yellow’ factors themselves are unstable and undergo changes of some sort. We are quite aware that the observations here presented are decidedly fragmentary in comparison with the more exhaustive investigations of many recent mendelian students. This has been due to several circumstances. In the first place, our studies of color mutations have throughout been regarded as incidental to our main program of work, which has concerned itself with the characters of ‘natural’ subspecies. In the second place, Peromyscus is not well adapted to experiments in which rapid breeding is an important consideration. Not only is it commonly impossible to obtain more than two or three generations in a year, but the cage-born mice show a considerable percentage of sterility, which may at any time bring some valuable descent line to a close. Questions which one could promptly settle by appropriate matings with more favorable material must await many months for an answer or must even remain unanswered. Finally, we must mention that these studies were interrupted when far from complete, and that a large part of the stock was killed at that time, although certain lines of experimentation were resumed later. This interruption was due to the absence of the senior author from La Jolla during the greater part of one year and to the junior author’s permanently severing his connections with the Scripps Institution in the fall of 1919. These explanations, we trust, will temper the criticisms of those who may be disposed to wonder why we have failed to settle certain obvious problems or even to attempt certain obvious experiments. The cuthors may fairly ask to be credited with COLOR MUTATIONS IN MICE OF PEROMYSCUS 291 having thought of some, at least, of the numerous lines of possible experimentation which will occur so promptly to the reader. In the ensuing pages the several color mutations will first be discussed separately. Later, the results of crossing these mutant stocks will be considered. THE ‘YELLOW’ COLOR VARIETY The earlier history of one of the ‘yellow’ stocks here consid- ered has been recorded in a previous paper (Sumner, 717), and a description of the hair has likewise been given (Sumner, 718). It is of some interest that this and five other independent out- croppings of ‘yellow’ in our stock have all been derived from the La Jolla strain of Peromyscus maniculatus gambeli. This may be due in part to the fact that more of these mice have been reared than those of any other local race—more, perhaps, than all of the others combined—and that the chances of encounter- ing such infrequent sports have thus been increased. On the other hand, it should be remarked that the ancestors of the ‘yel- lows’ were all trapped within an area of a few square miles, so that the outcroppings of this character may not have been wholly independent of one another. With the exception of a single aberrant individual,? which was probably a juvenile yellow, none of this color variety have been found by us among our wild stock, although we have trapped at least a thousand mice of this subspecies at La Jolla. In each case the yellows have appeared for the first time either in the first or the second cage-born generation. We do not, however, infer from this that the ‘mutation’ has been due to the artificial conditions of captivity. The probable occurrence of at least one wild yellow renders this unlikely, as also the fact that sev- eral wild specimens proved to be heterozygous (according to accepted standards) when mated with cage-bred yellows. 2 Also trapped at La Jolla (Sumner, 717). 292 F. B. SUMNER AND H. H. COLLINS The ‘a’ strain of yellows (fig. 2) The three chief independent strains of yellows will be desig- nated as ‘a,’ ‘b,’ and ‘c,’ respectively, according to the order in which they appeared in our stock. It is the ‘a’ strain which has been referred to in earlier publications. As already stated, this strain appeared among the offspring of two males and three females, these five being derived from a single pair of wild individuals (P2? 46 and P o& 16). The parents and grandparents were all of normal appearance. Unless the ‘mutation’ appeared for the first time in the germ- cells of the parent generation, these five parents of the original yellows must have all been heterozygous, since each gave rise to at least one yellow. The total number of their offspring (exclud- ing those dying very young) was 24, of which 8 (1 ¢#, 5 @ and 2 of unknown sex) were yellows, while 16 (8 #, 5 2, and 3?) were of the wild color. Furthermore, three of these parents, when mated later to yellows, gave rise to 5 yellows and 5 of the wild color. If they were not actually heterozygous in origin, they must, at least, have been producing ‘yellow’ and normal gametes in about equal numbers. Two matings of yellows of this strain resulted in 10 offspring, all yellow. For the most part, however, the matings of these mice were made with non- yellows or with yellows of another strain, as will be described below. As previously stated, these mice ‘are of a peculiar yellow- brown hue, probably lying between the ‘cinnamon buff’ and the ‘clay color’ of Ridgway, and not unlike the most highly colored parts of the hair in P. m. sonoriensis.”? The peculiar hue was attributed to two causes: 1) the larger number of banded | (agouti’) hairs, in proportion to the all-black, and, 2) the greater proportional extent of the yellow region on these banded hairs.‘ As regards the first point, it should be added that strictly ‘all- black’ hairs (i.e., those entirely devoid of a paler cross-band) Sumner (’17).—Such a comparison with any set of color standards is ad- mittedly extremely crude, since the pelage is very far from being a uniformly tinted surface. 4 Sumner (718). COLOR MUTATIONS IN MICE OF PEROMYSCUS 293 are nearly or quite lacking in some yellows, and that, when present, they are probably confined to the darker, middorsal region of the body. It is not improbable, also, that the yellow pigment of the agouti hairs is more abundant or more highly concentrated in this color variety. This is yet more likely in the case of the ‘b’ strain to be described next. As also stated in earlier papers, the ventral pelage of the ‘yel- lows’ is more intensely white than that of the ‘wild type.’ This is due to the greater length of the terminal pigmentless zone of the hairs on the under surface of the body. Indeed, in the mid- ventral line, the basal ‘plumbeous’ zone of the individual hairs is commonly quite lacking, the pelage being entirely white. There appears to be no reduction, however, in the depth of pigmentation of this basal zone throughout the body, nor do the tail, feet, ears, eyes, etc., appear to show any diminution in the amount of pigment normally present. Mice of this strain differ considerably from one another in shade. In some specimens the entire pelage, or certain areas, exhibits a decidedly ‘dusky’ hue, owing, apparently, to the pres- ence of a larger proportion of the all-black hairs or of hairs heavily tipped with black. Likewise, the richness of the color varies very much, the brighter specimens displaying considerable orange-yellow, the duller ones having a ‘washed-out’ appearance, suggesting that of jute or some similar vegetable fiber. These differences are probably due to the amount of the quality of the yellow pigment in the individual hairs, though no careful micro- scopic examination has thus far been made.® On the whole, the mice of the ‘a’ strain are of a decidedly less rich hue than are those of the ‘b’ strain next to be described. Only eight adult skins of the former strain are available for color tests with the tint-photometer. These give the following mean and extreme readings, in terms of the three ‘primary’ colors of the color screens. The computed proportions of black, 5 Studies of the quality and distribution of hair pigment, in relation to sub- species, color mutation, and behavior in hybridization, are being undertaken in this laboratory by Mr. R. R. Huestis. The matter has already been dealt with, to some extent, by Sumner (18). 294 F. B. SUMNER AND H. H. COLLINS white, and ‘color’ (in this case a yellow-orange) are likewise shown in table 1.° These figures may be profitably compared with similar ones giving results for a series of the normal wild gambeli race. Table 2 is based upon ten skins of adult individuals, five of which were trapped in December and January, five in June. Care was taken to select a representative series including lighter and darker specimens.’? The conspicuously darker shade of even the paler mice of the wild type is indicated by the higher per- centages of black, while the comparative lack of color is also obvious. ‘The ratio of red to green on the other hand (see below), is very close to that in the ‘a’ yellows, being 3.09, as compared with 3.17. Thus the difference between these ‘yellow’ mice and the normals appears to depend chiefly upon the relative proportions of black and of yellow pigment, not upon the char- acter of the latter. The ‘b’ strain of yellows (figs. 3, 4, and 8) | A single pair of normally colored wild mice (P @ 15 and P & 59) in the cultures of the junior author became the parents 6 These last figures represent the proportional magnitudes of three sectors on a color wheel which would combine to produce the shade in question, assuming that the black disk was of zero luminosity, and the white equal to that of the standard, and that the colored sector was of maximum saturation and intensity. As a matter of fact, the commercial cardboard disks are very far from fulfilling these conditions, so that large corrections (over 25 per cent in the case of ‘white’) must be made, in order that color wheel and photometer determinations shall agree. For reasons which cannot here be discussed, the proportion of black is regarded as equal to the difference between the highest color-screen reading and 100 per cent, the proportion of white being equal to the lowest color-screen reading, and the ‘color’ constituting the balance. In an earlier paper (Sumner, ’21) the pro- portion of black was computed differently, following the instructions contained in a pamphlet issued by the manufacturers. The values thus obtained were somewhat too high. The procedure here adopted has the authority of Mr. F. E. Ives, the inventor of the instrument, and, furthermore, the figures thus arrived at correspond fairly well with those obtained by the color-wheel method (due corrections being made). 7It is true that this series includes none as pale and as highly colored as certain extreme variants of the ‘normal’ stock. At least one of these last (result- ing from selective mating for two generations) resembles an average ‘yellow’ in appearance, though undoubtedly quite different genetically. COLOR MUTATIONS IN MICE OF PEROMYSCUS 295 of 22 cage-born offspring, of which 14 (5 # and 9 2) were of the wild type, while 8 (5 @ and 3 @) were ‘yellows.’ As in the case of the ‘a’ strain, the proportion of recessives among the - offspring of these presumably heterozygous parents was some- what too high, though, as before, the total number of individuals TABLE 1 ‘A’ yellows HIGHEST LOWEST MEAN Color-screen readings Bree es byt ces is Sire yatta ee 32.0 25.5 28.44 SOMA A eit RN. heady eat See 18.5 14.0 16.75 BUC =VI Ole beers reno cere cee cate ea eer 13.0 9.0 alesyé lsiele 3 502 AL Oe OSs. 74.5 68.0 71.56 \WA OVC Sip aero oie Seeks At Seen Paes 13.0 9.0 Leys (Chilo CARNE ce ee are 19.5 150 17.07 TABLE 2 Normal gambeli HIGHEST LOWEST MEAN Color-screen readings [RUG EI Sy Rw eles costa A separ een 19.0 14.5 16.15 NG LECTIC Se et Ne a orc eo ae 13.0 9.5 11.25 Biwe=vaio lets. sie ae eet 10.0 Ua 8.90 BIAGEE id ..1 5 uA baer eat eres ee etme iit 85.5 $1.0 83.85 LTC aeons nar uci, rie eet pene aeaee 10.0 7.5 8.90 | ST a nap a aoa ders rin eine eI eae hs 9.0 5.0 7.25 was not sufficient to justify any conclusions from this fact. It will be noted that in this case the proportion of males among the yellows was much higher than among the wild type, the re- verse being true of the first strain. Such differences are prob- ably accidental. 296 F. B. SUMNER AND H. H. COLLINS The female parent of these yellows (P 2 15) when mated to one of her yellow offspring bore one normal and three yellow -young. The male parent (P &@ 59) was mated to nine other females’ of the wild type, these being either wild mice or cage- bred ones, unrelated to the yellow stock. Eight of these matings resulted in the birth of twenty-four young, all of normal color. The other female (a wild specimen) gave birth to four normals and one yellow. She was evidently another heterozygous (or ‘mutating’?) individual, unrelated so far as we know, to the male. Matings between yellows of this strain yielded over fifty re- corded offspring, the sexes being represented in about equal proportions. All of these mice have been entered as ‘yellows,’ though this characterization is subject to the qualification to be discussed presently. Matings of yellows with those known (on the basis of parentage) to be heterozygous® gave 39 offspring, of which 21 are recorded as ‘yellows,’ 11 as ‘normal,’ and 7 as ‘doubtful.’ If those of the last class were all to be included among the ‘normals,’ we should have a reasonably close approach to the 50: 50 ratio. Otherwise, there are too many yellows. Only two matings are recorded between individuals, both known from their parentage to be heterozygous. These gave 7 normal, 1 yellow, and 1 of uncertain type. It was early noted that the ‘a’ and ‘b’ yellows differed quite perceptibly in respect to their mean color tone. The second strain is, on the average, of a richer color, there being fewer black hairs in the pelage, and the pigment of the ‘ticking’ being redder. Indeed, the term ‘yellow,’ as applied to the ‘0d’ strain, is in most cases a decided misnomer. ‘The brighter specimens 8 These figures relate only to fertile matings. Throughout this work many matings were made which yielded no results. 9 Excluding the large number of related individuals which we know to have been heterozygous only from the fact that they produced one or more yellow offspring. The inclusion of these would, of course, be unwarranted unless it were possible also to include such heterozygous individuals as gave rise to no recessive offspring. There is no way of identifying these. COLOR MUTATIONS IN MICE OF PEROMYSCUS 297 are not far from the ‘ochraceous tawny’ of Ridgway’s ‘Color Standards,’”’ while those of the ‘a’ strain approach more nearly the ‘clay color,’ though the latter comparison is quite misleading. It soon developed also that many of the ‘yellows,’ both of the ‘q’ and ‘b’ strains, were of a much less intense color and con- tained more black hairs than those which had been first examined (fig. 4). These latter have, for the sake of convenience, been termed ‘atypical yellows.’ This expression is quite arbitrary, however, since one may arrange among the offspring of ‘yellow’ parents a graded series between the most ‘typical’ yellows and specimens closely resembling the paler and more buff-tinted individuals of the wild type. Indeed, where we are dealing with the offspring of heterozygotes, it is not in every case possible to distinguish the pure recessive ‘yellows’ from the others. Regarding the genetic status of these ‘atypical yellows’ we are at present far from clear. That they are not heterozygous individuals, resulting from blended inheritance (imperfect domi- nance), seems certain. This we conclude both from the fact that such individuals have never resulted from the mating of yellows with pure dominants, and from the fact that two of these ‘atypical yellows’ have never produced offspring of the wild type.'” Unfortunately, the distinction between ‘typical’ and ‘atypical’ individuals was not always recorded in our earlier entries, and these cover perhaps the major part of our yellow stock. Our records seem to show, however, that whereas very clear (‘typical’) yellows tend to produce offspring like themselves, ‘atypical’ _ offspring have occasionally been born to two perfectly ‘typical’ parents. They have likewise resulted from the mating of a ‘typical’ yellow with a heterozygous animal whose yellow parent was also ‘typical.’ In a number of instances, too, it is known that ‘typical’ and ‘atypical’ mice have occurred among the off- spring of the same parents, and even within the same brood. That two ‘atypical’ individuals have ever produced ‘typical’ young we have no clear evidence. On the contrary, abundant 10 Unless we regard as atypical yellows certain of the ‘doubtful’ specimens which appeared in the F, generation of the yellow-pallid cross (see below). 298 F. B. SUMNER AND H. H. COLLINS records show that the offspring of two ‘atypical’ animals gener- ally resemble their parents in this respect.!! It is not impossible that we are concerned merely with differ- ences of the type which are commonly called ‘phenotypic’ or ‘somatic’ (i.e., non-hereditary). But such an explanation is hardly consistent with the facts just cited. It seems more likely that we have to do with the presence or absence of ‘modifying factors,’ or possibly even with the existence of unstable factors, or departures from the simple ‘factorial’ scheme of heredity. Table 3 gives the average and extreme values for the color determinations of twenty-four adult pelages of the ‘b’ yellows. About two-thirds of these were listed as ‘typical’ or ‘nearly typical,’ the others as ‘atypical.’ From tables 1 and 3 it appears that the ‘a’ strain shows a shghtly higher percentage of white, while the ‘b’ strain shows a slightly higher percentage of black, the values for ‘color’ being nearly the same for the two. The significance of these differences is doubtful. Of far more importance is the fact that the ratio of red to green” is distinctly higher for the ‘b’ strain than for the ‘a,’ the mean figure being 3.59 for the former and 3.17 for the latter. A study of the frequency distributions shows that this difference is probably a real one, and indeed a casual comparison of the skins reveals it to the eye. Further evidence of such a difference was derived from the examination of living specimens, many of which were not skinned. Hybrids between the ‘a’ and ‘b’ yellows Matings of these two strains resulted in all cases in offspring which were listed as ‘yellows.’ Three ‘a’ females were mated to two different males of the ‘b’ strain. The resulting twelve F, hybrids appeared, on the whole, as intermediate between 11The interruption of these studies above referred to is largely responsible for these uncertainties. 12 The values for red and green employed for this purpose are the excess of each that remains after deduction of the amount which combines with the other colors to constitute the ‘white.’ The lowest color-screen reading (in this case the blue-violet) also serves to indicate the amount of white, there being no ‘free’ blue-violet. COLOR MUTATIONS IN MICE OF PEROMYSCUS 299 the parents in their mean color tone, though they presented a considerable range of variability. A single mating between a ‘b> male and a heterozygous ‘a’ female led to the birth of one offspring of the wild color and two decidedly ‘atypical’ yellows. It is thus evident that these two shades of yellow do not result from the modification of independent genetic factors, as is the case with the other color mutations to be described presently. Assuming that the differences between the ‘a’ and ‘b’ strains are hereditary at all—which seems fairly certain—we may, on the one hand, have to do with a case of ‘multiple allelomorphs,’ the two ‘yellows’ representing slightly differing modifications TABLE 3 “B’ yellows HIGHEST | LOWEST | MEAN Color-screen determinations VERGO capeeatin ei Wa Sk gas piled in oyna cade ene B 30.5 22.5 Pipes reenter Ar Oh, Sean) 18.0 15 15.00 Ee VlOLe bares mete cree ee i tars, eo ES 10.27 Bee AL Paths Oe Ce Ne 77.5 69.5 72.85 Se et sae TLS VIMROLY Oss Ch 12.5 7.5 10.27 el. 2) BON ceeviy s bat ony: 19.5 | ‘alae 16.88 of the same color factor. Or the primary factor concerned may be the same in the two cases, the difference being due to the presence in one variety of a secondary ‘modifying’ factor. The mature pelages of twelve of these hybrid yellows were subjected to the color analysis. The mean values for black, white, and total ‘color’ are close to those for the two ‘pure’ strains, which, as stated before, agree closely with one another in these respects. Furthermore, these hybrids are strictly in- termediate as regards the spectral position of their yellow pig- ment, as is indicated by the ratio of red to green. This is true not only of the mean value of this ratio (3.33), but of its range. As already stated, this intermediate fcondition is apparent to the eye. 300 F. B. SUMNER AND H. H. COLLINS Further evidence for the same interpretation of these color varieties is derived from back-crosses. Matings between F, hybrids and ‘a’ yellows resulted in ten offspring. Five pre- pared skins are available from this lot. These give us 3.2 as the mean ratio of red to green—a figure lower than all but four of the twenty-four ‘b’ yellows and almost identical with the average of the ‘a’ race. Notes were made, furthermore, upon the other specimens, either when living or freshly killed. These indicate that they were, for the most part at least, of a dull yellow or buff appearance. In some cases they were expressly likened to the ‘a’ yellows. Matings of some of the foregoing back-cross individuals inter se—((a-b) - a) - ((a-b) - a)'%—resulted in the birth of thirteen young, all of which were listed as ‘yellows,’ though they are described in much the same terms as their parents. The ground- color was a very dull yellow or buff, darkened by a considerable admixture of black hairs. They resembled the duller specimens of the ‘a’ yellows, and in no-case approached the more ruddy hue of the ‘b’ strain. The other .back-cross (i.e., between the F, hybrids and the ‘hb’ yellows) gave a quite different result. Of the two mature skins which were preserved, both are closely similar to the bright- est ‘b’ yellows in appearance, giving red: green ratios of 3.5 and 4.0, respectively. These lie altogether outside the range of the ‘a’ series, and the larger figure almost reaches the extreme for the ‘b’ series. Furthermore, notes made upon the entire lot (thirteen in all), when living or freshly killed, show that at least six resembled the ‘b’ strain more nearly than the ‘a,’ while several others are listed as ‘intermediate.’ There is no record of a speci- men’s having the predominant appearance of the ‘a’ strain. 13 We have employed hyphens instead of multiplication signs in designating these various crosses, since reciprocal crosses were commonly made, and we wish to be non-committal as to which parent belonged to which race. When the multiplication sign is used, it is commonly understood that the female parent is named first. COLOR MUTATIONS IN MICE OF PEROMYSCUS aol Independent lines of ‘yellows’ As already stated, the presumably heterozygous male parent of the ‘b’ strain produced one yellow and four dark offspring when mated with one of the nine females (P 92 91) which were used in addition to the mother of the ‘b’ yellows. There is no record of any member of this brood having left any descendants, the skin of the yellow was not preserved. Three yellows and three normals were produced by the mating of a ‘b’ yellow (Ci &@ 78) with an unrelated wild-type mouse (C, 2 96), which was considerably paler than the average but was not regarded as a ‘yellow,’ even an ‘atypical’ one. These mice likewise left no descendants. Another independent outcropping of the ‘yellow’ mutation consisted of a single individual, which appeared in a brood of three, whose parents and grandparents were known to be of the ‘wild color.’ This chanced to occur in the course of an ex- periment in which paler and darker strains of the ‘normal’ mice were crossed. We are not, however, disposed to attribute any significance to the latter fact. As in the preceding cases, the parents of the yellow individual were doubtless heterozygous for the yellow factor, unless they were themselves producing ‘mutant’ germ-cells de novo. This strain, likewise, was not continued further. Yet another independent appearance of the ‘yellow’ color variety, which we may call the ‘c’ strain, occurred in a lot of La Jolla gambeli which are believed to have been trapped at least a year later than any of the preceding ones. A pair of wild mice (‘selection series’ P @ 118 and P &@ 22) gave rise to eight young, of which three were yellows (one of these being doubtful), the remainder being of the normal color type. The female was likewise mated to a yellow male of the ‘a-b’ lot, and the male was mated to three yellow females of the ‘a,’ ‘b,’ and mixed strains. Among the thirteen young thus produced, only three yellows appeared, instead of six or seven as would be the expectation from such a mating. As in the case of all of the yellows, subse- quent to the ‘a’ and ‘6’ strains, these lines were brought to a 302 F. B. SUMNER AND H. H. COLLINS close with the generations just referred to. No careful compari- sons were made between any of these mice and the ‘a’ and ‘b’ yellows, and only one of the skins was saved, so that it is now impossible to make sucha comparison. It is our recollection that the ‘c’ mice resembled the ‘b’ rather than the ‘a’ strain, though the single preserved skin is probably intermediate. It is worth noting, though perhaps not significant, that the heterozygous (?) parents of five out of six of our yellow strains gave numbers of yellows in excess of mendelian expectation. Of the 68 mice thus produced, 44 were normal, 24 yellow, giving a ratio of 1.8:1, instead of 3:1. Such a departure from the TABLE 4 | DARKER | LIGHTER | MEAN Juvenile ‘yellows’ BLES clo Ale Sogn Ses oe RA Oe A engi eae 84.5 80.0 82.25 NAG Wire ee Merce) EAR chee ala ere a re ante eR ar 120 16.0 14.00 Colors. cHes es Reha eas be lant he nea: Aaa oro 4.0 3.75 ‘Normal’ juvenile gambeli Bilacle eye eRe tases Borkah LL. eeee 91.0 85.0 88.00 Whe R eee: heecereta ney eet tone ee 8.0 12.0 10.00 COlOTRSa aE eae ee eee sae 1.0 3.0 2.00 ‘expected’ condition may well be accidental, however, particu- larly in view of the fact that the ‘heterozygous’ parents of the ‘a,’ ‘b,’ and ‘c’ strains gave an excess of wild-color offspring when mated with yellows (viz., 16:11). Whether these parents were in reality heterozygous, rather than original producers of mutant germ-cells, is not definitely shown by our records. The data given seem compatible with either interpretation. Juvenile yellows As stated in earlier papers, the yellow variety is nearly or quite as distinguishable in the juvenile pelage as in the mature (figs. 7 and 8). Table 4 gives the proportions of black, white, and color in 14 This apparent excess of yellows may be due to another cause (see Summary and Conclusions). COLOR MUTATIONS IN MICE OF PEROMYSCUS 303 two juvenile ‘yellows,’ a darker and a lighter specimen. In comparison with these are shown the corresponding values for darker and lighter specimens of the wild type, the latter extreme being taken from a selected strain of pale or ‘buff’ animals. The wide differences between both the mean and the extreme values of the two series are sufficiently obvious. On the other hand, there are but trifling differences between the darker ‘yellow’ and the paler ‘normal’ individual. THE ‘PALLID’ COLOR VARIETY These mice were first referred to as ‘partial albinos’ (Sumner, 17), but this designation was plainly at variance with cus- tomary usage, so that the non-committal term ‘pallid’ was later adopted. This ‘mutation’—if it did arise de novo during these experi- ments—appeared among a lot of F, hybrids between Peromys- cus maniculatus rubidus and P. m. sonoriensis. Four pallids and seven of the wild type resulted from the mating of an F, male and his two sisters, each of these last producing two pallids. It is certain that this was no simple segregation phenomenon, due to the recombination of factors regularly present in the two subspecies which were crossed. Up to the present time, more than 300 F, and F; hybrids between these two races have been reared and no other case of the pallid mutation has come to light. Since the pallid-color variety has been described in some de- tail in earlier papers (Sumner, ’17, 718), a brief account will suffice here. It is characterized primarily by the lack of most of the black (or sepia) pigment found in normal mice (figs. 5, 9). This lack appears in the absence of all-black (i.e., non-banded) hairs from the pelage, and the extreme reduction of pigment in the basal zone of the others. The latter is of a pale ashy hue in- stead of slate-colored. Furthermore, the eyes are dark red instead of black, the ears are not appreciably pigmented, and the dorsal tail stripe (normally due to dark hairs) is scarcely perceptible. A further peculiarity of this strain is the fact that the eyes are smaller, or at least less protruding, than in the wild type. The pallid mice are pale gray when young, developing a considerable admixture of yellow or orange when adult. 304 F. B. SUMNER AND H. H. COLLINS In a previous paper several peculiarities were pointed out in the microscopic appearance of the individual hairs: 1) A consid- erable proportion of these are practically devoid of pigment in the zone which is ordinarily yellow, the rest being normal in this respect; 2) the normally dark surface pigment of the terminal portion of the hairs is nearly or quite invisible; 3) in the basal zone, the normal black pigment bodies are represented by groups of small irregular granules or flocculent dark masses. While the range of variation in the pallids is not as great as that of wild mice belonging to some of the natural subspecies, well-marked individual differences are none the less present. Some specimens have a considerably greater amount of yellow in their pelage than others, presenting a richer color on this account. These are perhaps intermediate between the ‘cinnamon’ and ‘cinnamon buff’ of Ridgway. On the other hand, specimens have appeared in which the pelage is noticeably darker than the average. Between these two extremes all gradations may be found. Even wider variations have been met with in respect to the eye color of the pallid mice. In some individuals this is searcely darker than that of the eyes of true albinos. In at least two Specimens, on the other hand, it is nearly as dark as in normal animals of the wild type, though even here careful comparison reveals an undoubted difference. In the great majority the eyes may be characterized as dark red. Whether or not these individual differences in coat color or eye color are hereditary has not been determined." It is of possible significance that both of the dark-eyed variants arose as ‘ex- tracted recessives,’ after a cross with other mutants (albino and yellow). One of them possesses, in addition to dark eyes, the darkest pelage of any pallid yet noted. Aside from the occurrence of these variations of a possibly genetic nature, the pallid mutation has behaved, in every respect, as a simple monohybrid, recessive character. Despite the ex- istence of plainly darker specimens, no true ‘intermediates’ or 15 One of the two dark-eyed individuals referred to is apparently sterile; the other is not yet old enough for breeding purposes. COLOR MUTATIONS IN MICE OF PEROMYSCUS 305 doubtful cases have been encountered. It is not worth while to detail the numerous matings which have been made within the pallid stock. It should be pointed out, however, that the same excess of recessives (here four out of eleven) was found among the offspring of the original heterozygous (?) parents, as in the case TABLE 5 Adult pallids | HIGHEST | LOWEST | MEAN Color-screen readings i 2h 2 Ss Age 40.5 31.5 35.54 (GUST A 8 rr a a a Pk Sith Da 26.0 19.5 22.04 Blue-violeter. .s8 5220 ee eee eee 19.0 14.0 16.17 Equivalents in black, white, and color Blache. 58 oie Sate des «heehee BES ot 68.5 59.5 64.46 Vi UVES T ob RY SO eh et 0 teh Eee” 19.0 14.0 16.17 DMO Tyee ay hes tons cite a are & cee eats 26.0 15.0 19.37 TABLE 6 Juvenile pallids Color-sereen readings (mean) IRGO, ct Aa eG LSS OR AER ID > PS MRAR AY a no Tae UEC eC ema e Ve Ringe, £p FAs 31.75 (CGA 8 Ae ast ee tS PRE aS LOE Me, COP eO Re De Cy Uae cena A 25.00 Bineauroletrer tty. 22. eit. cath ids ca peers tae yan Eb 22.00 LBA OL easy ae ake RN Sele” et ites De elpoiale Dich 2 alata SL A iy MAERAR 68.25 Vaan c(h 4 ape et oS BO “aR Oia ae | SPAS OL od A ae ee Ae Oe 22.00 COLO eee ae ae ACE EES rE eee ieee UN | pies omskd a by 2) ORs of all but one of the strains of yellows. At best, however, these combined figures merely suggest a possibility. The following figures (tables 5 and 6) express the results of color determinations of twelve adult and two juvenile skins of the pallid mice (see also pl. 2). It is evident that the juve- nile animals surpass the adult in black and white (i.e., are more gray), being relatively deficient in color. The ratio of red THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 306 F. B. SUMNER AND H. H. COLLINS to green is 3.30 for the adult pelages, 3.25 for the juvenile. Thus the quality of the yellow pigment appears to be closely alike in the early and later pelages of the pallid mutant. Likewise, there are no very wide differences in this respect between the pallids, yellows and normal gambeli. TRUE ALBINOS True albinism has appeared but once in our cultures, although many thousand mice have been reared since the commencement of these studies. Two broods from a single pair belonging to the first cage-bred generaton of P. m. gambeli (La Jolla race) consisted of two albinos and six of the wild type. There was one albino of each sex, both being fertile. At least one of the normal color proved to be heterozygous; the others were either sterile or their condition was not ascertained. These albinos have, as was to be expected, behaved as simple mendelian recessives. A few matings between individuals known to be heterozygous (other than the parents of this strain) have given 7 normal and 6 albino young, an obvious excess of the latter. Matings between albinos and heterozygotes, on the other hand, gave 17 normal and 18 albino, which is as close an approach as possible to the ‘expected’snumber. The mice of this strain (fig. 6) are plainly complete albinos, comparable with ordinary white mice, which are albinic house- mice (Mus musculus). Indeed, the white Peromyscus are very similar to the latter in appearance, differing chiefly in having larger and more protruding eyes and somewhat longer ears. They likewise differ in being almost completely odorless, as are also normally colored specimens of Peromyscus.!7 As al- ready stated, the eyes of the albinos average much paler than those of the pallids, the former being pink, the latter commonly dark red. In both of these varieties, they are distinctly smaller (or at least protrude less) than in normal individuals. 16 We have had as many as 1500 mice at one time in our ‘murarium,’ most of these being cage-born. The experiments have lasted seven years. 17 A single cage of ordinary white mice will impart a pronounced ‘mousy’ scent to an entire room even when well ventilated. This is not true of a thousand specimens of Peromyscus. COLOR MUTATIONS IN MICE OF PEROMYSCUS 307 Two skins of the white race of Peromyscus have been tested with the tint-photometer. Although these pelages are probably as ‘white’ as those of any albino mammal, they are obviously not of so intense a white as the standard magnesium carbonate. Few persons, however, would probably expect to find such a considerable reduction in luminosity as actually occurs. The percentage of white proved to be only 75.7, as compared with the standard, leaving 16.7 per cent of black and 7.5 of ‘color,’ in this case a yellow (R:G = 1.58). Neither black nor yellow, however, are due to the presence of the ordinary hair pigments, since these appear to be entirely lacking. The black results chiefly from the loss of light which passes through and between the nearly colorless hairs, and is not reflected back to the eye. The proportions of the primary colors are not quite the same, however, as in the standard, owing doubtless to a faintly yellowish tint in the keratin of the hair. YELLOW-PALLID CROSSES Matings were made between eight yellows and three pallids, resulting in the birth of twenty-two young. These were all of the wild type, of a medium shade, and fairly uniform in color, presenting about the same appearance and range of variation as a similar number of local gambeli. The yellows employed were partly of the ‘a’ strain, partly crosses between the ‘a’ and ‘b’ strains. Of the F, generation, 5 males and 11 females were successfully mated, yielding 64 young. There thus chanced to be exactly four times the minimum number required for the proportional representation of all classes in a dihybrid cross. Of the 64 F, individuals, 36 were males, 27 females, and one of unknown sex. Since there were no significant differences in the distributions of genetic classes, according to sex, males and females will be combined in the treatment below. On the assumption that we had to do here with a typical di- hybrid cross, involving complete dominance, the most probable distribution of classes for this number of individuals would be: 308 F. B. SUMNER AND H. H. COLLINS 36 normals (wild type) 12 yellows 12 pallids 4 double recessives. Since the peculiarities of both the yellows and the pallids de- pend upon the absence of pigment, and since the former do not lack any pigment which is not likewise lacking in the latter, it would seem probable that the double recessives would be indistinguishable from ordinary pallids. The proportions above stated would accordingly become 36:12:16. The actual numbers! proved to be: 36 normals 6 yellows 7 doubtful (normals or yellows?) 15 pallids. The exact agreement of the first of these figures and the almost exact agreement of the last are sufficiently striking. If it were permissible to regard the ‘doubtful’ specimens as being geneti- cally ‘yellows,’ a single further transposition would bring these figures into exact conformity with ‘expectation.’ Unfortunately, the case is not as simple as this. The thirty-six ‘normal’ mice, while differing among themselves, presented no greater range of variability than would a similar random collection of wild gambeli, which race, indeed, they re- sembled pretty closely in appearance. In this respect, they agreed with their F, parents. Despite evident differences of color, likewise, the pallid specimens probably showed no greater range of variation than was to be found in the original pallid stock. The ‘yellow’ class in the list includes only those concern- ing which no doubt was felt. While none of the pelages were as rich in color as the brightest of the ‘b’ strain of yellows, they were probably an average lot. The difficulty here relates to the status of the ‘doubtful’ specimens, intermediate in appearance between yellows and normals. One of these died early, so that its status could not 18 The classification of individuals was made without any reference to these totals, which were not computed until later. COLOR MUTATIONS IN MICE OF PEROMYSCUS 309 be tested. Of the remaining six, two were entered as ‘probably yellows,’ though ‘atypical’ (p. 297), while four were entered as ‘probably not yellows.’ They were, however, recorded as being much paler and more buff than the average gambeli. These six specimens were mated in various combinations, and some twenty-five descendants were born.!® The latter were a rather nondescript lot. Five of them were pallids—a result which was not unexpected, since even some of the ‘normals’ were doubtless heterozygous for the pallid factor. It is of interest that these F; ‘pallids’ included the dark-eyed specimen, with darker pelage, referred to above (p. 304). The others, for the most part, differed rather widely from the average normal gambeli. A few might have passed for paler (more buff) repre- sentatives of the latter subspecies, and had nothing in their ap- pearance to suggest the ‘yellow’ variety. One or two looked as if they might be ‘atypical yellows.’ The majority, while not at all uniform, were of a curious, somewhat ruddy, appearance, having little resemblance to the typical yellows of either of the chief strains discussed above, but also probably unlike any wild mice which we have trapped. Even some of the descendants of the F, pair which had been listed as ‘probably yellows’ showed no nearer approach to the yellow type than the others. One of these, indeed, is entered as ‘medium gray.’ This case is, of course, complicated by the fact that we are dealing with a mongrel combination of three subspecies, as well as with two different color mutations. The ‘pallids,’ as already stated, sprang from a sonoriensis-rubidus cross, while the ‘yellows’ were of pure gambeli stock. This intermixture of subspecies does not, however, affect the clear segregation of the pallid and the albino factors. It is only the ‘yellow’ factor (or factors) concerning which there is any question. These difficulties cannot be cleared up until, 1) the genetic behavior of the ‘yellow’ color variety has been far more thoroughly 19 These births occurred during the protracted absence of the senior author. Upon his return, it was not in many cases possible to distinguish the children of a given pair from their grandchildren, or from broods resulting from the mating of parents and offspring. If the parent mice had been pure recessives, however, this fact would, of course, have made no difference in the result. 310 F. B. SUMNER AND H. H. COLLINS tested and, 2) the behavior of the color differences entering into subspecifie crosses has been determined with more precision. Investigations are under way which may settle some of these questions. ALBINO-YELLOW CROSSES Matings of three yellows and four albinos resulted in the birth of thirty F; offspring, of which twelve males and eleven females have lived to maturity. All are of the wild color, and would pass for normal gambeli. . Thus far, the F, generation consists of 83 individuals, of which 52 are normal (wild color), 13 yellow, and 18 albino. On the assumption that these factors are not linked, the ‘expected’ numbers are 47, 16, and 21, respectively. The departure from expectation is doubtless accidental. In any case, there is no evidence of linkage, the occurrence of which would have reduced the proportionate number of dark individuals, instead of increas- ing it. The number of F, albinos and yellows which have thus far been tested for linkage is very small, but it is of interest that the proportion of recombinations is even greater than would be expected from random assortment (Sumner, ’22). Thus the meager data at hand make it plain that no considerable degree of linkage, if any, exists between these factors. ALBINO-PALLID CROSSES Matings of five pallids and two albinos resulted in the birth of nineteen young (11 3,8 9@), all fully pigmented mice, having the appearance of ordinary normal gambeli. It is of interest that conclusive evidence was found for a high degree of linkage between the pallid and the albino factors (Sumner, ’22). Only sixteen F, young have thus far been reared, derived from simple F; xX F, matings, though a considerable number of a somewhat more complex pedigree were obtained. Of these six- teen, 9 were dark, 6 pallid and 1 albino. The abnormal propor- tions of the two recessive classes is doubtless a chance result due COLOR MUTATIONS IN MICE OF PEROMYSCUS Sia to inadequate numbers. Other types of matings give no grounds for expecting the number of albinos to be deficient here. The really important tests, as stated in another paper, have been made, 1) by mating ‘extracted’ albinos of the F, generation with ‘pure’ pallids (i.e., those known to be free from the factor for albinism); 2) by mating extracted pallids with pure albinos, and, 3) by mating extracted albinos with extracted pallids. There were likewise a number of matings in which the pedigrees were somewhat less simple than here indicated. Eighteen F, mice were involved in these tests. The total number of their offspring was 135, the number per pair rang- ing from three to twenty-six. Not all of these parents, taken singly, have thus far given birth to a sufficient number of young to prove their genetic composition with any certainty. But the cumulative testimony of all of these matings is overwhelming. Not a single pallid mouse and only two albinos have appeared among the 135 young which have thus far been born. Had there been a normal proportion of ‘carriers’ among the parents, these matings should have yielded fifty-five of the recessive types. That all of the offspring with two exceptions (these being sibs) _ were of the wild type is evidence of a high degree of linkage (in this case ‘repulsion’) between the albino and the pallid factors. SUMMARY AND CONCLUSIONS 1. Three distinct color ‘mutations’ have been described, which first appeared in captive stock of the commonest species of California deer-mouse, Peromyscus maniculatus. 2. Two of these, the ‘yellow’ and albino varieties appeared in cultures of P. m. gambeli, originally trapped in the vicinity of La Jolla. The third, or ‘pallid’ variety, first appeared in the F, generation of a cross between the subspecies rubidus and sonoriensis. 3. The albino and pallid varieties arose but a single time each in our cultures. Of the ‘yellows’ there were six independent outcroppings. 4. At least two of these independent yellow strains differed from one another in the mean color tone displayed, and this 312 F, B. SUMNER AND H. H. COLLINS difference proved to be, hereditary. Since the hybrid offspring of these two strains were all ‘yellows,’ and displayed, on the whole, an intermediate tint, we probably have to do either with a case of ‘multiple allelomorphs’ or a case in which one or more ‘modifying factors’ condition the difference. 5. In none of these cases is the evidence sufficient to show whether the actual mutation, or modification of a genetic factor, occurred in our own cultures, or whether the mutant factor had been present for many generations in a simplex condition. 6: The number of ‘mutants’ originally produced was consid- erably higher than would be expected, on the assumption that the parents were both heterozygous. This excess was found in five out of six of the independent outcroppings of yellow. It was also found in the pallid strain, but not in the very small number of original albinos. Combining all the offspring of these original heterozygous parents, we have 87 individuals, of which 57 were of the wild-type and 30 were mutants (recessives), giv- ing a ratio of 1.9:1, instead of 3.:1. The departure from the normal is not, however, of very probable significance.2° Fur- thermore, there is another possible interpretation of this excess of recessives. We are necessarily dealing only with the offspring of parents known to have produced some recessives. It may well be that there have been other pairs of heterozygous parents in the stock, which have not been recognized as such owing to their having produced only normal offspring. Inclusion of these last would increase the ratio of dominants to recessives. 7. All of these mutations, like the vast majority of those described by previous writers, plainly involve the loss of some-— thing normally present. In the case of the albinos, all pigment has been lost, both from the hair, the skin, and the retina. The pallid mice have lost most of their dark pigment, and probably some of their yellow, and here also the loss has been general, affecting all pigmented parts of the body. In the yellows there has been an almost complete suppression of the all-black (un- banded) hairs and a shortening of the basal, deeply pigmented zones of the others. In the ‘agouti’ hairs of these mice on the 20 The probability is only about nine out of ten. COLOR MUTATIONS IN MICE OF PEROMYSCUS ate other hand, the amount of yellow pigment has certainly been increased, so that there has been a partial compensation for the loss of dark pigment. The eyes, ears, and feet of the yel- lows are as dark as those of the normal. 8. These three mutant types are all recessive to the wild type. The albinos and pallids breed true and exhibit compara- tively little variability. Likewise they segregate clearly in crosses with the wild type or with one another. The yellows, on the other hand, exhibit a wide range of variability, intermedi- ates being found between the typical yellow and the normal condition. They also display other irregularities which will be discussed in another section. 9. These three mutations relate to quite distinct genetic factors. Any two, when crossed, give rise to the wild type in the F, generation. In the F, the wild type and two mutant types segregate clearly, except for certain irregularities with re- spect to the yellows. 10. Albino-pallid crosses reveal the existence of a high degree of linkage between these two factors. On the contrary, no link- age appears to exist between the albino and the yellow factors. The yellow-pallid cross was not tested in this respect. 11. In the F, generation of the yellow-pallid cross, the propor- tion of yellows proved to be considerably too small, though these discrepancies may perhaps be accidental. There were, in addi- tion to the true ‘yellows,’ about an equal number of ‘doubtful’ individuals, approaching the normal in appearance, which ap- peared to be genetically neither true yellows nor true normals. Likewise, in the original yellow cultures, there occurred, as stated above, many somewhat intermediate individuals, these being sometimes found in the same brood with typical ones. The genetic status of these ‘atypical yellows’ and other ‘doubtful’ individuals of the same stock has not yet been determined by us. We are certain, however, that they are not merely mice which are heterozygous for the yellow factor. Heterozygotes are commonly as dark as the ‘wild’ type of gambeli. 12. The ‘pallid’ mice, though far more regular in their genetic behavior than the yellows, nevertheless show a quite evident 314 F. B. SUMNER AND H. H. COLLINS variability in their coat color and an even wider variability in their eye color. In respect to the latter, they range from a pink only slightly darker than that of the albinos to a shade only slightly paler than the full black of the wild type. Indeed, both of these extremes have been found among the derivatives of an albino-pallid cross. Whether these differences are due to ‘modi- fying factors’ or to the ‘contamination of factors,’ or whether they are ‘purely somatic’ (whatever that may mean!) we are unable to conjecture at present. 13. There are included in the preceding pages the results of numerous color analyses made by the senior author with the aid of a Hess-Ives tint-photometer. The principal (macroscopic) differences between the ‘yellow’ and ‘pallid’ mutants and ‘wild- type’ mice of the subspecies gambeli were found to be due to different proportions of black, white and a ‘color’ of tolerably constant quality. The spectral position of this last, as judged by the red: green ratio, was found not to differ very widely in any of these forms. This may be taken as evidence of a consid- erable degree of uniformity in the ‘yellow’ pigment of the hair, though the latter is doubtless not the only factor concerned in the gross results. Small, though well-marked, differences were found, on the other hand, even in this red: green ratio, the most noteworthy case being that of the ‘a’ and ‘b’ strains of yellows. 14. To what degree the color mutations here discussed cor- respond with those which have been described for house-mice or other domesticated rodents, we cannot state with certainty. The authors have not had the opportunity to make the neces- sary comparisons either with living specimens or skins of such races. On first thought, it might seem that our ‘yellows’ are of the same type as the familiar recessive, black-eyed yellows, whose condition is attributed to the replacement of the ‘extension’ factor, ‘E’, by its recessive allelomorph, ‘e’ (Castle, ’20, p. 124). It must be recalled, however, that the black pigment in our races is far from being restricted to the eyes, but is present in full intensity in the bases of the body hairs, in the ears, feet, tail, and some other parts. COLOR MUTATIONS IN MICE OF PEROMYSCUS 315 Castle (’16, ’20) records that “the occurrence of yellow sports among wild meadow mice (Microtus) has been observed by Cole, Barrows, F. Smith and others,” and Dunn (1921) lists several wild rodents in which ‘restricted yellow’ is said to occur.”! Unfortunately, no further particulars are available relative to the pigmentation or genetic behavior of these. It is hardly necessary to point out that our ‘yellows’ have no relation to the race of dominant yellow house-mice, which has been so much discussed in genetic literature. ‘ Regarding the possible relationship of our ‘pallids’ with other described color varieties of rats and mice we can speak somewhat more definitely. The almost (though not quite) complete lack of black pigment, together with the presence of abundant yellow pigment, in the hair and the dark red color of the eyes would suggest that our ‘pallid’ race corresponds, in some sense, to the ‘red-eyed yellows,’ discussed by Castle (14 and later papers) and others for rats.22. This surmise is greatly strengthened by the fact, referred to above, that in both species there is a high degree of linkage between the factor for the red-eyed mutation and that for albinism (Castle, 714, ’16 a, 719; Castle and Wright, 5; Dunn, °20). The occurrence of true albinos among Peromyscus has already been recorded by Castle (12). Castle’s specimens belonged, however, to a different species from ours (P. leucopus novebora- censis), the progenitor of the strain having been sent him from Michigan. Doctor Castle kindly furnished us with a pair of these mice, but the latter, like all the surviving members of his strain, proved to be sterile. The ‘identity,’ in terms of the factorial hypothesis, between albinism in Peromyscus and that in the house-mouse cannot, of course, be taken for granted. Since the crossing of these genera seems to be impossible, this question can never perhaps be con- clusively: answered. 21 ‘Peromyscus maniculatus gambeli’ is included in this list. If Dunn here refers to the ‘yellows’ discussed in the present paper, and reported earlier by Sumner, he is probably not justified in assigning them to this class. 22 First referred to as ‘black-eyed yellows.’ 316 F. B. SUMNER AND H. H. COLLINS LITERATURE CITED Castie, W. E. 1912 On the origin of an albino race of deer-mouse. Science, vol. 35, pp. 346-348. 1914 Some new varieties of rats and guinea-pigs and their relation to problems of color inheritance. American Naturalist, vol. 48, pp. 65-73. 1916 Genetics and eugenics. Harvard University Press. 1916 a Further studies of piebald rats and selection, with observations on gametic coupling. Carnegie Institution Publication no. 241, pt. III, pp. 163-190. 1919 Studies of heredity in rabbits, rats and mice. Carnegie Institu- tion Publication no. 288, pp. 1-56. 1920 Second edition of Genetics and eugenics. Caste, W. E., AND Wricut, 8S. 1915 Two color mutations of rats which show partial coupling. Science, vol. 42, pp. 193-195. Dunn, L.C. 1920 Linkage in mice and rats. Genetics, vol. 5, pp. 325-343. 1921 Unit character variation in rodents. Journal of Mammalogy, vol. 2, pp. 125-140. Ripeway, R. 1912 Color standards and color nomenclature. Washington: published by the author. Sumner, F.B. 1917 Several color mutations in mice of the genus Peromyscus. Genetics, vol. 2, pp. 291-300. 1918 Continuous and discontinuous variations and their inheritance in Peromyscus. American Naturalist, vol. 52, pp. 177-208, 290-301, 439-454. 1920 Geographic variation and mendelian inheritance. Jour. Exp. Zo6l., vol. 30, pp. 369-402. 1921 Desert and lava-dwelling mice and the problem of protective coloration in mammals. Journal of Mammalogy, vol. 2, pp. 75-86. 1922 Linkage in Peromyscus. American Naturalist. Wrigut, 8. 1917 Color inheritance in mammals. II. The mouse. Journal of Heredity, vol. 8, pp. 373-878. III. Therat. ibid., pp. 426-430. PLATES 317 Bw DN Ee PLATE 1 EXPLANATION OF FIGURES Normal Peromyscus maniculatus gambeli of abouts medium shad Typical specimen of ‘a’ yellow. Typical specimen of ‘b’ yellow. ‘Atypical’ b yellow. 318 COLOR MUTATIONS IN MICE OF PEROMYSCUS PLATE 1 F. B. SUMNER AND H, H. COLLINS Io @) Jo) PLATE 2 EXPLANATION OF FIGURES Adult pallid, of about medium shade, Adult albino. Juvenile gambeli (‘wild type’). Juvenile yellow (from same brood as preceding). Juvenile pallid. 320 COLOR MUTATIONS IN MICE OF PEROMYSCUS PLATE 2 Fr. B. SUMNER AND H. H. COLLINS Abstracted by George Howard Parker, author Harvard University. The crawling of young loggerhead turtles toward the sea. Newly hatched loggerhead turtles find their way from their nests to the sea in consequence of at least three factors: first, positive geotropism, as shown in their tendencies to move down slopes; second, their response to their retinal images, in that they move toward regions in which the horizon is open and clear and away from those in which it is interrupted by complicated masses, and, third, their probable response to color, in that they move toward blue areas rather than toward those of other colors (Hooker). These animals are not appropriately described as phototropic, for they do not move either toward a source of light or away from it, but they are to be regarded as exhibiting a more complex condition in that they respond to the details of their retinal images rather than to these images as wholes. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JULY 24 THE CRAWLING OF YOUNG LOGGERHEAD TURTLES TOWARD THE SEA G. H. PARKER Zoological Laboratory, Harvard University It is difficult to imagine a more striking and invariable reaction among higher animals than the crawling of newly hatched logger- head turtles (Caretta caretta Linn.) toward the sea. For a long time this response has excited the interest of field naturalists, and within recent years it has been studied with especial care by Hooker (’08 a, ’08 b, ’09, 711) and commented upon by Mayer (09, p. 121). To see a dozen of these newly hatched creatures, that have had no previous experience with the ocean, scramble toward it, notwithstanding that it may not be within the range of their vision, is a sight never to be forgotten. Any attempt on the part of an observer to check them in their course seems only to excite them to further effort which does not cease till they have reached the water. To the observer they seem to be drawn toward the sea by an influence as mystical as it is impelling. On July 5th a considerable number of these turtles were hatched at the Miami Aquarium, Miami Beach, Florida, and it was here that I had an opportunity of studying their reactions not only on the day of hatching, but also over a subsequent period of a week orso. My thanks are due to the officers of the Miami Aquarium Association for the privilege of working at the aquarium labora- tory and for their generosity in providing me with all that was necessary for my investigations. After my attention had been called to the remarkable regularity with which the young turtles on the aquarium wharf went toward the water, I began some more or less systematic observations and experiments. A piece of smooth paper-board, about a meter square, was laid down horizontally on the wharf and surrounded on all four sides by a low wooden fence some 15 cm. high. At the 323 324 G. H. PARKER middle of the pen thus constructed turtles were liberated one at a time and with their heads pointed in sequence north, east, south, and west. The water and the late afternoon sun were both to the west and the turtles almost without an exception took that course. To ascertain whether the sun or the water was the effective element in the situation, I carried the paper-board and about a dozen turtles across the narrow key on which the town of Miami Beach is situated to the opposite shore. Here the water was to the east while the sun of course remained in the west. On re- setting the pen on the beach and liberating the turtles as before at its center, they were found to go as regularly to the east, i.e., toward the water, as they had previously gone to the west. It was therefore plain that the sun had no significant influence over their movements, but that these were related to the body of water. On my return from the ocean on the east side of the key to the bay on the west side I stopped in a field about midway between the two bodies of water, and having set up the pen here with its floor horizontal, I proceeded to a third set of tests. I was greatly interested to see that under these circumstances the turtles were not disposed to move much from the center of the board and that when they did move they were as likely to go in one direction as in another. During these tests the sun was still high in the western sky and the results showed again that this source of light was not a significant factor in determining the direction of motion. Not only did these observations demonstrate that the sun was ineffective, but they showed also that the water, at least at the distance of about a quarter of a mile, was also ineffective, for the turtles went as often to the north or the south as they did to the east or the west. Had they been under the influence of the water, they should have turned even in this intermediate position to the east or the west, but not to the north or the south. Apparently the middle of the key was a region in which the turtles were as in- different to the influence of the water as they were in all places to that of the sun. So far as the sun is concerned, my observa- tions agree entirely with those of Hooker, who declared both in CRAWLING OF YOUNG TURTLES TOWARD THE SEA a20 his preliminary (’09, p. 124) and in his final report (11, p. 70) that the sun has nothing whatever to do with the direction in which the turtles creep. During these preliminary trials it became perfectly evident that the young turtles were very responsive to the slope of the surface upon which they moved; in other words, that they were geotropic to a marked degree. Care was therefore taken that the paper-board on which the tests were carried out was always horizontal and attention was given to the geotropism of the turtles as such. In all my experience the young loggerhead turtle was always positively geotropic. It regularly goes down slopes, notwithstanding the fact that it has ample energy and strength to go up them, and it is responsive to even so slight an inclination to the horizontal as 10°. On more considerable inclinations the animals go down with a rush. Hooker (711, p. 72) states that ‘‘under ordinary circumstances the young turtles are nega- tively geotropic, but if the possible descents have been exhausted, they become positively geotropic.”” As a descent is evidence of positive and not negative geotropism, Hooker has apparently confused terms and, if this is so, his observations agree with mine except that I have never seen any evidence whatever of negative geotropism (movement against gravity). In all my tests of turtles from the time of their hatching till the end of their first week of life, they have been consistently positive in their geo- tropism. When placed on the natural slope of a beach, even though they cannot see the water, they travel downward at a considerable rate. How they escape from the shallow nest in which they are hatched I do not know, for I have never had the opportunity of studying them under these circumstances. Newly hatched turtles in the laboratory were from the beginning always positively geotropic, so that I have no reason to believe that there is any change in their geotropism. Such nests as I have seen were always shallow, and it is quite possible that turtles on hatching pass up their slopes and over their rims under other influences than those having to do with geotropism and thus escape. But concerning the details of this question I have no facts to offer. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 326 G. H. PARKER In all the tests I carried out, as already stated, the turtles were uniformly positively geotropic. Having determined that young turtles are positively geotropic, I next turned my attention to their responses to water. That they are not directed in their movements by water vapor in the air, by the smell of water, or of materials associated with it, or by the sound of waves, etc., has been abundantly shown by Hooker ('11), and on these points I can confirm practically all his statements. If next a course over which young turtles are making their way toward the sea a shallow vessel of water is placed so that they might enter it, they pass it by without showing even a deflection in their line of march. Had the water in itself possessed a quality attractive to the turtles, some change in their course would certainly have been evident. That water does not thus attract them is also evident from the fact that turtles liber- ated in the pen on the laboratory wharf in the darkness of night did not move toward the water as they did in the daylight, but crept about indiscriminately. . If water in itself does not influence the direction of movement of the young turtles and if sources of light, such as the sun, are equally ineffective in this respect, what is the factor that deter- mines the course that they take on a horizontal surface? When turtles were liberated at the center of the horizontal pen already described as set out on the laboratory wharf and their courses toward the water were closely observed, it was found that these courses were not directly toward the water, which was almost exactly west, but they were a little to the north of west. When the surroundings were inspected to ascertain what might be present to cause this peculiarity, it was seen that the main mass of the aquarium building loomed up a little to the south of east from the pen and that if a straight line was drawn from this mass through the center of the pen westward it would point to a very open unobstructed horizon. Such a line included the course taken by the turtles. It therefore seemed possible that the young turtles took a course which led away from the most considerable mass upon the horizon and toward the most unobstructed and open parts of that line. To test this view I tried an experiment CRAWLING OF YOUNG TURTLES TOWARD THE SEA 327 almost exactly like that described by Hooker (’11, p. 72) in that I set up the horizontal pen in an open field to the east of a low hedge west of which was a sea-wall and the bay. On liberating the turtles here they all took easterly courses away from the hedge, and incidentally away from the water. ‘Their courses were toward the open field. Evidently, the mass of the hedge and the open field influenced the direction taken much as the laboratory buildings and the open water had done, but in this instance it was perfectly evident that the water had no necessary part in determining this direction. These results agree exactly with those obtained by Hooker. I next set up the pen in a square open field, three sides of which were bounded by well-grown trees and the fourth fairly open. The turtles went with reasonable regularity toward the open side. I then transferred the pen to a very open sandy plane and piled up next one side of it a number of boxes and tin cans till a wall nearly a meter high was formed. From this the turtles regularly moved away. These various tests led me to conclude that any large mass interrupting the horizon forms a center from which young loggerhead turtles retreat. But these animals not only retreat from conspicuous masses on the horizon; they also move toward the open horizon with great certainty. I was persuaded of this by an accidental observation made while I was testing a very different matter. I had placed the horizontal pen between and in front of two well-grown bushes to ascertain whether the turtles would orient to one or other of these or give a combined reaction to both. Much to my sur- prise the turtles did not retreat from the bushes, but took a course in the opposite direction as though they were going directly through the opening between the bushes. On looking through this opening I observed what had escaped my attention before, a considerable stretch of free horizon. I repeated this test several times and always with the same result, the turtles took a course between the two bushes. Consequently, I concluded that though young loggerhead turtles move away from large interrupting masses on the horizon, they also move with great certainty toward a section of open horizon even though this may be relatively small. 328 G. H. PARKER In a final set of experiments designed to test the effects of the overhead and the horizontal fields of light, I had an excellent opportunity to observe the exact method of response of the turtles to their illuminated surroundings. Two large collecting tubs were placed upon a sea-wall, one right side up and the other bot- tom up. The tubs had a diameter of about 50 cm. and a height of a little over 30 cm. Five turtles were tested by being placed alternately on top of the inverted tub at its center and inside the upright tub likewise at its center. From the top of the inverted tub the turtle could see the whole landscape; from inside the upright tub it could see only the overhead sky. From both positions the turtles were free to move in any horizontal direction. In each set of tests a given turtle was headed successively north, east, south, and west. The sea-wall on which the tubs were set ran approximately north and south; to the west was open water; to the east a field with trees and shrubbery. In the twenty tests inside the tub the turtles remained stationary for five minutes in fourteen instances and in the remaining six they took various courses which may be roughly described as twice to the northwest, twice to the east, once to the northeast, and once to the south- west, showing that the overhead sky had no effect on their orientation. In the twenty tests on the outside of the tub the turtles went invariably to the west, away from a horizon inter- rupted by trees and shrubbery and toward one of open water. But the interesting part of these tests was not so much the direc- tion taken by the turtles as the way in which this direction was apparently discovered. When the young turtle was‘set at the middle of the inverted tub, it rested there quietly about half a minute, raised its head high in air, made a complete circle or more in a very restricted area, and then moved off immediately to the west. The preliminary circular movement, almost always a complete circle or more, was made irrespective of the position in which the turtle was set. To all appearances it seemed as though the animal tested first the whole horizon and then moved in a direction away from large masses and toward greatest openness. In describing the photic responses of the young loggerhead, Hooker uses the terms photophilism and phototropism (711, p. 71) CRAWLING OF YOUNG TURTLES TOWARD THE SEA 329 without, however, making very clear what is meant by these, and finally concludes that, though the animals are not influenced in their movement by the sun, they are nevertheless positively phototropie (11, p. 75). He compares them with certain posi- tively phototropic animals studied by Cole (’07) inwhich responses to the area of illumination rather than to the intensity of the light was the determining factor in their locomotion. In my own tests on the turtles I have never seen any evidence that they are, strictly speaking, phototropic. Thus I have never been able to get a turtle in a dark room to creep toward a light such as hap- pened with all the animals tested by Cole. Hence I think it improbable that the young loggerheads are correctly described as positively phototropic. To me they seem to be an example of a much more complicated set of relations than those seen in photo- tropism. Their retinal images are immensely complex as com- pared with those in many of the lower forms, and they respond more to the details of these images than to the images as wholes. That part of the image which represents the region of the horizon is much more importantin determining the direction of locomotion in the young turtle than any other part. If a portion of the horizon is interrupted by many masses rich in detail, such as trees, shrubbery, houses, etc., it may form a center from which the turtle will move away. If a portion of the horizon is un- interrupted and very uniform, as where sea and sky meet, that part may form a center toward which the turtle will go. These conditions indicate that the details of the retinal image in the turtle are the significant features in determining the direction of its creeping rather than the image as a whole and that conse- quently the turtle possesses a kind of vision more like that in the human eye than like that in the eye of purely phototropic animals, even if we include among these the peculiar instances pointed out by me (’03) and by Cole (’07) in which the size of the illuminated area is as significant as the intensity of the light. Hooker’s contention (’09; 711, p. 74), that turtles move toward blue rather than toward other colors and thus reach the sea, may perfectly well be correct. When I made my tests I unfortunately had no adequate means of experimenting with colors, and con- 330 G. H. PARKER sequently I am not in a position to add anything to this aspect of the subject. JI am nevertheless convinced that beside color the photic complexity and photic simplicity of the region of the horizon, as has already been detailed, are factors of first impor- tance in determining the direction of locomotion, for in a number of my tests, as for instance those in the open field, natural blues even in the sky were often absent. Hence I conclude that, quite aside from the effect of color, the direction of locomotion of young loggerhead turtles is significantly influenced by the interrupted- ness or openness of the region of the horizon. On beaches locomotion down a slope, away from an interrupted horizon and toward an open one, as well as toward masses of blue, would almost invariably lead to the sea. These doubtless are the chief factors that influence the course of the newly hatched loggerhead turtle whereby it reaches the ocean. Although water is the environment to be attained, water in itself plays no part in directing the movements of this animal which are indirectly influenced by those features of the environment just enumerated. It would be interesting to ascertain whether any of these factors affect Fundulus in its escape over the beach to the sea from small pools as described by Mast (715). CONCLUSIONS Newly hatched loggerhead turtles find their way from their nests to the sea in consequence of at least three factors: first, their positive geotropism as shown in their tendencies to move down slopes (Hooker); second, their response to their retinal images in that they move toward regions in which the horizon is open and clear, and away from those in which it is interrupted, and, third, their probable response to color in that they move _toward blue areas rather than toward those of other colors (Hooker). These animals are not appropriately described as either nega- tively or positively phototropic, but are to be regarded as exhibit- ing a more complex condition, in that they respond to the details of their retinal images rather than to these images as wholes. CRAWLING OF YOUNG TURTLES TOWARD THE SEA Stay k LITERATURE CITED Coz, L. J. 1907 Anexperimental study of the image-forming powers of various types of eyes. Proc. Amer. Acad. Arts Sci., vol. 42, pp. 335-417. Hooker, D. 1908 a The breeding habits of the loggerhead turtle and some early instincts of the young. Science, vol. 27, pp. 490-491. 1908 b Preliminary observations on the behavior of some newly hatched loggerhead turtles (Thalassochelys caretta). Yearbook Carnegie Inst., Washington, no. 6, pp. 111-112. 1909 Report on the instincts and habits of newly hatched loggerhead turtles. Yearbook Carnegie Inst., Washington, no. 7, p. 124. 1911 Certain reactions to color in the young loggerhead turtle. Papers Tortugas Lab., Carnegie Inst., vol. 3, pp. 69-76. Mast, 8. O. 1915 The behavior of fundulus, with special reference to overland escape from tide-pools and locomotion on land. Jour. Anim. Beh., vol. 5, pp. 341-350. Mayer, A.G. 1909 Ann. Rep. Director Dept. Marine Biol. Yearbook Carnegie Inst., Washington, no. 7, p. 121. Parker, G. H. 1903 The phototropism of the mourning-cloak butterfly, Van- essa antiopa Linn. Mark Ann. Vol., pp. 453-469. Abstracted by William M. Goldsmith, author Southwestern College, Winfield, Kansas. The process of ingestion in the ciliate, frontonia. The food of the ciliate, frontonia, is primarily diatoms, desmids, euglenas, filaments of oscillatoria, and various other microscopic plants. The mouth is normally very small, but may be expanded to approximately two-thirds the length of the body without injuring the organism. Five factors are involved in the process of ingestion of material longer than the expanded width of the body of the frontonia. A. Action of oral cilia: The cilia about the mouth of frontonia exert a direct pull upon the incoming food. B. Action of the locomotor cilia: The cilia of the body drive the organism forward and thus force the stationary food into the mouth. C. The rotation of the body axis: The end of the fiber usually enters the mouth and passes anterodorsally until it comes in contact with, and exerts a pressure upon, the aboral wall, after which the frontonia swings around and releases the tension. Points of contact between the ingested particle and the inner side of the body membrane are called tension points. D. Body contractions: A series of sharp contractions of the body wall assists in relieving certain other tension points. E. Cyclosis: Cyclosis probably aids by moving the end of the fiber around the wall. Unusual and fantastic figures are produced through the contortion of the organism by the ingested food. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JULY 24 THE PROCESS OF INGESTION IN THE CILIATE, FRONTONIA! WILLIAM M. GOLDSMITH Southwestern College, Winfield, Kansas TWENTY-FIVE FIGURES (THREE PLATES) INTRODUCTION The present paper is primarily a record of a series of observa- tions on the ingestion of various kinds of food by the ciliate, Frontonia leucas, and on the relation which this unusual method of ingestion bears to the variation in shape and habits of the organism. ‘The points of chief importance and interest in con- nection with the present problem are as follows: 1. Frontonia frequently takes food consisting of filaments many times longer than itself. The manner of ingestion of such food is explained in detail. 2. The food of frontonia, is primarily diatoms, desmids, euglenas, filaments of oscillatoria, and various other microscopic plants. Various indigestible particles may also be ingested. 3. The mouth of frontonia is normally very small as compared with that of Paramecium and other common ciliates. How- ever, it may be expanded to approximately two-thirds the length of the body without injuring the organism. 4, The normal shape of the body may be altered by certain characteristic contractions, by simple twisting and bending, and particularly by the presence of ingested materials. 1 These investigations were carried on in the Zodlogical Laboratory of the Johns Hopkins University during the year 1919-20, in connection with the regular laboratory course in animal behavior. The writer is indebted to Prof. S. O. Mast for many valuable suggestions during the progress of the work. He is also under obligation to Prof. Asa A. Schaeffer, of the University of Tennessee, for reading the manuscript. 333 334 WILLIAM M. GOLDSMITH 5. Food is not taken into the mouth by the usual ciliary action of the cytostome. A number of related factors are involved in this process. ‘These are considered in the text and are listed in the summary. GENERAL BEHAVIOR Under normal conditions, in a quiet culture, frontonias may be seen swimming slowly here and there near the substratum apparently in quest of food. Any slight disturbance, such as the jarring of the container or the addition of weak chemicals to the culture, causes the organisms to rise from the bottom and to swim around more rapidly. However, after the removal of the stimulus they soon settle down to the bottom and continue the slowly swimming movements. Schaeffer (‘‘Ameboid Move- ment,’ ’20) says: ‘‘Frontonia feeds mostly by ‘browsing,’ that is by eating particles lying on or against some solid support.” If an individual chances to come in contact with any object approximating the size of the food it is accustomed to eating, such as filaments of oscillatorias, diatoms, desmids, and various other microscopic plants, it usually pauses, places the oral opening near the object and proceeds to brush it with the oral cilia as though attempting to ascertain its nature (fig. 1). While the mouth is in close proximity with the object, the posterior part of the body frequently swings about this point as upon a pivot, sometimes turning through an are of 90° or even entirely around. It is not uncommon for the organism to leave the object, move off for a short distance, then turn slowly about and swim here and there over the same area, eventually coming in contact with the same object and repeating the process. THE MECHANICS OF INGESTION Although frontonias ingest food particles of various shapes, the process of feeding can be more successfully observed when the food is in the shape of a filament. Before actual ingestion is begun the organism usually moves over the food and slowly swings around until the longitudinal axis is parallel with the long axis of the object to be engulfed. It then moves slowly INGESTION IN THE CILIATE, FRONTONIA 300 forward with the oral cilia in contact with the object (fig. 1) until the end of the linear food particle is reached, when the mouth is slowly pushed over the end of the object and thus ingestion begins (fig. 2). Just as the oscillatoria filament is about to enter the mouth, the frontonia bends the anterior end downward as shown in figure 2. This brings the plane of the mouth perpendicular to the long axis of the fiber, and thus permits the fiber to enter with the least resistance. As the fiber slowly enters the body, the granules and food particles suspended in the endoplasm are pushed aside, leaving a clear space on either side of the entering food. This space usually presents the general appearance of the ectoplasm. From all indications it seems quite certain that a small amount of water is taken in with the fiber which adds to the transparency of the surrounding space. This clear area is not definitely set off from the endoplasm material as is the case during the last two or three hours of digestion. The protoplasmic granules and smaller food particles may pass from one area to the other, and often crowd in and at times, and at certain places, obliterate the transparent space. The movement of these granules in front of and to the side of (fig. 3) the incom- ing food fiber is quite characteristic of the movements accom- panying the entrance of any solid into a viscous medium con- taining particles in suspension. As will be suggested later, there is little streaming of the granular endoplasm unless there is first a movement of the incoming fiber or possibly a contrac- tion or other movement of the body wall. A. Ciliary action—first and second factors of ingestion The mechanics by which the oscillatoria filament (or any other material) is caused to enter the body of the frontonia is of vital interest in connection with the present investigations. At the outset it should be emphasized that the customary method employed by the ciliates, namely, the sweeping of food particles into the mouth in a current of water created by the cilia, is obviously out of the question, since the food is oftentimes much longer than the organism. First, the oral cilia may be in actual 336 WILLIAM M. GOLDSMITH contact with the food and exert a direct pull thereon and thus actually pull the object into the mouth. Secondly, the action of the cilia of the body may push the frontonia in the direction of the food and thus force the end of the object through the mouth opening. Prolonged study revealed the fact that both factors of ingestion are employed. The most conclusive evidence in support of the first possibility was found in the fact that the organism was oftimes seen to lie quietly while the food slowly entered the mouth; while, on the other hand, it was not uncom- mon for the oscillatoria fiber to remain comparatively still, while the frontonia slowly moved forward as the end of the fiber entered the mouth. This forward movement suggests that the frontonia either pushes the fiber into its body by swimming toward and around it or that the oral cilia, pulling upon one end of the stationary fiber, move the frontonia in the direction of the food. The fact that the ciliate at times moves forward when there is a concavity at the oral region (fig. 4) suggests that the push comes entirely from the locomotor organs. However, other situations are noted wherein the oral region moves along the fiber while the ciliate as a whole and the fiber itself are both stationary (figs. 10 and 11, h tol). Such observations would seem to es- tablish the fact that both the oral and locomotor cilia play a part in the mechanics of ingestion. B. Body movement—third and fourth factors tnvolved in the mechanics of ingestion; In case the food particle is no longer than the expanded width of the body of the ciliate, the two factors heretofore considered suffice to explain ingestion. In the case of a diatom, for exam- ple, the body cilia force the frontonia forward while the oral cilia pull the food into the mouth. However, when one end of the food body is forced against the aboral wall, as at a, figure 3, and the other end still protrudes from the mouth, continued ingestion, if no other factors entered, would cause a rigid fiber to be thrust firmly against the aboral wall. Further ingestion is impossible without the play of other factors, and these appear to result from the stimulation due to the pressure of the end of INGESTION IN THE CILIATE, FRONTONIA 300 the fiber against the body wall. It will be convenient to desig- nate the points where this occurs as tension points. Specifi- cally, however, the writer would define tension points as those points of contact between the ingested material and the body wall in which a sufficient pressure is exerted to be a stimulus. Such stimuli result in, (a) the changing of the angle between the body axis and the fiber, or in, (b) certain characteristic body ‘contractions. It will be shown later that cyclosis is also an important factor in relieving the stimulation at the tension points, especially when flexible fibers are being ingested. When the food fiber comes in contact with and protrudes the aboral wall, special effort seems to be exerted in an attempt to continue ingestion without altering the process. This outward pressure at the tension point seems to serve as a stimulus and to cause one or more things to happen. Sometimes partial or complete ejection of the food takes place, either suddenly by jerky movements, or slowly by reversing the ingestion process. Usually, however, the body of the frontonia goes through cer- tain squirming movements and straightens out more in line with the axis of the fiber, thus aiding in the continuation of the ingestion process, (fig. 4). It will be noted from figures 1 to 8 that the frontonia is now turned through an angle of 180° from the position at the beginning of ingestion. When in this position the incoming food meets the least resistance, as the end of the fiber must now travel posteriorly along the aboral wall (6, ¢ and d) rather than anterodorsally, as it did when it first entered the mouth. Thus, at the completion of this stage of ingestion the organism is turned completely around (compare fig. 1 and 8). It will be noted that during the early stages of ingestion the cil- iate is usually directly over the fiber with the anterior end bent downward so that the mouth will come in contact with the end of the food body. This being the case, the end of the fiber which is being ingested is raised from the substratum. The turning of the body, as shown in figures 3 to 6, causes the point of contact (fig. 3, a) of the end of the oscillatoria filament and the body wall to shift posteriorly (figs. 4, 5, and 6; b, ¢, and d). After the body reaches approximately the position 338 WILLIAM M. GOLDSMITH shown in figure 5, the usual method of ingestion (the pull of the cilia of the mouth and the push of the locomotor cilia) carries the end of the fiber along the aboral wall to the posterior end of the frontonia (fig. 7, e). The continued pressure exerted from within not only makes more pointed the posterior end, but also causes an elongation of the entire organism (fig. 8). The anterior end now moves along the fiber, thus causing the mouth to be drawn well toward the anterior end of the organism (fig.’ 8, f). As the mouth is pulled forward the pressure at the poste- rior end becomes greater and greater. This is the second tension point in the process. The stimulus causes the organism to again undergo sharp body contractions. As in the former case, if the pressure is not relieved the oral cilia are relaxed, causing the body again to shorten. With a whirling backward move- ment the frontonia ejects the food particle (fig. 12). However, if the fiber bends or breaks, normal ingestion continues. In the specific case under consideration, the oscillatoria fila- ment was bent as indicated (fig. 9) and the mouth continued to move along the fiber (fig. 10, h, 7, and 7), causing the posterior end to be drawn toward the mouth. The whole animal was bent upon itself like a hinge (fig. 11). Since under the given pull the ciliate had now reached its limit of expansibility and, fur- thermore, since the fiber did not bend again, further ingestion was impossible. Accordingly, the frontonia suddenly contracted, whirled about the oscillatoria filament, causing the mouth (m) to be pried wide open, and flung itself from the food (fig. 12). With reference to this particular method of ejection, Schaeffer says: ‘‘If there are several coils of a filament whose other end is fast, rolled up inside of a frontonia, the mouth sometimes stretches antero-posteriorly until the coil as a whole without unwinding is thrown out of the body.” Rigid fibers were used extensively as food for experimental purposes, as the organism would continue to draw in one of them as long as possible and then eject it, only to repeat the process time after time. Since the fiber was longer than the expanded length of the organism and not sufficiently flexible to be wound up inside of the ciliate, the repeated attempts at complete in- INGESTION IN THE CILIATE, FRONTONIA 339 gestion were, of course, futile, but the process was nevertheless normal. This repetition made it possible to observe in detail again and again all of the movements involved in the ingestion of the same fiber by the same frontonia. These observations were furthermore especially valuable, as the fiber bent at a weak place at a distance from the ingested end equal to the ex- panded length of the ciliate. This bending permitted each process to continue more than twice as long as it otherwise would have done, since it was possible for more of the fiber to be ingested. C. Cyclosis, the fifth factor of ingestion Schaeffer emphasizes the fact that ‘‘in Frontonia leucas, ro- tational streaming is under the control of the organism, and special use is made of it in feeding.’”’ Although it will be shown later that cyclosis is effective, and in some cases essential, dur- ing the ingestion of certain flexible fibers, observations show that with some food material complete ingestion takes place without this fifth factor. A reconsideration of figures 1 to 9 willillustrate the point under consideration. In this case the rigid alga fila- ment is forced into the body by the pull of the oral cilia and the push of the locomotor cilia of the body wall. The rotation of the body relieves the tension point (fig. 3, a) and permits the end of the fiber to pass down the aboral wall. Although there are slight indications of cyclosis during the ingestion of material of this type, careful observation makes it evident that rotational streaming is not essential. It might be concluded, then, that rigid material whose length is no greater than the expanded length of the frontonia can be, and is, ingested without the effec- tive play of cyclosis. VARIATION IN SIZE OF MOUTH Such observations on ejection of partly ingested material as those considered above revealed some interesting facts regarding the nature of the mouth of frontonia. In many cases specimens which had ingested a sufficiently long fiber to produce a coil inside of the body suddenly whirled about and caused the mouth 340 WILLIAM M. GOLDSMITH | to be expanded sufficiently wide for the spiral to pass out without being first unwound. In many instances the mouth was stretched almost the length of the body. However, the author would con- clude from his observations that the stretching of the mouth of frontonia is brought about by mechanical means through the twisting movements considered above rather than through being under the control of the organism itself. At first this unusual process was thought to be simply the breaking of the body wall, but by segregating individuals which had thus ejected rolls of oscillatoria filaments it was found that they were in no wise injured and that the stretching of the mouth was a normal process. Some of the unpublished notes of Schaeffer bear directly upon this point. He says in part: ‘‘In case the thread is too long, the coil does not always unwind but in nearly all cases, if the coil consists of many turns, the coil in its entirety comes out of the animal, the mouth apparently stretching for nearly the whole length of the animal. This is a normal proc- ess and does not hurt the frontonia.’”’ The mouth of the fron- tonia is not only expanded to an unusual width during ejection, but also many instances of ingestion, or attempted ingestion, have been noted in which the mouth was pushed open almost far enough to ingest objects as large as the animal itself. Figure 13 shows a frontonia attempting the ingestion of a mass of débris larger than its own body. Observations indicate that this enlarging of the mouth during ingestion is brought about by the play of the first two factors involved in ingestion, namely, by the pull of the oral cilia and by the forcing of the organism against and around the material being ingested by the action of the locomotor cilia of the body wall. INGESTION OF LARGE DESMIDS At times desmids which seemed to be ciliated (fig. 16) were seen to swim here and there through the frontonia cultures. Since the high power revealed a thin layer of protoplasm between the ciliated wall and the body of the desmid (Closterium), it was evident that these unusual organisms were frontonias which had engulfed desmids of more than twice their normal length. INGESTION IN THE CILIATE, FRONTONIA 341 Since Closterium was the largest rigid body known to be ingested by any frontonia, a study was made of the methods employed. In order to expediate observation, rich cultures of frontonia were deprived of food for a number of hours (from twelve to twenty-four), and were then removed to depression slides con- taining numerous specimens of Closterium. Under these condi- tions, the ciliates readily attacked the desmids until many fron- tonias attempting ingestion could be observed at the same time. In practically all cases ingestion was indeed only an attempt, as complete ingestion was of very unusual occurrence as com- pared with the number of trials. For example, on December 10, 1919, at 8:00 A.m., numerous specimens of Closterium were added to a rich culture of hungry frontonias in a Syracuse watch- glass, and the culture observed at brief intervals throughout the day. Although the ciliates spent the day in almost con- tinuous attempt at ingestion, only three could be found at 5:30 p.m. which contained closteria. The method of taking in these unusually large food particles was found to be almost identical with that involved in the eat- ing of oscillatoria filaments as recorded in the earlier part of this paper, except that, of course, the mouth was more expanded. The average limit of linear expansion is shown in figure 15. At this point the organism either suddenly jerked back, whirled about, and left the desmid, or allowed the mouth to recede slowly down the desmid and completely ejected same, or re- laxed as shown in A, figure 14, after which other attempts might be made before the food was completely ejected. INGESTION OF SMALLER BLUE-GREEN ALGA FIBERS, OSCILLATORIA PROLIFICA, ETC. The five factors considered in the earlier part of this paper are all noticeably effective during the ingestion of small flexible fibers. Figures 17 to 21 show the fibers being formed into a coil. The particular significance of this set of observations was in the further demonstration that cyclosis, regardless of the cause, is effective, if not essential, in some cases of ingestion. Without assuming that this is actually the case, it would be THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 342 WILLIAM M. GOLDSMITH very difficult to explain how the tension point at c, figure 19, could be relieved. Since the contractions, mentioned as the fourth factor, are direct compressions of the body wall, they would seem to force the end of the fiber through the body mem- brane. Therefore the movement produced by cyclosis would probably be the only factor which would alleviate this tension. When long fibers, as illustrated in figure 20, were completely ingested, the ciliate became very sluggish and discontinued the usual movements until digestion was nearly completed. EFFECT OF THE INGESTED MATERIAL UPON THE SIZE AND GENERAL APPEARANCE OF THE BODY Ingested food causes an unusual variety of shapes of the frontonia’s body. Cultures taken directly from the brook have been found to contain individuals of almost every imaginable shape. The various shapes, of course, depend upon the variety of food available. After a few weeks’ work the experimenter could cause to be produced many desired fantastic figures. For example, it was a very simple matter to produce the char- acteristic ‘half-moon’ frontonia shown in figure 23. This was done simply by cutting oscillatoria fibers into pieces slightly longer than the linear expanded length of the average frontonia. The imperfect ‘half-moon’ shown in figure 24 resulted from the ingestion of a longer fiber than was used in the case of the typical ‘half-moon,’ while the bow-and-arrow-like ciliate (fig. 22) is an unusual case in which a shorter piece of blue-green alga lodged perpendicular to the fiber which produced the ‘half-moon.’ The interesting case shown in figure 25 is simply a ‘half-moon’ frontonia in which the action of the digestive fluids caused the ends of the fiber to curl. Had the fiber given way in the center, the shape would have been markedly different. Since the food materia! includes not only hundreds of the smaller and more or less common fresh-water algae and the slowly moving protozoa, but also the limitless variety of foreign matter and débris which one finds in the sediment of a brook or which may be added to a culture, one is not surprised to find almost any imaginable shape. Moreover, the general appearance of the frontonia not INGESTION IN THE CILIATE, FRONTONIA 343 only varies with the shape, size, color, density, and flexibility of the ingested material, but also with the arrangement as well. Although material of almost any color might appear in the body of the frontonia, the more common colors, especially during the progress of digestion, are the various shades of brown, green, and blue. DIGESTION Although the problem of digestion need not necessarily be considered with the mechanics of ingestion, a number of simple observations were made along this line. As was suggested in the earlier part of this paper, the smaller solid particles of the cell are not usually found in contact with the long food fibers. This clear space forms the beginning of the future food vacuole. The digestive fluid attacks certain parts of the fibers, especially the ends, more readily than others, and this causes the replacing of the graceful curves by sharp bends, breaks, and general dis- tortion. The walls of the oscillatoria filament give way and after two or three hours of digestion the free ends usually begin to roll up. Later the fibers break at various points and the pieces roll up until only small spherical food vacuoles containing irregular masses remain. ‘The entire process consumes approxi- mately six hours. SUMMARY 1. Observations and experiments were made upon frontonias while these organisms were ingesting euglenas, diatoms, desmids, and oscillatoria filaments. 2. The ingestion of blue-green algae, especially oscillatoria filaments, furnished the most conclusive demonstrations of the method involved, as the process continued a greater length of time and involved more factors than did the ingestion of smaller organisms. 3. Five factors are involved in the process of ingestion of material longer than the expanded width of the body of the frontonia. In case of smaller particles, the third, fourth, and fifth factors mentioned below are not essential to ingestion. 344. WILLIAM M. GOLDSMITH A. Action of oral cilia. The cilia about the mouth of the frontonia exert a direct pull upon the incoming food. B. Action of the locomotor cilia. The cilia of the body in general drive the organism forward and thus force the stationary food into the mouth. C. The rotation of the body axis. The end of the fiber usually enters the mouth and passes anterodorsally until it comes in contact with and exerts a pressure upon the aboral wall (fig. 3), after which the frontonia swings around through an angle of almost 180°, using the mouth asa pivot. This change of position permits the fiber to pass dorsally along the aboral side of the ciliate. Through the play of either factor A or B, or both, in- gestion continues until the fiber exerts such a pressure on the body wall at the extreme posterior end that the organism is extremely elongated and pointed (fig. 8). Such pressure on the body wall acts as a stimulus, causing movements that re- lieve the stimulation. The rotation of the body axis assists in relieving the stimulation at certain of these tension points. D. Body contractions. A series of sharp contractions of the body wall assists in relieving certain other tension points. KE. Cyclosis. Cyclosis aids by moving the end of the fiber around the wall, thus making further ingestion possible (fig. 19). 4, Unusual and fantastic figures are produced through the contortion of the organism by the ingested food which varies in size, shape, density, elasticity, and color (figs. 21 to 25). PLATES 345 PLATE 1 EXPLANATION OF FIGURES 1 A normal frontonia approaching the end of an oscillatoria fiber prior to ingestion. 2 Oscillatoria fiber entering the body of the frontonia. Characteristic shape of the organism during the early stage of ingestion of linear objects. 3and4 Turning of the body of the frontonia in order to relieve the stimula- tion at the first tension point (fig. 3, a). 4to6 The body cilia is pushing the frontonia in the direction of the food and thus forcing the end of the object posteriorly along the aboral wall. 7 and 8 The fiber reaches the posterior end of the organism and causes an elongation of the body of the frontonia. 9 The fiber breaks and ingestion continues by the oral cilia pulling the mouth along the object. 346 PLATE 1 INGESTION IN THE CILIATE, FRONTONIA WILLIAM M. GOLDSMITH rere ac * i PLATE 2 EXPLANATION OF FIGURES 10 and 11 The mouth of the frontonia continues to be pulled along the fiber (h, i, 7, k, and l) until the body is stretched to its maximum. 12 Further ingestion being impossible, the mouth stretches anteroposteriorly while the entire organism whirls about and flings the fiber from the body. 13 # A frontonia attempting the ingestion of an object larger than its own body. 14 to 16 Ingestion of a large desmid (Closterium). 348 PLATE 2 eT ee - shat ¥: = Ty ecuca om aun et a2 Pat See ai Rags SNORE Serna ey tes INGESTION IN THE CILIATE, FRONTONIA GOLDSMITH WILLIAM M. 349 PLATE 3 EXPLANATION OF FIGURES The ingestion of flexible fibers (Oscillatoria prolifera) 17 to 20 Method by which a number of coils of an alga filament are rolled up inside of a ciliate. 21 Two frontonias attempting to ingest the same filament. In this particular instance the mouths met and the organism on the left slowly ejected the fiber as it passed into the mouth of the one on the right. 22 to 25 The ingested material alters the size, shape, and general appearance of the body of the frontonia. 300 PLATE 3 INGESTION IN THE CILIATE, FRONTONIA WILLIAM M. GOLDSMITH > Se ss Y 7 Abstracted by Donnell Brooks Young, author Carleton College, Northfield, Minnesota. A contribution to the morphology and physiology of the genus Uronychia. This paper records the results of regeneration experiments on the hypotrich Uronychia. It shows that in this form the power to regenerate parts lost by cutting or other injury is not dependent upon the presence of a micronucleus, although parts without a micronucleus do not divide. The ability to regenerate increases with age, being least just after division and greatest just before the process starts. The large cirri are so highly differentiated that injury to them alone does not result in their regeneration, although they do regenerate if the body of the animal is injured. Injuries to the micronucleus frequently result in the formation of monsters. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SHRVICH, JULY 24 A CONTRIBUTION TO THE MORPHOLOGY AND PHYSI- OLOGY OF THE GENUS URONYCHIA DONNELL BROOKS YOUNG Carleton College THREE TEXT FIGURES AND THREE PLATES (TWENTY-EIGHT FIGUR ES) CONTENTS Prmardactioi. 4040}. avian tek. doe. deed cemodccid, oer 353 Martens lean Gu IriethOG Sts scp tae anne segorheks pacha sce aahpict ciel ake ic AL ch reese 354 Wegeripiion, of the sens Oramyehia cates 3 =) ick caiene sicgm seus sie ahs aoe sree 355 Pescription of W transluga: epee. oes Coe sek eas Seems om 358 Dercnption ely setigerar se see. oo ek Ash a ED ae 359 Description of, U. binucleate... seuyyee) aces ooh: dere Pend oc Rg eee ear 360 Morma| cell iv Aston) TW. SO bMSEL A occ ..8 04 he aes «0,515 cae) sen s\acn Bue aes, ACRE 361 RPE EMELINE NR ao fo peta sical ee aides ele whan tis a aie ve oA ae Cee 362 Cells cut in first fifteen minutes after division.......................5- 365 Cells cut from fifteen to sixty minutes after division................... 369 Cells cut later than sixty minutes after division....................... 370 CellsveutrnrearlyxdivistOMs nnticrccnitlele cece citaae octets ce encima 312 Sells CULT OMIA GIVASTOB: «See ees 2 Oe Me ey eee tee Lee ee eee 375 @ellsieutrinidate divisionss4 43414. 9.) 1. WOR wha) Bee Ae 375 PA normal TE SENETAGION: 4... 3% 3 oe w/a d= co Gesd to cates A eras obs eee 376 TEV CUUCE Seer itrs stow derneaiikes tied o-sy erste ee fore Ee 379 SBE UMN CALE OMY 5 ON cece Pe eae Ree a Re OE Saale SRR EE SO rare, Pera 379 inne WM binucleate. .43 63 Re OS SO Ue 382 IDIscUSSIONP aad, CONCMISIONS: «<7 «excited = Paton wits rice einbhaies oops 383 ns EPEEEEE ERAS RNR ES RO aes circ a 2 Roa ire in atone ACP TC 388 NOTECL AUG UENCIUGG ies src s are cone Seen are cic ciel aa Ae Pensa CUM Mares eres ce, tio cere ater 389 INTRODUCTION In 1910, while working at the biological station at Roscoff, Dr. G. N. Calkins demonstrated that the power of regeneration in the European species of Uronychia, Uronychia transfuga, varied considerably according to the time which had elapsed since the last division. It was at his suggestion that these studies were repeated on the American species. The following work was done at the Marine Biological Laboratory, Woods 353 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 354 DONNELL BROOKS YOUNG Hole, during the summers of 1919 and 1920. I wish to take this opportunity to thank Doctor Calkins for the interest which he has shown and for the help and advice which he has given. I am indebted to my wife, Helen Daniels Young, for her pains- taking preparation of the figures. MATERIAL AND METHODS Uronychia is a common marine hypotrich found at Woods Hole. Practically every salt-water culture examined during the summer was found to contain it, although all the forms which were used for experimentation were collected from two limited regions. It soon became evident that more than one species was present in these cultures, and the first problem was to make an accurate study of them so that they could be distinguished from each other. This led to a study of the genus as a whole with the results given in the next section. No satisfactory culture medium was found, so that it was not possible to keep isolation cultures going for more than a few generations. However, for the purposes of this work an infusion made of eel-grass, flour, and a very little malted milk in sea-water was found to serve the purpose. Mass cultures were kept on hand, and the forms to be used for the various experiments were selected from these. The method of procedure was as follows: the mass cultures were examined under the low power (10 x oc. and 55 mm. obj.) of a binocular microscope, and those individuals which were seen to be in the process of division were picked out by means of a small pipette and isolated in culture dishes in a few drops of the culture medium mentioned above. Such dividing forms can be identified quite easily because of the cirri which form precociously on the daughter cells. Usually there was but little difficulty in finding as many such individuals as could be used at one time. These were kept under observation and the time of division noted. Then at the desired time after division one of the daughter cells would be isolated in another culture dish and cut... It was found that after a little practice the cutting could be done free-hand, in any plane desired, with a small sharp MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 39505 scalpel. Careful study of the fragments made the exact plane of cutting certain. In all cases the normal sister cell was kept as a control, and if for any reason it did not live and divide, the experiment was not listed as successful. Individuals experi- mented on were kept until they died or else divided. Drawings were made from life of some of the more interesting cases. Stained preparations were made for the study of nuclear conditions. Specimens to be stained were put upon a cover-slip which had been smeared with a small amount of egg albumen and then a drop of the killing fluid added. The best killing fluid was found to be a saturated solution of bichloride of mercury in absolute alcohol. As soon as the specimen was stuck to the cover-slip it was put into a dish of the killing fluid and left for five minutes. It was then transferred to 95 per cent alcohol and then to water. Most of the individuals were stained with Heidenhain’s haema- toxylin, the short method. This gave most excellent results, for it not only stained the nucleus clearly, but it demonstrated the cirri as well. No other stain showed the new cirri in divid- ing individuals as sharply. Acid fuchsin, methylene blue, and Mallory’s triple stain were used with indifferent success. For the study of sections the following method gave satis- iactory results. Uronychia transfuga was found in abundance in the zoogloea scum of old culture dishes. The only other hy- potrich of the same size found here was a Euplotes. The scum from one dish was removed by rolling it into a ball, which was then fixed. Flemming’s, Bouin’s, Zenker’s, and Schaudinn’s fixing fluids were used. In this way a number of Uronychia could be handled together. This zoogloea was then embedded in the usual manner and sectioned (5). Iron haematoxylin and Mallory’s triple stain were used. The best results were obtained with Zenker’s fixation and iron haematoxylin. DESCRIPTION OF THE GENUS URONYCHIA (STEIN) Diagnostic characters: medium-sized, colorless hypotrich with a constant body form; body oval, rigid, truncate anteriorly, somewhat rounded posteriorly ; peristomal fossa broad and deep, 356 DONNELL BROOKS YOUNG extending more than half the length of the body. The adoral zone is represented by cirrus-like membranelles which originate from pockets or pits on the anterior border. The mouth is situ- ated in the left posterior part of the peristome cavity. Three to six membranelles are present in the gullet. The peristome is bordered by preoral and endoral undulating membranes. Three bow-shaped cavities are hollowed out of the posterior end of the carapace, two ventral and one dorsal. Originating from the dorsal pit, which is situated on the right side, are three large cirri which curve toward the median line. Inserted in the right ventral pit are four or five straight cirri, while two sickle-shaped cirri originate from the left ventral pocket. Two distinct types of movement are observed, one a steady, forward swimming or crawling and the other a backward or somewhat sidewise jumping, darting, or spinning. In the first type only the cirri of the adoral zone and the undulating mem- brane are at first seen to be in motion. Careful observation shows, however, that there are some smaller cirri on the ventral side which are used for this ordinary swimming. At the slightest irritation the animal will vigorously contract one or more of the large posterior cirri and dart backward with surprising rapidity. Frequently these jumps are so rapid that the eye cannot follow and the animal seems to have vanished as if by magic. After a series of such jumps, the number and violence depending on the strength of the stimulus, the animal will come to rest again and resume its normal swimming. The nuclear complex varies. The macronucleus is usually broken up into two or more fragments. One or two micronu- clei may be present. A contracting vacuole has been described by Claparéde and Lachman as follows, ‘‘La vésicule contractile n’est point placé du cété droit mais du cdté gauche, immediatement en avant. des deux pieds dorsaux gauches.”’ Calkins states that in U. setigera the contractile vacuole lies between the two sets of posterior cirri. These observations have not been verified, and it is certain that in some species, U. binucleata and U. seti- gera, no contractile vacuole is present. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 357 Kent (81), following Stein (’59), defines the genus in these words: ‘‘ Body oval, encuirassed, turgid, the sides rounded, trun- eate in front with a prominent membranous upper lip. The hinder extremity having developed on the ventral side two con- verging bow shaped fissures into which the short claw shaped anal and marginal uncini or styles are inserted; ordinary ventral or frontal styles entirely absent; the peristomial evacuation pocket shaped, closing sphincter-wise at will, its inner or right hand border bearing a band shaped undulating membrane.” Although this description holds in general, it is not correct in some points. The membranous upper lip is not found in all species and Kent’s own figures (figs. 9, 10) fail to show it. At times the undulating membranes of the peristome protrude in front, and it may have been this which was seen. From the study of the living animal it is difficult to determine whether the posterior cirri are inserted in two ventral pockets or whether some are in a third pocket which is on the dorsal side. Stained preparations (fig. 14) confirm Maupas when he writes, ‘Stein place encore dans cette serie (i.e., with the ventral posterior cirri) les trois gros appendices de la région postérieure de bord droit de ces mémes Infusoires. Mais c’est ]& une erreur; car ces appendices sont séparés des cirres transversaux par une mince lamelle prolongement de l’extrémité postérieure ou caudale du corps par conséquent appartiennent a la face dorsale ainsi que Claparéde et Lachman lavaient déja bien reconnus.”’ The cirrus-like membranelles are somewhat differentiated into a central and two lateral groups. Ordinarily the central group is the one most easily seen. The latera] ones bend in towards the center and partially close the anterior end of the peristome. Oc- casionally they spread out, and it is their movement as they open and close over the end of the peristome that accounts for the appearance of the sphincter-wise closing of the peristome. Only two species have been described for the genus, Uronychia transfuga (O. F. M.) and U. setigera (C.). This first is defined by Kent (’81), following Stein (’59), thus, ‘‘Body ovate, trun- cate in front, slightly narrower posteriorly, more usually ob- liquely truncate, angular and bent toward the left, but somewhat 358 DONNELL BROOKS YOUNG evenly rounded, the lateral margins symmetrical; the surface of the dorsal region sometimes smooth, sometimes longitudinally ribbed; anal uncini and styles variable in character and number, usually from three to seven or eight recurved and occasionally fimbriated uncini inserted in the right posterior cleft but not more than two or three in the opposite one; each of the fascicles occasionally supplemented by one or two fine simple setae.” This description is a very inclusive one, and it seems probable that some of the variations mentioned are in reality separate species. Sufficient data are not given, however, to be sure of this. Unfortunately, no mention is made of the nuclear com- plex or of the exact arrangement of the cirri. An examination of the drawings which Wallengren (’02) (fig. 8) made in his studies of the regeneration of the cirri during division shows an animal which differs in shape and number and arrangement of cirri from that which Calkins used for his work at Roscoff (fig. 12). A detailed study of living material would probably show them to be different species. Three forms of Uronychia are to be recognized at Woods Hole. These differ in size, nuclear complex, and arrangement of cirri. The largest of the three corresponds most closely to Stein’s de- scription of U. transfuga, and is probably the same one which Calkins used at Roscoff. This species was seen only once during 1919, but was found in abundance in 1920. It appeared too late, however, for experimental use. It may be described as follows (fig. 7): Description of Uronychia transfuga Carapace 130 to 170 long by 100 to 120u wide and 25 to AOun thick. Anterior and posterior ends rounded. Carapace arched and smooth. On the posterior ventral border is a cavity in which are inserted two heavy curved cirri and a cluster of from five to seven small straight ones. From the right ventral pos- terior cavity originate four or five straight cirri. From the right dorsal posterior cavity project three heavy curved cirri. The anterior cirrus-like membranelles, seven to nine in number, are set in slight depressions on the anterior edge of the carapace. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 359 On the right and left sides of the carapace are other cirrus-like membranelles which are not as easily seen, but which are di- rected forward and which can be made to close over the open- ing of the peristome. ‘The peristome which extends back about half the length of the body is bordered by two undulating mem- branes of about equal size. The mouth is situated in the left posterior part of the peristome cavity and has membranelles init. ‘The nucleus is made up of a macronucleus which is broken up into from eight to fifteen fragments arranged in the form of a horseshoe with the open side toward the right. A small micronucleus is located between two of the posterior pieces of the macronucleus. Uronychia setigera (Calkins, ’02) (figs. 1, 2, 3) This species may be distinguished from the other species of the genus by the following characters: Carapace 40 to 50u long by 25 to 35y wide and 10 to 15u in thickness. Anterior truncate, somewhat rounded posteriorly, with three or four ridges on the dorsal surface, which is but slightly arched. The arrangement of cirri is practically the same as that described for U. transfuga. The following points should be noted, how- ever. In addition to the three heavy curved cirri which project from the right dorsal posterior cavity, there is also found a single slender straight one. Another very slender cirrus originates from the right ventral posterior cavity at the right of the four or five straight ones, and being as a rule at an angle to them, is difficult to see. On the left margin of the peristome are two very delicate, sickle-shaped membranelles. The peristome which is deep and extends backward for more than half the length of the body, is bordered by an undulating membrane on either side, the one on the left being the larger. Just anterior to the pocket-like mouth, which is situated in the left posterior portion of the peristome cavity, are two cirri which originate within the peristome. A third cirrus arises from behind the mouth and extends forward. Within the mouth are four mem- branelles (text fig. A). The nuclear complex is more or less 360 DONNELL BROOKS YOUNG band-like, with the macronucleus broken into two large pieces and the micronucleus situated between them (fig. 15). It is _ situated on the left side of the body. Uronychia binucleata (new species) (figs. 4, 5, 6) This species is distinguished from the others by the follow- ing characters: Carapace 60 to 80u long, 50 to 554 wide and 20 to 35. thick. Dorsal surface arched and marked with small pits. The anterior end more rounded than truncate. Text fig. A Outline drawing of U. setigera, showing the position and arrangement of the peristome cirri. From astained preparation. The posterior cirri differ from those of setigera in that never more than four are present in the right ventral pocket and that they are proportionately much shorter. No cirri originate in the peristome which, as in setigera, extends back for more than half the length of the body. The undulating membranes of the peristome are large and are seen frequently, as they are protruded balloon-like while the animal is swimming. Three of the deli- cate sickle-shaped membranelles are found on the left peristome border. The macronucleus is broken up into from three to five fragments and is in the characteristic position on the left side. Two micronuclei are present, one being located between the two MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 361 posterior parts of the macronucleus, and the other between the two anterior parts. In his study of U. setigera, Calkins (02) did not recognize more than one species, and therefore his description is a composite one. As he states that no stained preparations were made, in all probability the nuclear complex which he mentions, a spheri- eal macronucleus with a micronucleus beside it, is not nucleus at all but a food vacuole. NORMAL CELL DIVISION IN U. SETIGHRA (Figs. 16 to 21) Normal cell division was worked out in detail in U. setigera, both with living individuals and by means of stained specimens. Unusually fine material can be obtained, for not only do the nu- clear parts stain clearly, but the cirri, especially those just form- ing, hold the stain and can therefore be studied in detail. The results of this study confirm Wallengren’s observations (’02) in every respect. As is the case with all hypotrichs, all of the old cirri are absorbed and new ones, precociously formed, take their places. The following observations made in the laboratory show the processes. 8.15 a.m. Individual isolated in a hanging-drop. 11.15. One of the right dorsal cirri of the anterior cell shows. 12.45. Several of the other precocious cirri of the anterior cell seen. 1.10 p.m. Some of the posterior precocious cirri show. 1.20. First sign of constriction noted. 2.10. Precocious pos- terior cirri larger than the old. 2.30. Complete separation of the cells. From this it will be seen that the whole process of division after the first appearance of the precocious cirri lasts about three hours. From eighteen to thirty-six hours elapse normally between successive divisions. A study of stained individuals shows the process in more detail. Figure 17 shows a stained individual in the first stage of division. Precocious cirri can be seen just starting to develop a little above the middle of the body. The micronucleus has enlarged a little and is seen to be moving toward the outer edge of the cell. ‘This individual shows that the cytoplasmic changes, 362 DONNELL BROOKS YOUNG such as the formation of the precocious cirri, and the nuclear changes proceed together. Figures 16 and 18 show ventral and dorsal views of a somewhat later stage. The micronucleus has moved out from the macronuclear band and has begun its mi- totic division. The macronuclear fragments unite to form a band, but from these and many other preparations it is seen that there is considerable variation in this respect. Figures 19 and 20 show the precocious cirri still more advanced and the micronucleus separated into two parts which are, however, still connected by spindle fibers. Figure 21 shows the two cells almost ready to separate. From these drawings it will be seen that there is some variation as to time of absorption of the old cirri and as to the changes of the macronucleus. Calkins (11), in his figures of the division of U. transfuga, shows that the axes of the daughter cells change before the cells sepa- rate so that they are joined by a subterminal instead of a terminal protoplasmic connection. This causes all of the posterior cirri of the anterior cell to lie to the right of the uniting protoplasm. This shifting of axes was not observed in any of the forms studied at Woods Hole. EXPERIMENTAL WORK The following tables and summaries give the results of the various merotomy experiments. No mention is made of the controls, for unless these were normal the experiments were not recorded. ‘The results are listed in tables according to the time after division at which the cells were cut and to the proportionate sizes of the pieces. The various planes of cutting are shown in text figure B and in the tables the cuts are recorded as indicated there. Thus ‘transverse A’ means that the cut was in a trans- verse plane and anterior to the center of the cell. A complete record was not kept of the time which elapsed -before the division of the operated cell. However, it was noted that in many instances the control cell divided two and oc- casionally three times before the operated one, and in two cases the control cells divided in eighteen and twenty-three hours, while the cut cells required forty-two and fifty-seven hours. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 363 Thus the process of regeneration delayed the division rate. The only exceptions to this were those cells which were cut during division. In these cells division was completed before the regeneration of the missing parts and resulted in the formation of one normal cell and one which was abnormal in structure because of the cut. The normal cell divided again at the usual rate, while in the operated one division was delayed by the proc- ess of regeneration. Obl. 2 OBL B bl. C Ob1.D BAN Si Long.A Tons Long. Gg Text fig. B Diagram showing the planes of cutting as listed in the tables following. In order to describe the experiments it is necessary first to describe the way in which cells regenerate. The first evidence of recovery after cutting is the closing over of the cut surface. This is accomplished to some extent by the drawing in of the edges of the body much as Holmes describes for Loxophyllum (07). However, in Uronychia the cuirass or pellicle is so rigid that this process is not very extensive. Apparently a membrane is formed to cover the rest of the surface. During this process there seems to be a tendency for the body to assume normal pro- portions, but probably the drawing in of the cut surface is enough 364 DONNELL BROOKS YOUNG to account for this appearance. Whether regeneration proper ever begins or not, this closing in of the cut surface takes place if the cell lives for more than a very few hours. ‘The regeneration of the posterior end can be followed very easily because the cirri are so definite. At first they appear as swellings on the cut surface, but these elongate and finally show the characteristic shape. Frequently the normal cirrus apparatus does not develop, but lacks some of its parts or the parts are abnormally arranged. This is especially true of amicronucleate fragments. ‘The re- generation of the anterior end is not as easily studied, for the anterior membranelles are not as clearly seen as the larger pos- terior cirri. At times it is difficult to be sure that the undulating membrane, which so often is protruded in front of the body, is not mistaken for new membranelles. However, the first evi- dence of regeneration is the appearance of slight pits from which the membranelles later develop, and by looking for them it is possible to be sure that new membranelles are forming. When an animal was cut into two parts the power of coédrdinat- ing the movements of the motile organs was destroyed in both pieces. For some this lack of control was very apparent, and each of the cirri moved independently, frequently in opposition to each other. This resulted in a very irregular and erratic movement of the fragment. Usually in the course of an hour the fragment became quiet and in a few hours the motor organs worked in harmony again. In Euplotes, one of the closely related hypotrichs, Yocom (18) has found a well-organized ‘neuromotor’ apparatus, which he believes acts as a codrdinating center for the complicated sensory and motor organelles. If such a system should be found to exist in Uronychia, the reactions of the fragments could be easily explained. Although some time has been spent in a search for such a system, none has been seen as yet. As is discussed elsewhere, all that can be seen are fibers originating from the basal bodies stretching anteriorally. No center body or moto- rium is to be found (text fig. C, a and b). The loss of codrdination mentioned above could be accounted for by the severing of neuromotor fibers when an animal was MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 365 cut into two pieces, if such a neuromotor system exists in Urony- chia. As the immediate effects of the injury wore off, the cirri would become quiet and coédrdinated movements would again be possible with the reéstablishment of a neuromotor center. Text fig. C Drawings of two sections (a and b), showing the granular pro- toplasm, the basal bodies (b.b.), and the fibers which join with them (f). The protoplasm is so granular that it is impossible to trace the fibers to any central body or motorium. Cells cut in first fifteen minutes after division Summary of table 1. Thirteen individuals of the species U. setigera were cut into practically equal pieces during the first fifteen minutes after division. In only one case (no. 26) did both pieces die without dividing. In six cases parts which were supposed to be without micronucleus died without any trace of regeneration. ‘The pieces which were probably provided with a micronucleus regenerated and later divided. In five cases the fragment without a micronucleus showed slight regeneration. That is, the cut surface closed over and new cirri apparently began to form, but it is doubtful whether they would have com- pleted regeneration, for some lived three to five days without doing so. In only one case (no. 50) did the piece without a mi- cronucleus grow to be at all normal. This individual was cut a little to the left of the center in a longitudinal plane (long. B). The amicronucleate fragment was somewhat larger and must have had in it most of the posterior part of the macronucleus. 366 DONNELL BROOKS YOUNG It lived for four days and regenerated one of the two left posterior cirri. It may be that the cut left the base of this cirrus attached to the right piece, and if so this accounts for the subsequent re- generation. The cell never became normal. The length of time which pieces lived after cutting seemed to depend on the amount of injury done by the cut and on the amount of food present in the cell. Several instances were noted in which the knife crushed one part of the cell more than the other. This crushing sometimes resulted in immediate death and sometimes in merely delaying the process of regeneration. The amount of food present in a cell could sometimes be told by the number of food vacuoles visible. In one instance (no. 47) there was a large food vacuole, and the piece having it lived for five days as against two to four for the others. Even though in some cases a new mouth may have developed in the amicronucleate piece, ap- parently no new food was taken into the cell. Evidently the pieces died of starvation. Summary of table 2. Thirteen individuals of U. setigera cut into pieces of markedly unequal size during the first fifteen minutes after division are listed in table 2. In only two cases was any regeneration noted in the piece without a micronucleus. Both of these pieces had the posterior cirri attached and the anterior end not only closed over, but new anterior membranelles formed. These pieces lived more than twenty-four hours and shortly before death had the appearance of a very much trun- cated but otherwise normal individual. In ten cases no regen- eration was seen in the small fragment and in most of them death was immediate. Experiment no. 122 is especially interest- ing, for in this case, although the large piece lived for thirty hours, no regeneration took place. The fragment was then killed and stained. No micronucleus was found (fig. 22). The small piece died very soon after cutting, without any signs of regenerat- ing. The cut was recorded as transverse C. If the plane was a little farther forward than C, it seems probable that the mi- cronucleus was injured or destroyed. From experiments which were performed and which will be described later, this is the probable explanation of this case. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 367 TABLE 1 Uronychia setigera cut into practically equal parts during the first fifteen minutes after division MENT aa PLANE OF CUT an: SION Regeneration Fate Regeneration Fate min. 37 | ns. None Died at once Complete | Divided 121 oy |-Obl, B: Slight Died Complete | Divided 26 4 branss Fa. None Died Complete | Died 39 7 | Long. B. Slight Died Complete | Divided 40 @ |. Crans, B. Slight Died Complete | Divided 47 7 | rans, By Slight Died Complete | Divided 43 8 | Trans. B. None Died at once Complete | Divided 4{/ 10 | Long. B. Slight Died Complete | Divided Ato. 10; | ‘Trans. B: None Died Complete | Divided MOM 2) |e irans’ Be None Died at once Complete | Divided 109 | 13 | Obl. B. None Died at once Complete | Divided M5. 13... Trans, B. Slight Died Complete | Divided 50 | 15 | Long. B. Partial Died Complete | Divided TABLE 2 Uronychia setigera cut into unequal parts during the first fifteen minutes after division EXPERI-} TIME ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE ee Sua PLANE OF CUT NUM- DIVI- BER SION Regeneration Fate Regeneration Fate min. 44 Bra|) rans. Ax None Died Complete | Died 123 On| murans: vA None Died at once Complete | Divided 5 Bei Ob E. Slight Died Complete | Divided 70 ters Leans. oe None Died None Died 64] 10 | Trans. A. None Died at once Complete | Divided 125i HOY ODED: None Died Complete | Divided 119 | 10 | Obl. A. Slight Died None Died Ban elare i Obn es. None Died at once Complete | Divided 45. | (13<7| Obl. D: None Died at once Complete | Divided 117 | 14 | Obl. E. Complete | Divided None Died Gaul) ale # |) Drans. Complete | Divided None Died Peis 15. | Obl, Az None © Stained None Died faa) 15 | Trans. ©; Complete | Divided None Died ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE 368 DONNELL BROOKS YOUNG POSTERIOR OR LEFT PIECE Regeneration Fate Complete | Divided Complete | Divided Complete | Divided Complete | Divided Complete, | Divided Complete | Divided Complete | Divided Slight Died Complete | Divided Complete | Divided Complete | Divided Complete | Divided TABLE 3 Uronychia setigera cul into practically equal parts fifteen minutes to one hour after division EXPERI- TIME ANTERIOR OR RIGHT PIECE MENT AFTER PLANE OF CUT NO eae ee Regeneration Fate min, 111 16 Trans. B. None Died 118 16 Trans. B. None Died 112 19 Trans. B None Died 36 20 Long. B. None Died 133 20 Trans. B: Slight Died 42 22 Trans. B. None Died 135 22 Trans. B. Slight Died 134 ks i Desa Yl 3 None Died 116 25 Trans. B. None Died 130 28 Trans. B. Slight Died 28 55 Obl. B. Partial Died 110 60 Trans. B. Partial Died TABLE 4 Uronychia setigera cut into unequal parts fifteen minutes to one hour after division EXPERI-| TIME ANTERIOR OR RIGHT PIECE ont eatery PLANE OF CUT BER SION Regeneration Fate min. 1260 ol SialgranseeAL None Died at once 127 20 Trans. A. None Died 4G |’ 22 | Trans: A-D. None Died Go| eet Olen: Complete | Divided 128"\"F23; || Trans ©: Complete | Died 136) 9°24). |"EransC: Complete | Divided 62/25) | Ob: Complete | Divided 1200/95) “I Erans' ©: Complete | Divided WPA bye HCO}, Ae None Died 1940) eitranss:A\ None Died 27a OOn irate eA Slight Died Regeneration POSTERIOR OR LEFT PIECE Fate Complete | Divided Complete | Divided Partial Divided Cirri reg. after div. None Died None Died None Died at once None Died None Died None Died Complete | Divided Complete | Divided MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 369 Cells cut from fifteen to sixty minutes after division Summary of table 3. Twelve individuals of U. setigera were cut into practically equal pieces from fifteen minutes to one hour after division. In one case (no. 134) both pieces died without either completing regeneration. Although neither piece was stained, from the position of the cut probably the micronu- cleus was injured. No regeneration of the amicronucleate piece was noted in seven instances (nos. lil, 118, 112, 36, 42, 134, 116). Death of the amicronucleate fragment usually came with- in a few hours of merotomy. In three individuals (nos. 133, 135, 130) slight regeneration was seen. All of these were cut in a median transverse plane (transverse B). New posterior cirri appeared as small swellings, but never went further than this in their development. The anterior amicronucleate fragment of one animal (no. 110) cut an hour after division regenerated almost completely. The posterior cirri began to form, but were irregular in their arrangement and never grew to normal size. This fragment died at the end of two days, before the other part had divided. Summary of table 4. Eleven individuals were cut into quite unequal pieces from fifteen minutes to one-half hour after divi- sion. Both parts of no. 12 died almost at once. This was due to mechanical injury, for the larger piece was crushed and died before the smaller. One other experiment (no. 128) resulted in the death of both pieces, but before death the larger regenerated completely. The cause of death is not known. Regeneration in the small piece occurred in but one other instance (no. 27), but regeneration was incomplete, the posterior cirri appearing as tiny swellings; the small piece was from the anterior end. One very interesting experiment (no. 46) showed an unusual condition. The first cut failed to injure the body and removed only the last two-thirds of the posterior cirri. A second cut re- moved the anterior end of the cell. Regeneration was complete as far as the anterior membranelles were concerned, but the injured posterior cirri did not develop. At cell division the old, injured cirri were replaced by new ones as usual. This case will be discussed more fully in a later section. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 370 DONNELL BROOKS YOUNG Cells cut later than sixty minutes after division Summary of table 5. Twenty-three individuals of U. setigera were cut into practically equal parts from one hour after division up to the time of the following division. In one case (no. 106) both pieces died without dividing, but in this instance the pos- terior fragment completed the missing parts before its death and the anterior one grew to be nearly normal. It may be that the micronucleus was destroyed, for the plane of cutting was within the limits of the position of this organelle. The amicronu- cleate fragments regenerated only partially in four instances. TABLE 5 Uronychia setigera cut into practically equal parts later than one hour after division ae ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE MENT geese PLANE OF CUT ; ag Regeneration Fate Regeneration Fate hrs. min. 3O|aeal 50 | iranssS. Partial Died Complete | Divided 33; 1 15 | Long. B. Complete | Died Complete | Divided 49) 1 AD polar saab: Partial Died Complete | Divided 54) 2 00 | Trans. B. Complete | Died Complete | Divided 315], 00 | Long. B. Complete | Died Complete | Divided Sol 2 00 | Obl. E.-D Complete | Died Complete | Divided 53} = 2 15.) “Drans., B: Complete | Died Complete | Divided HA eo AQ) s| “Drans... Complete | Died Complete | Divided 16, 3 45 | Trans. B Slight Died Complete | Divided WA mene) 45 | Trans. B Complete | Died Complete | Divided 103) 3 50 | Long. B Complete | Died Complete | Divided 106| 4 00° | Trans. B Partial Died Complete | Died 108} 4 00 | Trans. B Complete | Died Complete | Divided 2 ee, 30 | Obl. E Complete | Divided] Partial Died 99, 4 Fae Obl Complete | Divided} Complete | Died 97| 5 00; | trans. B. Complete | Died Complete | Divided 101; 5 O00) | Trans. Bi: Complete | Died Complete | Divided 102| °° 5 1574) lb rans. 3: Complete | Died Complete | Div. un- equal 85} 10 30. |. Trans. B. Complete | Died Complete | Divided 86, 10 40 | Trans. B. Complete | Died Complete | Div. un- equal 891 10 45 | Trans. B. Complete | Died Complete | Div. un- equal SO 10) 45) dirans: 1B: Complete | Died Complete | Divided Oi) 10 745. | Trans. B. Complete | Died Complete | Div. un- equal MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 371 Three of these fragments were from the anterior end of the ani- mal, and it was easily seen that the posterior cirri which they formed were not normal. In at least one the full number of cirri did not form, and the arrangement was therefore unusual. The one posterior amicronucleate piece which did not complete regeneration died at the end of about twenty hours. This may have been the result of mechanical injury. Eighteen experi- ments showed complete regeneration in both fragments, although the ones without micronuclei invariably died without dividing. These pieces lived sometimes five days. The parts with mi- cronuclei completed regeneration and were, to all appearances, normal at the beginning of division. However, in four instances the division resulted in the formation of cells of unequal size. Summary of table 6. Nineteen U. setigera were cut into very unequal parts from one hour after division up to the time of the following division. In four of these experiments both parts died, but not until after regeneration had begun in all. The plane of cutting in each instance was such that it probably in- jured or destroyed the micronucleus. In four other cases the smaller parts died very soon after the cutting, evidently be- cause of the mechanical injury. In all of these instances the larger part regenerated completely and divided. In eight other experiments the smaller piece did not regenerate. The larger part invariably did regenerate and divide. In only two cases did the smaller parts regenerate completely, but these parts did not divide. In both of these the individuals formed from the small pieces were minute (in length only 10 to 15,), but nor- mal in every respect as far as could be seen. In three of the cases mentioned above, the division of the part with micronu- cleus resulted in the formation of two individuals of unequal size, just as was observed in those instances listed in table 5. This unequal division indicates that a ‘division zone’ is formed during the growth of the cell long before the actual process of division begins. 372 DONNELL BROOKS YOUNG Cells cut in early division Summary of table 7. Nine individuals of U. setigera were cut in various planes during the early stages of division. At this period the micronucleus has not begun to elongate in a spindle, and any plane passing just above or just below the center leaves it uninjured. Two such cuttings were made. In experiment no. 3 the anterior end of the dividing cell was removed. Figure 23 was drawn from the stained preparation of the regenerated amicronucleate anterior fragment. Aside from the lack of a mi- cronucleus the cell is complete. The posterior fragment com- pleted its division before the missing parts were regenerated. This process took about ten hours and the complete sister cell divided twice before the regenerated one did once. In experi- ment no. 74 the animal was cut so that, as in experiment no. 3, the posterior part contained the micronucleus. However, in this case regeneration was completed before division. At the time of cutting the process of division had not progressed as far as in the first case. Evidently up to a certain stage in divi- sion the process can be stopped for the time being, but beyond that stage the momentum of division is sufficient to carry through that process regardless of other influences. ‘Two individuals, nos. 34 and 65, were cut exactly in the center, and although the pieces regenerated in but little longer than it would have taken for the cells to have completed the division which they had begun, they never divided. It is quite certain that the micronuclei were destroyed. Figure 24 shows one of these cells, the anterior from experiment no. 65, which was killed and stained as soon as its sister cell died. No micronucleus was present. In the case of three individuals only small fragments were removed. In these division progressed normally and the lost parts were replaced only after the completion of the division. The time taken for regeneration was about the same as that which was needed for the regeneration of a similar part when the cut was made on a cell within the first few minutes after division. One (no. 83) was cut in a longitudinal plane. Both parts com- pleted division and the two cells with micronuclei regenerated. TABLE 6 Uronychia setigera cut into unequal parts later than one hour after division ional ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE eee basse tag PLANE OF CUT ; j a Regeneration Fate Regeneration Fate hrs. min. 31 120 Obl. D. None Died Complete | Divided 6 1 40 Obl. C. None Died Complete | Divided 51 2 1 Trans. A. | None Died at | Complete | Divided once 141 2° 15 Trans. D. | Complete | Divided | None Died 59 2 30 Trans. A. | Slight Died Complete | Divided 18 3 45 Obl. F. Complete | Divided | Slight Died 104 3 50 Obl. A. Complete | Died Complete | Died 105 3 50 Trans. A. | Slight Died Complete | Divided 107 4 00 Long. C. Partial Died Complete | Divided 100 5 00 Obl. A. Complete | Died Partial Died 98 5 00 Trans. C. | Complete | Died Complete | Died 24 5 30 Obl. D. Complete | Died Complete | Divided 94 7 00 Obl. A-B. | Partial Died Complete | Div. un- equal 96 7 00 Trans. A. | Slight Died Complete | Div. un- equal 87 10 45 Trans. C. | Complete | Died Complete | Died 88 10 45 Trans. A. | Partial Died Complete | Divided 92 10 45 Trans. A Complete | Died Complete | Div. un- equal 7 19 00 Trans. C Complete | Divided | Slight Died 8} 119 00 Trans. C Complete | Divided | None Died at once TABLE 7 Uronychia setigera cut in early division ae ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE MENT PLANE OF CUT plea Regeneration Fate Regeneration Fate 3 | Trans. A-B Complete | Stained After div.| Div. ant. cell small o4 | Trans. B. Complete | Died Complete | Died 65 | Trans. B. Complete | Stained Complete | Died (4 APObIEB. Complete | Died Complete | Div. after 24 hours 83 | Long. B. None Div. and died| After div. | Divided 68 | Trans. A. None. Died at once} Afterdiv.| Div. ant. cell small er Obl Fr. After div. | Divided None Died BP) | (Oa Dy Slight Died After div.| Div. ant. cell small 78 | Trans. A and C. | Small pieces both died Complete | Div. after 12 no regeneration hours both were normal BY is 374 DONNELL BROOKS YOUNG The two amicronucleate pieces died without regenerating. One individual (no. 78) was cut into three pieces. The two end parts which were small died very soon. The center piece, however, completed its division and the halves regenerated in about twelve hours. TABLE 8 Uronychia cut in mid-division EXPERI- ANTERIOR OF RIGHT PIECE POSTERIOR OR LEFT PIECE MENT PLANE OF CUT |i el SWEAR Regeneration Fate Regeneration Fate 19 | Trans. A. | Slight Died After div. | Div. ant. cell small “| Obl. C. | None Died | After div. | Div. ant. cell small fe Trans. A. | Complete | Divided) Complete | Divided 69 | Long. B. Slight after} Divided| After div. | Divided div. io 79 | Trans. C. | After div. | Divided} Slight Died 80 84 | Obl. E. After div. the post. | After div. the post. cell reg. com- cell died without pletely, the ant. cell died with- reg. while the out reg. ant. cell reg. complete. TABLE 9 Uronychia setigera cut in late division EXPHRI- ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE MENT PLANE OF CUT aS ORL ESBOEE Regeneration Fate Regeneration Fate 20 | Trans. C. | After division! Divided None Died 35 | Trans. C. | None Div. and post. | Slight Died died 113 | Long. B. Slight after Div. and Complete after Divided division died division 131 | Long. B. Abnormal Died Complete after Divided division 132 | Obl. B. None Div.and died | Complete after Divided division we | ‘Obi SE: Div. ant. cell died without Div. ant. cell reg. and reg., post cell reg. and died div., post cell died without reg. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 375 Cells cut in mid-diviston Summary of table 8. Ten individuals of U. setigera were cut at about the middle of the division process. At this time the micronuclei were somewhat separated and a single cut could not destroy both unless it was made in a longitudinal plane. Two individuals were cut in the plane of division. The resulting cells developed to be normal animals just as if the usual division process had been completed. In six cases where either the pos- terior or anterior end of the dividing animal was removed, divi- sion was not disturbed, but was completed before any regenera- tion took place. The small amicronucleate parts were probably injured by the cutting in one or two cases, for no regeneration was recorded for two of them and in the other four only slight regenera- tion was listed. The large pieces completed division in the usual time. Afterwards the injured cells replaced the missing parts. In one case (no. 69) where the cut was in the median longitudinal plane (long. B), division was completed in both fragments, but only those of the left side, the ones with micronu- clei, regenerated. Experiment no. 84 is particularly interesting, for the cut was oblique E. This divided the animal so that when division was completed there was a small and a large cell originat- ing from both the posterior and the anterior half. Neither of the small pieces regenerated. Both of the large pieces reformed the missing parts, but only one of them divided. Probably the micronucleus of the anterior half was destroyed, for the cut was about in its region. Cells cut in late division Summary of table 9. Six individuals of U. setigera were cut in late division stages. In general the results correspond to those obtained from cells cut slightly earlier. When cut trans- versely division proceeded normally and the injured cells re- generated after division. This transverse cut was made in two experiments (nos. 20 and 35). The amicronucleate parts cut off died very soon, in one case without any, in the other with slight regeneration. Two individuals were cut in the median 376 DONNELL BROOKS YOUNG longitudinal plane (long. B). In one case, experiment no. 113, division was completed in both parts. Those with micronuclei regenerated in ten to eighteen hours and later divided. The other two pieces died without regenerating completely. In the other case where a longitudinal cut was made, the micronucleate part divided and regenerated, but the amicronucleate piece instead of dividing, doubled on itself and formed a monster which died in about twenty-four hours. In experiment no. 132 the cut was oblique (oblique B) and evidently passed in front of the micronuclei, for the anterior part which contained also a small piece from below the division plane divided and both pieces thus formed died without. regenerating; while the posterior part divided and both cells regenerated completely and later divided. In experiment no. 77 the animal was cut much as was the one in experiment no. 84, table 8. The results were the same. The anterior part divided and the anterior cell thus formed lived, regenerated, and later divided while the posterior one died with but slight regeneration. The posterior fragment of this experiment also divided, but the resulting anterior part died, while the posterior part lived and later divided. Abnormal regeneration Abnormal regeneration—table 10. It sometimes happened that an attempt to cut a cell was not successful and that the in- cision did not separate the two halves. Usually in such cases the wound healed in a few hours and the animal was normal. Individual no. 114 was in an early stage of division when it was injured while attempting to cut it. In this case the plane of the cut was transverse in the center of the body. Instead of dividing normally, the growth of the precocious cirri became abnormal and the cut surfaces fused together so that a monster was formed. This lived for five days and on the fourth had the appearance shown in figure 25. In all probability, in this in- stance the micronucleus was injured by the cut. Two other experiments (nos. 32 and 72) in which abnormal development occurred may be accounted for by this explanation. ‘The first of these was a cell cut twenty-three minutes after division and MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 377 the other was operated on at a mid-division stage. In the for- mer the cut was made in a longitudinal plane between the pos- terior cirri and extended a little to the left. The pieces were united by a strand of protoplasm and seemed to rotate on each other, so that when reunited they had the appearance as shown in figure 26. In an attempt to determine whether a cut in the region of the micronucleus would produce abnormalities, sixteen cells were operated on. Four of these were successful. Two were cut in early division. One of these did not complete division, but developed as has been described for individual no. 114. TABLE 10 Uronychia setigera. Abnormal regeneration due to incomplete cutting EXPERI- MENT TIME OF CUTTING PLANE OF CUT RESULTS NUMBER 114 | Early div. z Trans B. from | Did not complete div. but formed a left side monster 32 | 23 min. after | } Long. B. from | Lived three days but was never nor- div. post. mal 72 | Mid-div. Trans: B, Ant. cell died. Post. abnormal, never diy. 146 | Early div. % Obl. D. Div. ant. cell died, post. abnormal 145 | Early div. 3 Obl. A. Did not divide. Abnormal 147 | Late div. 4 Obl. D. Div., ant. abnormal, post. normal 148 | Mid-div. 4 Obl. A. Div., ant. normal, post. abnormal The other completed division, but the anterior cell soon died and the posterior became abnormal, although it lived for four days. In the case of a cell operated on in mid-division the posterior cell was injured, and although the anterior cell was normal, the cirri of the injured part developed to at least twice their normal length. In the fourth case the individual had al- most completed division when the anterior part was injured. Division was soon finished, but the cirri of the injured cell were long and very irregular. It is doubtful whether abnormalities resulting from incomplete cuts near the center of the body can be accounted for on the basis of an injury to the neuromotor system, even if we assume 378 DONNELL BROOKS YOUNG that such a system exists in Uronychia. If such an explana- tion is correct, any cut severing the fibers connecting the moto- rium with the cirri should produce the same results. This is not the case, for only when the cut was in the region where the micronucleus is usually found, did monsters develop. TABLE 11 Experiments on starved cells of Uronychia setigera. Cells starved two days EXPERI- MENT TIME AFTER PLANE OF CUT pee + a Hote ke nay Fate Regeneration Fate hrs min. 170 10) } Trans®.A. None Died at | Complete | Divided once 171 i pe fed Ds ern ots By None Died at | Complete | Divided once 172 1 OO Obie Dp: Slight | Died Complete | Divided 173 3 45> trans: Be Slight | Died Complete | Divided 174 5 30 | Trans. A. None Died Complete | Divided 7s {| 10) 00 | Trans. B. None Died Complete | Died tee LO 00 | Trans. B. Shght | Died Complete | Divided 178 | 14 00 | Trans. B. Slight Died Complete | Divided OY 12 OO} «| Brags: Slight | Died Complete | Died TABLE 11A Uronychia setigera starved four days esa seme aay ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE NUM- DIVISION ILENE NOR NCAT BER Regeneration Fate Regeneration Fate hrs. min. 179 20/ jr Obl. ¥ Complete | Died None Died at once 180 30 | Obl. E. Complete | Died None Died at once 181 45 | Trans. C. | Complete | Divided | None Died at once 182 1 45 | Trans. B. | Slight Died Complete | Died 183 2 30 | Trans. C. | Complete | Died None Died at once 184 4 00 | Trans. C. | Complete | Died Slight Died 185 a 00 | Trans. B. | Partial Died Slight Died 186 a 00 | Trans. C. | Complete | Divided | Slight Died ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 379 Starved cells Experiments on starved cells—table 11. When starved by being placed in a poor food medium, U. setigera grows smaller, but the protoplasm does not become vacuolated as does that of Paramecium. Individuals will live for some time and even divide if put into filtered sea-water, although the interval between divisions is greatly increased. Experimental cuttings were made on individuals starved two and four days, although not enough data were obtained to fur- nish absolute evidence of the effects of starvation. It was soon found that if put back into plain sea-water cut cells died with- out regeneration, while if transferred to food media some would live and divide, so this method was used in the experiments performed. In all, thirty-seven cuttings were made. In twenty of these experiments both fragments died at once. These are not listed in table 11. Nine individuals were cut after two days of starvation. Both pieces died in two of these experiments, although regeneration took place in the larger micronucleate fragments. In the other seven the parts with micronucleus regenerated and divided. The amicronucleate parts died with no regeneration or with very little. Hight individuals were cut after four days of starvation. In five of these the small parts died at once or within a few hours without regeneration, while the larger micronucleate fragments regenerated even though they died without division. In one case both fragments showed some regeneration, but died before it was completed. In two experiments the larger pieces com- pleted regeneration and divided, while the small parts died without regeneration. Cutting cirri only In experiment no. 46, listed in table 4, by accident the posterior cirri were cut off without injuring the body. Later the cell was cut in the desired plane. However, it was noticed that 380 DONNELL BROOKS YOUNG the posterior cirri never regenerated, but were replaced by new ones at the following division. This suggested that possibly the cirri differed from the rest of the cell in their power to re- generate, and this led to further experimentation. In all, eight attempts were made to remove part of the cirri without injuring the cell. It was possible to tell whether or not the body was injured, for when the cell was cut the cirri were held together by the protoplasm removed with them. Six of the eight attempts were successful. Without the posterior cirri the animals swam about normally, but did not dart and jump. In no ease did regeneration of the missing cut cirri take place, even though the cutting was done from twenty-two minutes to four hours after division. Figure 27 represents a cell whose cirri were removed two hours and forty-five minutes after divi- sion. The drawing was made from life twelve hours later. In this, as in the other five of the six successful experiments, the new cirri formed at the division period to replace the muti- lated ones. Stained preparations, both of total mounts and of sections, of normal individuals show that the cirri have a well-developed plate of basal granules imbedded in an area of dense protoplasm, as Maier (’02) has demonstrated for the cirri of Stylonychia histrio. If this basal plate is removed or injured, new cirri will form, but if the cirri themselves are cut without touching the basal granules no new growth takes place. The basal gran- ules therefore do not have the power in themselves to reform the cirri. New basal granules can be formed from which new cirri will develop, but the old basal granules apparently have no power to replace lost parts of cirri. Evidently the development of the cirri is dependent on the activity of the basal granules and once a basal plate has formed a cirrus, its power of causing further growth ceases under normal conditions. In experi- ments in which the micronucleus was injured or destroyed dur- ing the division of the animal, as for instance experiment no. 114, figure 25, the cirri did not stop growing when they had reached normal length and so became abnormally long. This may indicate that the micronucleus has some influence over the activity of the basal granules and that if it is injured during the MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 381 formation of new cirri, the growth will continue as long as the animal lives. If, however, the cirrus has completed its growth, the lack of a micronucleus does not result in any further changes. Entz (09) and Collin (’09) claim a nuclear origin for these basal granules. In the normal division of Uronychia the first evidence of their appearance is at the time of the formation of the precocious cirri, and these arise at the surface of the cell, quite close to the spot which they will occupy in the adult. If they do have their origin from the micronucleus it must be that the substance from which they are formed is set free during the process of normal cell activity and not just at the time of the formation of the cirri, for the above experiments show that new cirri with basal granules develop in amicronucleate fragments. Collin claimed that in Anoplophyra branchiorum the basal granules developed in connection with the macronucleus. This may be the case in Uronychia, for no experiments were made which would be conclusive in regard to this point. However, the observations of normal growth as well as of regeneration after injury are in closer agreement with Maier’s view that the basal granules arise as ‘“‘cytoplasmic bodies at the surface of the cell.” He believes, furthermore, that they serve merely as anchors or supports for the cirri and are not to be regarded as kinetic in function. Observations on Uronychia agree with Yocom’s work (18) on Eupictes patella. He describes a basal plate made up of basal granules imbedded in a dense substance which serves as a support for the cirri. He adds, ‘‘this is in agree- ment with Maier who considered the basal plate as the means of support for the cirrus but it is to be remembered that in Euplotes the function of supportisto be attributed only to the dense opaque protoplasmic plate in which the basal granules are imbedded and that the basal granules themselves are given an entirely different function.”’ In Euplotes patella Yocom demonstrated that the basal granules are in direct contact with the fibers of the neuromotor apparatus and he believes, therefore, that ‘this forms a basis of attributing to the basal granules the function of receiving stimuli. Such impulses received cause a contraction of the central contractile axis of each component cilium of the cirrus thus causing a lashing of the whole organ.” 382 DONNELL BROOKS YOUNG Cutting Uronychia binucleata Experiments on Uronychia binucleata—table 12. It was noticed very early in the course of this work that regeneration occurred in both fragments of some individuals regardless of the time which had elapsed between division and cutting. Furthermore, both pieces divided. ‘This was the first evidence showing that more than one species was being used. When it was discovered that two micronuclei were present in one species, while the TABLE 12 Experiments on Uronychia binucleata Paes TIME AFTER | 5 avn orcur ANTERIOR OR RIGHT PIECE POSTERIOR OR LEFT PIECE NUM- DIVISION BER Regeneration Fate Regeneration Fate Ars min 150 5 | Long. C. | Slight Died Complete | Divided 151 12 Obi": None Died Complete | Divided 153 TSS RObE Complete | Divided None Died 61 2ONs|OblD» None Died at Complete | Divided once 155 25 | Long. C. | Slight Died Complete | Divided 159 45 | Long. B. | Complete | Died Complete | Divided 161 1 00 | Obl. A. Complete | Divided Partial Died 13 3 OM ObEKC: Partial Died Complete | Divided In the experiments listed below, the cut was transverse or but slightly oblique and near the center of the cells. The time after division varied from five minutes in the case of no. 149 to twenty hours after division in the case of no.9. In all cases both fragments regenerated and divided. Nos. 149, 152, 2, 154, 156, 57, 48, 158, 10, 11, 160, 162, 163, 164, 165, 57, 50, 166, 13, 14, 15, 167, 95, 93, 169, 9. other had but one, conflicting results obtained by cutting were explained. Even before it was known that two species were being used for experimentation, a record was kept of the relative size of the individuals used and it was noticed that the large individuals showed greater powers of regeneration. Thirty-four experi- ments were performed on this species. Hight cases were recorded in which one part died, while the other fragment regenerated completely and divided. The amount of regeneration in the eight fragments depended on the size of the piece, the time after MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 383 division of the cut, and the amount of mechanical injury done by the cut. For instance, the smaller fragment in experiment no. 61 was killed by crushing. In experiment no. 13 the cut was made three hours and ten minutes after division and the smaller parts constituted nearly one-fourth of the whole cell, yet regeneration was only partial. While in the case of the smaller part of no. 159, which constituted nearly half of the cell, regeneration was complete, even though the cut was made an hour after division. Twenty-six individuals were cut through the middle of the cell in such a way that one micronucleus was present in each fragment. In all of these cases, regeneration was complete and division followed. It is not known what nuclear changes are involved in regeneration and subsequent division. DISCUSSION AND CONCLUSIONS The study of the three species of Uronychia found at Woods Hole, both of normal individuals and of those which were used for experimental purposes, seems to point to rather definite con- clusions as to the function of certain cell organs. Because of the difference in nuclear structure in these species it is possible to use one as a control for the others. Various functions have been ascribed to the micronucleus of the ciliates, including its activity in connection with regenera- tion. Gruber (’85) thought that in Stentor, at least, the mi- cronucleus was not as important for regeneration as the macronu- cleus. He based this conclusion on the fact that in fragments of conjugating Stentor no regeneration takes place until one of the micronuclei takes on the form of the macronucleus. Stevens (04), on the other hand, found that in Lichnophora no regenera- tion takes place unless both the macronucleus and micronucleus are present, and even then only slightly. Lewin (10) agreed with Gruber and did not believe that the micronucleus is needed for regeneration or even for growth and division in Paramecium caudatum. He based his conclusions on the fact that he found a monster with the nuclear elements unequally distributed which, he claimed, produced on division a race of amicronucleate indi- 384 DONNELL BROOKS YOUNG viduals. His published evidence, however, seems somewhat inconclusive. Calkins (’11) found that only a small percentage of Paramecia would regenerate regardless of the position of the plane of cutting. However, both nuclear elements were always present in all those which did regenerate. Evidently there is great variation in different species of Protozoa in regard to the ability to regenerate, and it is possible that the micronucleus functions differently. The micronucleus has long been recognized as a diagnostic characteristic of the group of ciliates. In some instances the micronuclei cannot be found during the vegetative stages and becomes separated from the macronucleus only during conjuga- tion, as Calkins (12) demonstrated for Blepharisma undulans. In Opalina, Metcalf (’09) showed that when syngamy takes place the nuclei show two types of chromatin comparable to macro- and micronuclei. Dawson (’20) has described an Oxytricha with no micronucleus, and he has followed the life-cycle sufficiently to show that the sexual phases are abortive. Here evidently is a ciliate without one of the most important organelles. This Oxytricha is able to live and divide without a micronucleus, but such a case certainly is the rare exception. In Uronychia it is clear that regeneration can and does take place under certain conditions without the presence of any micro- nucleus. Stained preparations fail to show that micronuclei have formed from the macronucleus, as Lewin (11) suggested might be the case. Usually the amicronucleate pieces became abnormal if they lived for more than three or four days, so it might be said that for perfect regeneration the micronucleus is essential. These amicronucleate pieces apparently starved to death for, as far as could be discovered, no food was taken in or assimilated. In many cases no evidence was seen to indicate that a mouth was formed. Stained preparations do not show one, but as the mouth is not always demonstrable even in normal individuals, this is not conclusive. The micronucleus is necessary for normal growth and division. In no ease in U. setigera with its one micronucleus did both pieces divide, even though they did regenerate, while in binucleata, cut MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 385 under the same conditions and differing only in the possession of two micronuclei, both pieces regularly did regenerate and divide. It is not easy to cut Uronychia in such a way that either piece will be free from a part of the macronucleus. In fact, it would be impossible to be sure that some of the macronucleus was not included, for there is so much variation in that structure. These experiments do not offer any evidence of its function. The fact that various monsters have been produced by in- juring the cells in the region of the micronucleus indicates that it regulates the growth processes. In those experiments in which the micronucleus was destroyed during the division process (cf. table 12) the process of regeneration of new cirri did not stop at the usual time and monsters resulted. Even in the regenera- tion of amicronucleate pieces resulting from cuts not made dur- ing division, it was noted occasionally that the cirri continued growth throughout the life of the cell. Usually the cell died from starvation before striking abnormalities were produced. This function of the micronucleus might be compared with the regulating action of the endocrine organs in the metazoa. In summarizing his work on Uronychia transfuga, Calkins wrote as follows: ‘“‘The results might be interpreted by the as- sumption of a specific substance, possibly enzymatic in nature which accumulates with the age of the cell until a condition analogous to saturation is reached. With the formation of the new cell organs this substance, it may be further assumed, is exhausted and regeneration is impossible save with the full com- plement of cell organs.” These experiments seem to bear out such conclusions. A comparison of these results with those obtained from the experiments on U. transfuga seems to show that this substance accumulates in the protoplasm of the cell at an earlier period in the American species. For instance, Calkins found that regeneration of fragments without a micronu- cleus was seldom completed if the cutting was done before the animal had begun its division. In U. setigera, however, after five or six hours had elapsed since division, amicronucleate pieces almost always regenerated the lost parts and assumed a more THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 3 386 DONNELL BROOKS YOUNG or less normal form. Therefore, such a substance is not formed by the micronucleus and stored in it until the time of division when, by the changes in the micronucleus, it is liberated. Rather it indicates that such an hypothetical substance formed while the micronucleus is present accumulates in increasing amounts in the protoplasm up to the time of division when it is used up in the regeneration of those parts which have to be formed anew. In starved cells it would appear that this substance is not formed as rapidly as in normal individuals, indicating that it depends on the taking in and assimilation of food. Just what the nature of this hypothetical substance is is doubt- ful. Meyer (04) described a substance which he discovered first in Spirillum volutans and which takes nuclear stains, al- though it is not chromatin. He believes this substance, which he calls volutin, to be a reserve of nuclear material. This has been found in many other bacteria, in yeast, and in the group of the flagellates. Reichenow (09) demonstrated that volutin is a nucleic-acid combination and that as the chromatin in the nucleus increases, the volutin decreases in the cytoplasm; also that when the nucleus is not growing the volutin increases. I have not been able to demonstrate the presence of volutin in Uronychia, but it or some similar substance would explain the action of regeneration; that is, the nuclear reserve would be used up during cell division, and as a result the ability to regen- erate would be at a low ebb immediately after division. As the growth processes go on, a new supply of the reserve substance would form, and thus the power of regeneration would increase. Lund (18) has carried out a series of experiments showing that the resistance of Paramecium caudatum and Didinium nasutum to KCN varies according to the age of the cell. She states that the resistance of Paramecium to KCN ‘‘when allowed to feed on bacteria, showed a marked increase, and when fed on yeast the resistance increases to a smaller degree, from the time of division up to the following division.’”’ “When Para- mecium and Didinium are prevented from obtaining food the resistance to KCN gradually decreases below its value at the completion of division.” These differences in resistance are MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 387 explained by assuming that they are due to changes in permea- bility of the cell. There is clearly a close resemblance between the curve of variations in the resistance of Paramecium to KCN and the curve of regenerative power of Uronychia. Both are due to changes in the cytoplasm, and it may be that the sub- stances which make the cells less susceptible to the action of KCN are the same as those which increase the regenerative power. It is possible that the change is a physical one, as Lund sug- gests. Instead of a volutin-like substance being formed, the cytoplasm changes with cell age, the age at which the changes occur varying with different species, until a certain physical state is reached in which enzymes originating from the micronu- cleus are activated. These would be liberated as formed and would not be stored in the micronucleus to be set free only at division. The results on the whole seem to bear out the state- ment of Morgan (’01) when he said that ‘“‘the nucleus supplies certain products of metabolism that must be present before the protoplasm can successfully carry out its innate tendency to complete the typical form.” In Paramecium Calkins (11) found that ‘‘there is strong evi- dence of a division zone which lies in the center of the cell. If the cell is cut anterior or posterior to this zone the fragment divides in the original plane into a truncate abnormal form and anormal form. ‘The truncate form may divide again not through its center but through the center of the cell were it perfect.” Lewin (710) finds a somewhat similar condition in his merotomy experiments on Paramecium, for he states that ‘‘since under normal conditions no two sister merozoites were found to divide there is a suggestion that possibly there exists in the cell a lo- calized division center which passes on sectioning to one mero- zoite leaving the other incapable of division.’’ According to these conclusions, the reason why division does not take place in parts of protozoan cells is not necessarily the lack of one of the cell components, such as the micronucleus, but rather that in a protozoan cell there is a potential plane of division, and when the cell is cut this zone can be present in only one of the frag- 388 DONNELL BROOKS YOUNG ments. If this were the case, it would appear that a cell cut below the center would lead to division of the anterior part only, the posterior part not having the division plane present. In Uronychia, however, in some cases the smaller posterior piece was the one to divide, while the anterior part with the so-called division zone died without division after it had regenerated. That a division zone does exist in Uronychia is indicated by those cells which were cut from five hours after division up to the time of the next division. In many cases the subsequent division, after regeneration, of these individuals resulted in the formation of two unequal cells, and in every case the smaller cell was from the side which had been injured by the cutting. This division zone is not as marked in Uronychia as that which Cal- kins describes for Paramecium and it is not developed until a few hours after division. This is shown by the fact that the size of the daughter cells resulting from a regenerated indi- vidual did not differ to any appreciable extent unless the cutting had been done five or more hours after division. SUMMARY 1. Three species of Uronychia are found at Woods Hole, one of which has two micronuclei, while the others have but one. 2. In Uronychia all the old cirri are absorbed during the divi- sion of the animal and new ones are formed precociously. 3. The power to regenerate parts lost by cutting or other injury is not always dependent upon the presence of a micro- nucleus. 4, The ability to grow and divide is dependent on the presence of a micronucleus. 5. The power to regenerate lost parts varies with the age of the cell, being least shortly after division and increasing up to the next division, being best developed at the start of division. 6. In division, nuclear changes and cytoplasmic changes pro- egress together, and it is difficult to tell which starts the process. 7. In Uronychia the large cirri are highly differentiated, in that they do not regenerate unless the body is injured. The formative agency of the cirri lies in the body protoplasm, namely, MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA 389 the basal bodies. Simply cutting the cirri without injuring the basal bodies does not result in regeneration. 8. Abnormalities may be produced in Uronychia if the micro- nucleus is injured. Such forms die without dividing. 9. There is evidently a division plane established at a fairly early period in the cell, and this is not altered by cutting the cell. LITERATURE CITED Bitscuur, O. 1885 Bronn’s Klassen und Ordnungen des Thier-Reichs. Bd. 1, Abth. ITI. CLAPAREDE ET LACHMANN 1856-57 Etudes sur les Infusoires et les Rhizopodes. Inst. Nat. Génevois, T. 4-5, pp. 184. Cauxins, G.N. 1904 Studies on the life history of Protozoa. IV. Death of the A series. Jour. Ex. Zoél., vol. 1, p. 422. 1911 Regeneration and cell division in Uronychia. Jour. of Ex. Zool., Feb., p. 95. 1911 Effects produced by cutting Paramecium cells. Biol. Bull., vol. PALS 10s, X05 1901 Marine Protozoa of Woods Hole. U. 8S. Fish Commission Bulletin. 1912 The paedogamous conjugationof Blepharisma undulans St. Jour. Morph., vol. 28. Couns, B. 1909 La conjugaison d’Anoplophyra branchiarium., Arch. de Zool. Exp. et Gen., T. 1, ser. 5. Dawson, J. A. 1920 An experimental study of an amicronucleate Oxytricha. Jour. Exp. Zo6l., vol. 30. DorteIn, F. 1916 Lehrbuch der Protozoenkunde, Vierte Auflage. Jena. Entz, G. 1909 Studien itiber Organization und Biologie der Tintinniden. Arch. fiir Prot., Bd. 15. GruBER, A. 1883-85 Ueber kiinstliche Theilung bei Infusorien. Biol. Centralb., Bdi3;, 5, 5095.bd.,4, S.7175d.5,,5: 137. Homes, 8. J. 1907 The behavior of Loxophyllum and its relation to regen- eration. Jour. Exp. Zodl., vol. 4, p. 399. Kent, W.8. 1881 Manual of the Infusoria. LeDantec La régénération du micronucleus chez quelques Infusoires ciliés. ©; KR. Acad, Set. Paris, 2.125. Lewin, K. R. 1910-12 Nuclear relations of Paramecium caudatum during the asexual period. Camb. Phil. Soc. Proc., vol. 16, p. 39. 1911 The behavior of the infusorian micronucleus in regeneration. Roy. Soc. Proc., Ser. B., vol. 84, p. 382. Lunp, B. L. 1918 The toxie action of KCN and its relation to the state of nutrition and age of the cell. Biol. Bull., vol. 35, p. 207. Maier, H. N. 1903 Der feinere Bau der Wimperapparate der Infusorien. Arche tur Eroterodaemon tot Mavpas, E. 1883 Contributions a 1’étude morpholigique et anatomique des Infusoires ciliés. Arch. de Zool. Exp., 2Ser., T. 1. 390 DONNELL BROOKS YOUNG Mercaur, M. Opalina: its anatomy and reproduction with a description of in- fection experiments and a chronological review of the literature. Arch. fiir Protistenk., Bd. 13. Meyer, A. 1904 Orientierende Untersuchungen iiber Verbreitung, Morpho- logie und Chemie des Volutins. Botan. Ztg., Bd. 62,8. 113. Mincutn, B.A. 1912 Anintroduction to the study of the Protozoa. London. Moraan, T. H. 1901 Regeneration of proportionate structures in Stentor. Biol Bull voles pralle ReicHenow, E. 1909 Untersuchungen an Haematococcus pluvialis nebst Bemerkungen iiber andere Flagellaten. Arb. a. d. Kais. Ges.-Amte, Bd. 33. 8. 1. Stevens, N. M. 1904 Further studies on the ciliate Infusoria Lichnophora. Arch. fiir Protist., Bd. 3. Stein, F. 1859 Der Organismus der Infusionsthiere, I: Abtheilung. WALLENGREN, H. 1902 Zur Kenntnis des Neubildungs- und Resorptions pro- cesses bei der hypotrichen Infusorien. Zool. Jahr. Abt. fiir Anst. der Thiere, Bd. 15, 8. 1. Yocom, H. B. 1918 The neuromotor apparatus of Euplotes patella. Univ. of Cal. Pub. in Zo6l., vol. 18. PLATE 1 EXPLANATION OF FIGURES Uronychia setigera. From life. Dorsal view. U. setigera. From life. Ventral view. .setigera. From life. Side view. U. binucleata. From life. Ventral view. U. binucleata. From life. Side view. 6 U. binucleata. From life. Dorsal view. 7 U.transfuga. Dorsal view of a stained preparation. oF WN re S| MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA PLATE DONNELL BROGKS YOUNG 39] PLATE 2 EXPLANATION OF FIGURES 8 U. transfuga. From Wallengren, ‘‘Zur Kenntnis des Neubildungs- und Resorptions-processes bei der Theilung der hypotrichen Infusorien,”’ fig. C. 9 U.transfuga. From Biitschli, plate LX XII, fig. 4a. 10 U.transfuga. From Biitschli, plate LXXIT, fig. 4b. 11 U.setigera. From Calkins, ‘‘Marine Protozoa of Woods Hole,”’ fig. 55. 12 U.transfuga. From Calkins, ‘‘ Regeneration and Cell Division in Urony- chia’ fig. 1 13. U.binucleata. Dorsal view of a stained preparation. 14 U.setigera. Side view of astained preparation. 15 U.setigera. Ventral view of a stained preparation. 16 U.setigera. Ventral view of an early stage in division. From a stained preparation. 17 U. setigera. Ventral view of the first stage in division. From a stained preparation. MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA DONNELL BROOKS YOUNG 393 PLATE 2 PLATE 3 EXPLANATION OF FIGURES 18 U. setigera. Dorsal view of about the same stage as shown in figure 17. From a stained preparation. 19 U. setigera. Dorsal view of a mid-division stage. From a stained prepa- ration. 20 U. setigera. Ventral view of a mid-division stage. From a stained preparation. 21 U. setigera. Ventral view of the daughter cells just ready to separate. From a stained preparation. 22 U. setigera. An individual which was cut in such a way that the micro- nucleus was destroyed. The specimen was stained thirty-six hours after cutting. 23 U.setigera. An individual which was cut in a median transverse plane at an early division stage. The micronucleus was evidently destroyed. Regenera- tionwascomplete. Experiment no. 3. 24 U. setigera. Another individual cut as in the case of experiment no. 3. Experiment no. 65, 25 U.setigera. A monster resulting from a cell injured in an early division stage. Experiment no. 114. 26 U. setigera. An abnormal cell which developed as the result of a cut which almost separated the two pieces. They rotated on each other and fused as shown. 27 U. setigera. A cell which had the posterior cirri cut and which did not regenerate them until division. Thissketch was made twelve hours after the cirri were cut. 28 U. setigera. A monster which developed from one of the amicronucleate cells of experiment no. 131. 394 MORPHOLOGY AND PHYSIOLOGY OF GENUS URONYCHIA PLATE 3 DONNELL BROOKS YOUNG pA \ 395 PROMPT PUBLICATION The Author can greatly assist the Publishers of this Journal in attaining prompt publication of his paper by following these four suggestions: 1. Abstract. Send with the manuscript an Abstract containing not more than 250 words, in the precise form of The Bibliographie Service Card, so that the paper when accepted can be scheduled for a definite issue as soon as received by the Publisher from the Editor. 2. Manuscript. Send the Manuscript to the Editor prepared as described in the Notice to Contributors, to conform to the style of the Journal (see third page of cover). 3. Illustrations. Send the Illustrations in complete and fin- ished form for engraving, drawings and photographs being pro- tected from bending or breaking when shipped by mail or express. 4. Proofs. Send the Publisher early notice of any change in your address, to obviate delay. Carefully correct and mail proofs to the Editor as soon as possible after their arrival. By assuming and meeting these responsibilities, the author avoids loss of time, correspondence that may be required to get the Abstract, Manuscript and Illustrations in proper form, and does all in his power to obtain prompt publication. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 4 NOVEMBER, 1922 Resumen por el autor, W. W. Swingle. Experimentos sobre la metamorfosis de los anfibios neoténicos. 1. El autor ha alimentado Necturus adultos con grandes cantidades de tiroides, en plena actividad fisiol6gica, y lébulo anterior de la glaindula pituitaria, transplantando simultdnea- mente la tiroides delarana. Los resultados han sido negativos al cabo de cuatro meses, a pesar de la enorme cantidad de tiroides ingerida. 2. Las tiroides de Necturus normales fueron trans- plantadas en larvas j6venes de Rana clamata, desprovistas atin de miembros. Al cabo de diez a catorce dias aparecieron los sintomas de hipertiroidismo, tales como el desarrollo de los miembros y atrofia casi completa de la cola. A pesar de sus caracteres larvarios, Necturus posee glandulas tiroideas activas y parece haber perdido su capacidad de transformarse bajo el estimulo de la alimentacién tiroidea. 3. La tiroides de un ajo- lote de 14.25 pulgadas de longitud y por lo menos de cuatro ahlos de edad fué transplantada en larvas de R. clamata, des- provistas atin de miembros. La glandula era grande, vascular y las vesiculas estaban distendidas por el coloide. Se corté en seis pedazos, que fueron injertados en otras tantas larvas. Un renacuajo murié; los restantes presentaron la reaccién hipertiroidea tipica a los ocho dias. Dos semanas después del injerto las larvas presentaban patas y marcada reabsorcién de la cola. De este modo, una sola tiroides de ajolote contiene bastante cantidad de hormoén activo para metamorfosear prac- ticamente a cinco renacuajos, pero cuando se la deja persistir dentro del ajolote no puede iniciar la metamorfosis. La neotenia del ajolote parece depender aparentemente de la incapacidad de la glandula tiroides para verter su hormén, completamente formado y fisiol6gicamente activo, en la sangre. Un factor que permite la excreccién falta en este caso. Los ajolotes se met- — amorfosean rapidamente cuando se les alimenta con grandes can- tidades de tiroides. Experimentos semejantes fueron llevados a cabo con la tiroides de anuros neoténicos. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 2 EXPERIMENTS ON THE METAMORPHOSIS OF NEOTENOUS AMPHIBIANS W.W.SWINGLE Osborn Zoélogical Laboratory, Yale University TWO TEXT FIGURES AND TWO PLATES (EIGHT FIGURES) The discovery of thecausal relationship existing between certain endocrine secretions and amphibian metamorphosis has served to stimulate anew the interest of investigators in the problem of neoteny and paedogenesis as presented by various species and genera of this vertebrate group. The retention of the larval form either permanently or for periods far beyond the normal time required for metamorphosis is known as neoteny or neotenie (Kollmann, ’82). This author, who has studied the problem carefully, distinguishes between partial neoteny, where the animal is simply retarded in meta- morphosis beyond the normal time and passes the winter as a tadpole, and total neoteny in which case the animal retains its gills and other larval characters becoming sexually mature in this condition. Partial neoteny is quite common in such frog species as R. clamata and R. catesbeiana as there is a pronounced tendency of these forms to prolong the larval life an extra six or eight months beyond the usual one- and two-year period. Indeed, the larval life of Rana catesbeiana may extend to the period of sexual maturity in some tadpoles in so far as the possession of ripe spermatozoa is concerned, and this appears to be the explana- tion of the second larval sexual cycle described by the writer in a previous paper (721). The classical example of total neoteny is axolotl, the paedo- genetic larva of Amblystoma tigrinum. The researches of Duméril (’65), Chauvain (’75) and others on the metamorphosis of axolotl have resulted in the more or less current belief that the 397 398 W. W. SWINGLE peculiar group of aquatic amphibians known as perennibran- chiates (Necturus, Proteus, Typhlomolge, etc.) are permanent larval forms capable of reproducing the species. The group is supposed to represent a sort of retrograde evolution from an originally terrestrial life to permanent aquatic existence by suppression of metamorphosis. The evidence for this view is suggestive; briefly stated it is somewhat as follows: 1. It is.generally conceded that the perennibranchiates do not form a natural group, but are to be regarded as a hetero- geneous assembly; various genera are undoubtedly represented in the group. These animals probably became neotenic at a phylogenetically old stage and are hence the oldest and not the youngest members of the present-day urodeles. 2. Various anatomical features of the group, such as the pentadactyloid limb, presence of lungs, suppression of internal gills, and connection of the pelvic girdle with the vertebral column, point to a terrestrial existence somewhere in the history of the group. 3. Perhaps the most suggestive line of evidence for the view that the perennibranchiates are permanent larval forms is the occurrence of neoteny and paedogenesis as aberrations of develop- ment in semiterrestrial species of urodeles. For instance, it has long been known that the larvae of certain European salamanders fail to undergo metamorphosis and occasionally attain the size of 80 mm., whereas the normal size at transformation is 40 mm. Larvae of Triton have been reported 80 to 90 mm. long with functional gills and sexual organs fully developed. DeFilippi (61) found in one locality in Lombardy sexually mature larvae. According to him, such gill breathing, sexually mature specimens occur constantly in a small lake in the province of Ossola in the Italian Alps. Many other cases have been reported, and the classical example of the axolotl is well known. It will be recalled that this creature was classified by systematists as a distinct species of perennibranchiata until Duméril described its meta- morphosis into Amblystoma tigrinum. 4. Lastly, it has been repeatedly stated in the literature that one of the perennibranchiates, Typhlomolge, is hereditarily METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 399 lacking in the thyroid apparatus, which, if true, accounts for the suppressed metamorphosis. It will be recalled that it is possible to induce neoteny in anuran larvae by thyroid extirpation. This evidence is suggestive, and taken in conjunction with what we know of the thyroid gland and its relation to metamor- phosis suggests that the thyroid mechanism of forms such as Necturus, axolotl, and neotenous anuran larvae is defective and incapable of bringing about transformation. EXPERIMENTS ON PERENNIBRANCHIATES (NECTURUS MACULATUS) A group of adult necturus were obtained from the Ohio Valley and repeatedly injected with thyroid extract, and at the same time forcibly fed large quantities of the desiccated commercial preparation by means of a long glass pipette thrust down the throat. This procedure was repeated several times with negative results, despite the fact that the thyroid dosage was relatively enormous. ‘The physiological activity of the thyroid prepara- tion was tested by feeding small amounts to larvae of R. clamata, averaging 50 mm. total length, but without hind legs. ‘These animals promptly showed marked indications of metamorphosis within eight days from the date of feeding (fig. 7). The iodine content of the desiccated tissue was given on the label of the bottle as 0.21 per cent by weight and the analysis made by the chemists of Parke, Davis & Company, Detroit. It is obvious that the thyroid preparation used in the experiment was not responsible for the negative results. One animal was injected twice intraperitoneally with 10 mg. of thyroxin iodine obtained from the laboratories of EK. R. Squibb & Sons, and then forcibly fed large quantities of desiccated thyroid and anterior pituitary lobe substance. The physiological activity of the thyroxin iodine was tested by placing two young specimens of. Amblystoma punctatum in a 1 to 50,000 solution, whereupon they metamor- phosed within two weeks. None of these agents, singly or taken together, produced the slightest indications of metamorphosis, nor, I may add, appeared to harm the Necturus in any way. As a last resort, three thyroid glands of newly metamorphosed 400 W. W. SWINGLE Rana clamata frogs were engrafted subcutaneously in one animal. Four months later, no metamorphic changes had appeared. The experiment was abandoned, with the conviction firm in the writer’s mind that, although adult Necturus may possibly be induced to metamorphose, thyroid tissue alone is not the agent that will accomplish the transformation. Axolotls readily re- spond to thyroid administration by metamorphosis according to Laufberger (713) (cited by Adler, ’16), Huxley (’20), and others. Examination was made of the thyroids of untreated Necturus, but nothing unusual was observed, except that in some animals the glands are small and the vesicles more or less isolated from each other, while in other animals the glands may be large. The blood supply appeared normal in those individuals with compact glands. The thyroid of one animal consisted of but four to six extremely large follicles on either side, in others the gland was larger and comprised ten to sixteen large follicles, while in one animal the glands consisted of twenty-one or twenty-two follicles on each side. In the last-mentioned case the animal had been treated with thyroid and thyroxin iodine several weeks previous to examination. In order to test the physiological activity, a series of hetero- plastic thyroid transplantation experiments were made. In the first set of experiments one-half of the thyroid gland (from one side only) was engrafted intraperitoneally into immature Rana clamata tadpoles averaging 52 mm. total length, with hind-leg buds 2.7 mm. long, but undifferentiated. Within eight days the tadpoles which received the graft showed all the symptoms of hyperthyroidism. The animals were greatly emaciated, with protruding eyes; the hind legs increased in length from 2.7 mm. to 8mm. with complete differentiation. In two animals the right fore leg had appeared and the tail atrophied to half its original length. At the end of the eighth day from the date of trans- plantation the larvae measured 26 mm. total length, whereas the week previous the same individuals averaged 52 mm. It was impossible to keep the tadpoles alive until they completely metamorphosed, so great was the acceleration of metabolism and metamorphic change. Death in most cases was apparently METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 401 due to respiratory difficulties. Several tadpoles were kept alive until tail resorption was nearly complete by placing them in shallow containers and passing a stream of compressed air through the water. In a later series of experiments the thyroids were cut into small pieces and each part transplanted separately into immature tadpoles without hind limbs. One animal received three large colloid-filled vesicles dissected out of the gland; another received four follicles; the remainder received seven large follicles each. The grafts were made July 26th, and on July 30th, when ex- amined, several engrafted individuals showed evidences of hyperthyroidism, such as emaciation and limb development. By August 2nd all of the animals showed a marked reaction to the graft; emaciation was very marked, the eyes protruded, and the legs had greatly increased in length. One animal had the right fore leg through the skin, and the remainder showed autol- ysis of the skin in the region where the fore limb later appears. The control animals remained unchanged (figs. 3 to 6). August 7th, when the experiment was discontinued, all engrafted animals were in advanced stages of metamorphosis. During the course of the experiment the animals were fed quantities of Spirogyra. ° They fed very little after the first three or four days, and not at all following the onset of marked metamorphic change. This experiment shows clearly that the thyroid glands of adult Necturus are highly active metamorphosis-inducing agents. It is reasonable to assume that if they are capable of producing marked metamorphic changes when transplanted into immature anuran larvae within eight days, they are potentially competent” of doing likewise in Necturus if this animal still retained any capacity to transform. It will be shown later that the relation of the thyroids to metamorphosis in Necturus is quite different from the situation existing in axolotls and neotenous anurans. While writing this paper the writer came upon a statement by Uhlenhuth (21) that Jensen (’14) had subjected Proteus to the action of thyroid substance, but did not get any demonstrable results. Uhlenhuth’s comment upon this experiment is interest- ing. He says: ‘‘Many causes may have been responsible for this 402 W. W. SWINGLE failure (i.e., Jensen’s), in particular the fact that the animals were too old when they were subjected to the thyroid feeding.” And elsewhere the same author states “that nothing is known of the endocrine system of Proteus.” The writer ventures to predict that if the thyroid glands of an untreated Proteus are engrafted into anuran larvae, the latter will react similarly to those engrafted with Necturus glands. These experiments negative any such assumption as Uhlenhuth’s (21, page 201) that, “if the thyroid substance is capable of causing the development of the characters of a terrestrial amphib- ian, the administration of thyroid substance should cause the metamorphosis of Proteus anguineus.’”’ This writer apparently holds the view that any perennibranchiate will, if fed thyroid, metamorphose, and that retention of larval characters in these forms is due to absence or defect of the thyroid mechanism. The present experiments lend no support to any such hypothesis. In regard to the statement that the endocrine system of Proteus is unknown, it is interesting to note that Franz Leydig in 1853 (p. 62), describes the thyroids of this animal. According to his description, they apparently do not differ greatly from those of Necturus. Uhlenhuth lays great emphasis upon the fact that one of the perennibranchiates, Typhlomolge rathbuni, is said to lack the thyroid gland, and states in effect that the reason for the retention of the larval characters is due to the thyroid absence. Perhaps this is true, but it strikes the writer as being rather odd that the retention of larval characters of this perennibranchiate should depend upon the absence of the thyroid function, and that such characters are retained in another form (Necturus) in spite of the presence of a most potent and active thyroid, in spite of feeding, injecting, and engrafting of thyroid substance. There is another point of interest in connection with the dis- cussion of Typhlomolge, and that is the apparent absence of thyroid glands in adults of this peculiar animal. Emerson (’05) studied the general anatomy of two specimens preserved in 4 per cent formalin, and merely mentions in the course of her dis- cussions, ‘‘Sections of the head reveal the presence of a thymus METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 403 gland, but I do not find thyroids.’’ This is the only mention made of the thyroids in her paper. Recently, through courtesy of Prof. H. H. Wilder, the writer had an opportunity of examining Miss Emerson’s material, consisting of serial sections through the head of one animal. No trace of a thyroid was observed, but it should be stated that some of the epithelial structures had disappeared. The writer has carefully examined three adult specimens of Typhlomolge and failed to find any trace of a thyroid. The entire lower-jaw region, back to and including the heart, was dissected under a high-power binocular microscope, and some tissue sectioned, but with negative results. However, the failure to find the glands does not necessarily mean that they were not present or had not been present at some period, because the animals had been preserved over fifteen years in alcohol and many of the epithelial structures had undergone disintegration during this interval. If the thyroid mechanism of Typhlomolge is congenitally lacking, then this amphibian is the only vertebrate known in which the gland is normally absent. It has been stated in the literature that Typhlomolgeis only the neotenic larva of Spelerpes, but it is well known that Spelerpes larvae possess thyroids. It seems probable that the Texas cave salamander has a thyroid, but that it develops as a diffuse aggregation of follicles, somewhat similar to the condition known to exist in teleosts. At any rate the question of the presence or absence of the gland deserves further investigation before it can be accepted as an established fact, because the history of vertebrate morphology is replete with descriptions of forms supposedly anomalous for the lack of certain structures, only to be later shown to possess them. To sum up, it may be said that these experiments indicate that Necturus and probably other perennibranchiates have per- manently lost their ability to metamorphose into terrestrial forms under the stimulus of thyroid administration alone: our experi- ments indicate that the thyroid apparatus of these animals is highly active and potent despite their larval characters. 404 W. W. SWINGLE These experiments, however, do not rule out the possibility of inducing the metamorphosis of perennibranchiates by other means than that of thyroid feeding or transplantation. The cause of the non-metamorphosis of these forms may be pluri- glandular in origin, and a result of defective interrelation of various endocrine glands. It should be added that the writer fed one Necturus small quantities of desiccated ovarian, testicular, ad- renal, and anterior pituitary lobe tissues, along with large quanti- ties of thyroid extract, but without avail. It is probable that transplants of these various glands simultaneously would have had more effect than feeding the desiccated substances, in case the animal possessed the capacity to transform. Administration of endocrine secretions, no matter in what quantity given, can give positive results only when acting upon an appropriate hered- itary substratum. The indications are that the hereditary factors concerned in the metamorphosis of Necturus have become so modified that the appropriate substratum is lacking, thus render- ing the thyroid hormone powerless. It is obvious that hereditary conditions in the perennibranchiates are quite different from those in axolotl, in regard to metamorphosis, since the latter readily respond to thyroid feeding and the former do not. EXPERIMENTS WITH AXOLOTL THYROIDS Through the courtesy of Prof. Henry Laurens, of the Depart- ment of Physiology, the writer obtained a very large specimen of Axolotl mexicanum (neotenic larva of Amblystoma tigrinum) for thyroid transplantation work. The animal was a very large one, measuring 14.25 inches from snout to tail-tip. The exact age of the specimen is unknown, as it was obtained, along with several others, from Albuquerque, New Mexico. When first brought to the laboratory the animal was about 8 inches long, and hence presumably about two years of age at the time; it was kept under laboratory conditions for two more years, thus making four years the animal’s approximate age when used by me. This specimen was the only one of the lot that failed to metamorphose within a few months following removal from its native habitat to New Haven. METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 405 The thyroid glands were compact and large—larger than the glands of newly metamorphosed R. clamata frogs—and made up of large and small follicles filled with colloid. The blood supply to the glands was rich, apparently much more so than is the case with larval anurans; only a superficial examination was made to test this point because of the lack of sufficient material. The pituitary gland was also examined; the pars anterior seemed normal or possibly rather small when the relative sizes of all the lobes are considered. The thyroids were dissected out and each gland cut into three pieces of approximately equal size. Each piece of tissue was then transplanted intraperitoneally into immature Rana clamata larvae averaging 51.5 mm. total length with hind-leg buds 2.2 mm. undifferentiated. A few hours following grafting one tadpole jumped out of the container and was found dead, leaving but five transplanted tadpoles. Eight days after transplanting the pieces of axolotl thyroid, the engrafted tadpoles showed all the characteristic features of hyperthyroidism, such as cessation of growth, marked acceleration of limb development, tail atrophy and resorption, and body emaciation. August 20th, or twelve days after transplantation, four of the engrafted animals were found dead (figs. 8 to 10). The photo- graphs show very well the marked tail atrophy and resorption and the fore-leg development. The early death of the animals was due to the great acceleration of metabolism and metamorphic change. Undoubtedly, smaller pieces of the axolotl thyroid would have had the same effect upon metamorphosis without the too destructive rise in katabolic activity. The amount of tail resorption can be judged by the fact that during the twelve days of the experiment the average total length of the tadpoles decreased from 51.5 mm. to 29.6 mm. The control animals remained unchanged.! 1 Since this was written one hundred and nine large axolotls were obtained by Professor Harrison from Albuquerque, New Mexico, and given to me for experi- mentation. This experiment was repeated on a large scale with identical results. Mr. Carl Mason, of this laboratory, metamorphosed thirteen normal, thyroidless, and pituitaryless R. sylvatica tadpoles by transplanting pieces of the thyroid of a single 14-inch axolotl. 406 W. W. SWINGLE The pituitary gland of the axolotl was also engrafted into an immature larva of R. clamata, measuring 45 mm. total length, hind limbs 2.5 mm. long and differentiated into thigh and shank, but without toe points. All three lobes of the gland were trans- planted together. ‘The success of the graft was attested by the color change in the larva induced by the expansive action of the pars intermedia secretion upon the melanophore system. Eighteen days after the graft was made the animal had not changed in any way either in regard to limb development or growth, so the experiment was abandoned. Examination of the implanted gland showed it to be mostly resorbed. The negative result following grafting of the axolotl pituitary is in striking contrast to that obtained when the pituitary of a newly metamorphosed frog is transplanted into immature anuran larvae. However, it must be remembered that in the latter case we are dealing with a homoplastic graft and in the former with a heteroplastic one, and a single transplant at that. It is un- fortunate that a larger amount of axolotl material was not available, for it is of importance to know whether or not the pituitary gland of the axolotl is active, and some idea of its potency can be obtained by testing its effect upon limb develop- ment of anurans. If the results are consistently negative, then it is probable that the gland is defective in so far as its relation to the thyroid mechanism is concerned. The results obtained by grafting portions of axolotl thyroids, are clear-cut and admit of but one interpretation: namely, that the thyroid apparatus of this animal is highly active and potent in inducing marked metamorphic change when transplanted into immature anurans, but is apparently incapable of initiating metamorphosis when left unmolested in its normal place. This experiment seems to rule out the idea that the axolotl’s thyroid secretion is defective. If the thyroid glands of a single axolotl when cut into six fragments are capable of initiating metamorpho- sis in five anuran larvae grafted with a single fragment (the sixth animal died) within ten to fourteen days, surely we may safely assume that the same glands, entire, contain enough of the active hormone to initiate metamorphosis in the single axolotl of which METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 407 they originally formed a part. The metamorphosis of an anuran larva involves much more fundamental transformation and reorganization of tissues and organs than the same process in axolotl. Our experiment suggests four possible factors to account for this anomalous situation: 1) Possibly the blood supply taking the hormone away from the gland is defective and the thyroid consequently unable to release its secretion. 2) The thyroid is able to collect, store, and transform the incoming iodine taken from the food and water into the physiologically active hormone, but owing to defective nervous stimulation the gland is unable to release the secretion into the blood stream. 3) The secretion, though perfectly formed, is unable to escape from the gland, owing to some defective interrelation between the pituitary and thyroid, or pessibly some other endocrine gland which supplies the necessary stimulus to the thyroid, thereby acting as the releas- ing factor. 4) The blood and tissues of neotenous forms may contain substances that neutralize or render impotent the meta- morphosis-inducing agent of the thyroid hormone. If it is true, however, that the thyroid hormone is unable to escape because of defective outlet through the blood stream, or because of defective interrelationship between various com- ponents of the endocrine system, how is it that axolotls usually promptly metamorphose when taken from their native habitat or when subjected to sudden environmental changes, such as a change from New Mexico to New Haven?? Furthermore, aside .from the nervous system, there are few anatomical or physio- logical mechanisms which hold such power over other structures as to permit a gland like the thyroid to manufacture and store in large quantities a highly complex substance, but apparently prohibits its release. When we consider the thyroid glands of an axolotl filled to capacity with highly active secretion, as our experiments clearly show, yet apparently unable to release the 2 Of the 109 axolotls received from Albuquerque, only those that were thyroid- ectomized failed to metamorphose spontaneously a few weeks after removal from their native habitat. Left unmolested in the New Mexican environment, the animals may remain permanent larvae and grow to a length of 14 inches. 408 W. W. SWINGLE hormone into the network of capillaries surrounding the gland in sufficient quantities to induce transformation, we are led to the conclusion that in the last analysis the crux of the problem is defective stimulation (perhaps inhibition) of the gland by that portion of the animal’s nervous system responsible for the flow of secretion under normal conditions. How else can one reduce to harmony the multiplicity of factors that have been invoked to explain axolotl metamorphosis, save by reducing them all to a common factor: 1.e., agents which pro- duce their effect by subjecting the organism to more or less violent changes of the environment thus acting as a constant ‘nervous stimulant? For example, a few of the agents (aside from thyroid or iodine feeding)’ that have served to initiate axolotl metamor- phosis are: sudden changes in food supply, drying of swamps or pools in which the animals live, changes in the temperature of the water (Shufeldt, ’85); forcing the animals to breathe air, insuf- ficiently aerated water (Chauvin, ’75, ’77); administration of salicylic acid (Kaufman, 718); shifting of the animal from its normal habitat to other districts, such as from New Mexico to New Haven. A glance at this list of factors indicates that their varied nature alone negatives the idea that any one of them can be the real causative factor in axolotl metamorphosis. However, all can be classed as shocks to the organism, and it may possibly be that such more or less constant excitation may bring about nervous stimulation to the thyroid sufficient to overcome the inhibiting influence and release the stored secretion, thus initiating meta-. morphosis. The nature of the inhibiting factor is of course the crux of the problem, and in the last analysis is probably of endo- crine origin acting through the intermediation of the nervous 3 Apropos of Uhlenhuth’s claims that iodine has nothing to do with axolotl metamorphosis, the recent papers of Jensen and Hirschler are of interest. Jensen (Compt. Rend. Soe. de Biol., T. 85, 1921) metamorphosed axolotls by injections of iodocasein, iodoserumglobulin, and iodoserumalbumin. Also by feeding with an organic iodine compound—iodo-thyrosin. Hirschler (Arch. Entw. Mech., 1922 metamorphosed axolotls and anuran tadpoles by feeding elemental iodine in various forms. These investigators worked on strains of axolotls which do not spontaneously transform. METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 409 system. On theoretical grounds, the writer believes that elec- trical stimulation, thyroid puncture, and extirpation of the thyroid and reimplanting it into the same individual will metamorphose axolotl, but to date has been unable to obtain sufficient animals for experimentation along the lines indicated. If such procedure should cause metamorphosis, then it is clear that the physiologically active hormone is not released from the thyroid in sufficient quantity to induce transformation. From the evidence at hand it seems to the writer that such is probably the case. Axolotls readily respond to thyroid feeding or to in- jections of iodothyrine by transforming, and the amount of thyroid substance required is not excessive. If the assumption were correct that the blood and tissues of this neotenic form con- tained substances which neutralized or rendered impotent the thyroid hormone, thus preventing metamorphosis, why should small amounts of thyroid substance, when fed, be able to pro- duce an effect? The evidence obtained from thyroid transplanta- tion experiments with neotenous anurans is interesting in this connection. EXPERIMENTS WITH THE THYROIDS OF NEOTENOUS ANURANS The larvae of the green frog, Rana clamata, have a larval period of approximately one year: i.e., 370 to 400 days from the date of egg deposition. The animals attain a length of about 65 mm. at metamorphosis, which occurs in late July and early August. However, it has been repeatedly observed by the writer that many larvae fail to transform at the usual time and remain an extra year as tadpoles. Such animals are typically neotenous forms and, as they continue growing throughout the larval period, they ultimately reach a size considerably in excess of that generally exhibited by the species at metamorphosis. Larvae measuring 75 to 90 mm. total length, with differentiated hind legs varying from 4 to 20 mm., have been captured in the months of November, December, and January from various pools in the vicinity of New Haven. The tendency of the species is to prolong rather than curtail the span of larval existence. Because of this fact, a series of thyroid-transplantation experi- 410 W. W. SWINGLE ments were carried out with these neotenous individuals, in the hope of determining the endocrine locus responsible for the failure to metamorphose at the proper time. The first procedure was to- test the physiological activity of the thyroid apparatus of such animals by transplanting the glands into immature larvae of the same species with undifferentiated legs and noting the effect upon metamorphosis. December 17, 1920, three immature larvae, averaging 33.5 mm. total length, without hind limbs, except undifferentiated epithe- lial buds, were engrafted with the thyroid glands of 80-mm. neotenous larvae with hind legs 11.6 mm. long. The engrafted - forms were the smallest obtainable at this season of the year and not over six months of age. It is unfortunate that alarger number could not have been used, but the results are clean-cut, as will be seen later, when the experiment was rechecked with another group of animals in midsumnier. One animal was in advanced stages of metamorphosis on January 17th, just one month from date of grafting. The right fore leg was through the skin and the left fore leg appeared the day following (January 18th). For a week previous to the first appearance of the fore limbs the engrafted animal showed marked signs or hyperthyroidism, such as emaciation, protrusion of the eyes, slight tail atrophy, and autolysis of the skin over the region of the fore legs. The control larvae at this time averaged 34 to 38 mm. in length and showed no change in regard to leg devel- opment from the condition when the experiment was started. The other two larvae of the engrafted culture developed fore limbs January 22nd and typical frog mouth. All of the animals died before tail resorption was complete. Figure 1 is a drawing of an engrafted larva and its control. Figure 2 shows the neotenous type of tadpole from which the thyroid glands were taken. Generally the limb development is less marked than the drawing indicates. When the drawings were made the animals were nearly a year past their normal time of metamorphosis. Briefly summarized, the experiment shows that transplantation of the thyroid glands of neotenous larvae 80 mm. total length, with differentiated hind limbs 11.6 mm., into immature tadpoles METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 411 33.5 mm., without limbs, brings about very marked metamorphic changes within thirty days, although the animals could not be reared to the stage of complete tail resorption. It was observed that in the engrafted animals autolysis of the skin over the region of the fore limbs occurs independently of limb development as a distinct phenomenon of anuran meta- morphosis. Years ago Braus (’06) described similar phenomena in developing tadpoles after extirpation of the limb bud. I men- tion it here because of the remarkable autolysis which is sometimes observed in transplanted larvae; the fore limbs may be small, whereas the skin area destroyed may be very large indeed com- pared with limb size. It should be remembered, however, that in anurans the fore and hind legs tend to keep pace with one another in development, only the fore legs are not visible because of the opercular covering. The chief point of interest, however, is the odd fact that much greater metamorphic change follows transplantation of the thy- roids of 80-mm. neotenous larvae with hind legs 11.6 mm. into immature larvae without limbs than occurs in control animals 80 mm. with legs 11.5 mm. In other words, the grafted glands wrought far greater changes in the same time interval (approx- imately one month) when transplanted into immature larvae than when left undisturbed in the mature forms. This result, so curious and at variance with what one might expect, led to a repetition of the same experiment the following summer, from a different angle. Immature larvae averaging 40 mm. total length with hind leg buds 0.5 mm. were transplanted with the thyroids of mature though not neotenous tadpoles 68 mm. total length with hind legs 11.5 mm. The results were similar to those of the earlier experiments, though not so striking as one would expect, because the mature control animals (not neotenous) in this experiment were approaching metamorphosis at the end of the experiment, whereas in the previous experiment the neotenous controls showed no change, and in fact passed the winter in the laboratory as tadpoles. The experiments indicate that the thyroids of extra-season, neotenous anuran larvae with hind limbs 11 mm. long are physio- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, No. 4 412 W. W. SWINGLE logically active and capable of inducing metamorphosis, pro- vided their contained secretion could get into the blood stream. Like the thyroid mechanism of axolotl, the glands of these larvae seem to be rendered more or less functionless by an inhibiting factor which prevents secretion of the hormone into the circula- tion. Following transplantation, the inhibition apparently is overcome by the acquisition of a new blood and nerve supply A Fig.1 Immature R. clamata larvae engrafted with the thyroids of extra large neotenous tadpoles with hind limbs 11.6 mm. long, B, immature control animals of Agroups. X 2. in the new environment, because the absorbed secretion induces metamorphosis. A modification of the experiment just recorded was attempted; the thyroids of large neotenous larvae were engrafted intra- peritoneally into other extra-season animals of the same size and developmental stage. The idea was, that since the glands of such individuals are physiologically active and capable of METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 413 inducing marked metamorphic changes when transplanted into immature animals, they would probably produce similar effects in neotenous larvae while undergoing resorption in the foreign Fig. 2. Neotenous R. clamata tadpoles. Numbers of this type of larvae, measuring 75 to 90 mm. total length, with hind limbs 5 to 25 mm. long, pass an extra year beyond their usual period of metamorphosis in this stage. Drawing natural size. environment because inhibiting factors could not prevent the release of the contained secretion. The experiment is briefly presented in the following tables: 414 W. W. SWINGLE TABLE 9 December 22, 1920 ANIMALS FROM WHICH GLANDS = WG 7 Dy Ss ENGRAPFTED WERE TAKEN CONTROL Total length Hind legs Total length Hind legs Total length Hind legs mm. mm. mm. mm. mm. mm. 80 8.5 81 8.5 81 8.5 82 10.0 82 8.5 82 9.0 83 9.0 81 9 80 8.0 80 8.5 80 10 79 8.5 81 8.5 83 8 81 10.0 82 9.0 79 8.5 83 8.5 81.3 8.9 80.7 8.8 81 8.8 TABLE 10 January 80, 1921 ENGRAFTED CONTROL Total length Hind legs Total length Hind legs 80 . 25 80 9.5 82 24 81 10.0 80 24 82 10.0 83 23 81 10.5 82 26 83 10.0 81.4 | 24.4 81.4 10.0 Note: One engrafted animal died January 23rd. TABLE 11 March 1, 1921 ENGRAFTED * CONTROL Total length Hind legs Total length Hind legs mm, mm, mm, mm. 83 31 82 133 82 32 83 13 82 29 81 14 84 30 82 WPS 82.7 30.4 82 13.4 METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 415 One engrafted animal died February 25th. The experiment was abandoned March 10th, as some of the transplanted individ- uals had fore limbs. The data clearly indicate that the thyroid glands of neotenous tadpoles with hind legs 8 to 10 mm. are physiologically active and capable of inducing acceleration of limb growth and development when transplanted into other neotenous animals of similar size and developmental stage. However, metamorphic change following grafting is not so rapid or marked as when the same type of gland is implanted in im- mature (non-neotenous) larvae. But in either case the absorp- tion of the secretion of the grafted gland induced a greater reac- tion than the same gland is capable of producing (in the same time and under similar conditions) when left unmolested in the neotenous animal of which it was originally a part. SUMMARY OF CONCLUSIONS 1. Adult Necturus were fed, injected, and engrafted with physiologically active thyroid substance with negative results. The injection of 20 mg. of thyroxin iodine had no effect upon metamorphosis, whereas a 1 to 50,000 solution of this compound readily metamorphosed immature larvae of Amblystoma. The experiment indicates that perennibranchiate amphibians are permanent larvae and have lost the ability to transform under the stimulus of thyroid treatment. 2. Necturus differs markedly from the axolotl in its reaction to thyroid administration, as the latter readily metamorphoses when fed or injected with this substance. 3. The thyroid glands of the perennibranchiate Necturus are quite variable in size, and in some animals may occur as large vesicles more or less isolated from one another. In other in- dividuals the glands are rather small and may consist of but four to six extremely large vesicles. In Necturus the thyroids are generally located near the apex of the triangle formed by the geniohyoid and external ceratohyoid muscles. 4. Despite its larval characters, Necturus possesses thyroid glands of great physiological activity, as shown by heteroplastic transplantation into immature anuran larvae. 416 WwW. W. SWINGLE 5. A giant axolotl, 14.25 inches long, several years of age, was found to have a highly active metamorphosis-inducing thyroid apparatus. The thyroid of a single specimen cut into six pieces and transplanted metamorphosed five immature anuran tadpoles within two weeks. The sixth animal died following the operation. 6. The axolotl’s thyroid is normal in appearance and of large size, consisting of numerous large vesicles filled with colloid. The gland is surrounded by a rich network of capillaries. 7. The failure of the axolotl to metamorphose appears to be due to the inhibition or the defective development of some un- known factor which normally serves to release the fully formed hormone from the thyroid into the blood stream. It is suggested that defective nervous stimulation or perhaps inhibition is the immediate cause of retention of the secretion within the thyroid vesicles, but that in the last analysis some defect of interrelation of the various components of the endocrine system is probably responsible for the nervous inhibition or lack of normal stimulation. 8. Experiments on large neotenous anuran tadpoles indicate that the failure of these animals to metamorphose at the proper time probably is due to the same causes responsible for axolotl neoteny: 1.e., the thyroid glands apparently do not secrete their fully formed hormone into the blood stream because of some unknown inhibiting influence. The thyroid inhibition seems to be less marked in anurans than in the axolotl, since neotenous tadpoles eventually metamorphose if given sufficient time. 9. The next step in the analysis of amphibian neoteny is to determine the nature of the factor responsible for the failure of the thyroid to release its hormone (or at any rate to render it impotent in so far as metamorphosis is concerned). Is this unknown factor hormonal, or nervous, or both? 10. In the older work on amphibian neoteny too much stress was laid upon the exogenous factors as causative agents, and too little, if any at all, upon endogenous factors, and heredity. METAMORPHOSIS OF NEOTENOUS AMPHIBIANS 417 ADDENDUM Uhlenhuth’s claim that urodeles differ from anurans in that their metamorphosis is independent of iodine and influenced only by the thyroid hormone itself is rendered invalid by the following experiment: The thyroid glands of large axolotls (seven inches total length) were extirpated and the animals kept for five months in the laboratory following the operation, then injected twice with strong doses of tyrosine in which two atoms of iodine had been substituted for two hydrogen atoms of the molecule forming the compound 3-5 diodotyrosine. The animals metamorphosed within seventeen days following injection. Control thyroidless axolotls injected with equal quantities of pure tyrosine and later with large amounts of 3-5 dibromtyrosine showed no evidences of metamorphosis. Further evidence that Uhlenhuth’s claim is not valid is fur- nished by Huxley and Hogben who metamorphosed Salamandra and Triton larvae by immersion in dilute solutions of iodine (Proc. Roy. Soc., vol. 98, 1922); by Hirschler, who metamor- phosed axolotls and tadpoles by administration of elemental iodine and iodoform (Arch. Entw. Mech., 1922), and lastly by Jensen who metamorphosed axolotls by injections of iodized casein, iodized serum globulin and iodized serum albumen (Compt. Rend. Soc. de Biol., 85, 391-392, 1921). BIBLIOGRAPHY ADLER, Leo 1916 Untersuchungen iiber die Entstehung der Amphibieneotenie, Pfliiger’s Archiv, Bd. 39. Bravs, H. 1906 Vordere Extremitit und Operculum bei Bombinatorlarven. Morph. Jahrbuch, Bd. 35, Heft 4. vy. Cuauvin, Marte 1875-76. Uber die Verwandlung des mexikanischen Axolotl in Amblystoma. Zeitschr. f. wissensch. Zool., Bd. 25, Suppl., und Bayz. De Fitiprr 1861 Sulla larva del Triton alpestris. Arch. per la Zool. e per VAnat. Comp. Genova (quoted from Gadow). Dumsérit, Aucust 1865 Nouvelles observations sur les axolotlsnés a la menag- erie. Comp. Rend., T. 61. Emerson, E. T. 1905 General anatomy of Typhlomolge rathbuni. Proc. Soc. Nat. History, Boston, vol. 32. Gapow, H. 1909 The Cambridge Natural History, vol.8. 418 W. W. SWINGLE Huxtry, J. 1920 Metamorphosis of axolotl by thyroid feeding. Nature, vol. 104, no. 2618. Jensen, C. O. 1916 Ved Thyroiden—praeparater fremkald Forwardlung tros Axolotll’en. Oversigt. Klg. Danske Vidensk., Selsk. Forhandl., Copenhagen (cited by Uhlenhuth, 721). Kaurman, L. 1918 Researches on the artificial metamorphosis of axolotls. Bull. Acad. Se. Cracon, Ser. B., 32 (cited by Uhlenhuth). Kotimann, J. 1884 Das Uberwintern von europiiischen Frosch und Triton- larven und die Umwandlung des mexikanischen Axolotl. Verhandl. d. Naturh. Gesellsch. Basel. LAursEerGER, V. 1913 Ovzbwzeni metamorfos axolotlIn Krmenim zlazon stit- non. Biologicke Lysty (cited by Adler, 716). Leypia, Franz 1853 Anatomisch-physiologische Untersuchungen iiber Fische und Reptilien. Berlin. SHureLtpT, R.W. 1885 Mexican axolotl and itssusceptibility to transformation, Science, vol. 6. Swinetr, W.W. 1921 The germ cells of anurans. I. The male sexual cycle of Rana catesbeiana larvae. Jour. Exp. Zodl., vol. 32, no. 2. 1922 The thyroid glands of the perennibranchiate amphibians. Anat. Rec., vol. 23, no. 1, p. 100, Proc. Am. Soc. Zool. 1922 Experiments with necturus and axolotl thyroids. Anat. Rec., vol. 23, no. 1, p. 106, Proc. Am. Soe. Zool. Untenuuta, E. 1921 Internal secretions in growth and development of am- phibia. Am. Nat., vol. 55, no. 638. PLATE 1 EXPLANATION OF FIGURES 3 Larva grafted ten days with small piece of Necturus thyroid, and control. 4and5 Larvae engrafted eight days with pieces of Necturus thyroid. 6 Larva grafted six days with Necturus thyroid, and normal control. 7 Larva fed mammalian thyroid tissue (desiccated) eight days. Control animal same as in figure 6. METAMORPHOSIS OF NEOTENOUS AMPHIBIANS PLATE 1 W. W. SWINGLE PLATE 2 EXPLANATION OF FIGURES 8 Immature R. clamata larvae ten days following transplantation small _ pieces of axolotl thyroids. 9 Same as figure8. Ventral view. 10 Control larva for animals shown in figures § and 9. 420 METAMORPHOSIS OF NEOTENOUS AMPHIBIANS PLATE 2 W. W. SWINGLE 421 Resumen por el autor, H. P. Kjerschow Agersborg. Algunas observaciones sobre los estimulos cualitativos qui- micos y fisicos en los moluscos nudibranquios, con especial mencién del papel de los ‘‘rinéforos.”’ Hermissenda responde a los estimulos tactiles aplicados sobre cualquier parte del cuerpo. Los tentaculos dorsales producen la respuesta mis efectiva. La cabeza y los tentaculos dorsales son mis sensitivos a los acidos y sales en solucién que cualquier otra parte; el extremo de los tentaculos dorsales produce la respuesta mas efectiva. Los tentaculos orales son casi tan sensi- tivos como los dorsales a la accién de los diferentes estimulos quimicos; pero los orales poseen en adicién una funcidn selectiva en el sentido de que cuando reciben un estimulo de algtin ali- mento sabroso puede obligarse al animal a moverse en la direc- cidn del estimulo. Los tentaculos orales producen una reaccién positiva definida hacia los estfimulos alimenticios; la reaccién hacia los estimulos de distancia es menos definida que la de los estimulos de contacto. Los tentaculos dorsales (‘‘rin6foros’’) no dan ninguna muestra de sentido olfatorio. Las reacciones de Dendronotus no son marcadas, en general. Melibe selec- ciona su alimento con ayuda de sus tentsculos orales (cirros) ; los tenticulos dorsales, aunque estan ricamente inervados por nervios de la region anterior del cerebro (‘‘ganglios olfatorios’’) no parecen funcionar como Organos olfatorios. La reaccién hacia las corrientes de agua no se altera cuando se extirpan los tentaculos dorsales. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 2 SOME OBSERVATIONS ON QUALITATIVE CHEMICAL AND PHYSICAL STIMULATIONS IN NUDIBRANCHI- ATE MOLLUSKS WITH SPECIAL REFERENCE TO THE ROLE OF THE ‘RHINOPHORES’! H. P. KJERSCHOW AGERSBORG University of Nebraska, Lincoln, Nebraska TWO FIGURES INTRODUCTION The purpose of the investigation upon which this paper is based was to determine the exact function of the dorsal tentacles which have come to be considered as organs of smell, and are generally called ‘rhinophoria.’ Karly writers on nudibranchiate mollusks, Alder and Hancock (45, p. 19), Hancock and Embleton (’52, p. 242), Jeffreys (’69), ascribed to the dorsal tentacles the function of olfaction, and Bergh (79), agreeing with these authors, employed the term rhinophoria. In fact, Tapparone-Canefri (’76) suggested this term for Melibe papillosa De Filippi, calling the ‘tentacula’ rhynophoria. Also later writers, Fischer (’87), Pelseneer (’06) (Prof. E. Ray Lankester’s ‘‘A Treatise on Zoology’’), seem to agree on this point. Hescheler (’00), however, uses the term ‘Kopftentakel’ (Prof. Arnold Lang’s ‘‘Lehrbuch der vergleichen- den Anatomie der wirebellosen Thiere”). Copeland (’18) thinks that the snails Alectrion obsoleta and Busycon canaliculatum are as successfully directed toward distant food by means of an olfactory apparatus consisting of a single organ of smell, asso- ciated with a siphon terminating in a shifting ‘nostril’ for sampling the surrounding water and its contents, as animals with paired olfactory organs and fixed nostrils. But Arey (’18) disagrees with ‘From Puget Sound Biological Station, Friday Harbor, Washington; and contribution from the Zodélogical Laboratory, of the University of Illinois, under the direction of Henry B. Ward, no. 206. 423 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, No. 4 424 H. P. KJERSCHOW AGERSBORG Copeland on this point. He finds that the ‘rhinophores’ of Chromodoris elegans and C. zebra have nothing in connection with olfaction. Alder and Hancock, Hancock and Embleton, Jeffreys, Tapparone-Canefri, Bergh, Fischer, et al., based their opinion, relative to the function of the dorsal tentacles, on morphological data. ‘The findings of Arey are somewhat in agreement with my own observations (vide infra). And, as we will see, it may not be well to designate a specific function to these organs, even though their response may be similar to that of vertebrates with definite localized and known sense organs relative to their specific function. Thus, even though the so- called rhinophorium is highly innervated with nerves from the anterior part of the brain (Hancock and Embleton), it may not be a good criterion at all by which to judge its function, for the simple reason that the brain of invertebrates is not analogous to the brain of vertebrates. In fact, Minnich (21) has shown that the organs of taste in the butterflies Pyrameis atalanta Linnaeus and Vanessa antiopa Linnaeus are located in the tarsi. He demonstrated that tarsal chemoreceptors are present in all four tarsi of the walking legs. The removal of the antennae labial palpi, and rudimentary fore legs in Pyrameis does not affect in any significant way the responses produced through contact chemical stimulation of the tarsi. From the anatomy of Lepidoptera it is seen that the pedal nerves are innervated from the thoracic ganglia, and not from the brain. Chemo- receptors, according to Minnich, may be divided into two classes: first, those affected in general by volatile materials, the source of which may be more or less remote from the receptive surface; second, those affected in general by non-volatile materials, the source of which must be in intimate contact with the receptive surface. The former serve as distance chemoreceptors; the latter, as contact chemoreceptors. The above distinction, how- ever, is far from being an absolute one, but is merely useful as the best single condition by which the two groups of sense organs may be conveniently differentiated. For, it is contended, in the last analysis both are stimulated by a solution of the exciting material, the solvent consisting, at least in part, of the secretion present on the sensitive surface. STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 425 DISCUSSION The following data were collected from experimental studies of Hermissenda opalescens Cooper, Dendronotus giganteus O’ Donoghue, and Melibe leonina Gould. Type 1—Hermissenda opalescens Cooper This species has been accurately described by O’Donoghue (’21); it is, however, necessary to give a brief account of certain morphological structures because of the variance in the nomen- clature employed by many writers relative to certain points. Gasteropods have commonly one or two pairs of tentacles on the head. The anterior pair is frequently spoken of as oral tentacles or buccal or inferior (Fischer), vordere Fiihler (Claus and Grob- ben, ’10), or Mundtentakeln (Lang and Hescheler), and the posterior as dorsal tentacles, hintere Fiihler (Claus and Grobben), or Kopftentakeln (Lang and Hescheler), or rhinophores by other writers, notably by Bergh. In some species (Melibe Rang, Dendronotus Ald. & Hance.) the oral tentacles may be lacking, as a distinct pair, or may consist of one or more rows of varied sized cirrhi around the fringe of the hood (Kjerschow Agersborg, "19, 721, ’21 a, ’22), and in that way the oral tentacles sometimes may not be readily recognized. ‘The dorsal tentacles may also be modified so as to make them at first sight quite indistinguishable from the papillae (dorsal cerata), as in Dendronotus. In this paper I will treat of the rhinophores as dorsal tentacles (not ‘head tentacles’ (Kopftentakeln), as they are sometimes situated on the neck, e.g., Hermissenda, etc.), and the cerata as papillae (Kjerschow Agersborg, ’22). The anterior or buccal tentacles may most appropriately be called oral tentacles not only because of their proximity to the mouth, but also because of the common usage of the term in this connection. The oral tentacles (O’ Donoghue,’ 21) in Hermissenda opalescens consists of one pair, situated anterolaterally on the head. They. are lanceolate, tapering gently to a point. In a specimen which measured 40 mm. from the anterior end to the tip of the foot, they were 12 mm. or a little less than twice the length of the dorsal 426 H. P. KJERSCHOW AGERSBORG tentacles, the anterior quarter being white. In normal specimens they are in constant motion, being generally directed from the head at an angle to each other of about 65°. When the animal moves, they are directed forward and sideways in a lashing fash- ion, the tips being the most motile parts. The dorsal tentacles are situated on the top of the neck, one on each side of the red median dorsal line, and 1.5 to 2 mm. back of the anterior border of the foot. They are about one-half the length of the oral tentacles and they are directed almost vertically from the body, or sometimes at an angle to each other from 0° to ca. 23°. In external structure, they differ from the oral tentacles, which are smooth, in that they are supplied with a series of prominent annulations. Like the former, they are white at the ends. For experimental purposes, I selected at random a number of large and small individuals, which I paired into a number of sets, each consisting of a small and a large individual (vide ut tabula). Tactile stimulation. The entire surface, including the papillae, the parts between the papillae, the foot including the lanceolate posterior prolongation which extends back of the papillae com- monly up to one-half the length of the body, the oral and dorsal tentacles, and the anterolateral prolongations of the foot, is sensitive to touch. The tentacles are more sensitive to touch or tactile stimuli than are any other parts of the body. It is hard to tell which of the tentacles are the most sensitive to tactile stimulation, because the oral tentacles are frequently in constant motion, but judging from the response obtained when the two are touched similarly, it appears that the dorsal tentacles are the more sensitive of the two, at least to tactile stimuli. That is, an equally gentle touch to them gives a more effective response in the dorsal tentacles, in that they not only contract by shortening relatively more, but also by remaining retracted longer than the oral tentacles. The oral tentacles may shorten a little, but their main response to tactile stimulation consists in being jerked away from the stimulus and then be put back at once to their former or similar position. STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 427 Chemical stimulation. As shown per the accompanying tables (pp. 433-438), a number of chemical solutions of different con- centrations were applied to the various parts of the body by the capillary-pipette method. ‘This pipette was sealed at one end and insulated around the middle by a cork, for manipulation purposes. The same amount of the particular chemical used (unless stated differently) was applied each time. ‘The pipette was rinsed exter- nally each time after refilling to avoid diffusion of the chemical so- lution in the medium of the specimen experimented on and in order to secure as nearly as possible the normal condition of the sea- water. Sixty-two animals were used. Each one was placed in a finger-bowl half filled with fresh sea-water, and the pipette then applied with the chemical solution so as not to touch the body with the pipette, but to allow only the chemical solution in question to pass onto the desired part of the body. Solutions heavier than sea-water were allowed to flow from above; those lighter than sea-water were applied to the animal when it was crawling on the surface film of the water or on the side of the vessel. The following signs represent the relative degrees of modes of response to the stimuli: the negative sign represents an attempt to avoid the stimuli. There are five of this kind, three positive and three others as explained below: 1. —g, means general but a slow reaction with an attempt to avoid the stimulus. 2. —, means a more definite negative response. 3. ——, means a still more definite negative response. 4. ——-—, means explosive negative response. a. ——-—-—, means explosive negative response, violent con- tractions and twistings of the body or parts of it. 6. The plus signs, +, ++, and +++, mean various degrees of positive response with an attempt to move in the direction of, or toward the stimuli. 7. 0, means no reactions. 8. ©, means indefinite reaction which shows no particular effort to avoid the stimulus. 9. ©, means indefinite reaction but rather with opposite direction to @. 428 H. P. KJERSCHOW AGERSBORG In general, the head is the most sensitive part of the body both to tactile and chemical stimulation. The effect of acid gives an almost constant result. Comparatively, the dorsal tentacles are more sensitive than the oral tentacles to acids, and the tips of the dorsal tentacles respond more definitely to this stimulus than do the tips of the oral tentacles. Hermissenda opalescens gives a positive reaction toward 2 M ethyl alcohol and a negative reaction to2Mmethyl. That is, the organism would even suck the ethyl- containing pipette, but avoid the methyl-containing one. Speci- mens would eat readily cucumarian gonads treated with ethyl aleohol and show no ill ‘effects. One specimen which ate 10 mm. of cucumarian gonads. treated for 40 min. in 2 M methyl, died before the next day. It was found that this species would eat very readily various kinds of animal matter: jelly-fish tentacles, gonads of sea-urchins (Strongylocentrotus drébachien- sis), sea-cucumber (Cucumaria japonica), and of the sand-dollar (Echinarachinus excentricus), etc. ‘The gonads of Cucumaria are filamentous in nature and are readily measured quantitatively.? I treated, therefore, strings of gonads of Cucumaria japonica in various chemical solutions: M 0.25 lithium chloride; M to M 0.5 sodium chloride; M 0.05 magnesium sulphate; M 0.10 sodium salicylate; sat. sol. quinine sulphate; 2 M glycerol; 2 M ethyl, and methyl from 5 min. to ca. 6 hrs. Hermissenda showed a marked difference in response to food thus treated in comparison of the reaction to normal food, i.e., food which had not been treated. And this difference corresponded somewhat to the reaction when these chemicals were applied onto the surface of the body. For example, the response to M 0.25 LiCl. is 0 to all parts of the body. When presenting food to the animal treated in this solution, it was found that the animal took to it as readily as to untreated food. The response to M NaCl is—(see ex- planation of sign); food treated in this solution was taken by the animal from 0 to 50 mm. in one minute. The exact results were: Out of ten specimens, five of which were given food treated ten 2 These strings (filaments) were on the whole ca. 1 mm. in diameter; the exact volume was not computed. The amount later is indicated in mm. of the gonadic filament. STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 429 to fifteen min. ate as follows. Ten min. treatment, 25 mm.; 15 min, treatment: 17, 19, 20 and 25 mm. The other five had their food treated for 1 hr. and 40 min. in the same solution and the amounts these ate, respectively, were: 0, 12, 17, 20 and 50 mm. The same ten specimens, on another day, ate as follows, the food being treated for 15 min. in M 0.05 MgSO: mm. mm. mm. mm mm. mm. mm. mm. mm. mm. ta htt iii: Nace 50} 20] 30] 50] 50] 50] 50] 30] 20] 20 aaa mpitont 15| 15| 20] 35] 22] 50| 30] 10] 30] 32 Total in day......... | 65| 35| 50| 85] 72|100| 80] 40| 50] 52 It was noted that the response of the organism when stimulated with M magnesium sulphate was 0. That is, it notices the stimulus, but does not attempt to avoid it definitely. One might, therefore, expect a result as noted above which is nearly similar to the results obtained from the control (see the table below). Fed on gonads treated with 0.1 M sodium salicylate for 10 min. the result was that all moved away from it at first. Three min. later, one ate 30 mm. which had been treated for 30 min., one nibbled at it and bit it into parts, but did not eat, it. The others moved away from it. ‘The reaction of the organ- ism to M sodium salicylate is from — — to ———-—-; it was, therefore, no surprise that the organism did not want to feed on this food even though it was treated in a much more dilute solu- tion as compared with those mentioned above. The Nudibranch ate food treated for 6 hrs. in glycerol: 40, 40, 40, 40, and 50 mm. The reaction to glycerol was 0. The same individuals at the same hour also ate, respectively, 50, 17, 25, 17, and 50 mm., treated in 2 M ethyl alcohol. The average amount of food taken at this time was: 90, 57, 65, 57and 100mm. The response to 2 M ethyl was © or nearly +. The other five would not take food treated in 2 M ethyl, but when it was treated in M ethyl they ate: 50, 50, 50, 50, and 40 mm., respectively. The amount of food taken, which was treated in sat. sol. of quinine sulphate, was as follows: a, bit at it and spat it out; b, do. do.;e, ate 25 mm.;d, ate 50 mm. at once, and e, avoided it altogether. 430 H. P. KJERSCHOW AGERSBORG The chemical response to quinine sulphate is shown in the table to be from —g to —. The same individual which ate 50 mm. treated with quinine, ate 20 hrs. earlier 25 mm. food treated with 2M NaCl for 15 min. That is, my data show this animal ate on the average for six days 51 mm. per day, the food being treated in various ways; or, it ate ca. 3.23 mm. more than the average of five individuals, but its records show it ate some each day, Le., from 25 to 85 mm. each day during the week. It may be, there- fore, that specimen d was in a better physiological condition at the time when it took a full meal of food, e.g., 50 mm., which the others would not do. An individual which in a morning did not eat from the food treated with M ethyl ate 10 mm., in the after- noon, of food treated with 2 M methyl; on the next morning it was dead, however. The chemical response to methyl was — (vide ut infra tabula). It is then seen there is a definite relation between the sensitivity of the external parts of the organism to certain chemical solutions, and the taste of the organism, e.g., quinine sulphate gives a response from —g to —, the organism is careful about eating food treated in suchasolution. If hungry, it may eat it at once; if not hungry, it may bite at it (taste it?) and then either eat only a little, void it, or avoid it altogether. (N. B.—It is to be noted that the food was left with the individual up to one minute, after which time it was removed in all cases.) The most interesting results were obtained by using broth of various animals, e.g., of jellyfish, sea-urchin’s gonads, etc., and oils. I also used an emulsion prepared from raw jellyfish and from the gonads of sea-urchins. The reaction was positive for all of these, but more so for the cooled broth. Applying the emulsion or broth with a pipette on the dorsal tentacles, Her- missenda turns round apparently in search for the stimulus; applying the stimulus in front, it makes progressive movements toward the stimulus and bites in the pipette and works the oral apparatus continuously. When broth of the sea-urchin’s gonads is applied onto the top of the head or on the dorsal ten- tacles the response is not really definite, the organism seems to be confused; but when the same amount of broth is let a short distance (5 mm.) in front, it moves toward the same and starts STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 431 working the oral apparatus sucking in the mixture. Applying broth on the back it turns around, as if searching for the stimulus. The reaction for broth of sea-urchin gonads is twice as strong as for that of jellyfish. The response was from + to ++4, but most effective for the sea-urchin’s. The oral tentacles showed a most definite positive reaction toward the stimulus. Touching the tentacle on the left or right side, I could lead the animal at will from one side to the other; it always turned after a short interval of time toward that side which tentacle was stimulated. This is equally definite when using a piece of solid food which it eats readily, e.g., gonads of sea-cucumber or sea-urchin. The reaction as shown by Hermissenda toward distance stimuli is less definite than that toward contact stimuli. Receptors for distance stimuli seem to be present in the oral tentacles; but receptors for contact stimuli, judging by the mode of response, are more specific than are the distance receptors. Contact receptors are present all over the body, perhaps also chemoreceptors, but the latter are specialized in the oral tentacles, as they seem to be used in discriminating between foods. They may, therefore, be gustatory in function, because the animal may be led about, from side to side, in the dish by touching the oral tentacle by some palatable food; that is, a food which the organism feeds on readily. No such results are effected by treating the dorsal tentacles, the so-called rhinophores, in the same way. The head, in general, seems to be most sensitive to stimuli, tactile or otherwise. When food stimuli are applied to the head, the animal turns in various directions so as to search for them. The dorsal tentacles do not respond to distance stimuli; their response to a food stimulus is similar as that given to a tactile stimulus, e.g., a clean glass rod. Hermissenda does not have the ability to locate solid foods; it really seems that the animal comes upon the foods accidentally. It is apparent that the animal can taste food in solution with most parts of its external surface, and particularly that of the head and the oral tentacles; but it does not look as though the organism is capable of scenting its food. I am not able to say that the dorsal tentacles are used for this purpose. I repeatedly found hungry animals within ca. 1 em. from food they readily ate when 432 H. P. KJERSCHOW AGERSBORG the same was placed in front of them so as to be touching the lips. In fact, the animals would then make a swift nip for such foods with the jaws. If an animal, which had not fed for twelve hours, was at rest next to a piece of gonads (the food it ate the most readily) it would immediately become active by being touched on the oral tentacles by the food, and then begin to crawl about. If it came upon the food, feeding started at once. Touching the animal similarly gently on other parts of the body did not result in the active moving about. This may show that the chemo- receptors, as stated above, are better organized or specialized in the oral tentacles. During such forced movements, the animal moved practically in a straight line (in a circle in the finger-bow]), but it bent the head, now and then, from side to side, and was constantly lashing the oral tentacles as if it were feeling its way. I repeated Arey’s experiment by holding a drop of various kinds of oils between the ‘rhinophores’ without allowing the oils to come in contact with them, but I did not get any response. Allowing a drop of oil to come onto the body, the oral tentacles only gave definite response. The response was — to saffrol, and — — to bergamot oil. The dorsal tentacles, for the same oils gave 0. The rest of the body gave —, or 6. The animal does not show any awareness to clove oil when a drop of it is suspended free between the dorsal tentacles. Allowing the same to touch any part of the body the response is —. The response to organnum oil is © for the head and 0 for the rest of the body (vide ut infra tabula). To cedar-wood oil, the response is either © or 0. And to orange flavor it is 0. The following tables show the exact data relative to the number of specimens used, the size and condition of the specimens, the stimuli employed, the parts stimulated, the relative response, etc., of Hermissenda opalescens Cooper (tables pp. 433-438). Arey quotes Crozier as thinking that the dorsal tentacles of Chromodoris are rheotropic, their prime importance being in effecting orientation to the water current. But the dorsal tentacles of Hermissenda do not seem to have a rheotropic func- tion, because specimens with one or both of the dorsal tentacles removed oriented as easily and moved against the current as did 433 STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS ee ee eee qgueulaAoul sdoys yoo} aOlojue ye poyddy qusWoA OUI PIVMIOJ YUaT[ooxo ‘OOF jo di aor1eysod ye porddy asuodsal ou “WOTyN{OSs J OT, asuodsoai ou “WOTyN{OS JT OT, esuodsai ou ‘UOT}NIOS JT OY, esuodsar ou ‘MOT4ZNTOS JT OT, asuodsar Ou ‘UOTYNIOS JT OT, 4X0} 9} 90s ‘SuSIS JO UOIzeUL|dxe 10,7 SHUVNAY aS 400} Jo quy, 1S oa a 4ooj jo e1PPIN Sa— — — ——— = — oa — ae ee lo eee ee — S=818 = | 3 —s/——5 400. ; sayo cone Apog | _ ale ee -uy eed [esi0q dg Ly TAWics sLuvd T WIdaVv.L soo -8]U94 [RIO IOBN ‘IN Z IOBPN ‘WW IOBN ‘WG IOBN ‘W IO®N ‘ING IO®BN ‘IN IOBN ‘IN Z 1O®N ‘W IOBN ‘IN 3 1IO®N ‘W IOPHN ‘ING IOPHN ‘WZ IOVHN ‘WZ IOFHN ‘WZ IOPHN ‘WZ peoFy qqaso ITAWLIS GHG “urut 03 “D “uIUL OZ “OD “WUE OF ‘YsIssn[g “Wl OF ‘ystssn[g “uu Gz ‘XD “UIU GZ “*) "mul GZ “4 “UIUL GZ “Y) "url OZ “*) “ut OZ “4D “uu 0% ‘5D "ul OZ “4D ‘uu OZ ‘D "wu GZ ‘5 “UU OF poor) NOILIGNOOD aNV a@ZIS Pv ON § ‘ON Z ON 12./&/8 T ‘ON aivd @HLaNV IVOCIAIGNI 70 UGAWON P. 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ITAWIES pie al sae Ge E 3 - -WON adaiLVTOWILS SLuVd panuyuoo—se ATAVL 437 STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS © 8 (S) NS) i) ic) i) = 8 © ic) oad pejovryu09 yOOF ay} Jo dt} 04) TIM | O 0 07) 0 o}j,0dId oY} syons ey ‘uryoIn-ves jo yyorg | + 9}40dId oy} syons ey suryoim-ves jo yyorg | + + elie SN[NUIT}S 94} 197/o SuINy IFT BIO} -o1A elienby ysyAqTial JO a}jyedId q[nq-reqqna yAIMA | —— | —— |——|—— e77edId q[nq-reqqna yyIAA | —— | —— |——|—— sd30 SuuteyT | 3 — -- — js — x Sa SS Se awe es SS EEE EE ES EE EE eee eee ee ee ++ +4 4p ++ | [ro © | “AM Iepep — | orpeg = [Org es LOART 0 | osuvI() | OAT 0 osUBI() | Yjoaq ae es sey) yor +++] pafoop YpOrq “tr ts perso) | Yyorq ++ | — pajoop —— Oca paincr == "| Oe OseF | NOH — | 4ue0 10d [ | NOH — | 4u00 10d [ IOH — | quod aod [ IOH —— | 9ua0 tod [| “UIUL CF [BULLON “UIU OF [wuIL0 N “WU CF [BULLON “UU OF [BULLON “UIUL CP [BULLON “UIUL OF [BULLON “WU GE [BULLION “UIUL OE [BULLON, “UIUL GE [BULLION “WU ZG [BULLION “WIUL GE [BULLON “UU GE [BULLION “UIUL CF [BULLION “UU 0Z [wulto N “UIUL GE [vuIoN M d P. KJERSCHOW AGERSBORG 1s 438 pepuedsns st doip UdYM SSoUsIVMG OU SMOYY [lO YIM yowqju0o ul uoyM ATUO BAT}ISUIG psey ey} wo ATUO DAT}ISUIG SaUVANaY 0 0 0 i) ® onlideg @) yooy Jo dry, ® 700} JO e[PPIN ® yoo} IoLeyuWy 0 0 0 0 0 0 0 0 = fy 0 = r=) 0 ) je] 4 | g) ao is} a 2a oD Oo na nQ g g 2, Bi + + 5 iS S 1 ' sopoequo} [esIoq cP) soloeyUo} [BIO adaLvIoOWiis sLuvd papnpuoj—e HTAV.L prox | O&O ee) [ECReAT uINUUBSIIC CINUUBS LIC) ‘O youres. log, ‘O youres.1log [fe TASER SD ITOWILS “UU 2 [BULION “ULUT OG [BULLON “ULUL OF [BULION “UIUL JF [BULLION “UIUL ZF [BULION “UU ZG [BULLION “UU GE [BULLON NOILIGNOO aNvV aZIs aLva anv ctce:e -WON STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 439 the normal individuals. That is, I placed a number of normal specimens in a narrow trough ca. 60 cm. long, 5 em. wide, and 6 cm. deep, and tilted it at one end ca. 3°, allowing a gentle stream of sea-water to pass in at the raised end and out in the opposite end through a sieve. Everyone oriented himself toward the current and moved first against it until arriving at the end of the trough. A number of individuals in which the dorsal tentacles had been removed, either partly or totally, either only one or both, behaved in the same manner as the non-mutilated ones, save two of the ones operated on, which seemed to be a little disturbed at first. Otherwise it did not seem to make any difference whether the ‘rheotropic’ tentacles were present or not. The function of the dorsal tentacles, therefore, does not seem to be ‘rheotropic’ in Hermissenda. They are tactile organs, how- ever, and may also be slightly gustatory in function. Type 2—Dendronotus giganteus O’ Donoghue I collected a single specimen of the wonderful species Den- dronotus giganteus from between the logs of the floating dock at the station. It measured 140 mm. in length, 60 mm. deep, and the foot was 90 mm. long and 40 mm. wide. (For a complete description, vide O’Donoghue, ’21.) This species is much more sensitive to tactile stimuli than is Hermissenda, and is, therefore, not so easily experimented on. The slightest disturbance of the water caused some local contraction of the body. However, the following data were collected, and it may be of interest to study them in comparison with those on Hermissenda (table 3, p. 440). This table (table 3) shows graphically that the dorsal tentacle, the ‘rhinophore,’ of Dendronotus is on the whole more sensitive to contact stimuli whether physical or chemical. Type 8—Melrbe leonina Gould One specimen of this remarkable species was given to me by Dr. Elmer Lund, who found it at the floating dock. The speci- men measured 65 mm. in length; the height was 10 mm., and the KJERSCHOW AGERSBORG 1ek Vale 440 epuasstulloy LOJ sv poyyepy | — dJOYA B Sv VAOUL you pI, | 0 asfOYM BSB vAOW JOU piq |s —|8s —|s —|s8 —| — - [OBN 0} popesolo00v ole SyuNMMOAOTT | 0 apnulUL T— OJ MaIpYZIM sopovjzus} [esiop oy, :Bo1e UdATS UO 19yvM Ul [OFHIN ‘IV Z Jo doap ouo Surddoap Ag poyyout oyyodid Aavyidey | 0 yoo} jo dry, | S SHUVNGU 3003 Jo arp | WS o o 4Oo} 1OlI0jUy i=) j=) i=) o7ey ——|-908 WIIpos JT [ord A419 “TW E OSOLONS ‘TA as07yOVR] “FAT G'O ION ‘We IOV W 920 [HN ‘IW IOTHN ‘W@ OTB L Apog avpideg sa[oejU94 [vsIOg auLvTOWILS sLuvd € WIAVL S9[9BIU94 [BIO Bern ITOWLLS poo “UIUL OET poor “ULUL OFT poo) “ULUL OFT peep “ULUL OST poop “UIUT OPT poor) “UUL OST poor) “ULL OFT poop “ULUL O§T pea) “ULUL OET NOILIGNOD GNYV @ZI8 12./¥/8 1./¥/8 12./¥/8 1Z./¥/8 12./¥/8 13. /¥/8 1Z./¥/8 1Z./¥/8 SnoyUBsIs snyouoipusd GLYa GNV saiouds 10 ANVYN STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 441 width 8 mm. The hood 30 mm. in diameter in either way; the foot 35 mm. in length and on the average ca. 5 mm. wide. As in other species of this genus, the hood is fringed with rows of cirrhi; in this species it is fringed with two rows, the outer of which is five times larger than those of the inner row. But the cirrhi of the inner row are twice the number of the outer, uniform in size and arrangement. That is, there is usually one small cirrhus at the base of each large, and one between. The large Fig. 1 Ventral view of the hood of Melibe leonina Gould drawn from life by the author. Bc., body cavity; Db., base of the stalk of the left dorsal tentacle; F., foot; L., left lip of the mouth; Lc., large cirrhus; M., mouth; Sc., small cirrhus. Fig. 2 Ventral view of the hood, contracted, as in the process of swallowing; note the dorsal tentacles (‘rhinophores’) are not contracted. Drawn from life by the author. Dt., dorsal tentacle (left); Lc., large cirrhi; F., foot; Rh., rim of the hood. (Note that the anterior part of the hood is brought caudad, e.g., to bring the food near to the mouth so that the lips can take hold of it; compare the position of the dorsal tentacles with that in figure 1.) cirrhi terminate ca. 4 mm. on each side from the midventral line of the caudoventral aspect of the rim of the hood. This species is highly sensitive to tactile stimuli. The very slightest ripple on the water disturbs it. Figure 1 shows the position in which the hood is held in life; the large cirrhi (lc.) are stretched outward and the small cirrhi (sc.) inward. When a glass rod is introduced into the water, even with the utmost care, and at ca. 20 cm. away from the animal, the hood con- 44°? H. P. KJERSCHOW AGERSBORG tracts and the organism becomes restive. Complete contraction of the hood is effected by introducing the rod within the area of the hood. When a crustacean, ca. 10 mm. or more (I got it to take two Amphipoda, a Cammarus ca. 15 mm. long andaCaprella ca. 20 mm.) is dropped within the rim of the hood, it closes up the hood firmly (fig. 2) without contracting the tentacles on the back of the hood, opens the mouth (while the hood is yet con- tracted), and passes the food into the digestive tract. It also ate a strip of cucumarian muscle ca. 60 cu. mm. but its favorite food seems to be Crustacea (Kjerschow Agersborg, ’21). Its favorite position is on the surface film with the back inverted. The cirrhi seems to be receptors of tactile stimuli. The animal may try to swallow anything that comes within the rim of the hood, but it does not swallow everything; it actually tries to eject solids which have come within the rim of the hood and which it cares not toeat. In life an inner axial white rod is seen through the wall of the cirrhic cone. This axial rod, as I have shown be- fore (22, in press), consists of nervous tissue; fine fibers radiate from it to the periphery. The dorsal tentacles are not more sen- sitive to tactile stimuli than are the cirrhi. But when they are touched with a glass rod they contract within the sheath of their stalk and remain contracted for a short time. SUMMARY 1. Hermissenda opalescens Cooper responds to tactile stimuli applied to any part of the body: the head, the oral and dorsal tentacles, the body, the various parts of the foot, and the papillae. The dorsal tentacles give the most effective response to a tactile stimulus, such as the end of a glass rod. 2. The head and the dorsal tentacles are most sensitive to acids; but of the two, the latter are more sensitive to acids and salts in solution, the tips giving the most effective response. 3. The oral tentacles are almost as sensitive to stimuli as are the dorsal tentacles, but in addition to a general response of this nature, the oral tentacles also have a selective function in that when they are stimulated by some palatable food, the animal may be made to move in the direction of the stimulus; if the STIMULATIONS IN NUDIBRANCHIATE MOLLUSKS 443 animal responds to a stimulus negatively, the appliance of it in front of a progressively moving animal may bring it to a halt; the directions of its movements may also be changed. 4. The following table shows the comparative response of the oral and dorsal tentacles of Hermissenda to a piece of gonads of Cucumaria japonica Semper. The difference is practically 100 per cent. TABLE 4 ORAL | DORSAL mpivipvan |TPNT-|TENTA-| Toon REMARKS akc + 0 152s 2e— lib" + 0 15°C, one a 0 15eGs ; 4 = 2a =: +? 15°C. || Allowing food totouch the right or left ten- 2D ® rs) 15°C. tacle, the organism turns or does not re- 6.=2e = 0 15°C. spond, in the direction of the stimulus 7, — sb oo ven wll Oe Si )8e 7 [ice trel) Gye ASP. | Oi—sa0. +e 0 | T5c€ If this table is compared with the results as given in table 2, Uu-Zz, pp. 487-488, it is seen that the oral tentacles have the power of discrimination between certain substances, such as food and odorous oils, while the dorsal tentacles lack this power for the same substances. There is no evidence that the ‘rhinophores’ are olfactory in function. 5. The dorsal tentacles of Dendronotus giganteus, like those of Hermissenda, are the most sensitive parts of the body to tactile stimuli (table 3). 6. In Melibe leonina Gould the cirrhi are more sensitive to tactile stimulus than are the dorsal tentacles. 444 H. P. KJERSCHOW AGERSBORG LITERATURE CITED AqgrErsBorG, H. P. Kuerscoow 1919 Notes on Melibe leonina Gould. Pub. Puget Sound Biol.Sta., vol. 2, no. 49. 1921 Contribution to the knowledge of the nudibranchiate mollusk, Melibe leonina Gould. Amer. Nat., vol. 55, May-June. 1921 a Onthe status of Chioraera Gould. Nautilus 35: 50-57. 1922 The morphology of the nudibranchiate mollusk Melibe leonina Gould. Jour. Morph. (in press). ALDER, A., AND Hancock, A. 1845 A monograph of the British nudibranchiate Mollusca. The Ray Society, London, ArEy, Lestie B. 1918 The multiple sensory activities of the so-called rhino- phores of nudibranchs. Amer. Jour. Physiol., vol. 46, no. 5. Brereu, R. 1879 On the nudibranchiate gasteropod Mollusca of the North Pacific Ocean, with special reference to those of Alaska. Proc. Acad. Nat. Sci. Phila., vol. 31, 3d ser. 9. Cravs, C., AnD Grosppen, Kart 1910 Lehrbuch der Zoologie. Marburg in Hessen. CopeLanp, Manton 1918 The olfactory reactions and organs of the marine snails Alectrion obsoleta (Say) and Busycon canaliculatum (Linn.). Jour. Exp. Zodl., vol. 25, no. 1. O’Donocute, Cuas, H. 1921 Nudibranchiate Mollusca from the Vancouver Island region. Trans. Royal Canad. Inst., Toronto, vol. 13, no. 1. FiscHer, Paut 1887 Manuel de Conchyliologie et de Paléontologie Con- chyliologique, Paris. Hancock, A., AND EMBLETON, D. 1852 Onthe anatomy of Doris. Phil. Trans, Royal Soc., London, part 1, vol. 142, JEFFREYS, J. G. 1869 History of naked marine Gastropoda. Brit. Conchol., vol. 5. Lane, Arnotp 1900 Lehrbuch der vergleichenden Anatomie der wirbellosen Thiere. Mollusca. Jena. LANKESTER, E. Ray 1906 A treatise on zoology, part 5, Mollusca. London. Minnicu, DwieotE. 1921 Anexperimental study of the tarsal chemoreceptors of two nymphalid butterflies. Jour. Exp. Zoél., vol. 33, no. 1. TAPPARONE-CANEFRI 1876 Genere Melibe Rang. Melibe papillosa De Filippi. Memorie della Reale Accademia delle Scienze di Torino, Ser. 2, T. 28, pp. 219-220. ae bi ea 7 “0 Hin pom “<4 if | a arse balsiicicien ‘dieb ery en Vi anane sayy a0, ae aeete al ae aaeraoite gb ehaciveloe . TIVE paddle boa ei Bas ¢ Atte ye et ee mi? ACNE ‘ake, Woes Ball duh Bibeln tg (ales. od “wien UA 4 Lear a Wide it0D yes Hovis, foe’. mtunles la digeery'T axodnen Be pe risus ial. Sip, Kp THN} dig aus aly sur U vot tin Gens) rejac 4) al moi) ane 2 meet LE ‘Bol iy ebadeirahtasty. oe Molt were’ ‘nAtoplos ate too O1seas 1S nen ty Ba. 14 wacrian 9b BS EiBeipibuo ie un scurgly wbkaler vais whined eTquisia. avidin Fe nessaal val de tsbeoqeet widuast sheng lanine 17 Jovi idae fee | uy ob ehyqeatiins losualoa o7at, ,tihaliteah aid, ip nine . ron rbd dalod ite lol taid wb whe CON emegti ‘ ns aqpiadl ... nleatiegge ; af otosmetetbooud sdiiee plas ob Aa ayoq NOL sls obute 5 it it ) BATS Aredia .Getinnees 7) aaniiBios (1g i wren oT Atirrtos ela coissest ob adam EA apes inind low nie kasataaoqnuivsh obstaie peice hdyeh: cae aie arantddiracsook wapiatwie wba inthe eOtwtasondy al ape a5 oh eke iain be udtshetideeriay “):, 4% olee) done rei yest uatt) Jehah iottatbou cl armotnuriie)S ich. walter gis ar a Bs wp : a he eatyi ss Jia aby een | 0, COROER DY veer aioe, i, AA aw! hi SESW IPRS ts ig shit NR sidonidenbyry coenengaen ls harass pti 9 foEty! ns ais wot . WORF Noe Y CMa) a (ats aves OMS aes pda dina 39 AE TA: tidy otatlve ides ab igure fis (Sh wrisd ab ryt nas) at, DE: aly “ai wreclereairnn [a vigil phy gate a, aerius? + chee weKAND raat ajatks etiecvicineti 6 A oe a Tat lagi! . ne nae Hinson qdllenelaliiencambdnel al Teale SUMED. 102 iiiverty Ma: afi el tetal embullatasatrhe MBAR’ MWe AMA ea LA 38 atl is Amie aocbcatialhcot gel eal Wi eh drilonl le qi) Ose as yy “9 i, me ne he eRe! a as Wipe? ON Ole A> Rn - fy ' , ; er . =A i 4 | adil pry ; ie LAT Ad oral Woo teae Weciahe 1! , hs é . Ti id ayy ‘ar Vid - \ a , } ' a Fi mt. , * i. Ay af oe a) | 7s & . ., > q a a) ® cael —t- ‘A _- } » s] bp : P ia Pa i " f cs A dud ie i . es ny ¥ Resumen por el autor, D. E. Minnich. Un estudio cuantitativo de la sensibilidad tarsal a las soluciones de sacarosa en la mariposa Pyrameis atalanta L. El autor ha demostrado previamente que los tarsos de la mariposa Pyrameis atalanta L. son sensitivos en contacto con la estimulacién quimica. Una de las substancias que la mariposa puede distinguir claramente con los tarsos es una solucién IM de sacarosa. Si se ponen en contacto con esta solucién, Pyra- meis siempre responde, sin relacién alguna con su condicion nutritiva. El animal puede también responder si los tarsos tocan el agua destilada, pero solamente después de un periodo mds o menos prolongado de inanicién total. Ademas, la inges- tién de agua inhibe inmediatamente la respuesta. Es posible, por consiguiente, mantener a Pyrameis en un estado de 100 por ciento de reaccién a la solucién IM de sacarosa, pero solamente en uno de O por ciento de respuesta al agua sola. Bajo estas condiciones, la concentracién minima de sacarosa necesaria para producir una respuesta, esto es, la concentracién del umbral de respuesta a la sacarosa, varia directamente con la condicién de la nutricién. Durante low periodos de inanicién de sacarosa el umbral decrece gradualmente, y puede alcanzar niveles tales como M/3200, M/6400 y ain M/12800. Pero con la iniciacién de un periodo de dieta a base de sacarosa el umbral se eleva subitamente a un nivel generalmente de M/10, que se conserva proximamente constante mientras la dieta continua. Cuando se compara con la de otros animales, la sensibilidad de Pyrameis a la sacarosa est’ muy desarrollada. Este hecho esta sin duda relacionado con el hecho de que los azticares forman el alimento principal de este insecto. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 2 A QUANTITATIVE STUDY OF TARSAL SENSITIVITY TO SOLUTIONS OF SACCHAROSEH, IN THE RED ADMIRAL BUTTERFLY, PYRAMEIS ATALANTA LINN. DWIGHT E. MINNICH Department of Animal Biology, University of Minnesota ONE FIGURE CONTENTS An fro cate hlO NA! kyy« ch lac,..0. oe ee oe eck HE es oer OE ion ertrraectens 445 ATES GINO CLS esc ecru cs oe OP dah ee eee HE ER st aes Gs ciaiep Shei Obele oars sure 446 NESTS oe ae ere as RES Pe RIOR OS Oe Pom Smee eG a Sretetmianmae saints sere 448 Conclusions eve Ase ho eH oe ee nee Aono ee idettas seis a ele 456 Bila Rap iy tess. Set ebsgs ara citpOew Seite sire crue s shoe eects or eer troetanes ees Saba seep 457 INTRODUCTION It is well known that many insects are extremely sensitive to distance chemical stimulation. The males of certain Lepi- doptera, in particular, exhibit at the time of mating a degree of sensitivity probably unique in the animal kingdom. Thus Riley (94, p. 39) describes an experiment in which a male of Philo- samia cynthia Drury was successful in seeking out a female a mile and a half away, and Fabre (’79-’04), in his classic experi- ments upon several other species of Lepidoptera, reports equally remarkable results. But while we do know something of the degree of olfactory sensitivity possessed by insects, we know absolutely nothing concerning their acuity of taste or contact chemoreception. This is due to the fact that the only organs of taste heretofore described were located on the mouth parts or within the buccal cavity, where experimentation was virtually impossible. Recently, however, I have shown (Minnich, ’21) that the nymphalid butterflies, Vanessa antiopa Linn. and Pyrameis atalanta Linn., possess taste organs or contact chemo-recep- tors on their ambulatory tarsi. In a more detailed study of 445 446 DWIGHT E. MINNICH Pyrameis alone (Minnich, ’22) I have shown further some of the substances which can be differentiated through the tarsal organs. These substances consist of distilled water and several aqueous solutions, including a solution of 1M saccharose. In the course of this investigation two facts were discovered which have made possible a quantitative study of the tarsal sensitivity to saccharose solutions. First, contact of the four ambulatory tarsi with a 1M saccharose solution will always effect an extension of the proboscis, irrespective of the nutritional condition of the animal. From the time of its emergence from the pupa until its death, whether starved or abundantly fed, the butterfly will continue to respond with the utmost constancy to this stimulus. Second, contact of the tarsi with distilled water alone will also effect an extension of the proboscis, but only after a prolonged period of inanition with respect to water. Thus, a butterfly deprived of water for a sufficient number of days, generally four to seven, will finally become 100 per cent responsive to water. This response, however, ceases at once if the animal be allowed to drink water. By the mere administration of water, therefore, Pyrameis can be maintained in a state of 0 per cent responsiveness to water, but of 100 per cent responsiveness to 1M saccharose. Under these conditions, the minimal concen- tration of saccharose necessary to effect a response is readily determined. It is the purpose of the present paper to report the data obtained in this fashion. METHODS All butterflies were hatched in captivity. Upon hatching they were kept without food or water until they became 100 per cent responsive to water. They were then placed on a diet of distilled water which was administered morning and evening. Although some individuals appeared quite weak after the period of total inanition, they generally revived after their first access to water, and for some days the water diet was sufficient to maintain them in a normal condition. Sooner or later, however, the butterflies began to show signs of weakness which gradually increased until further trials became impossible. At this point 1M saccharose TARSAL SENSITIVITY IN PYRAMEIS 447 was substituted for distilled water, and again the animals generally revived. The diet of saccharose was continued for three days, after which the animals were subjected to a second period of total inanition, followed by a second period of water diet, followed in turn by a second period of 1M saccharose diet. Those individuals which survived the entire experiment were thus carried through six nutritional periods: a first period of total inanition, a first period of water diet, a first period of 1M saccharose diet, a second period of total inanition, a second period of water diet, and a second period of 1M saccharose diet. The method employed in making trials was the following. The animal was placed in a holder manipulated by hand, and was held with its four ambulatory tarsi in contact with a flat layer of absorbent cotton contained in a Syracuse watch-glass and saturated with the stimulating substance. Each trial with a given concentration of saccharose was immediately preceded by a trial with distilled water alone. If there was any evidence of response in the preliminary trial the saccharose test was abandoned, thereby avoiding any possibility of misinterpretation. No visible movement of the proboscis during one minute con- stituted a no response, while extensions of the proboscis during the same interval, whether partial or complete extensions, con- stituted a response. After each trial with saccharose the feet were carefully rinsed in distilled water and dried by contact with clean filter-paper. Excepting the first three to five days after hatching and days of responsiveness to water alone, an attempt was made to deter- mine the threshold concentration of saccharose for each animal daily. In general, this was accomplished by means of four determinations, although occasionally as many as six or seven were made. These determinations were made at least one hour apart. Between them, however, tests with other substances, viz., 1M saccharose, 2M NaCl, and M/10 quinine hydrochloride, were being made at minimal intervals of fifteen minutes. As far as I was able to observe, the trials with these various sub- stances in nowise affected the threshold of response to saccharose. 448 DWIGHT E. MINNICH In selecting the concentrations of saccharose for making deter- minations I followed no fixed, uniform procedure. Instead, I was guided by the immediate reaction of the individual together with its previous behavior. In general, the first day of experi- mentation was begun with a concentration of M/10 or M/100, while each day thereafter was begun with the concentration equal to or just below the threshold concentration of the previous day. If this concentration produced a response, lesser concen- trations were tried; if it produced no response, greater concentra- tions were tried. This general procedure is brought out clearly in column IV of table 1, where the concentrations used on but- terfly no. 13 are given in the order tried, with the reaction indicated in each case. The quality of saccharose employed in making solutions was U. 8. P. Stock solutions of M/10 and M/200 were made up from time to time during the experiments, while the other dilu- tions employed were prepared fresh daily from these stock solutions. The present experiments were carried out at the same time and on the same animals as the experiments described in a pre- vious paper (Minnich, ’21). For a more detailed account of general methods than the one given here the reader is referred to that paper. RESULTS A total of seven butterflies was experimented upon. ‘Three of these died quite early in the experiment, probably as a direct result of starvation. Of the remaining four, two survived to within a few days of the end of the experiment, while two others not only survived the entire experiment, but were still in vigorous condition several days later when they were killed and preserved for further study. The most complete data were obtained on butterfly no. 13, and, since they are typical of the results obtained on the ani- mals as a whole, they are presented in full in table 1. It will be noted that during the first three days after hatching no trials were made. The first determination was thus made on the 4th day when the threshold concentration was found to be M/200. TARSAL SENSITIVITY IN PYRAMEIS 449 TABLE 1 Showing the responses of butterfly no. 13 to distilled water and to various concentra- tions of saccharose, under different nutritional conditions. Changes in nutri- tional conditions were always made in the evening after the trials of the day were completed. This is indicated by a repetition of the day number followed by (p.m.). Threshold concentrations are indicated in italics I II III IV ie ata Sis a Y phd Sena eneree oe RESPONSE TO SACCHAROSE SOLUTION 1 Not tried Not tried 2 Not tried Not tried 3 Not tried Not tried 4 No response M/10 Complete extension No response M/100 Complete extension No response M/200 Complete extension No response M/400 No response Given neither wa- 5 Complete extension | Not tried * ter nor food Complete extension | Not tried Partial extensions Not tried Partial extensions Not tried 6 Complete extension | Not tried Complete extension | Not tried Complete extension | Not tried Complete extension | Not tried 7 Complete extension | Not tried Complete extension | Not tried 7 (p.m.) 8 No response M/200 Complete extension No response M/400 Partial extensions No response M/200 Complete extension No response M/400 No response Offered distilled 9 No response M/400 Complete extension water morning No response M/800 Partial extensions and evening No response M/1600 No response No response M/800 No response No response M/400 Partial extensions 10 No response M/800 Complete extension No response M/1600 No response 450 DWIGHT E. MINNICH TABLE 1—Continued I II Ill IV paren ag aS ear TILLED | RESPONSE TO SACCHAROSE SOLUTION ( 10 No response M/800 Partial extensions No response M/400 Partial extensions Offered distilled water morning til No response M/1600 Complete extension and evening No response M/3200 Complete extension No response M/6400 No response No response M/3200 No response 11(p.m.) 12 Not tried Not tried 13 No response M/200 No response No response M/100 No response No response M/50 No response No response M/10 Complete extension Offered 1M sac- charose morn- 14 No response M/50 No response ing and evening No response M/10 Complete extension No response M/50 No response | No response M/10 Complete extension 15 No response M/50 No response No response M/10 Complete extension No response M/50 No response No response M/10 Complete extension 15(p.m.) 16 - | No response M/50 Partial extension No response M/100 No response No response M/50 No response No response M/10 Complete extension Ui No response M/100 Complete extension No response M/200 No response Given neither wa- No response M/100 Partial extensions ter nor food No response M/200 No response 18 Complete extension | Not tried Partial extensions Not tried No response M/200 No response No response M/100 No response 19 Complete extension | Not tried Complete extension | Not tried TARSAL SENSITIVITY IN PYRAMEIS 451 TABLE 1—Continued I II Ill IV CONDITIONS OF DAY OF RESPONSE TO DISTILLED 3 NUTRITION AGE WATER RESPONSE TO SACCHAROSE SOLUTION (| 19 Complete extension | Not tried No response M/100 Complete extension Given neither wa- ter nor food 20 Complete extension | Not tried Complete extension | Not tried Complete extension | Not tried Complete extension | Not tried 20(p.m.) 21 No response M/1600 No response No response M/400 No response No response M/200 No response No response M/100 Partial extensions No response M/50 Partial extensions 22 No response M/200 No response No response M/100 Partial extensions No response M/50 Complete extension No response M/200 Partial extensions No response M/400 No response Us No response M/400 No response | No response M/200 No response | No response M/100 No response Offered distilled No response M/50 Complete extension water morning No response M/100 Partial extensions and evening 24 No response M/100 Complete extension No response M/200 No response No response M/100 Complete extension No response M/200 No response 25 No response M/200 Complete extension No response M/400 Complete extension No response M/1600 No response No response M/800 No response No response M/400 Partial extensions No response M/800 No response 26 No response M/800 Complete extension No response M/3200 Partial extensions No response M/6400 Complete extension ase cn bo DWIGHT E. MINNICH TABLE 1—Concluded I II III IV eee ade © eee Fee ON NER YY» | RESPONSE TO SACCHAROSE SOLUTION Offered distilled ( 26 No response M/25,600 No response water morning( No response M/12,800 No response and evening ( No response M/6400 No response 26(p.m.) AT No response M/100 No response Offered 1M sac- No response M/50 No response charose morn- No response M/10 Complete extension ing and evening No response M/50 No response No response M/10 Complete extension 28(a.m.)| Animal found dead The 5th, 6th, and 7th days, no trials were made with saccharose since the animal responded to water. During the 7th day the animal became so weak that further experimentation was impossible. Accordingly, trials were suspended, water was administered, and the butterfly was allowed to recuperate. On the following morning, viz., the morning of the 8th day, the butterfly had entirely recovered, its responsiveness to water had disappeared, and trials with saccharose were resumed. As the period of water diet continued, the threshold of response to saccharose gradually fell from M/400 on the 8th day to M/3200 on the 11th day. At the close of the 11th day the animal again evinced signs of weakness, and it was necessary to change the diet to 1M saccharose. Absence from the laboratory prevented any trials on the 12th day. When experimentation was renewed on the morning of the 13th day, however, the butterfly had completely recovered its normal vigor. But the threshold of response had risen from M/3200 to M/10, and at this level it steadily remained through- out the entire period of saccharose diet, viz., the 13th, 14th, and 15th days. Following the first period of saccharose diet came the second period of total inanition, which lasted from the 16th to the 20th days, inclusive. During the first two days of this period, the TARSAL SENSITIVITY IN PYRAMEIS 453 16th and 17th days, the threshold of response to saccharose again fell to M/100. On the 18th, 19th, and 20th days the respon- siveness to water gradually reappeared and prevented any conclusive determinations with saccharose. At the close of the trials on the 20th day the second period of water diet was begun, and on the following morning the respon- siveness to water had completely disappeared once more. The threshold concentration of saccharose on this the 21st day was still M/100. The water diet was continued until the close of the 26th day, the threshold concentration of saccharose gradually falling meantime until it reached M/6400. In the last two trials of the 26th day the butterfly had become extremely weak—a fact which may account for the negative results obtained. The physical condition of the animal on the evening of the 26th day necessitated the administration of saccharose, and accordingly the second period of saccharose diet was begun. On the morning of the 27th day the butterfly appeared greatly revived. But tests with saccharose showed that the threshold of response had again risen to M/10, precisely as it had done at the beginning of the first period of saccharose diet. On the evening of the 27th day the animal seemed quite as vigorous as usual, but on the morning of the 28th day it was found dead. In figure 1 the threshold concentrations of saccharose for butterfly no. 13 are represented graphically. A glance at the curve shows that during periods of total inanition followed by periods of water diet, i.e., during saccharose inanition, the threshold of response gradually fell, while immediately a period of saccharose diet was begun the threshold abruptly rose. Two facts are thus brought out clearly. First, the minimal effective concentration of saccharose for the tarsi varies directly with the nutritional condition of the animal. During periods of saccharose diet the threshold remained at M/10; during periods of com- plete inanition and water diet, i.e., saccharose inanition, the threshold gradually fell to M/3200 or M/6400. Second, the minimal effective concentration after prolonged inanition with respect to saccharose may be extremely low. At the close of the first period it was M /3200; at the close of the second, M /6400. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL, 36, NO. 4 454 DWIGHT E. MINNICH In table 2 I have summarized the results obtained on all seven butterflies. While the results there presented exhibit rather wide individual differences, they show the same general features which are brought out in the behavior of butterfly no. 13. Thus the threshold of response in all animals fluctuated directly with the nutritional condition. Furthermore, the minimal effective concentration after prolonged inanition with respect to saccharose was very low in general. The extremely low thresholds, M /3200, M_/6400, and M /12,800, which were observed in four of the seven animals, are particularly noteworthy. TABLE 2 Showing the threshold concentrations of saccharose solution, under varying nutri- tional conditions, in seven butterflies. An asterisk signifies the death of the butterfly within twenty hours after the determination indicated ANIMAL pai a FIRST PERIOD Neerae OF Searop ee SECOND PERIOD seerOniGe NUMBER ANGE OF WATER DIET | SACCHAROSE TOTAL OF WATER DIET | SACCHAROSE DIET INANITION DIET 11 Not tried | M/100 M/10 M/50 M/12, 800 | - M/10 12 Not tried | M/200 M/10 M/50 M/400* 13 M/200 | M/3200 M/10 M/100 M/6400 M/10* 22 Not tried | Not tried M/100 M/400 M/3200 M/100 23 Not tried | M/100* 24 Not tried | M/12, 800* 25 Not tried | M/400* This responsiveness to very dilute solutions of saccharose affords further evidence of a point previously made by me, viz., that the nature of tarsal stimulation is not osmotic, but chemical (ef. Minnich ’21, p. 198). Throughout the present experiments all animals were tested four times daily with several solutions other than those mentioned in this paper. Among these were 1M saccharose and 2M NaCl. On the days when the four butter- flies mentioned in the foregoing paragraph were responding to such dilutions of saccharose as M/3200, M/6400, and M/12,800, three of the four gave no response whatever to 2M NaCl. Yet the NaCl solution was vastly the more effective osmotically. It might be objected that the salt solution produced such a powerful osmotic effect as to inhibit response, whereas a more 455 TARSAL SENSITIVITY IN PYRAMEIS “a[qISssOduII 910M BSOIBYIOVS YIM SUOT]BUIULIOJEp YOTYA Burunp ‘ouoje 10yvM 0} SsoUBAT}ISUOS JO SpOlled o}eoIpuUl Soul] UoYOIq Ul UMOYS BAIND OT} JO SUOTAOT ‘“WOT}BIZWOOUCD oY} JO pBozSUT pozjoTd UdeEq SBY WOr}VI}MI9MOD OY} JO wyyWUeso] oy} ‘voeds O[QUIIVAG OY} UI SAINO oY} JO SoANywoJ JUBOBTUSTS oY} JNO SulIq OF Topo UT “ET “OU Api} nq ur spotsed [BUOT}II} NU SHOWA SulINp asorvyoo"S JO SUOT}VIJUSDUOD PLOYsery} FO uoryeyuosoider oder [ “Shy Ge ee de Gene te ie eae Wie Oc 20) Be) Scio) <6] ISS SAI Ols O28) Za. Gar £ «if SF VM, {3/Pp yoodes J}9/P wojyem uollueul )eJO} JaIpP ‘yooes joIp ywayem uvoljrueul JO Poldad Pug JO poldad Pug JO Poldad PuUZ jo Pol4ed js JO Posed js] J2}O} JO POl4ed 4ST skeg ul ody UONBUJUSOUOD pjoyseuy], Jo wiyjiuego 456 DWIGHT E. MINNICH dilute solution would have produced a response. But this ob- jection is at once ruled out by the fact that a 1M solution of saccharose always yielded a 100 per cent response. The tarsal sense organs, therefore, appear to be quite unaffected by osmotic pressure and must be regarded as specific chemoreceptors. The tarsal sensitivity of the red admiral to saccharose is quite remarkable when compared with the sensitivity of other animals to the same substance. Among the marine invertebrates sac- charose appears to have little or no stimulating power apart from the osmotic effect of high concentrations. The same holds true for the lower aquatic vertebrates. According to the table given by Parker (12, p. 228), Amphioxus, Ammocoetes, Mustelus, and Amiurus are entirely unresponsive to saccharose. Moreover, the weakest solution which the human tongue can detect is but M/50. But as we have seen in two of the seven butterflies tested, nos. 11 and 24, the tarsi were able to discriminate an M/12,800 solution. In other words, the tarsi of these two butterflies were 256 times more sensitive to saccharose than the human tongue. The sensitivity of Pyrameis to saccharose is thus a highly specialized one. The reason for this is not far to seek. Pyrameis feeds on nectar, exuding sap, and juices of ripe and decaying fruit. In all of these substances sugars are found. In the laboratory I have kept the butterfly alive and in good condition for thirty days on 1M saccharose solution. Sugars thus appear to be the most important food of this insect, and the highly developed sensitivity to saccharose is doubtless directly correlated with this fact. Like the organs of olfaction, therefore, the organs of taste may be very highly developed in certain lepidopterous forms. CONCLUSIONS 1. In Pyrameis the threshold of response to saccharose solutions varies directly with the nutritional condition of the individual. During periods of total inanition followed by periods of water diet, i.e., during saccharose inanition, it gradually falls. But with the initiation of a period of saccharose diet, it rises abruptly to a level which remains approximately constant throughout the remainder of the period. TARSAL SENSITIVITY IN PYRAMEIS 457 2. After prolonged inanition with respect to saccharose the threshold concentration may fall as low as M/3200, M/6400, or even M/12,800 in some individuals. The tarsal sensitivity of Pyrameis to saccharose may thus be as much as 256 times that of the human tongue. 3. The highly developed sensitivity to saccharose is doubtless correlated with the fact that sugars form the chief food of this insect. BIBLIOGRAPHY Fasre, J. H. 1879-1904 Souvenirs entomologiques. me serie, nos. XXIII, XXIV, XXV. 8meedition. Paris. Minnicu, D. E. 1921 An experimental study of the tarsal chemoreceptors of two nymphalid butterflies. Jour. Exp. Zoél., vol. 33, pp. 173-203. 1922 The chemical sensitivity of the tarsi of the red admiral butter- fly, Pyrameis atalanta Linn. Jour. Exp. Zoél., vol. 35, pp. 57-81. Parker, G. H. 1912 The relation of smell, taste, and the common chemical sense in vertebrates. Jour. Acad. of Nat. Sciences of Phila., vol. 15, second series, pp. 221-234. Ritry, C.V. 1894 Thesenses of insects. Insect Life, vol.7, pp. 33-41. Resumen por el autor, Stefan Kopeé. Relacién mutua entre el desarrollo del cerebro y los ojos de los Lepidépteros. 1. Los ojos de la mariposa nocturna Lymantria dispar L., se desarrollan con completa independencia del cerebro y ganglio subesofdgico. El cerebro solamente ejerce una influencia regula- dora sobre la direccién de las fibras nerviosas que van desde la retina del ojo al ganglio 6ptico. 2. El gérmen de los ojos, ex- tirpado de la cabeza de una oruga e injertado sobre el abdomen, se desarrolla normalmente en la nueva posicién, a pesar de la ausencia de conexiones con la cadena nerviosa. 3. La extirpacién de los gérmenes oculares imaginales de las orugas impide el desarrollo de las capas externas del ganglio 6ptico del adulto y produce cambios en la estructura de ciertas capas internas, suponiendo que tenga lugar la regeneracién del gérmen del ojo. 4. El ganglio subesofagico de los ejemplares desprovistos de cerebro se desarrolla menos que en los individuos normales. La extirpacién del ganglio subesofdgico de la larva no ejerce in- fluencia visible sobre la formaci6n del cerebro imaginal. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 23 MUTUAL RELATIONSHIP IN THE DEVELOPMENT OF THE BRAIN AND EYES OF LEPIDOPTERA! STEFAN KOPEG Government Institute for Agricultural Research, Pulawy, Poland ONE PLATE (SIX FIGURES) THE DEVELOPMENT OF IMAGINAL EYES IN BRAINLESS INSECTS The correlation of the components of the eye in the ontogenetic development of vertebrates has been studied. The experiments undertaken by Spemann on the development of the lens and by Lewis on the development of the cornea and continued by Bell, Le Gron, Diirken, Ekmann, Fischel, King, Wachs, and others proved that while in certain amphibians the lens depends on the optic cup and the cornea on the lens, in others such a dependence cannot be ascertained. The mutual relationship between the development of the nervous system and the onto- genetic formation of the eyes in invertebrates has, so far as I know, not yet been studied. The investigations of Herbst on Crustacea (’96-"16), Carriére (’80) and Hanké (14) on the Mol- lusca, since they refer only to the processes of regeneration of the eyes, do not come into consideration here. In my experiments on insects I endeavored to study these relations by examining the development of the eye in moths the caterpillars of which had been deprived of the brain, and the development of the imaginal brain in animals whose caterpillars had been deprived of the eyes. I removed the whole brain (ganglion supraoesophageale) from several caterpillars of Lymantria dispar L after their last moult. 1 Paper from the Embryologic-Biological Laboratory, Jagellonian University, Cracow, Poland, presented in the Acad. of Sc., Cracow (ef. Bull. intern. Acad. d. Se. Cracovie, 1917). 459 460 STEFAN KOPE(G In this manner I also deprived the caterpillars of the larval optic ganglia (as to method of operation, ef. Kopeé, 718). Some of the caterpillars underwent pupation and developed to adult forms of moths. In all the specimens of these moths, or of their pupae, the eyes were quite normally developed macroscopically (ef. fig. 1, representing the eyes of brainless moth). In dissections of eyes which developed without the brain, only a more or less distinct wrinkling of the surface of the eye may often be noticed. I consider this deformation to be due only to the mechanical difficulties experienced by a brainless animal in drawing its injured head from the skin of the caterpillar or pupa during its metamorphosis. . Also microscopical researches have demonstrated the normal structure of ‘brainless’ eyes (cf. figs. 2. and 3, representing dissec- tions of eye which developed without the brain, with figs. 4 and 5 giving analogous dissections of normal eyes). Both the number of ommatids and the size and form of all components of the compound retina were in no wise different from those observed in normal circumstances. The arrangement of the pigment is somewhat different in figure 3 of the ‘brainless’ eye and in figure 5 of a normal one. But also in normal specimens we may ob- serve individual fluctuations in this arrangement of the pigment, due probably to the time at which they were killed. On the interior surface of the normal eye I always found the layer which is called by Berger (78) ‘the nerve-bundle layer’ (Nervenbiindelschicht) to be well developed. In this layer I have discerned two distinct parts: an external layer of single bundles, consisting of a relatively small number of nerve fibers coming directly from the retina of the eye (fig. 4, s.n.b.) and an internal one, which I might call the layer of compound bundles (c.n.b.). The compound bundles are made up of single bundles which are in both layers distinctly separated from one another and run regularly to the interior of the head, radiating in the direction of the optic ganglion. The arrangement of the single bundles does not evidence any changes in the brainless insects (fig. 2, s.n.b.), while the compound bundles always behave with remark- -able irregularity. These latter bundles have an abnormal BRAIN AND EYES OF LEPIDOPTERA 461 arrangement (fig. 2, c.n.b.). We get the impression that, in the ontogenetic development of the eye, the brain or the optic gan- glion has a determining influence on the direction of these nervous bundles. These anomalies might, however, just as well be attributed to different modes of growing together, which took place.during cicatrization of torn larval nervous bundles. The crucial fact here was the behavior of moths deprived of the germs of their imaginal eyes, these germs being subsequently regenerated. However, in these cases the larval nerve-bun¢cles underwent analogous tearing or concrescenses during operation, yet the arrangement of the single as well as compound bundles was quite normal. Consequently, during the formation of the imaginal eye the brain (or the optic ganglion) has an influence on the direction of the nerve-bundles proceeding from the retina to the optic ganglion. The other layers situated under normal conditions between the layer of nerve-bundles and the imaginal optic ganglion, which are considered by Berger to be a modified part of the optic gan- glion, arise through transformation of the larval optic ganglion which was removed together with the larval brain. In view of such anatomic-histological conditions it becomes quite clear that only the nerve-bundle layer described above is present besides the eye sensu stricto in the brainless moth. The simultaneous removal of brain and of the suboesophageal ganglion has convinced me of a quite similar independence of the evolution of the eye from the second ganglion situated in the head. Stress must be laid on the fact that in no case was I able to detect any processes of regeneration of the removed brain or suboesophageal ganglion. Thus the development of the imaginal eye of moths is quite independent of the presence of the brain and the suboesophageal ganglion. 462 STEFAN KOPECG THE DEVELOPMENT OF IMAGINAL EYES THE GERMS OF WHICH HAVE BEEN TRANSPLANTED ON THE ABDOMEN OF CATERPILLARS The transplantation of the eyes in vertebrates has been ac- complished by Uhlenhuth (12, ’13), who grafted larval eyes of salamanders and newts on other specimens of the same species. He convinced himself that the eyes undergo further evolution after certain transitory processes of atrophy. In order to render the independence of the development of the insectal eye still more striking, I attempted the transplantation of the germ of eyes to the abdomen, 1.e., to surroundings which are heterogeneous from an anatomical point of view. . I deprived seventy-five cater- pillars of the whole lateral part of the head, on which all their ocelli are situated. Johansen (’92) has shown that the material for the imaginal eye is contributed by the hypodermis between the larval eyes, which are to be found on the chitinous plate removed. I then inserted this plate into a large wound made on the fourth abdominal segment of the same caterpillar by the amputation of one of the paired orange-yellow warts. After a few hours the plate had been well fixed in the new position by means of coagulated blood. Out of forty-one pupae obtained from. this series of experi- ments, fifteen had distinctly demarcated, tiny hill-like con- vexities in the place where the plate had been grafted. These convexities differ from the integument of the other segment in that they have a more glossy and deeper hue. In thirteen of the moths which emerged from these pupae, a very distinct imaginal eye, in most cases of normal size, was found on the corresponding abdominal segment (fig. 6, tr. e.). The eyes were more or less hemispherical, sometimes somewhat wrinkled and divided by fur- rows. ‘The histological structure of these eyes differs in no way from that of normal eyes. We need only note that the height of the ommatidia was reduced and that there was a proportional shortening of their components. This detail was no doubt connected with the abnormal tension and pressure in the new surroundings of the eye which influence the developing organ. Here also the grafted eyes had a well-developed ‘nerve-bundle BRAIN AND EYES OF LEPIDOPTERA 463 layer’ running abnormally. I was, however, not able to find any nerves radiating from the eye deeper into the interior of the body. THE DEVELOPMENT OF THE BRAIN IN INSECTS DEPRIVED OF THE GERMS OF EYES In the following series of experiments I removed the above- mentioned plate, which contained the material for the eye of the moth, from one side of young caterpillars after the second or third moult, and without depriving the animals of the brain. I examined its behavior in cases when the removed hypodermis did not undergo regeneration and thus did not form an imaginal eye. At the same time the non-operated side of the head could be used asa standard. It was my intention to ascertain whether the morphological and histological metamorphosis which the larval brain undergoes when metamorphosing into the imaginal organ, is in any way dependent on the development or presence of the eye. In its principal part the brain of the moth is composed of the large optic ganglia resulting from the metamorphosis of those external parts of the larval brain, which form the optic ganglia of the caterpillar (cf. Johansen, ’92, and Bauer, ’04). In all the specimens which did not regenerate their eyes the imaginal optic ganglion was more voluminous, though shorter on the operated side than on the normal one. The granular, as well as the molecular layer and the ganglion-cell layer (Kérner, Moleku- lar and Ganglienzellenschicht of Berger), was never developed. It follows from the paper of Berger that the tissue which gives rise to these layers is situated in the external parts of the larval brain. This tissue remained intact in the head of the caterpillar operated upon: this I conclude from the fact that I never ob- served any injuries of the larval brain when, to verify the results of operation, I examined the microscopical sections of several specimens directly after operation. In other words, the absence of this layer of the optic ganglion in specimens which had no regenerated eye can only be interpreted by the absence of some developmental stimuli, derived from the normal eye in course of 464 STEFAN KOPE(C development or, as I had several times observed, from the re- generated eye. That is to say, when the eye is regenerated all the layers of the imaginal optic ganglion layers are developed quite normally also. The layer of the external chiasma (dussere Kreuzung) never was found afterward in the operated specimens. The layer of the internal chiasma and the external medullary layer (innere Kreuzung and jusseres Marklager of Berger) were often but slightly developed. The internal medullary (inneres Marklager) was thicker along the longitudinal axis of the brain, thinner along the transverse axis. These anomalies set in a still clearer light the influence exerted by the eye on the development of the imag- inal brain or sensu stricto, of its optic ganglion during its metamorphosis. In one of my previous papers I already urged this opinion, but was unwilling to consider it as proved on account of the small amount of experimental material then at my disposal (cf. Kopeé, ’13, p. 457). My former conclusion as to the dependence of the formation of the brain on the formation of the imaginal eye in insects is now well founded and it finds perfect confirmations in the important investigations of Herbst (716) on changes in the structure of the optic ganglion in certain crustacea deprived of the eyes. In connection with the results discussed here I wish to remark briefly that in the development of both ganglia of the head a certain mutual dependence may be observed. In microscopical sections of brainless specimens we are at once struck by the ex- ceedingly meager development of the suboesophageal nervous ganglia. Its normal development is evidently correlated to some extent with the formation of the brain. The removal of the larval suboesophageal ganglion, on the contrary, has no visible effect on the formation of the imaginal brain. SUMMARY 1. The eyes of the moths develop in complete independence of the brain and the suboesophageal ganglion. The brain exerts only a regulating influence on the direction of the nerve fibers going from the retina of the eye to the optic ganglion. BRAIN AND EYES OF LEPIDOPTERA 465 2. On the other hand, if the imaginal eye, the germ of which had been removed in the caterpillar, is absent, the external layers of the optic ganglion do not develop at all, and certain internal layers show changes in their structure. 3. The germs of mature eyes grafted from the head of the caterpillar on its abdomen develop normally, notwithstanding the absence of any junction with the nervous chain. 4, In specimens deprived of brain the suboesophageal ganglion develops to a markedly less degree than in normal specimens. The removal of the larval suboesophageal ganglion has no visible effect on the formation of the imaginal brain. BIBLIOGRAPHY Bauer, V. 1904 Zur inneren Metamorphose des Centralnervensystems der Insecten. Zool. Jahrb., Abt. f. Anat. u. Ontog, Bd. 20. Bercer, E. 1878 Untersuchungen iiber den Bau des Gehirnes und der Retina der Arthropoden. Arb. d. Zool. Inst. Wien, Bd. 1. CARRIERE, J. 1880 Studien iiber Regenerationserscheinungen bei Wirbellosen. Wiirzburg. Hank6, B. 1914 Uber das Regenerationsvermégen und die Regeneration ver- schiedener Organe von Nassa mutabilis. Arch. Entw.-Mech., Bd. 38. Hersst, C. 1896 Uber die Regeneration von antennenihnlichen Organen an Stelle von Augen. I. Mitteilung. Arch. f. Entw. Mech., Bd. 2. 1900 III. Mitteilung. Ibidem, Bd. 9. 1916 VII. Meitteilung. Ibidem, Bd. 42. JOHANSEN, H. 1892 Die Entwicklung des Imagoauges von Vanessa urticae L. Zool. Jahrb., Abt. f. Anat. u. Ontog., Bd. 6. Korxc, St. 1913 Untersuchungen iiber die Regeneration von Larval-organen und Imaginalscheiben bei Schmetterlingen. Arch. f. Entw. Mech., Bd. 37. 1918 Lokalisationsversuche am zentralen Nervensystem der Raupen und Falter. Zool. Jahrb., Abt. f.allgem. Zool.u. Physiol., Bd. 36. UntenuutH, E. 1912 Die Transplantation des Amphibienauges. Arch. f. Entw. Mech., Bd. 33. 1913 Die synchrone Metamorphose transplantierter Salamanderaugen. Ibidem, Bd. 36. *pedojeaep A][vur.tou ‘efo poquvpdsuviy “a 47 [yuewses [VUIWIOpge YAALNOJ 9Y} UO poyuR[dsuesy Useq pey oAa [VUISVUIT OY} JO WIS OY} YOTYM Jo Av] [Id104v9 oY} WOIJ YJOU BJO UNWIOpqy 9 "Eg dINSY Ul SB SUOTYVIAIIGG’ OUIeS oY} puv UOI}VOYIUSvU oUIVS OY], ‘poylusvur o10ur ‘aXo owes OY} JO WAV G “UOT[Sues 014d0 04} pAvMO} SUIZVIpes ‘Sa_punq sNoAJoU punodur0d 94} JOJUSWIOSUVIIG[VULION *Z OINSY Ul sv SUOTZBIADIQG’ OUIeS OY} pus UOlyVoylusvUISUIBSOyT, ‘YJOUL[VUILOU vB JO 9Aa poqoosSsiIp oY} JO Yavd QuOoI F ‘ovlnuryod “4 {seuod ouly[eyskio “49 fsasuey “7 Speytuseur o1ou ‘oo owes OY} JO ye ¢& *IaA¥B] O[pUNq-sAr1oeu punodwiod “q'u'd !1aAV] o[pUN-dAdoU O[SUIS “qu'sieipryeuruo “0 fsesue] “7 “4,NOUT 4SVl OY} 194Jv UIvIq oY} JO poATidop useq pey YoIyM jo rvT[Id104v0 044 ‘qZour v JO ofa poyoossIp 94} Jo yavd yuoI Z *podojoagp [[oM A]JooJ10d SoA OFT] [VAIL] JO 99%4S JS¥[ OY} SULINp UIvIg JO poAlidep YOU s[vUIZJ vB JO YAvd IOTIOJUY [ "TT avdstp BVIIYUBWIATT Y}OUI OY} JO SoBvIS UMOIS-[[NJ VY} 0} 1oJor sydvasojyoyd oy} [TV SHuUnSIat AO NOILVNVIdxXa l ALVId 466 T ALVId OAdOM NVAIGLs VUALdIOGIdAT AOISAAM GNV NIVUE 467 Resumen por el autor, Stefan Kopeé. Autodiferenciacién fisiol6gica de los gérmenes de las alas injertados en orugas del sexo opuesto. El] gérmen de las alas imaginales injertado en ejemplares de orugas de Lymantria dispar L. del sexo opuesto contintia desar- rollandose y las alas diferenciadas a sus expensas presentan su tinte dimérfico propio, en vez de exhibir el del individuo sobre el cual se desarrollan. Los pigmentos de la escama no proceden, por consiguiente, directamente de la sangre desecada, sino que son el producto de ciertos cambios quimicos, los cuales, segun Mayer, tienen lugar en la sangre bajo la influencia de substancias especificas contenidas en las células formadoras de las escamas. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 23 PHYSIOLOGICAL SELF-DIFFERENTIATION OF THE WING-GERMS GRAFTED ON CATERPILLARS OF THE OPPOSITE SEX? STEFAN KOPEG Government Institute for Agricultural Research, Pulawy, Poland The development of the pigment in the wings of moths has not yet been sufficiently studied; there is, however, no longer any doubt that the chief ingredient in the formation of the pigments is the blood, or the so-called haemolymph of the animal. How this formation takes place has not yet been ascertained; some authors believe that the pigment is directly caused by the drying up of the haemolymph. On the contrary, according to Mayer (96, ’97), there are certain ferments in the scale-forming cells which render the formation of the pigment possible. On this view the pigment is the product of certain changes which occur in the insect’s blood, under the influence of special ferments. Crampton (00) is inclined to Mayer’s opinion, but I think that his notable experiments are not sufficient to demonstrate it. Crampton united various parts of the pupae of the moth Callo- samia promethea by means of paraffin, so that the front part of the body obtained belonged to one sex, the hind part to the other. These bodies developed further until the stage of the adult moth, when each part of the artificially united body showed its specific and dimorphic color, different in front and behind. “We must conclude, therefore,’ says Crampton, ‘‘that the production of the sexually-different ground-colors of the adult moths is determined by some ‘ferment’ factors which differ in the two sexes, and that the difference in the adult colors is not due to a difference between 1 Paper from the Embryologico-Biological Laboratory, Jagellonian University, Cracow, Poland, presented in the Acad. of Se. Cracow. (Cf. Bull. Acad. d. Se. de Cracovie, 1917.) 469 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 36, NO. 4 470 STEFAN KOPEC the respective haemal fluids of the two sexes.’”’ Crampton’s investigation itself, however, does not justify our drawing such a conclusion; at the same time it is quite true, as we shall soon convince ourselves. First, the united parts were relatively large bodies, very thoroughly and abundantly supplied with blood, this being different in each part; it seems to me very probable, therefore, that in the cases given there was no thorough mingling of the two haemolymphs in the artificially joined organism; since it is quite possible that these did not mix at all, Crampton’s conclusion does not seem to me to be proved. Secondly, the connection of the chrysalides was possibly made at too late a. stage for this supposed influence of one part on the other to be visible. I tried, therefore, to verify Mayer’s theory by a some- what different method. At the same time my experiments were intended to show the morphological and physiological self- differentiation of the insect wing still more distinctly than it has been shown in the experiments on the castration of cater- pillars. (See below.) I removed the germs of the first right wing from male cater- pillars of Lymantria dispar L. after their last moult, and in their place I grafted a similar germ from a female caterpillar, and vice versa. It was necessary that both caterpillars should be of about the same age, so that the time of pupation of the one operated upon would coincide most strictly with the time at which the implanted wing of the opposite sex should attain the stage of metamorphosis. Naturally, the fulfillment of this condition was extremely difficult, and on the other hand the metamorphosis of the grafted wing always occurs quite inde- pendently of the organism on which it has been implanted. Therefore, if the condition mentioned has not been fulfilled, the implanted wing undergoes metamorphosis at a different time than the caterpillar on which it is grafted and cannot easily come forth and develop subsequently. If we take into considera- tion also that the delicate germ was often injured during trans- plantation, it is not astonishing that out of 120 specimens oper- ated upon, the implanted germ developed into the pupal and imaginal wing only in two cases. In these two cases we had to DIFFERENTIATION OF GRAFTED WING-GERMS 471 do with male wings developed on female organisms. The grafted wings were somewhat folded and could not be drawn out of the pupal integument by the moth alone. During the extraction of these wings the scales, especially from the upper surface, nearly all remained in the pupal skin, from which they could very easily be removed uninjured. ‘The form and color of the scales were afterward studied exactly under the microscope. In one case the form of these scales was quite normal, in the other the scales were less deeply dentated than in normal con- ditions, which seemed to show that they were not completely developed. The color of these wings corresponds to the dimor- phic hue which the transplanted wing would have had if it had remained intact in the previous organism; therefore, the grafted wing was in both cases otherwise colored than the normal left wing of its foster-mother. The wings of the male, which had been transplanted on the female, had more or less dark gray or dark brown scales, in contrast to the female wings, which were for the most part white. I think the results of these experiments certainly speak in favor of Mayer’s theory, as it turned out that the cells of the wing germs were able to collect the respective dimorphic pig- ments in their scales, forming them from the heterogeneous blood of the other sex in the new surroundings. Thus the pigments of the scales are not directly derived from the desiccation of the haemolymph, but must be the outcome of certain chemical proc- esses occurring in the insect blood under the influence of sub- stances formed in the scale-producing cells, and considered by Mayer to be ferments. Owing to these substances, which are different in different parts of the imaginal wing, various pig- ments are formed in the blood of the insect, and this results in the production of complicated figures on the wings. ‘These substances develop by means of physiological self-differentiation, and their formation is outside the influence of the haemolymph of the other sex. This fact is unexpected, since the investigations made by Dewitz (709~16b), Steche (’12a,b,) and Geyer (13, 714) have demonstrated that the blood in insects of one and the other sex is not the same, but shows great differences in the 472 STEFAN KOPECG chemical qualities as well as in its coloring. It happens that the differences are not important enough to have a decisive in- fluence on the hue of the insect wing. Consequently, the real cause of the color of the wings of dimorphic moths, being distinct for the two sexes, is not the difference of the blood, but the difference of the substances which are present in the cells of the germs of the wings. The physiological self-differentiation of the corresponding cells takes place early, even before the pupation of the caterpillar. In this stage the germ of the yet unformed wing seems to resemble in some degree an exposed but as yet undeveloped photographic plate. The self-differentiation of the wing of moths, as I have stated it, is sufficient to interpret the known results on the castration of caterpillars, which does not lead to even the slightest changes in the dimorphic coloring of the wings of the adult moth (compare the experiments of Oudemans, 799; Kellogg, ’04; Meisenheimer, 07, .’09;. Kopeéé,;’08,'711, 713, and Geyer, 713). Prell (15'a), having castrated caterpillars of the moth Cosmotriche potatoria L., observed, on the contrary, a much larger variability in the direction of a lighter hue in the wings of castrated males than in normal specimens. Prell does not question the negative results of the experiments on the castration of moths hitherto performed, and obtained chiefly on the species Bombyx mori L. and Lyman- tria dispar L., but he believes that the results ought not to be extended to all forms of moths. It is possible that further in- vestigations made in this direction would lead us to distinguish between those forms of moths which react and those which do not react after castration by showing a change of color of the wings. But the experiments hitherto made by Prell are too small in number to draw any certain conclusion from them. In Prell’s investigations we also miss standard experiments, such as might exclude the possibility of the influence of the operation itself, whether the sexual glands were removed from the insect body or not. According to Prell’s own words, the castrated cater- pillars refused food, and they had also to be far more abundantly sprinkled with water. If we bear in mind that they were sub- jected to a powerful ether narcosis and that they often lost a DIFFERENTIATION OF GRAFTED WING-GERMS 473 large quantity of blood during the operation, we can readily understand that they might have been much weakened by the operation and have responded more readily to all changes of external conditions, such as temperature, moisture, etc., even when these were imperceptible to us. In contrast to Lymantria dispar, Cosmotriche potatoria belongs to those species of moths whose dimorphic wing-colors undergo distinct changes under the influence of cold; to this difference Prell ascribed his results, which he considers different. The later investigations of Prell (15b) on the castration of various Vanessae, the classical material for the study of the influence of temperature, do not seem to support this opinion. Castrated Vanessae as well as castrated females of Cosmotriche potatoria undergo no changes after castration, in contrast to males of the latter species. In this behavior of the Vanessae, Prell (15 b) sees a proof that in the experiments on males of Cosmotriche the change in hue was not excited by the operation itself, but that we have here to do with the effect of the removal of the sexual glands; for if the lighter hue of the wings of the males of] Cosmotriche were to depend on the operation itself, it would have to be admitted, according to Prell, that similar changes would appear also among the Vanessae operated upon, as they are even more sensitive to the influence of external conditions. I believe, however, that we might just as well suppose a different reaction power of the moth to castration, possibly different in the two sexes or in various forms of moths, as a different behavior of various organisms, in respect to their change of color, affected by debility and super- sensitivity resulting from the operation. In this way the results obtained by Prell may be made to accord with the results of the researches of Oudemans, Kellogg, Meisenheimer, and of my own. While some authors have seen certain contradictions in the results of different investigations, which, according to them, prove the influence of castration on the dimorphism of the moth, others have drawn the conclusion, from all these experiments, that the germ of the wings is already differentiated early in the larval life, and hence the removal of the gonad or the implanta- tion of glands of the other sex cannot change anything in the 474 STEFAN KOPEC coloring of the insect wing. The results of my experiments on the transplantation of the wing-germs confirm the latter conclu- sion, showing at the same time the principle in virtue of which these animals have quite a different position in regard to the development of their dimorphic secondary characters from that of vertebrates. Steche (12,a,b) and Geyer (713), who relied on their experiments on the dimorphic differences of the insect blood, expressed the opinion that the fundamentally different behavior of insects and vertebrates after castration is caused by the fact that the whole body (soma) of the former undergoes sexually dimorphic differentiation from the beginning of life. In the light of my own experiments described in this paper this hypothesis gains a new and important confirmation. SUMMARY The germ of the imaginal wings grafted on specimens of cater- pillars of Lymantria dispar L. of the opposite sex continues de- veloping, and the differentiated wings have the dimorphic hue proper to them, and not to the specimen on which they develop. The pigments of the scale therefore do not proceed directly from the desiccated blood, but are the product of certain chemical changes which, according to Mayer, occur in the blood under the influence of specific substances contained in the scale-forming cells. BIBLIOGRAPHY Crampton, H. E. 1900 An experimental study upon Lepidoptera. Arch. f. Entw. Mech., Bd. 9. DewiTz, J. 1909 Die wasserstoffsuperoxydzersetzende Fihigkeit der minn- lichen und weiblichen Schmetterlingspuppen. Zentrbl. f. Physiol., Bd. 22. 1912 Untersuchungen iiber Geschlechtsunterschiede. II. Ibidem, Bd. 26. 1916a Idem., III. Zool. Anz., Bd. 47. 1916 b Idem., IV. Zool. Jahrb., Abt.f.allg.Zool.und Physiol., Bd. 36. Gryer, K. 1913 Untersuchungen iiber die chemiche Zusammensetzung der Insectenhaemolymphe und ihre Bedeutung fiir die geschlechtliche Differenzierung. Zeitschr. f. wiss. Zool., Bd. 105. Ketioaa, V. L. 1904 Influence of the primary reproductive organs on the secondary sexual characters. Jour. Exp. Zoél., vol. 1. DIFFERENTIATION OF GRAFTED WING-GERMS 475 Korré, Sr. 1908 Experimentaluntersuchungen iiber die Entwicklung der Geschlechtscharactere bei Schmetterlingen. Bull. Acad. Sc.,Cracovie. 1911 Untersuchungen iiber Kastration und Transplantation bei Schmetterlingen. Arch. f. Entw. Mech., Bd. 33. 1913 Nochmals iiber die unabhingigkeit der Ausbildung sekundarer Geschlechtscharactere von den Gonaden bei Lepidopteren. Zool. Anz., Bd. 43. Mayer, A. G. 1896 The development of the wing scales and their pigment in butterflies and moths. Bull. Mus. of Comp. Zo6l. of Harvard Coll., 29. 1897 On the color and color-patterns of moths and _ butterflies. Ibidem, 30. MEISENHEIMER, J. 1907 Ergebnisse einiger Versuchsreihen iiber Exstirpation und Transplantation der Geschlechtsdriisen bei Schmetterlingen. Zool. Anz., Bd. 82. 1909 Experimentelle Studien zur Soma- und Geschlechtsdifferenzie- rung. Erster Beitrag. Jena: Fischer. OvupEemans, J. Tu. 1899 Falter aus kastrierten Raupen, wie sie aussehen und wie sie sich benehmen. Zool. Jahrb., Abt. f. Syst., Bd. 12. Pretut, H. 1915a Uber die Beziehungen zwischen primiren und sekundiren Sexualcharakteren bei Schmetterlingen. Zool. Jahrb., Abt. f. allg. Zool. und Physiol., Bd. 35, S. 183. 1915 b Ibidem, S. 593. Srrcun, O. 1912a Die ‘sekundiren’ Geschlechtscharaktere der Insekten und das Problem der Vererbung des Geschlechts. Zeitschr. f. ind. Abst. und Vererbungslehre, Bd. 8. 1912b Beobachtungen iiber Geschlechtsunterschiede der Haemo- lymphe von Insektenlarven. Verh. d. Deutch. Zool. Ges. Resumen por el autor, E. J. Lund. El control experimental de la polaridad orgdnica por medio de la corriente eléctrica. II. La polaridad eléctrica normal de Obelia. Una prueba de su existencia. En el tallo de la colonia de Obelia existe una diferencia definida de potencial eléctrico, tal que la regién apical de crecimiento es electronegativa con respecto a la regién media o mas basal. Esta polaridad eléctrica esta asociada con el tejido viviente del cenosarco y no se origina en ninguna otra estructura del tallo, porque no existe diferencia de potencial en: a) los tallos que se abandonan para que mueran y se maceran en agua de mar; b) los tallow desprovistos mecdanicamente del tejido vivo, y c) los tallos en los cuales el tejido vivo muere mediante la accién del cloroformo. La magnitud del descenso de potencial varia en trozos de tallos de diferentes colonias. También varia a lo largo del tallo de la misma colonia, siendo mayor en la regién apical en vias de crecimiento activo. La conclusién general derivada de los experimentos es que, puesto que las diferencias normalmente inherentes del potencial eléctrico tienen lugar en el tallo de Obelia y est’n asociadas con el crecimiento apical, antonces debiera ser posible inhibir 0 modificar el proceso del desarrollo por una aplicacién apropiada de una E.M.F. de origen externo. En un trabajo precedente el autor ha demostrado la posibilidad de esta modificacion. Translation by José F. Nonidez Cornell Medical College, New York AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 23 EXPERIMENTAL CONTROL OF ORGANIC POLARITY BY THE ELECTRIC CURRENT Il. THE NORMAL ELECTRICAL POLARITY OF OBELIA. A PROOF OF ITS EXISTENCE E. J. LUND The Puget Sound Marine Biological Laboratory and the Department of Animal Biology, University of Minnesota TWO FIGURES In the previous paper (Lund, ’21) it was shown that by passing an electric current of proper density lengthwise through an isolated internode from the stem of Obelia, it was possible to inhibit polyp formation on the end turned toward the cathode, while under these same conditions of current density a normal polyp formed on the end of the internode which was turned toward the anode. It was also shown that in order to inhibit regeneration of polyps on apical internodes, it was necessary to use a higher-current density than that which was necessary for inhibition of polyp formation in internodes from basal levels of the stem. The final general conclusion drawn from the experi- ments was, that since it was possible in this way to inhibit selectively polyp formation, the electromotive force applied from an external source probably acted upon some kind of system in the regenerating internode which was electrical (ionic) in its nature and which in all probability was closely associated with the mechanism which determines the polarity of the re- generating tissue. This conclusion did not rest alone upon the evidence from the experiments reported in the preceding paper, for several investigators have reported more or less convincing evidence that differences of electrical potential occur in hydroid stems of Tubularia, Pennaria, and Campanularia (see, e.g., Mathews, 703). More attention seems to have been paid to these normal continuously existing potential differences in plants than in animals (Buff, 54; Kunkel, ’81; Elfving, ’82; Burdon- Sanderson, ’82, and especially Miiller-Hettlingen, ’83). 477 478 E. J. LUND In the present paper evidence will be presented, which it is believed amounts to a proof of the existence of a normal difference of electrical potential in the living tissue of the stem of Obelia, the occurrence of which was suggested by the experiments in the previous paper. EXPERIMENTAL The galvanometer which was used to detect the currents ob- tained by leading off from different levels of the stem, was a Leeds Northrup instrument of somewhat more than one thou- sand megohm sensitivity.!. Under the conditions of the proce- dure in the experiments, it was difficult or impossible to make non-polarizable Zn-ZnSO, electrodes which maintained a per- fect iso-electric condition. Consequently, the simple arrange- ment of a copper wire inserted in a bent tube filled with sea- water, as shown in figure 1, was used as a readily washable electrode. The two electrodes used gave a sufficiently constant difference of potential for the purpose of the present experiments. The same pair of electrodes were used in all the experiments.? Suppose now that the electrodes are placed at a definite dis- tance apart and a piece of stem is placed upon them such that the ends rest in the drops of sea-water as shown in figure 1. The galvanometer will show a certain deflection due to the P.D. between the electrodes. If, now, the stem is reversed in its position and the galvanometer deflection is identical with the first, then obviously the current is the same. But, if the de- flection is less, then either one of two possibilities exists: 1) An inherent P.D. exists in the stem such that it opposes the P.D. between the electrodes or, 2) the ohmic resistance is greater in the latter position than in the first position. If the first of these possibilities is the correct interpretation, then obviously the deflection with the stem in the first position is greater than would occur if no P.D. inherent in the stem existed between the ends of the stem. The correct measure of the current due to the 1 The instrument was kindly loaned by Prof. H. L. Brakel, of the Department of Physics of the University of Washington. 2 It is apparently impossible to reproduce a Cu-CuS0O, electrode. ELECTRICAL POLARITY IN OBELIA 479 P.D. inherent in the stem is therefore represented by one-half the difference between the deflections. By proper calibration of the galvanometer, it therefore would be possible to determine the value of the current due to the P.D. between the ends of the stem, and by direct measurement of the resistance of the stem the P.D. inherent in the stem would of course be known. Figure 1 The second interpretation of the difference in deflection, when the stem occupies the two positions on the electrodes, would be that the current can pass more readily in one than in the opposite direction through the stem. The properties of the stem would therefore partially resemble the behavior of a rectifier toward an alternating current. That this possibility should be taken 3 According to F. Elfving (Bot. Zeit., Bd. 40, 1882, S. 257-263), Kunkel (Arbeit d. bot. Inst. Wurzburg, Bd. 2, Heft 2) found that the resistance of the shoot (seed- lings) when a weak ‘current’ was passed from base to apex through the shoot was less than the resistance of the same shoot when the ‘current’ was reversed. No experiments on the root are mentioned, but Elfving briefly states that an electric 480 E. J. LUND into consideration will be clear from such experiments as those reported by Bayliss (11) on certain colloidal salts when uniform distribution in a solvent is prevented by means of a membrane permeable to the anion, but impermeable to the cation. How- ever, it seems that in most if not all such cases a P.D. exists between the outside and inside of the membrane, so that in either case we should have to account at least in part for the difference in deflection by an inherent P.D. in the stem. In order to make clear the peculiarities of the records given in the tables, a sample series of tests upon an apical piece of the main stem of a colony is given as follows: APICAL END OF STEM PLACED ON DIFFERENCES BETW EEN TEST . SUCCESSIVE DEFLECT fort eta pce | mut ceed, Dele | cy area left scale left scale 1 21.0 <— 22.5 —1.5 2 20.0 i ea | ZOnD —6.5 3 17.5 <——-;—-—— 24.0 —6.5 4 170 <——| 23.0 —6.0 5 15.0 <—— apd As) —7.0 6 14.0 <—— 20.0 —6.0 The numbers represent the magnitude of the galvanometer deflections in millimeters on the scale. The direction of the deflection was the same in all the tests given in this paper. The arrows indicate the sequence of the tests; thus the first test with apical end of the piece on the right electrode gave a defiec- tion of 22.5 mm. The ‘position of the piece on the electrodes was then reversed. The deflection was now 21 mm. in the same direction on the scale. Returning the piece to its first orienta- tion gave 26.5 mm., and again placing the apical end on the left electrode gave 20 mm. The tests were repeated in this way until six pairs of readings were obtained in serial order. The duration current, sufficiently weak to be practically harmless when passed through the root of a seedling from base to tip, produced marked injury when passed through the root in the opposite direction. Recent experiments in this laboratory by Mr. Emmett Rowles on the growth of roots through which an electric current was passing seemed to indicate a difference in the effect on growth when the current passed in opposite directions. ELECTRICAL POLARITY IN OBELIA 481 of six pairs of tests was approximately ten minutes. It will be noted that the magnitude of the deflections decreased as time went on, but the differences between the deflections were about the same after the first test. The decrease in deflection is caused by the increase in resistance of the stem due to evaporation of the trace of water on its surface after removal from sea-water. A proof that this is the correct explanation will be given later when the tests on dead stems are considered. ‘The stems were manipulated by means of a wet camel’s-hair brush. In a series of tests on the same piece the distance between the electrodes was necessarily always the same. In the above tests it was 24 mm. In the last column of the table above are given the differences between the successive galvanometer deflections. The — sign indicates that the apical end is electronegative to the basal end and, as in the tables which follow, the + sign indicates that the apical end of the piece is electropositive to the basal end.‘ Since, for lack of space, it is impossible to publish the actual readings of the galvanometer deflections, the differences only between successive galvanometer deflections are given in the tables to follow. One important thing to be noted is, that since the deflections typically decreased in magnitude in successive tests on the same stem, then if, as sometimes happened, the difference of potential in a stem was small and at the same time the fall in total deflection due to drying was rapid, the differ- ences between such successive deflections would have a + sign, and therefore apparently indicate that the apical end was elec- tropositive to the basal end. Such a condition sometimes oc- curred in the tests in the tables given below. Therefore, the occasional occurrence of a + sign opposite a small (0.5 to 1.5 mm.) difference in deflection in some of the following tables does not necessarily indicate that the apical end was electropositive. Only a consistent occurrence of the + sign in successive large (2 to 10 mm.) differences between deflections can be taken as evidence that the apical end is actually electropositive. The ohmic resistance of equal lengths of stem from different colonies varies. This is also true of pieces of equal length from - 4 This refers to the current inside the stem. 482 Ey 7.) LUND different levels in the same stem. ‘The resistance of pieces of living stems 25 mm. in length varied, in a considerable number of determinations, from about 180,000 to 280,000 ohms, depend- ing upon the diameter of the stem and certain other conditions. The resistance of the galvanometer was 1000 ohms and that of the copper—sea-water electrodes was 5300 ohms. Constancy of the deflection when the electrodes are constant will therefore largely depend upon the constancy of the resistance of the stem. In table 1 A are given five series of tests upon the main stems of five different colonies designated by the numbers at the top of the table. The apical three-fourths of the main stem was isolated from its branches in each case. Immediately after removal of the branches, the tests were made in the manner described above. It will be seen by comparing the differences between deflections that a definite and marked difference of electrical potential occurs. The average differences between the deflections in the last five tests in A were 6.1, 8.1, 3.8, 9.6, 8.6 mm. ‘There is a perfect uniformity in direction of the P.D. inherent in the stem, such that the apical end is electronegative to the basal end. These pieces of stem were now placed in finger- bowls in fresh sea-water and allowed to regenerate for twenty- four hours. The tests were then repeated with the results given below in table 1 B. Comparison of the differences between deflections will show that these have decreased markedly during the preceding twenty-four hours. The differences are, however, quite definite and generally in the same direction as in the pre- vious tests. Two more experiments were carried out in which a total of ten stems were used. The period of regeneration between the tests was forty-eight hours. A marked decrease in the P.D. occurred in nine of the stems, but the original direction of the P.D. was apparently retained. One stem showed no P.D. between its ends at the end of the forty-eight-hour period. The following tentative explanation of this decrease in P.D. is offered at the present time. From the above experiments and others to follow it is easily shown that the apical end which is the actively growing region of the stem is electronegative to ELECTRICAL POLARITY IN OBELIA 483 the older basal end. Now, after isolation of the piece of the stem, the regeneration process sets in at the basal as well as the apical end and also at the cut ends of the branches. Usually TABLE 1 Electrical difference of potential between ends of apical three-fourths of the main stems of Obelia colonies; the latter are designated by the numbers at the top of the table. Numbers represent differences between successive galvanometer deflections on the scale. A gives the tests on the main stem made immediately after removing branches. JB, tests made twenty-four hours after removing branches, t.e., at the end of twenty-four hours’ regeneration. The — sign indicates that the apical end is electronegative to the basal end. The + sign may or may not indicate the opposite condition. See text for explanation DIFFERENCE BETWEEN SUCCESSIVE GALVANOMETER DEFLECTIONS 1 | Sm) aonnd © vo | leet ArH 100 > | (or) _ Average...... — SS lete st — tse ll | eS = bb co Co OO QoL oS oo eo 2 3 4 5 =e) a) Bs 7/10 0.0 —6.0 0.0 JENS) 2 a5 5 248) oT) —10.0 ens 85 a5 10-0 —9.5 2) alles = 820 = org ae) 0 BO ie) TO <0) 224'RU5 =—8.1 eens = OG =aeeG 0.0 eBid ul A. 0) 0.0 SE ae es 0.0 =. 5 eet) SS wi) =n amt 0 =f0 ee; 0.5 HON) =p 5 =. 250 4 120 Sos Z 356 180 EU) +3.0 0.0 EA) SANs 220) ile =r 16 a} (0) SEEDS peal) 233-250 —=—2h0 ey cael =30 mAs ef =A, Aah = 1G Average...... — 1.5 polyps appear at all these places. This means of course that after twenty-four hours of regeneration of the basal end the tissue has taken on the rdle and properties of a growing tissue similar to that at the apical end, and consequently its electrical 484 BH. WO ALUND potential should approach that of the apical end, with the result that the potential difference between the basal and apical ends should tend to disappear, which it does. PROOF THAT THE DIFFERENCE OF ELECTRICAL POTENTIAL IS ASSOCIATED WITH THE LIVING TISSUE IN THE STEM Differences in electrical potential such as described above for Obelia and originally for Tubularia, Pennaria, and Campanu- laria by Mathews (’03) might readily be conceived to originate elsewhere than in the living tissues of the stem. It is therefore necessary to remove completely any doubt on this point before we proceed with the analysis of the problem. Three different methods of testing the question will be given. Dead stems. Table 2 shows the results of tests on stems iso- lated from colonies left to die and macerate in sea-water in the laboratory for several weeks. It is clear from a comparison of the differences between deflections that no detectible P.D. exists between the ends of the stem. Living stems from which the tissue has been removed mechanically. The most striking proof of an inherent P.D. in the coenosare of the stem is given in table 3. Stems isolated from five different actively growing colonies were tested immediately. The results are given in table 3 A. All the tests show that a marked P.D. exists between the ends of the stem and that the apical end of the stem is electronegative to the basal end. After the tests in A were completed, the coenosare was mechanieally removed by rolling the round end of a glass rod along the stem. The perisare tube is elastic and returns perfectly to its original shape, auto- matically becoming washed free from cells and finally filled with sea-water instead of the living tissue. In this way an ideal con- trol for a crucial test is provided. Table 3 B gives the results of the tests on the perisare tubes filled with sea-water. No potential difference is evident. But, as was explained above in connection with table 1, the deflections in successive tests on the same peri- sare tube decreased. ‘This is due to an increase in ohmic resist- ance of the stem perisarc, caused by evaporation. ‘The results ELECTRICAL POLARITY IN OBELIA 485 in table 1 should be judged in the light of the results brought out in tables 2 and 3, and the experiments given below. TABLE 2 Showing absence of an electrical difference of potential in stems from two colonies which had been allowed to die in sea-water. A gives the tests on the main stem immediately after removing branches. B, tests on same stems after washing in fresh sea-water Nee eee — DIFFERENCES BETWEEN GALVANOMETER DEFLECTIONS 1 2 —1.0 +1.5 +1.0 +1.0 +0.5 +0.5 —1.0 0.0 —0.5 —0.5 A ; —0.5 21100 | +1.0 —0.5 +0.5 —0.5 —0.5 —0.5 +0.5 +0.5 —0.5 +0.5 IV ETHOCS te canta eee + —0.05 +0.1 | —1.5 —0.5 —0.5 0.0 —0.5 —0.5 —1.0 0.0 0.0 0.0 —0.5 —0.5 B 0.0 0.0 —0.5 —1.5 —1.5 —0.5 —0.5 MV CTERE Jac cisases = Ses toa —0.7 —0.2 See ae ae Oe Fae epee nes. Stems killed in chloroform. The apical halves of the main stem of five different colonies were isolated and the apical inter- node removed from each one. The pieces were tested im- mediately. In table 4 A are given the results. It will be noted 486 E. J. LUND that all but stem no. 3 show a definite P.D. in the same direction as that in the previous experiments. The question naturally arises, why did not stem no. 3 show a more definite and larger P.D.? Some light on this question will be given later. TABLE 3 Showing that the electrical difference of potential in a living stem disappears after the living tissue (coenosarc) in the stem has been removed mechanically and sea- water substituted for itin the perisarc tube. A, tests on living stem with coenosarce. B, tests on same stems after mechanical removal of coenosarc. Numbers at top of table refer to main stems of five different colonies. The — sign indicates that the apical end 1s electronegative to the basal end DIFFERENCES BETWEEN SUCCESSIVE GALVANOMETER DEFLECTIONS 1 2 3 4 5 | ERIE 250 —5.0 = 150 2545 7,0 —8.0 —5.5 a7 55 A ’ —5.0 80 mar) ral = 615 (eG =380 || =S-5 Baas el 55 —9.0 55 —10.0 S100 —6.0 Los —6.5 105 =o Average...... —5.0 —8.0 —5.4 — 8.0 — 7.3 +4.0 350 41.5 0.0 0.0 —=1.0 15 = 150 0.0 0.0 | 0.0 0.0 0.0 0.0 0.0 a 0.0 05 0.0 20005 0.0 —0.5 5 =rKO 0.0 01S 0.0 = 0 =055 40.5, + 0.5 || 055 05 Average...... +0.4 —1.5 —0.1 — 0.1 | 0.0 After the tests in A were made, all the pieces were treated with chloroform-saturated water for thirty minutes, then quickly rinsed in fresh sea-water and tested again. The tests after this chloroform treatment are given in B. It is clear that the P.D. has practically entirely disappeared. The differences in stems nos. 1 and 4 are small but definite. Retreatment of stems 1 and 4 obliterated all trace of P.D. The average differences between the deflections as given in the table are somewhat misleading, ELECTRICAL POLARITY IN OBELIA 487 for they are the averages of all the tests rather than the averages of the later tests in each series, which, as explained above, are the most significant, e.g., stem no. 3 A. In general, differences between galvanometer deflections of 1 mm. on the scale proved to be significant whenever the electrical resistance of the stem TABLE 4 Showing that treatment of the living stem with chloroform-saturated sea-water causes the disappearance of the normal difference of potential in the stem. A, tests made on living stems immediately after removal of branches. B, tests on the same stems after treatment in chloroform-saturated sea-water DIFFERENCES BETWEEN SUCCESSIVE GALVANOMETER DEFLECTIONS 1 2 3 4 5 970) =. 42.5 +0.5 —3.0 =A 15 a 0.0 —7.0 0.0 = 20) 0.0 —=9 0) = aug nl 0.0 = 350 = iG 2 =8.0 —4.0 —5.5 SO ee ay (as ase iis =F 0) =F i) 855 6:0 pero as =H 6 —5.0 Average...... —2.2 —2.9 —0.3 —1.6 —6.3 ess 0.0 3.0 oes 0.0 170 ETO ae 7 0.0 0.0 0.0 —0.5 STE 0.0 —0.5 B 0.0 0.0 =0)5 aie =055 —0.5 0.0 0.0 mee Se EtG 0.0 =-5 SBS Average...... —0.07 —0.1 +0.9 —0.3 —0.4 remained approximately constant. The limit of error is therefore to be judged to some extent from the conditions of the experi- ment and nature of the results. All the stems were now placed in fresh sea-water. No re- generation occurred, showing that loss of the normal P.D. in the stem was associated with death of the living tissue. A few experiments were carried out in which various concentrations of ether were used. The results indicate that a definite decrease 488 E. J. LUND and removal of the P.D. can be brought about by this anaesthetic. The complete investigation of the effect of anaesthetics upon the P.D. will be left for later consideration. The purpose of the previous experiments is simply to furnish a conclusive demon- stration of two facts: 1) A normal difference of electrical poten- tial exists along the stem of Obelia. 2) This potential difference is associated with the living condition of the tissue of the stem. TABLE 5 A represents a series of tests on apical pieces of the main stems of five different colo- nies. B represents a series of tests on the corresponding basal pieces of the stems of the same colonies. The lengths of apical and basal pieces of the same stem are equal. Note that the potential differences in the apical and basal pieces are oppo- site in direction DIFFERENCES BETWEEN SUCCESSIVE GALVANOMETER DEFLECTIONS 1 2 3 4 5 —3.0 —2.5 —2.0 —7.5 —9.0 mn —3.5 —1.5 —3.5 —5.5 —8.5 —3.0 —1.5 —4.5 —5.5 —7.5 —2.5 Average...... —3.0 —1.8 —3.3 —6.8 —8.3 +5.0 Zo +3.0 +4.5 +6.5 B +5.5 +1.5 +2.0 | +3.5 +6.5 +5.0 +1.0 +2.0 0.0 +5.5 +0.5 Average...... +5.1 +1.8 +2.3 +2.1 +6.1 THE DIRECTION AND MAGNITUDE OF THE FALL OF ELECTRICAL POTENTIAL ALONG THE STEM Because of the nature of the method used in the preceding and following experiments, it should be clear that the results are not what would be desired in a quantitative investigation of this problem. But, nevertheless, as will be indicated in the following two experiments, some idea may be gained of the magnitude and direction of the fall of electrical potential along the stem by comparing the magnitude of the differences in deflection produced by apical and basal halves or quarters of the same stem. ELECTRICAL POLARITY IN OBELIA 489 During the work represented in part by the experiments given above, the impression was gained that the amount of the P.D. in comparable pieces from different stems was distinctly different. In some pieces of stem very little, if any, difference could be detected, for example, table 4 A, stem 3, while in other pieces it would be relatively large. Therefore it was decided to compare ina preliminary way the magnitude and direction of the differences between deflections when pieces from the apical and more basal regions of the same stem were used. ‘Three experiments were carried out, in each of which five main stems of actively growing colonies were used. In the first experiment two pieces from the same stem were compared. The total length of each of the stems x ny Z : m n : Apex Al A2 Bl B2 Base Figure 2 numbered 1 to 5 in table 5 were, respectively, 110, 80, 100, 110, 115 mm. Three cuts were made, as shown in figure 2, 2, y, and z. The lengths of the pieces included between x and z in stems 1 to 5 were, respectively, 75, 70, 64 and 66mm. The cut at y was made exactly in the middle so as to give two pieces A and B of equal length. The tests on the A pieces are given in table 5 A and the tests on the corresponding B pieces in B of the same table. There is a distinct P.D. in the A pieces such that the apical ends are all electronegative to the basal end of the same piece. While in the B pieces the direction of the P.D. is reversed. The magnitude of the P.D. varies but is quite distinct in every case. A second experiment similar to the first except that the cuts y and 2, figure 2, were made proportionately nearer the apical end of the stem, again showed that the apical ends of the A pieces were electronegative and that in three of the B pieces the basal end was electronegative to the apical end. The other two B pieces showed no significant P.D. 490 E. J. LUND This peculiar opposed direction of fall of potential in the apical and basal pieces of the same stem suggested a further test by the procedure in the following experiment. Five actively grow- ing colonies, the main stems of which are numbered 1 to 5 in table 6, were used. The total length of the stems were, respec- tively, 100, 85, 75, 115, and 95 mm. The piece from each stem included between the cuts x and z in figure 2 was tested first. The lengths of the pieces from stems 1 to 5 were, respectively, 64, 57, 57, 83 and 70 mm. ‘The results are given in table 6 W. It will be noticed that while the apical ends in four of the stems are electronegative to the basal ends, still the differences are small. In the piece from stem no: 1 no P.D. could be detected. Each one of the five pieces was now cut at y, figure 2, into two pieces of exactly equal length and then tested immediately. The results from the apical pieces are given in table 6 A, while the corresponding tests on the B pieces are given in the same table in B. It will be observed that the P.D. in every one of the A pieces is quite large, the apical end again being electronegative to the basal end. All the B pieces show a marked P.D. in the opposite direction; this difference is, however, not as large in the B pieces as in the corresponding A pieces. Each one of the A and B pieces was now cut into two equal parts at m and n, figure 2, and tested at once. ‘The pieces from the same stem are numbered A!, A2, B!, B2, as in figure 2. It will be seen that the A! pieces show an unmistakable electro- negative condition of the apical end. Pieces A? of stems 1, 4 and 5 show the same condition as the A pieces, while the A? pieces of stems nos. 2 and 3 are doubtful. The B! pieces also show individual differences, while the B? pieces of stems nos. 1, 3, 4, and 5 show a quite distinct P.D. in the opposite direction. Piece B? of stem no. 2 is doubtful. From the experiments above it appears that the apical or erowing end of the stem shows the most distinct difference of potential. 'The middle pieces vary more or less, depending upon the individual stem, while the basal pieces tend to have the fall of potential reversed. * TABLE 6 W represents a series of tests wpon the ‘whole’ (see text) stem of five different colonies. A represents corresponding tests on apical half and B a corresponding series of tests wpon the basal half of the ‘whole’ stem after cutting the latter into equal halves. Note the small P.D.in W and the opposite direction of the potential in the A and B pieces. Aland A? represent tests wpon apical and basal halves, respectively, of the A pieces. B' and B* represent tests upon apical and basal halves, respectively, of the B pieces. Note the definite difference of potential in the apical pieces A} and the marked tendency to reversed direction of fall of potential on the basal pieces B? DIFFERENCES BETW EEN SUCCESSIVE GALVANOMETER DEFLECTIONS 1 2 3 4 5 0.0 —1.0 — 0.5 — 0.5 —s1e() Ww 0.0 —0.5 — 1.0 — 1.5 — 1.5 ) 0.0 —0.5 — 1.0 — 2.5 — 2.0 { —1.5 Average...... 0.0 —0.8 — 0.8 — 1.5 — 1.5 — 3.5 —6.5 — 3.0 — 6.0 — 8.5 A — 4.0 —5.0 — 2.5 — 7.0 — 8.5 — 5.0 —4.5 — 4.5 — 7.0 =" 5.5 Average......] — 4.2 —5.3 — 3.3 — 6.6 — 7.5 + 0.5 +2.5 + 2.0 + 2.5 + 2.0 B + 2.0 +5.5 + 2.0 7+ 4.5 + 3.0 + 3.0 +7.0 + 2.0 + 4.0 + 3.0 {} +1.5 Average...... + 1.7 +5.0 + 2.0 + 3.6 + 2.6 — 2.0 —6.0 — 8.0 — 5.0 —10.0 Al — 9.5 —5.0 —10.0 — 5.0 — 9.5 — 6.0 —3.0 —12.5 — 6.5 — 9.0 Average...... — 5.8 —4.6 —10.2 — 5.3 — 9.5 — 4.0 +3.0 — 0.5 — 6.5 — 8.5 A? — 5.0 —0.5 — 2.5 — 6.5 — 3.5 — 6.0 +3.5 0.0 —11.0 — 6.5 Average...... — 5.0 +2.0 — 1.0 — 8.0 — 6.1 —10.0 —6.5 + 2.0 + 0.5 — 5.5 = — 4.5 =10 + 4.5 49.5 aries — 5.5 +1.0 + 3.0 + 1.5 — 2.5 inet 5L0 ei5 Average...... — 6.2 —2.1 + 2.5 + 1.5 — 3.0 — 3.0 —2.5 + 3.5 + 2.5 + 2.0 Be + 4.0 —9.5 + 1.5 + 3.0 + 3.5 + 3.0 +3.0 + 4.0 + 4.0 + 4.5 Wik 225 Average...... + 1.6 ? + 3.0 + 3.1 + 3.3 492 E. J. LUND All that the writer desires to show by these experiments is: 1) that the magnitude of the fall of electrical potential differs along the length of the stem and, 2) that under the conditions of these experiments, the direction of the fall of potential may be different in more basal regions than in the apical part of the same stem. This latter result appears somewhat surprising, in view of the general morphological similarity of the different levels of the stem. It appears that the small potential difference (or absence of P.D., table 6 W, 1) shown by the whole pieces between cuts x and z was due to the fact that the apical and basal ends of these pieces were both electronegative to the middle region of the piece. This result suggests a similarity to the results obtained by Miiller- Hettlingen (’83) on growing seedlings. CONCLUSIONS It is evident that the observations reported in this paper appear complementary to the results reported in the first paper on the effects of an electric current on the regeneration process in the internodes of Obelia. Now, if differences of electrical potential are inseparably associated with structural polarity and apical erowth, then it follows that the stem or certain regions of the stem of Obelia—and also very probably each branch—serves as a conductor of a continuous electric current of perhaps varying intensity, which passes within the tissues of the stem and com- pletes the circuit in the surrounding sea-water. If the basal region is electronegative to the middle region of the stem, then of course the direction of the current would be the opposite in the lower parts of the stem to that in the apical region of the stem. In any case, if these constant bioelectric currents are inseparably associated with apical or basal growth, it is logical to expect that if they be appropriately opposed or augmented by the application of an external source of E.M.F., then the normal growth processes should show the effects of such interference. If this reasoning is correct, then an appropriate application of an external electromotive force to regenerating and growing tissues should be a unique instrument for the investigation of the causes and conditions of structural polarity and symmetries ELECTRICAL POLARITY IN OBELIA 493 which are everywhere a common fundamental characteristic of the individual in organic nature, whether it be a cell, group of cells, or whole organism. There is another significant point to which I wish to eall atten- tion. If cells, cell groups, and individuals, e.g., polyps in a structurally and physiologically correlated system like Obelia, possess an inherent mechanism for establishing directed electro- motive forces within themselves in relation to their structural or functional polarities, then it will be clear that here we may have a normal electrical mechanism which may serve as a de- termining factor in the process of orientation of cells with re- spect to one another during embryogeny and regeneration. I do not wish to carry this line of reasoning farther in this paper than to suggest the possibilities. In following papers will be presented experimental evidence relative to this question. Because of the nature of the experimental method employed in the experiments above, it was necessary to manipulate the stems and pieces in air. This obviously limited the degree of accuracy of the results A further detailed and quantitative study of the phenomena will be undertaken when appropriate and sufficiently accurate methods for measurement have been devised. SUMMARY 1. A definite difference of electrical potential occurs in the stem of the colony of Obelia. This confirms the conclusion arrived at by means of a different method in the preceding paper, that an electrical polarity is one primary associated condition for the development of morphological polarity. 2. The electrical polarity in the stem of Obelia is associated with the living tissue of the coenosare and does not originate in any other structure of the stem for no potential difference occurs in the following: a) Stems left to die and macerate in sea-water; b) stems from which the living tissue has been removed mechani- cally, and, c) stems in which the living tissue has been killed by chloroform. 3. The potential difference in the stem is not associated directly or simply with the mechanical injury due to cutting, and there- 494 E. J. LUND fore is not identical with the current of injury in, for example, muscle, nerve, and other tissues. 4. The magnitude of the fall of potential varies in pieces of stems from different colonies. It also varies along the length of the stem of the same colony, being greatest in the apical actively growing region. 5. A limited number of experiments indicated that the direc- tion of the fall of potential in basal regions of the stem may be opposite to that in the apical region. 6. The difference of electrical potential between apical and basal ends of pieces of stem after twenty-four or forty-eight hours of regeneration was less than the potential difference in the same pieces immediately after isolation. A tentative explana- tion of this result is given in the text. 7. The general conclusion from the experiments is, that since normally inherent differences of electrical potential occur in the stem of Obelia and are associated with apical growth, then it should be possible to inhibit or modify developmental processes by appropriate application of an E.M.F. of external origin. This was shown to be possible in the previous paper. LITERATURE CITED Bayuiss, W. M. 1911 IIJ. The osmotic pressure of electrolytically dissociated colloids. Proc. Roy. Soc. Lond., vol. 84 B, pp. 229-253. Burr, H. 1854 Ueber die Electricititserregung durch lebende Pflanzen. Anna- len d. Chem. u. Pharm., Bd. 89, S. 76-89. BURDON-SANDERSON 1882 On the electromotive properties of the leaf of Dionea in excited and unexcited states. Phil. Trans., vol. 173, pt. I, pp. 1-56. ELFvinG, F. 1882 Ueber eine Wirkung des galvanischen Stromes auf wachsende Wurzeln. Bot. Zeit., Bd. 40, 8. 257-264. Kunkxeu, A.J. 1881 Electrische Untersuchungen an pflanzlichen und tierischen Gebilden. Pfliig. Arch., Bd. 25, 8. 342-379. Lunp, E. J. 1921 I. Effects of the electric current on regenerating internodes of Obelia commissuralis. Maruews, A. P. 1903 Electrical polarity in the hydroids. Am. Jour. Physiol., vol. 8, pp. 294-299. MiLier-HetTriincEN, J. 1883 Ueber galvanische Erscheinungen an keimenden Samen. Pfliig. Arch., Bd. 31, 8. 193-214. SUBJECT AND AUTHOR INDEX DMIRAL butterfly, Pyrameis atalanta Linn. A quantitative study of tarsal sensitivity to solutions of saccharose in EHO red bodeeists ob ole eee ie le a sete 445 Aarrspore, H. P. Kierscnpow. Some ob- servations on qualitative chemical and physical stimulations in nudibranchiate mollusks with special reference to the réle Op the “rhinaphoresiamse ae. oes ates 423 Amphibians. Experiments on the meta- morphosis of neotenous...... . 397 Analysis of the factors underlying ‘suscep- tibility to transplantable tumors. A REVEUMO Rs a naience nee Mae en ska 67 Anesthetics. The control of head formation juPlanaria by means Ofy..asdse.coenesl ee 1 ACTERIAL content. The growth of Paramecium in infusions of known.... 135 Baup1, Epgarpo. Studi sulla fisiologia del sistema nervoso negliinsetti. II. Ricerche sui movimenti di maneggio provocati nei Coleotterta yr oni. aca ee ne al 211 formation in Planaria by means of anes- ehetics esa ee hs had oA Neale ee ey Ad CS eee of the opposite sex. Physiological self-differentiation of the wing-germs grafted on.................. 469 BER Aeon liiiaisloisicl-rit anes cout oes weereetncree ne 185 Chemical and physical stimulations in nudi- observations on qualitative............... 423 Ciliate, Frontonia. The process of ingestion SUSE LE Sy, rere actos eee ita ae ae 333 Coleoptera. Studi sulla fisiologia del sistema nervoso negli insetti. II. Ricerche sui movimenti di maneggio provocati nei... 211 Coutts, H. H., Sumner, F. B., anv. Fur- ther studies of color mutations in mice the-elecinid.29 5.0 = see a ee ene 477 Eh eee ae current. II. The normal elec- trical polarity of Obelia. A proof of its existence. Experimetal control of or- ganic polarity by the..................... 477 yes of Lepidoptera. Mutual relationship in the development of the brainand........ 459 495 ORMATION in Planaria by means of anesthetics. The control of head....... 1 Fowl, with remarks upon secondary sex characters. A case of true hermaphrodit- ISTHE Neneh enre eetey ht at debe eee 185 Frontonia. The process of ingestion in the CHIALO NS eee ers inc oeelas canis 333 ENETIC analysis of the factors under- lying susceptibility to transplantable tumors. Goupsmirg, Wiru1Am M. The process of in- gestion in the ciliate, Frontonia.......... 333 Growth of Paramecium in infusions of known bacterial content. ‘Dhewseees n..2e eee: 135 AMILTON, Witu1am F., Harrman, Cart G., AND. A case of true hermaph- roditism in the fowl, with remarks upon secondary sex characters............ 185 Harrman, Cart G., anp Hamiuton, WIL- LIAM F. A case of true hermaphroditism in the fowl, with remarks upon secondary Bex. Characters; 5 saee ee ee eee ee 185 Head formation in Planaria by means of - anesthetics. The control of.............. 1 Hermaphroditism in the fowl, with remarks upon secondary sex characters. A case Of tees ee ae eS ir ee eee 185 NFUSIONS of known bacterial content. The growth of Paramecium in............ 135 Ingestion in the ciliate, Frontonia. The DIGCESS) Of: eee See oc e eee es ees 333 Insects. II. Ricerche sui movimenti di maneggio provocati nei coleotteri. Studi sulla fisiologia del sistema nervoso negli.. 211 OPEC, Sreran. Mutual relationship in the development of the brain and eyes OL henidopterarccnc Mee ee eter eenine 459 Kopré, Steran. Physiological self-differen- tiation of the wing-germs grafted on cater- pillars of the opposite sex................. 469 EAPING of the stromb (Strombus gigas - Line) nes. ate reece 205 Lepidoptera. Mutual relationship in the development of the brain and eyes of... 459 Locomotion of certain marine polyclads. The role of the nervous system in the........ 57 Loggerhead turtles toward the sea. The Crawling of young. 7 eee eeL ee ee 323 Lunp, E. J. Experimental control of organic polarity by the electric current. II. The normal electrical polarity of Obelia. A proof ofats existence... 140.9 1-2 6-40 477 ARINE polyclads. The réle of the nerv- ous system in the locomotion of certain. 57 Metamorphosis of neotenous amphibians. Me Perinencts! ON Hess ss. een aeicee cele 397 Mice of the genus Peromyscus. Further studies of color mutations in............. 289 Minnicu, Dwieut E. A quantitative study of tarsal sensitivity to solutions of sac- charose in the red admiral butterfly, Pyrameis atalanta, linn... sissscers. ere cs 200 496 Mollusks with special reference to the réle of the ‘rhinophores.’ Some observations on qualitative chemical and physical stimulations in nudibranchiate........... 423 Morphology and physiology of the genus Uronychia. A contribution to the....... 353 Mutations in mice of the genus Peromyscus. Further studies of color................... 289 EOTENOUS amphibians. Experiments on the metamorphosis of............... 397 Nervous system in the locomotion of certain marine polyclads. The réle of the........ 57 Nervous system in the regeneration of poly- clad Turbellaria. The réle of the.. 49 Nervous system negli insetti. II. Ricerche sui movimenti di maneggio provocati nei coleotteri. Studi sulla fisiologia del...... 211 Nudibranchiate mollusks with special refer- ence to the rdle of the ‘rhinophores.’ Some observations on qualitative chemi- eal and physical stimulations in.......... 423 BELIA. A proof ofitsexistence. Experi- mental control of organic polarity by the electric current. II. The normal elec- trical polanityol sk saa le soas sone eae 477 OtmstepD, J. M. D. The role of the nervous system in the locomotion of certain mMmanmnespolyelads seas. ssa Neen Nee een: 57 OumstrepD, J. M. D. The réle of the nervous system in the regeneration of polyclad Dimbellantaey ees i eg ee 2 eee 49 Organic polarity by the electric current. Il. The normal electrical polarity of Obelia. A proof of its existence. Experi- mental icontroliofstceeeeeae aoe eae 477 ARAMECIUM in infusions of known bac- terial content. The growth of........... 135 Parker, G. H. The crawling of young log- gerhead turtles toward the sea. 323 Parker, G. H. The leaping of the stromb (Strombus PIQHS IL aTNe) sean cte sie see 205 Peromyscus. Further studies of color muta- tions in mice of the genus... Puituirs, RutH L. The growth of Para- mecium in infusions of known bacterial GCODTCII SS ay pone oer ein as eo ee 135 Physical stimulations in nudibranchiate mol- lusks with special reference to the rdle of the ‘rhinophores.’ Some observations on qualitative chemical and.............. 423 Physiological self-differentiation of the wing- germs grafted on caterpillars of the op- OSIUEISES ster eet... hitan ae aeeer 469 Physiology del sistema nervoso negli insetti. II. Ricerche sui movimenti di maneggio provocati nei coleotteri. Studi sulla.. 211 Physiology of the genus Uronychia. A con- tribution to the morphology and......... 353 Planaria by means of anesthetics. The con- trol of head formation in.............- 1 Polarity by the electric current. II. The normal electrical polarity of Obelia. A proof of its existence. Experimental con- trol GOLOreanics: Gen see ae ee eins 477 Polyclad Turbellaria. The role of the nery- ous system in the regeneration of. . re ao Polyclads. The réle of the nervous sy stem in the locomotion of certain marine......... 57 Pyrameis atalanta Linn. A quantitative study of tarsal sensitivity to solutions of saccharose in the red admiral butterfly. 445 UALITATIVE chemical and _ physical stimulations in nudibranchiate mol- lusks with special reference to the réle of the ‘rhinophores.’ Some observations INDEX Quantitative study of tarsal sensitivity to solutions of saccharosge in the red admiral butterfly, Pyrameis atalanta Linn. A... 445 R=. admiral butterfly, Pyrameis atalanta Linn, A quantitative study of tarsal _ Sensitivity to solutions of saccharose in thet eh ees ee ee eee 45 Regeneration of polyclad Turbellaria. The role of the nervous system in the......... 49 Relationship in the development of the brain and eyes of Lepidoptera. Mutual....... 459 ‘Rhinophores.’ Some observations on quali- tative chemical and physical stimulations in nudibranchiate mollusks with special reference to the réle of the................ 423 ACCHAROSE in the red admiral butter- fly, Pyrameis atalanta Linn. A quanti- tative study of tarsal sensitivity to solu- tions’Of:. S350 :35 |. ee ee eee 445 Sea. The crawling of young loggerhead turtles!toward the sss. a emer ee nen 323 Self-differentiation of the wing-germs grafted -aterpillars of the opposite sex. Physi- ICAL. See ee ee eee 469 Sensiuvity to solutions of saccharose in the red admiral butterfly, Pyrameis atalanta Linn. A quantitative study of tarsal.... 445 Sex characters. A case of true hermaphrodit- ism in the fowl, with remarks upon Secondary. 336. s235nja dle nee 185 Sex. Physiological self-differentiation of the wing-germs grafted on caterpillars of the OPpOSsitiesa ec. sso) cracks doe oe eee 469 Stimulations in nudibranchiate mollusks with special reference to the role of the ‘rhinophores.’ Some observations on qualitative chemical and physical....... 423 Stromb (Strombus gigas Linn.). The leaping Of the-i..5) teen. odes ee eee ee 205 (Strombus gigas Linn, ). The leaping of the Strom biterees 2s. she le cee 205 Srrone, Leonetyt C. A genetic analysis of the factors underlying susceptibility to transplantable tumors!s.cac. assent 67 SumMNER, F.B., anp Cotuins, H. H. Further studies of color mutations in mice of the genus: Peromyscuss..c4s29- 20. ee cee 289 Susceptibility to transplantable tumors. A genetic analysis of the factors underlying. 67 Swineir, W. W. Experiments on the meta- morphosis of neotenous amphibians...... 397 ARSAL sensitivity to solutions of sac- charose in the red admiral butterfly, Pyrameis atalanta Linn. A quantita- tive study Ofc: eh. be cee eee 445 Tesneu les eile tumors. A genetic analysis of the factors underlying susceptibility to. 67 Tumors. A genetic analysis of the factors underlying susceptibility to trans- planta ble iin: xdsir. s Ssles sen ee epee ae 67 Turbellaria. The réle of the nervous system in the regeneration of polyclad........... 49 Turtles toward the sea. The crawling of young) loggerhead oni cnrer uisiels <2 - eee 323 RONYCHIA. A contribution to the morphology and physiology of the _ enus.s2.0% Pee ee ne Pee nee eee 353 bY, fay fies grafted on caterpillars of the opposite sex. Physiological self- differentiation of the........%..-..-.-- 469 you NG, Donnett Brooks. A_ contri- bution to the morphology and ae i ology of the genus Uronychia.. be . Bb HOI Library - Serials ti WAI rietese! 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